Patent application title: BINDING MOLECULES BINDING PD-L1 AND LAG-3
Inventors:
IPC8 Class: AC07K1628FI
USPC Class:
1 1
Class name:
Publication date: 2022-06-16
Patent application number: 20220185894
Abstract:
The present invention relates to antibody molecules which bind to
programmed death-ligand 1 (PD-L1) and lymphocyte-activation gene 3
(LAG-3). The antibody molecules preferably comprise a CDR-based antigen
binding site for PD-L1, and a LAG-3 antigen binding site which may be
located in two or more structural loops of a CH3 domain of the antibody
molecule. The antibody molecules of the invention find application, for
example, in cancer therapy.Claims:
1.-39. (canceled)
40. An antibody molecule which binds to programmed death-ligand 1 (PD-L1) and lymphocyte-activation gene 3 (LAG-3), wherein the antibody molecule comprises: (i) a CDR-based antigen binding site for PD-L1; and (ii) a LAG-3 antigen binding site located in a CH3 domain of the antibody molecule, wherein the LAG-3 binding site comprises the amino acid sequences WDEPWGED (SEQ ID NO: 1) and PYDRWVWPDE (SEQ ID NO: 3).
41. An antibody molecule according to claim 40, wherein the LAG-3 antigen biding site further comprises the amino acid sequence SNGQPENNY (SEQ ID NO: 2).
42. An antibody molecule according to claim 40, wherein the LAG-3 antigen binding site comprises the amino acid sequence set forth in SEQ ID NO: 1 in the AB loop and the amino acid sequence set forth in SEQ ID NO: 3 in the EF loop of the CH3 domain.
43. An antibody molecule according to claim 41, wherein the LAG-3 antigen binding site comprises the amino acid sequence set forth in SEQ ID NO: 1 in the AB loop, the amino acid sequence set forth in SEQ ID NO: 2 in the CD loop, and the amino acid sequence set forth in SEQ ID NO: 3 in the EF loop of the CH3 domain.
44. An antibody molecule according to claim 40, wherein the antibody molecule is an immunoglobulin G1 (IgG1) molecule.
45. An antibody molecule according to claim 40, wherein the antibody molecule comprises the CH3 domain set forth in SEQ ID NO: 5, 10, or 20.
46. An antibody molecule according to claim 41, wherein the antibody molecule comprises a CH3 domain, wherein the CH3 domain comprises the sequence set forth in SEQ ID NO: 5, 10, or 20, and wherein the CH3 domain sequence further comprises a lysine residue (K) at the immediate C-terminus of the sequence shown in SEQ ID NO: 5, 10, or 20.
47. An antibody molecule according to claim 46, wherein the antibody molecule comprises the CH3 domain set forth in SEQ ID NO: 135.
48. An antibody molecule according to claim 46, wherein the antibody molecule comprises the sequence set forth in SEQ ID NO: 6, 7, 11, 12, 21, or 22.
49. An antibody molecule according to claim 46, comprising the complementarity determining regions (CDRs) set forth in SEQ ID NOs: 86 to 91.
50. An antibody molecule according to claim 49, wherein the antibody molecule comprises the VH and/or VL domains set forth in SEQ ID NOs: 92 and 93.
51. An antibody molecule according to claim 50, wherein the antibody molecule comprises the heavy chain sequence set forth in any one of SEQ ID NOs: 94 to 97, 100, or 101.
52. An antibody molecule according to claim 51, wherein the antibody molecule comprises the light chain sequence set forth in SEQ ID NO: 116.
53. An antibody molecule according to claim 40, wherein the antibody molecule is conjugated to an immune system modulator, cytotoxic molecule, radioisotope, or detectable label.
54. A nucleic acid encoding an antibody molecule according to claim 52.
55. A vector comprising the nucleic acid of claim 54.
56. A recombinant host cell comprising the nucleic acid of claim 54.
57. A method of producing an antibody molecule, comprising culturing the recombinant host cell of claim 56 under conditions for production of the antibody molecule.
58. A pharmaceutical composition comprising an antibody molecule according to claim 52 and a pharmaceutically acceptable excipient.
59. A method of treating cancer in a patient, wherein the method comprises administering to the patient a therapeutically effective amount of an antibody molecule according to claim 52.
60. A method according to claim 59, wherein the cancer is selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma, ovarian cancer, prostate cancer, colorectal cancer, fibrosarcoma, renal cell carcinoma, melanoma, pancreatic cancer, breast cancer, glioblastoma multiforme, lung cancer, head and neck cancer, stomach cancer, bladder cancer, cervical cancer, uterine cancer, vulvar cancer, testicular cancer, penile cancer, leukemia, multiple myeloma, squamous cell cancer, testicular cancer, esophageal cancer, Kaposi's sarcoma, and central nervous system (CNS) lymphoma, hepatocellular carcinoma, Merkel cell carcinoma, nasopharyngeal cancer, and mesothelioma.
61. An antibody molecule which binds to programmed death-ligand 1 (PD-L1) and lymphocyte-activation gene 3 (LAG-3), wherein the antibody molecule comprises the light chain sequence set forth in SEQ ID NO: 116, and the heavy chain sequence set forth in SEQ ID NO: 94.
62. An antibody molecule which binds to programmed death-ligand 1 (PD-L1) and lymphocyte-activation gene 3 (LAG-3), wherein the antibody molecule comprises the light chain sequence set forth in SEQ ID NO: 116, and the heavy chain sequence set forth in SEQ ID NO: 94, and wherein the CH3 domain sequence in the heavy chain has the sequence set forth in SEQ ID NO: 135.
Description:
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser. No. 16/311,604, filed Dec. 19, 2018, which is a national stage filing under 35 U.S.C. 371 of International Patent Application Number PCT/EP2017/065073, filed Jun. 20, 2017, which claims the benefit of U.S. Provisional Application No. 62/352,482, filed Jun. 20, 2016. The entire contents of each of these applications are incorporated herein by reference in their entirety.
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB
[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 18, 2021, is named F083170002US01-SEQ-ZJG and is 328,745 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to antibody molecules which bind to programmed death-ligand 1 (PD-L1) and lymphocyte-activation gene 3 (LAG-3). The antibody molecules preferably comprise a CDR-based antigen binding site for PD-L1, and a LAG-3 antigen binding site which may be located in two or more structural loops of a CH3 domain of the antibody molecule. The antibody molecules of the invention find application, for example, in cancer therapy.
BACKGROUND TO THE INVENTION
[0004] Lymphocyte Activation Gene-3 (LAG-3; CD223) is a member of the Ig superfamily, and is genetically and structurally related to CD4 (though with only 20% sequence identity). Like CD4, LAG-3 binds to MHC class II molecules but with higher affinity than CD4 (K.sub.D=60 nM). LAG-3 is expressed on activated T cells, NK cells, pDCs, B cells, y.delta. T cells and participates in immune suppression, particularly through persistent strong expression in a percentage of regulatory T cells (Tregs) (Liang et al, 2008).
[0005] The LAG-3 gene is located on human chromosome 12, adjacent to the CD4 gene, and spans 8 exons. There are five alternative transcripts, two of which generate protein products: a full length transmembrane protein and an alternatively spliced soluble monomeric form. The full-length transcript encodes a 525 amino acid protein with a molecular weight of 70 kDa and has functional activity, while the soluble form appears not to bind MHC class II molecules and its function is unknown. Human full-length LAG-3 protein has 93% sequence identity to Macaca fascicularis (cynomolgus monkey) LAG-3 and 70% sequence identity to Mus musculus (house mouse) LAG-3.
[0006] LAG-3 is a transmembrane protein with four extracellular Ig-like domains (D1-D4), and a cytoplasmic portion responsible for LAG-3 signalling. The cytoplasmic domain has an EP (glutamic acid/proline) motif that associates with LAG-3-associated protein (LAP) as well as a KIEELE motif thought to be required for LAG-3 modulation of T cell function. Reports on the role of the EP motif suggest that it may be responsible for trafficking of LAG-3 to the T cell surface membrane (Bae et al, 2014), or may be directly responsible for modulating downstream signalling of STAT5 during T cell activation (Durham et al, 2014), or possibly both.
[0007] The immuno-suppressive mechanism of LAG-3 on T cells is thought to be driven by cross-linking of LAG-3 on activated T cells resulting in decreased calcium flux and IL-2 release during T cell activation (Huard et al, 1997). On Antigen Presenting Cells (APCs), binding to MHC II molecules by LAG-3 positive regulatory T cells causes decreased IL-12 secretion and down regulation of CD86 (Liang et al, 2008), a "secondary signal" of activation, resulting in T cell anergy from improper activation and/or reduced antigen presentation by the APCs. LAG-3 knock out mouse models are viable, with only mild lympho-hyperproliferation (Workman et al, 2003), indicating that LAG-3 acts as a modest immune "brake".
[0008] This suppressive interaction between LAG-3 and MHC class II has also been proposed to occur between Tregs and CD4 positive T cells (Sega et al, 2014). Tregs supress the immune response either by release of suppressive cytokines (such as IL-10 and TGF.beta.), manipulation of inflammatory metabolism (such as CD73 catabolised adenosine), regulating APC maturation, or direct interaction between regulatory T cells and effector T cells. There is evidence in humans that MHC class II positive Tregs are more suppressive than MHC class II negative Tregs (Baecher-Allen et al, 2006) and actively suppress the immune response through direct interaction with LAG-3 expressed on effector T cells. While LAG-3 negative Tregs can suppress conventional T cell proliferation, LAG-3 negative CD4 and CD8 T cells are resistant to Treg immune suppression. This process was described to occur between human T cells through a process known as trogocytosis (Sega et al, 2014) whereby Tregs not only prevent APC maturation but also acquire MHC class I to suppress primed LAG-3 positive CD4 T cells.
[0009] LAG-3 expression is also a marker of repeated antigen stimulation. In cancer, T cells commonly adopt an "exhausted" phenotype, involving expression of immuno-suppressors such as PD-1, CTLA-4, TIM-3, and LAG-3 (Wherry et al, 2011), where the cells have a general inability to properly proliferate and secrete chemokines in response to antigen. Inhibition of these immune-suppressors lowers the immune threshold and (re-)enables a proper anti-cancer response by the T cells. In preclinical models, this has been borne out using antagonist antibodies against LAG-3, CTLA-4 and PD-1 where a decrease in tumour burden was seen. LAG-3 inhibition by antagonistic antibodies is thought to reactivate the immune response in the tumour microenvironment, where expression of LAG-3 on CD4 positive T cells and CD8 positive T cells is associated with an exhausted phenotype, and LAG-3 expression on Tregs is associated with potent immuno-suppressive capabilities. Antibodies blocking LAG-3 increase T effector cell proliferation, cytokine production, cytotoxicity, and decrease Treg suppressor activity leading to a decrease in tumour growth.
[0010] In human tumours, increased expression of LAG-3 was found on tumour-infiltrating lymphocytes (TILs) from human renal cell carcinomas and other tumours, such as melanomas and lymphomas (Demeure et al, 2001; Wolchock et al, 2013). Importantly, LAG-3 is also closely correlated with T cell dysfunction in patients with chronic viral infection (Workman et al, 2005) and cancer (Workman et al, 2003). LAG-3 has also been identified as a surface marker for tumour-infiltrating Tregs in a variety of human cancers (Camisachi et al. 2010; Gandhi et al, 2006).
[0011] Monoclonal antibodies to human LAG-3 are in clinical development to abrogate immune suppression and potentially enhance antigen presentation in cancers (solid and haematological malignancies).
[0012] LAG-525 and IMP-701 (Novartis AG), are human antibodies against LAG-3 and have advanced to Phase II and I clinical studies, respectively, in kidney cancer (Renal Cell Cancer); Non-Small Cell Lung Cancer (NSCLC); Nasopharyngeal Cancer; Colorectal Cancer; Melanoma; Gastric Cancer and Adenocarcinoma of the Gastroesophageal Junction.
[0013] Anti-LAG-3 antibody BMS-986016 (Bristol-Myers Squibb Company), is currently in Phase I clinical testing for Ovarian Cancer; NSCLC; Colorectal Cancer; Cervical Cancer; Melanoma; Gastric Cancer; Bladder Cancer; Head And Neck Cancer Squamous Cell Carcinoma; Renal Cell Carcinoma and in Phase II studies in NSCLC; Relapsed Chronic Lymphocytic Leukemia (CLL); Refractory Chronic Lymphocytic Leukemia (CLL); Melanoma; Non-Hodgkin Lymphoma; Hodgkin Lymphoma; Diffuse Large B-Cell Lymphoma; Indolent Lymphoma; Mantle Cell Lymphoma; Refractory Multiple Myeloma; and Relapsed Multiple Myeloma as either monotherapy or as part of combination therapies.
[0014] Further antibodies against LAG-3 are also in preclinical development.
[0015] Programmed cell death 1 (PD-1) and its ligands PD-L1 (CD274, B7-H1) and PD-L2 (B7-DC) deliver inhibitory signals that regulate the balance between T cell activation, tolerance, and immunopathology. PD-L1 is transiently expressed on all immune cells and some tumour cells.
[0016] PD-L1 is a type I transmembrane protein with two Ig-like domains within the extracellular region, a transmembrane domain and a short cytoplasmic domain. The cytoplasmic domain has no known signal transduction motif suggesting that there is no signalling by PD-L1 on interaction of the ligand with its receptor. Its molecular weight is 40 kDa (290 amino acids) and it is encoded by the CD274 gene on mouse chromosome 19 and human chromosome 9, respectively. PD-L1 is a member of the B7 protein family and shares approximately 20% amino acid sequence identity with B7.1 and B7.2. Human PD-L1 shares 70% and 93% amino acid identity with the murine and cynomolgus orthologs of PD-L1, respectively.
[0017] PD-L1 binds to its receptor PD-1 with an affinity (K.sub.D) of 770 nM. PD-1 is expressed on activated T cells, B cells, and myeloid cells, and modulates activation or inhibition of cellular immune responses. Binding of PD-L1 to PD-1 delivers an inhibitory signal, reducing cytokine production and proliferation of T cells. Consequently, PD-L1 expression by cells can mediate protection against cytotoxic T lymphocyte (CTL) killing and is a regulatory mechanism that dampens chronic immune responses during viral infections. Cancer, as a chronic and pro-inflammatory disease subverts this immune-protective pathway through up-regulation of PD-L1 expression to evade the host immune response. In the context of an active immune response, IFN.gamma. also upregulates the expression of PD-L1.
[0018] PD-L1 also mediates immune suppression through interaction with another protein, B7.1 (also known as CD80), blocking its ability to deliver one of the secondary signals of activation on T cells through CD28. In terms of PD-L1 expression on tumour cells and its engagement with B7.1, the relevance of this specific interaction in tumour immune resistance is still unclear.
[0019] PD-L1 expression has been shown in a wide variety of solid tumours. Of 654 samples examined in one study, spanning 19 tumours from different sites, 89 (14%) were PD-L1 positive (.gtoreq.5% frequency). The highest PD-L1 positive frequencies were seen in head and neck (17/54; 31%), cervical (10/34; 29%), cancer of unknown primary origin (CUP; 8/29; 28%), glioblastoma multiforme (GBM; 5/20; 25%), bladder (8/37; 21%), oesophageal (16/80; 20%), triple negative (TN) breast (6/33; 18%), and hepatocarcinoma (6/41; 15%) (Grosso et al, 2013). Tumour-associated expression of PD-L1 has been shown to confer immune resistance and potentially protect tumour cells from T cell mediated apoptosis.
[0020] Therapies targeting PD-L1 have shown excellent results in murine in vivo studies. In the B16 murine model of melanoma, treatment with anti-PD-L1 combined with either GVAX or FVAX vaccination strategies resulted in a significant effect both on survival (30 days for control vs 52 days for PD-L1-treated) and percentage of tumour-free (5%) animals upon conclusion of the study (Curran et al, 2010). Anti-PD-L1 therapy has also been used to study the mechanism of immune suppression in the P815 murine mastoma model. P815 cells injected into mice normally trigger a strong immune response, which results in their rejection. When PD-L1 is expressed on P815 cells, these cells escape immune attack, which in turn can be negated through administration of anti-PD-L1 antibodies (Iwai et al, 2002). It is evident that targeting the PD-1/PD-L1 axis in immunogenic human cancers (Herbst et at, 2014) results in a survival advantage through stimulation of an anti-cancer immune response (Wolchock et al, 2013; Larkin et al, 2015).
[0021] Atezolizumab (MPDL3280A, RG7466, TECENTRIQ) is a humanized IgG1 antibody which binds PD-L1. It is in clinical trials as a single agent and also in combination with other biologic and/or small molecule therapies for treatment of solid cancers, including colorectal cancer, breast cancer, non-small-cell lung carcinoma, bladder cancer, and renal cell carcinoma. Treatment with atezolizumab resulted in objective response rates (ORR) of 23% in NSCLC, 36% melanoma, 33% bladder, 14% in RCC, and 13% in head and neck cancers (Herbst et al, 2014; Powles et al, 2014).
[0022] Avelumab (MSB0010718C) is a fully human IgG1 antibody which binds to PD-L1 and is undergoing clinical testing in a number of cancers including bladder cancer, gastric cancer, head and neck cancer, mesothelioma, non-small-cell lung carcinoma, ovarian cancer, renal cancer and Merkel-cell carcinoma.
[0023] Durvalumab (MED14736) is a human IgG1 antibody which binds to PD-L1 and is being tested in clinical trials alone or in combination with tremelimumab in non-small-cell lung cancer, squamous cell carcinoma of the head and neck, bladder cancer, pancreatic cancer and with other biologic and small molecules in trials for additional solid cancers such as gastric cancers, melanoma and unresectable hepatocellular carcinoma.
[0024] Further anti-PD-L1 antibodies have been tested in clinical trials, including BMS-936559 and others are in preclinical testing.
[0025] However, few anti-LAG-3 therapies are currently in clinical testing and none have been approved for therapy so there remains a need to develop additional molecules which target LAG-3. Whilst there are some anti-PD-L1 therapeutics in development, current data shows that overall treatment with anti-PD-L1 monotherapy results in a response in less than 50% of cancer patients. Thus, there remains a need in the art for additional molecules which can target LAG-3 and/or PD-L1 and which find application in cancer therapy.
STATEMENTS OF INVENTION
[0026] Anti-PD-1 and anti-PD-L1 antibodies are predominantly involved in breaking immune tolerance and activating an anti-tumour immune response. LAG-3, expressed on T cells following activation, and constitutively expressed on exhausted T cells, further maintains these cells in a suppressive state. Blockade of LAG-3, when employed in combination with other established immune suppressive molecules (i.e.: PD-1, PD-L1) has also been shown to provide a synergistic improved immune response in murine tumour models (Woo et al, 2012). The present inventors postulated that therapies targeting both of these pathways simultaneously will directly address mechanisms which promote and maintain T cell exhaustion. In addition, the inventors expect that targeting LAG-3 may suppress antigen presentation through the action of LAG-3 expressing regulatory T cells on APCs and the published research documenting down-regulation of CD86 (Grosso et al, 2013). Blocking this interaction is expected to maintain antigen presentation, while blocking PD-L1 signalling is expected to break tolerance, resulting in a significant anti-tumour response when both pathways are inhibited at the same time.
[0027] Published data on anti-LAG-3 and anti-PD-L1 antibody combinations is limited though there are some results from preclinical syngeneic mouse tumour and viral load models. In a murine model of myeloma, a combination of anti-PD-L1 and anti-LAG-3 blocking antibodies was administered following low dose whole body irradiation and improved survival rates to greater than 80% (Jing et al, 2015). No evidence of systemic or organ-specific autoimmunity was observed. LAG-3 and PD-1 knock-out mice showed markedly increased survival from and clearance of multiple transplantable tumours (Woo et al, 2012).
[0028] The present inventors postulated that bispecific antibodies which bind to both LAG-3 and PD-L1 would confer a number of advantages over the combination of monoclonal antibodies against these antigens, including:
[0029] 1. Directed Therapy
[0030] Activated T cells express LAG-3 in the lymph nodes. One part of the anti-LAG-3/PD-L1 bispecific antibody targets primed LAG-3-positive T cells in the lymph node, which then traffic to the site of the tumour, transporting the bispecific antibody. Once within the tumour microenvironment, T cells carrying the bispecific antibody are able to immediately engage and block PD-L1 on tumour cells via the anti-PD-L1 portion. Consequently, all T cells traveling to the tumour site would be resistant to both LAG-3 and PD-L1/PD-1 signalling.
[0031] 2. Bridging
[0032] Primed CD8 positive T cells encounter tumour antigens within the tumour microenvironment, where they respond by killing the tumour cell in the absence of suppressive signals. Bispecific antibodies are expected to be superior over combinations of individual monoclonal therapies by maintaining or prolonging this contact between T cells and tumour cells. Signal strength in the activation of T cells is essential, which in the case of presented antigen in cancers may be key (Engels et al, 2013) and the presence of a bispecific anti-LAG-3/PD-L1 antibody bound to targets on APC or cancer cells is expected to increase the time in which the T cells can successfully recognise antigen and become activated.
[0033] 3. Localisation
[0034] In areas of inflammation and ongoing immune responses, PD-L1 expression is significantly increased because of localised IFN-.gamma. release. This is true whether on target cancer cells, tumour-associated macrophages (TAM), or repeated stimulation of T cell populations. A bispecific antibody that antagonises PD-L1 and LAG-3 is expected to localise and concentrate to the areas of greatest PD-L1 expression in the tumour whilst allowing the anti-LAG-3 portion to bind and prevent LAG-3-mediated suppression of T cells.
[0035] Following an extensive screening and affinity maturation programme, the present inventors were able to identify ten specific binding members comprising a binding site specific for LAG-3 in the CH3 domain of the molecule. These molecules were shown to have a high affinity for both human and cynomolgus LAG-3. The high affinity for human LAG-3 is expected to be advantageous in the treatment of e.g. cancers containing tumour-infiltrating lymphocytes (TILs) expressing LAG-3 in human patients, while the high affinity for cynomolgus LAG-3, which is comparable to the affinity for human LAG-3, is expected to be useful in the evaluation of the properties of the specific binding members in cynomolgus monkey disease models. The reason for this is that the results obtained are more likely to be predictive of the effects of the specific binding member in human patients than when a molecule which has a higher variability in its affinity for human and cynomolgus LAG-3 is tested in cynomolgus monkey models.
[0036] The specific binding members were also shown to have high activity in a T cell activation assay, which is expected to be predictive of improved efficacy in human patients through enhanced inhibition of LAG-3.
[0037] The present inventors further combined these specific binding members comprising a binding site specific for LAG-3 in the CH3 domain with an antibody Fab domain comprising a CDR-based antigen binding site for PD-L1 to create bispecific antibody molecules comprising binding sites for both LAG-3 and PD-L1, which are expected to have the advantages detailed above. Surrogate murine versions of these antibody molecules which bind to murine LAG-3 and murine PD-L1 were also prepared by the inventors and shown to be capable of significantly inhibiting tumour growth in syngeneic mouse models of cancer. In particular, use of these surrogate murine molecules demonstrated there is a synergistic effect on tumour growth suppression when an antibody molecule comprising binding sites for both LAG-3 and PD-L1 is administered to mice in the mouse models tested. Based on the similar mechanism of action of mouse and human LAG-3 and PD-L1 in the tumour environment, murine studies that show efficacy in diminishing tumour burden are expected to translate into clinical therapeutic benefits in human cancer patients. Based on these results, it is therefore expected that the antibody molecules of the invention will show a superior effect in the treatment of cancer in human patients, in particular in supressing tumour growth, than, for example, administration of two separate molecules which bind LAG-3 and PD-L1, respectively.
[0038] Thus, in a first aspect the present invention provides an antibody molecule which binds to both to PD-L1 and LAG-3. Specifically, these antibodies comprise:
[0039] (i) a CDR-based antigen binding site for PD-L1; and
[0040] (ii) a LAG-3 antigen binding site located in, or engineered into, two or more structural loops of a CH3 domain of the antibody molecule. The LAG-3 binding site preferably comprises the amino acid sequences WDEPWGED (SEQ ID NO: 1) and PYDRWVWPDE. (SEQ ID NO: 3).
[0041] The antibody molecule preferably comprises the amino acid sequence set forth in SEQ ID NO: 1 in the AB loop, and the amino acid sequence set forth in SEQ ID NO: 3 in the EF loop of the CH3 domain.
[0042] Thus, in a first aspect, the present invention provides an antibody molecule which binds to programmed death-ligand 1 (PD-L1) and lymphocyte-activation gene 3 (LAG-3). The antibody molecule preferably comprises (i) a CDR-based antigen binding site for PD-L1; and (ii) a LAG-3 antigen binding site located in a CH3 domain of the antibody molecule.
[0043] The LAG-3 binding site preferably comprises the amino acid sequences WDEPWGED (SEQ ID NO: 1) and PYDRWVWPDE (SEQ ID NO: 3). The amino acid sequence WDEPWGED is preferably located in a first structural loop of the CH3 domain and the amino acid sequence PYDRWVWPDE is preferably located in a second structural loop of the CH3 domain.
[0044] For example, the LAG-3 antigen-binding site may be located in a structural loop region of a CH3 domain of the antibody molecule, wherein the structural loop region preferably comprises two or more structural loops, and wherein the LAG-3 binding site preferably comprises the amino acid sequences WDEPWGED (SEQ ID NO: 1) and PYDRWVWPDE (SEQ ID NO: 3).
[0045] As a further example, the LAG-3 antigen-binding site may be engineered into two or more structural loops of a CH3 domain of the antibody molecule, wherein the LAG-3 binding site preferably comprises the amino acid sequences WDEPWGED (SEQ ID NO: 1) and PYDRWVWPDE (SEQ ID NO: 3).
[0046] As mentioned above, the sequences of the LAG-3 binding site are preferably located in two or more structural loops of the CH3 domain of the antibody molecule. In a preferred embodiment the LAG-3 antigen-binding site comprises the amino acid sequence set forth in SEQ ID NO: 1 in the AB loop, and the amino acid sequence set forth in SEQ ID NO: 3 in the EF loop of the CH3 domain.
[0047] The amino acid sequence set forth in SEQ ID NO: 1 is preferably located at residues 11 to 18 of the CH3 domain; and/or the amino acid sequence set forth in SEQ ID NO: 3 is located at residues 92 to 101 of the CH3 domain; wherein the amino acid residue numbering is according to the ImMunoGeneTics IMGT) numbering scheme.
[0048] The LAG-3 antigen-binding site of the antibody molecule may further comprise one of the following sequences, preferably in the CD loop of the CH3 domain of the antibody molecule:
TABLE-US-00001 (i) (SEQ ID NOS 2, 8 and 18) SNGQPENNY; (ii) (SEQ ID NO: 13) SNGQPEDNY; (iii) (SEQ ID NO: 23) SNGYPEIEF; (iv) (SEQ ID NO: 28) SNGIPEWNY; (v) (SEQ ID NO: 33) SNGYAEYNY; (vi) (SEQ ID NO: 38) SNGYKEENY; (vii) (SEQ ID NO: 43) SNGVPELNV; or (viii) (SEQ ID NO: 48) SNGYQEDNY.
[0049] Preferably, the LAG-3 antigen-binding site of the antibody molecule further comprise one of the following sequences, preferably in the CD loop of the CH3 domain of the antibody molecule: the amino acid sequence set forth in SEQ ID NO: 2, 28, or 38 in the CD loop of the CH3 domain. More preferably, the LAG-3 antigen-binding site of the antibody molecule further comprises the amino acid sequence set forth in SEQ ID NO: 2 in the CD loop of the CH3 domain
[0050] The amino acid sequence set forth in SEQ ID NO: 2, 8, 13, 18, 23, 28, 33, 38, 43, or 48 is preferably located at residues 43 to 78 of the CH3 domain of the antibody molecule, wherein the residues are numbered according to the IMGT numbering scheme.
[0051] The sequence of the CH3 domain of the antibody molecule, other the sequences of the LAG-3 antigen-binding site, is not particularly limited. Preferably, CH3 domain is a human immunoglobulin G domain, such as a human IgG1, IgG2, IgG3, or IgG4 CH3 domain, most preferably a human IgG1 CH3 domain. The sequences of human IgG1, IgG2, IgG3, or IgG4 CH3 domains are known in the art.
[0052] In a preferred embodiment, the antibody molecule comprises the CH3 domain set forth in SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50, more preferably the CH3 domain set forth in SEQ ID NO: 5, 30, or 40, most preferably the CH3 domain set forth in SEQ ID NO: 5. Alternatively, the antibody molecule may comprise a CH3 domain with an amino acid sequence which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50, preferably SEQ ID NO: 5, 30, or 40, more preferably SEQ ID NO: 5.
[0053] The antibody molecule may further comprise a CH2 domain. The CH2 domain is preferably located at the N-terminus of the CH3 domain, as in the case in a human IgG molecule. The CH2 domain of the antibody molecule is preferably the CH2 domain of human IgG1, IgG2, IgG3, or IgG4, more preferably the CH2 domain of human IgG1. The sequences of human IgG domains are known in the art. In a preferred embodiment, the antibody molecule comprises an IgG CH2 domain with the sequence set forth in SEQ ID NO: 53 or SEQ ID NO: 54, or a CH2 domain with an amino acid sequence which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 53 or SEQ ID NO: 54.
[0054] In a preferred embodiment, the antibody molecule comprises the sequence set forth in SEQ ID NO: 6, 7, 11, 12, 16, 17, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 46, 47, 51, or 52, or a sequence which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 6, 7, 11, 12, 16, 17, 21, 22, 26, 27, 31, 32, 36, 37, 41, 42, 46, 47, 51, or 52. More preferably, the antibody molecule comprises the sequence set forth in SEQ ID NO: 6, 7, 31, 32, 41, or 42, or a sequence which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 6, 7, 31, 32, 41, or 42. Yet more preferably, the antibody molecule comprises the sequence set forth in SEQ ID NO: 6 or 7, or a sequence which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 6 or 7.
[0055] In a preferred embodiment, the antibody molecule is a human immunoglobulin G molecule, such as a human IgG1, IgG2, IgG3 or IgG4 molecule, more preferably a human IgG1 molecule. The sequences of human immunoglobulin G molecules is known in the art and introducing a CH3 domain or CH3 domain sequence as disclosed here into such a molecule would not present any difficulty to the skilled person.
[0056] The antibody molecule preferably comprise the CDRs of the VH and/or VL domains set forth in SEQ ID NOs 92 and 93. Methods for determining CDR sequence in a given VH or VL domain are known in the art and include the Kabat and IMGT numbering systems. More preferably, the antibody molecule preferably comprise one or more, two or more, three or more, four or more, five or more, or all six of the complementarity determining regions set forth in SEQ ID NOs 86 to 91. Preferably, the antibody molecule comprises the VH and/or VL domains set forth in SEQ ID NOs 92 and 93, respectively.
[0057] In a preferred embodiment, the antibody molecule comprises the heavy chain sequence set forth in any one of SEQ ID NOs: 94 to 113, or a heavy chain with an amino acid sequence which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 94 to 113, provided the VH domain of the heavy chain sequence remains unchanged. More preferably, the antibody molecule comprises the heavy chain sequence set forth in any one of SEQ ID NOs: 94, 95, 104, 105, 108, and 109, or a heavy chain with an amino acid sequence which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 94, 95, 104, 105, 108, and 109, provided the VH domain of the heavy chain sequence remains unchanged. Yet more preferably, the antibody molecule comprises the heavy chain sequence set forth in SEQ ID NO: 94 or 95, or a heavy chain with an amino acid sequence which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 94 or 95, provided the VH domain of the heavy chain sequence remains unchanged.
[0058] In a further preferred embodiment, the antibody molecule may in addition, or alternatively, comprise the light chain sequence set forth in SEQ ID NO: 116, or a light chain with an amino acid sequence which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 116, provided the VL domain of the light chain sequence remains unchanged.
[0059] The antibody molecule is preferably capable of simultaneously binding to PD-L1 and LAG-3. The PD-L1 and LAG-3 may, for example, be present on two different cells. Without wishing to be limited by theory, it is thought that this results in cross-linking between cells and internalization of PD-L1 and/or LAG-3 making them unavailable for stimulation.
[0060] The present inventors have shown that an antibody molecule comprising (i) a CDR-based antigen binding site for PD-L1; and (ii) a LAG-3 antigen binding site located in a CH3 domain of the antibody molecule, in accordance with the present invention, FS18-7-9/84G09, surprisingly mediated Complement Dependent Cytotoxicity (CDC) of PD-L1 expressing cells but not of LAG-3 expressing cells, even when a mixture of LAG-3 and PD-L1 expressing cells were present in the sample. This property is expected to be useful where, as is the case with FS18-7-9/84G09, the CDR-based antigen binding of the antibody molecule targets a tumour cell and the binding site located in a constant domain of the antibody molecule targets an immune cell, as the immune cell would be protected from CDC mediated by binding to the antibody molecule, while tumour cells would be subject to CDC.
[0061] Thus, in a further embodiment, the present invention relates to an antibody molecule which binds to tumour antigen and an immune cell antigen, wherein the antibody molecule comprises:
[0062] (i) a CDR-based antigen binding site for a tumour antigen; and
[0063] (ii) an antigen binding site for an immune cell antigen located in a constant domain, preferably a CH3 or CH2 domain, more preferably a CH3 domain, of the antibody molecule,
[0064] wherein the antibody molecule does not mediate, or does not mediate significant, complement dependent cytotoxicity of an immune cell comprising said immune cell antigen when said immune cell is bound by the antibody molecule.
[0065] Preferably, the antibody molecule further mediates complement dependent cytotoxicity of a tumour cell comprising said tumour antigen when said tumour cell is bound by the antibody molecule.
[0066] Methods for measuring CDC of an antibody molecule are known in the art and described herein.
[0067] The present inventors have further shown that an antibody molecule comprising (i) a CDR-based antigen binding site for PD-L1; and (ii) a LAG-3 antigen binding site located in a CH3 domain of the antibody molecule, in accordance with the present invention, FS18-7-9/84G09, surprisingly mediated low Antibody Dependent Cellular Cytotoxicity (ADCC) of LAG-3 expressing cells compared with ADCC of PD-L1 expressing cells. This property is again expected to be useful where, as is the case with FS18-7-9/84G09, the CDR-based antigen binding of the antibody molecule targets a tumour cell and the binding site located in a constant domain of the antibody molecule targets an immune cell, as the immune cell would be subjected to lower ADCC than a tumour cell bound by the antibody.
[0068] As explained herein, mutations for reducing or abrogating ADCC activity of antibody molecules are known in the art. One such mutations is the LALA mutation described herein. It was unexpectedly found that FS18-7-9/84G09 has low ADCC activity towards LAG-3-expressing cells. Where it is not necessary to completely abrogate ADCC activity, this may represent an advantage.
[0069] Thus, in a further embodiment, the present invention relates to an antibody molecule which binds to tumour antigen and an immune cell antigen, wherein the antibody molecule comprises:
[0070] (i) a CDR-based antigen binding site for a tumour antigen; and
[0071] (ii) an antigen binding site for an immune cell antigen located in a constant domain, preferably a CH3 or CH2 domain, more preferably a CH3 domain, of the antibody molecule,
[0072] wherein the antibody molecule causes less ADCC with respect to immune cells comprising said immune cell antigen when said immune cells are bound by the antibody molecule than of tumour cells comprising said tumour antigen when said tumour cells are bound by the antibody molecule. Preferably, the antibody molecule does not mediate, or not mediate significant, ADCC of an immune cell comprising said immune cell antigen when said immune cell is bound by the antibody molecule. The antibody molecule may further not mediate, or not mediate significant, complement dependent cytotoxicity of an immune cell comprising said immune cell antigen when said immune cell is bound by the antibody molecule and/or may mediate complement dependent cytotoxicity of a tumour cell comprising said tumour antigen when said tumour cell is bound by the antibody molecule.
[0073] Methods for measuring ADCC of an antibody molecule are known in the art and described herein.
[0074] Various tumour antigens and immune cell antigens are known in the art. The tumour antigen and immune cell antigen are preferably cell-surface antigens. The immune cell antigen is preferably an antigen present on tumour-infiltrating lymphocytes.
[0075] The antigen binding site for the immune cell antigen preferably comprises one or more modifications in one or more structural loops of the constant domain of the antibody molecule, such as the AB, CD, and/or EF loop of the constant domain. For example, the binding site may be a LAG-3 binding site as herein described.
[0076] The antibody molecule of the invention may be conjugated to an immune system modulator, cytotoxic molecule, radioisotope, or detectable label. The immune system modulator may be cytotoxic molecule is a cytokine.
[0077] The present invention also provides a nucleic acid encoding an antibody molecule of the invention, as well as a vector comprising such a nucleic acid.
[0078] A recombinant host cell comprising a nucleic acid or the vector of the invention is also provided. Such a recombinant host cell may be used to produce an antibody molecule. Thus, also provided is a method of producing an antibody molecule of the invention, the method comprising culturing the recombinant host cell under conditions for production of the antibody molecule. The method may further comprise a step of isolating and/or purifying the antibody molecule.
[0079] The antibody molecules of the present invention are expected to find application in therapeutic applications, in particular therapeutic applications in humans, such as cancer treatment. Thus, also provided is a pharmaceutical composition comprising an antibody molecule according to the invention and a pharmaceutically acceptable excipient.
[0080] The present invention also provides an antibody molecule of the invention, for use in a method of treating cancer in a patient. Also provided is a method of treating cancer in a patient, wherein the method comprises administering to the patient a therapeutically effective amount of an antibody molecule according to the invention. Further provided is the use of an antibody molecule according to the invention for use in the manufacture of a medicament for the treatment of cancer in a patient. The treatment may further comprise administering an anti-tumour vaccine and/or a chemotherapeutic agent to the patient.
BRIEF DESCRIPTION OF THE FIGURES
[0081] FIGS. 1A-1B: FIG. 1A shows a sequence alignment of the nine Fcabs identified following the second affinity maturation, FS18-7-32; FS18-7-33; FS18-7-36; FS18-7-58; FS18-7-62; FS18-7-65; FS18-7-78; FS18-7-88; and FS18-7-95, against the parental Fcab, FS18-7-9. FIG. 1B shows the percentage sequence identity of each of these Fcabs with the sequence of the parental Fcab, FS18-7-9.
[0082] FIGS. 2A-2F show the results of a T cell activation assay. Specifically, FIGS. 2A-2F show representative plots of IL-2 release, which is indicative of T cell activation, in the panel of cell lines treated with mAb.sup.2 FS18-7-9/84G09 (FIGS. 2A-2C), or FS18-7-62/84G09 or FS18-7-78/84G09 (FIGS. 2D-2F), and control antibodies. FIG. 2A: The assay with LAG-3 and PD-L1 required inhibition of both targets (FS18-7-9/84G09LALA, FS18-7-9/4420LALA+84G09, 25F7+84G09LALA or 25F7+S1 LALA) to have activation. FIG. 2B: The assay with LAG-3 only required inhibition of LAG-3 for activation (FS18-7-9/84G09LALA, FS18-7-9/4420LALA+84G09, 25F7+84G09LALA, 25F7+S1 LALA, 25F7, FS18-7-9/4420 LALA). FIG. 2C: The assay with PD-L1 only required the inhibition of PD-L1 for activation (FS18-7-9/84G09LALA, FS18-7-9/4420LALA+84G09, 25F7+84G09LALA, 25F7+S1 LALA, 84G09LALA). FIG. 2D: The assay with LAG-3 and PD-L1 required inhibition of both targets (FS18-7-62/84G09LALA, FS18-7-78/84G09LALA, FS18-7-62/4420LALA+84G09, FS18-7-78/4420LALA+84G09, 25F7+84G09LALA or 25F7+S1 LALA) to have activation. FIG. 2E: The assay with only LAG-3 required inhibition of LAG-3 for activation (FS18-7-62/84G09LALA, FS18-7-62/4420LALA, FS18-7-78/84G09LALA, FS18-7-78/4420LALA, FS18-7-62/4420LALA+84G09, FS18-7-78/4420LALA+84G09, 25F7+84G09LALA or 25F7+S1 LALA). FIG. 2F: The assay with only PD-L1 required the inhibition of PD-L1 for activation (FS18-7-62/84G09LALA, FS18-7-78/84G09LALA, FS18-7-62/4420LALA+84G09, FS18-7-78/4420LALA+84G09, 25F7+84G09LALA or 25F7+S1 LALA).
[0083] FIGS. 3A-3C show that FS18-7-9/84G09 was able to induce T cell activation, as indicated by IL-2 release, in the presence of both cLAG-3+cPD-L1 and cLAG-3 or cPD-L1 alone showing functionally cynomolgus cross-reactivity.
[0084] FIG. 4 shows a representative plot from the SEB assay. LAG-3/PD-L1 mAb.sup.2 and the combination of the LAG-3/4420 mAb.sup.2+84G09LALA showed greater activation that the 84G09LALA mAb alone, whereas the LAG-3/4420 mAb.sup.2 or 4420 mAb did not show significant activation.
[0085] FIG. 5 shows the end tumour weights on day 20 in the non-established MC38 syngeneic tumour model. LAG-3/PD-L1 mAb.sup.2 (FS18-29/S1) treated mice had end tumours with significantly smaller weights than those treated with a combination of benchmark mAbs, C9B7W and S1.
[0086] FIG. 6 shows growth curves of the non-established MC38 syngeneic tumour model. LAG-3/PD-L1 mAb.sup.2 (FS18-29/S1) treated mice had smaller tumours than those treated with a combination of benchmark mAbs, C9B7W and S1, or those treated with S1 alone. The LAG-3/4420 mAb.sup.2 and benchmark anti-LAG-3 mAb had little impact on tumour growth.
[0087] FIG. 7 shows the end tumour weights on day 24 in the established MC38 syngeneic tumour model. LAG-3/PD-L1 mAb.sup.2 (FS18-29/S1) was just as effective in suppressing tumour growth as the combination of benchmark antibodies, C9B7W and S1. FS18-29/4420 alone had no noticeable impact on tumour growth and S1 and C9B7W both had a mild effect on resulting tumour growth.
[0088] FIG. 8 shows tumour growth curves of the established MC38 syngeneic tumour model. LAG-3/PD-L1 mAb.sup.2 (FS18-29/S1) treated mice had tumours volumes similar to those treated with a combination of benchmark mAbs, C9B7W and S1. Mice treated with S1 or C9B7W alone showed intermediate tumour volumes whereas LAG-3/4420 mAb.sup.2 treatment had no impact on tumour growth.
[0089] FIG. 9 shows the end tumour weights on day 20 in the non-established CT26 syngeneic tumour model. LAG-3/PD-L1 mAb.sup.2 (FS18-29/S1 and FS18-35/S1) supressed tumour growth to a greater extent than combination of benchmark antibodies, C9B7W and S1.
[0090] FIG. 10 shows tumour growth curves of the non-established CT26 syngeneic tumour model. There was a demonstrated statistically significant difference in FS18-35/S1 versus IgG control in suppressing tumour growth. Such a statistically significant difference was not observed with the combination of benchmark antibodies, versus the IgG control group.
[0091] FIG. 11 shows the end tumour weights on day 22 in the non-established MC38 syngeneic tumour model used to compare the effect of the LALA mutation in the mAb.sup.2 on tumour growth inhibition. There was no statistically significant difference in end tumour weight between mice treated with mAb.sup.2 with and without the LALA mutation.
[0092] FIG. 12 shows tumour growth curves of the non-established MC38 syngeneic tumour model used to compare the effect of mAb.sup.2 with and without the LALA mutation on tumour growth inhibition. There was no statistically significant difference in the tumour growth curves between mice treated with mAb.sup.2 with and without the LALA mutation, however there was a trend toward increased tumour growth inhibition by molecules containing the LALA mutation.
[0093] FIGS. 13A-13C show the effect of mAb.sup.2 treatment on T cell LAG-3 expression. LAG-3 expression on CD8 (FIG. 13A), CD4 (FIG. 13B) and FoxP3 (FIG. 13C) tumour infiltrating lymphocytes (TILs) treated with mAb.sup.2 FS18-29/S1, FS18-29/4420, S1, FS18-29/4420 and S1, or control antibody 4420 is shown at day 19 and 23 after tumour inoculation, corresponding to days 3 and 7 after the last mAb.sup.2/antibody dosing, respectively. LAG-3 expression was decreased after treatment with mAb.sup.2 FS18-29/S1 at day 19 and 23. Animals given a combination of FS18-29/4420 and S1 also showed a decrease in LAG-3 expression but the effect was delayed until day 23, while FS18-29/4420 or S1 administered individually resulted in little to no decrease in LAG-3 expression.
[0094] FIGS. 14A-14B show the percentage of lysis of PD-L1 and LAG-3 expressing Raji cells following various antibody/mAb.sup.2 treatments using a lactate dehydrogenase (LDH) release CDC assay. FIG. 14A and FIG. 14B show CDC-mediated lysis of PD-L1 and LAG-3 expressing Raji cells, respectively. Cells were incubated with anti-LAG-3 antibody 25F7, anti-PD-L1 antibody 84G09, anti-PD-L1 antibody 84G09 comprising the LALA mutation, anti-CD20 antibody Rituximab, mAb.sup.2 FS18-7-9/84G09, mAb.sup.2 FS18-7-9/84G09 comprising the LALA mutation, or Rituximab comprising the LALA mutation. Release of LDH was measured 4 hrs after treatment with baby rabbit complement and expressed as percentage compared to total lysis. The concentration of the antibody/mAb.sup.2 treatment is indicated on the X-axis. A shared value was set for the slopes of all curves in order to be able to compare across the curves.
[0095] FIG. 15 shows the percentage of dead PD-L1 and LAG-3 expressing Raji cells following various treatments using a flow cytometry based CDC assay. A mixture of differentially fluorescently labelled PD-L1 and LAG-3 expressing cells were incubated with control antibody 4420, anti-CD20 antibody Rituximab (RIT), a combination of anti-LAG-3 antibody 25F7 and anti-PD-L1 antibody 84G09, or mAb.sup.2 FS18-7-9/84G09. Cells were then treated with baby rabbit complement and stained using a dye only staining dead cells. The percentage of dead cells in the two cell populations was evaluated as a percentage of total cells. The cell type (PD-L1 expressing or LAG-3 expressing, identified by the differential fluorescent labels) evaluated following each treatment is indicated in FIG. 15 after the name of the relevant treatment, see e.g. 4420 PDL1, which refers to evaluation of viability of PD-L1 expressing Raji cells following treatment with control antibody 4420. The concentration of the antibody/mAb.sup.2 treatment is indicated on the X-axis
[0096] FIGS. 16A-16B show the percentage lysis (cytotoxicity) of PD-L1 and LAG-3 expressing Raji cells following various treatments using a lactate dehydrogenase (LDH) release ADCC assay. Cells were treated with anti-CD20 antibody Rituximab, anti-PD-L1 antibody 84G09, mAb.sup.2 FS18-7-9/84G09, Rituximab comprising the LALA mutation, mAb.sup.2 FS18-7-9/84G09 comprising the LALA mutation, anti-LAG-3 antibody 25F7, anti-PD-L1 antibody 84G09 comprising the LALA mutation, control antibody 4420, or control antibody 4420 comprising the LALA mutation. Treated cells were then co-incubated with primary NK cells and specific LDH release was measured as a percentage compared to total lysis of target cells. The concentration of the antibody/mAb.sup.2 treatment is indicated on the X-axis.
DETAILED DESCRIPTION
[0097] The present invention relates to antibody molecules which bind both to PD-L1 and LAG-3. Specifically, the antibody molecules of the present invention comprise a CDR-based antigen binding site for PD-L1 and a LAG-3 antigen binding site located in a constant domain of the antibody molecule. The terms "PD-L1" and "LAG-3" may refer to human PD-L1 and human LAG-3, murine PD-L1 and murine LAG-3, and/or cynomologus monkey PD-L1 and cynomologus monkey LAG-3, unless the context requires otherwise. Preferably the terms "PD-L1" and "LAG-3" refer to human PD-L1 and human LAG-3, unless the context requires otherwise.
[0098] The term "antibody molecule" describes an immunoglobulin whether natural or partly or wholly synthetically produced. The antibody molecule may be human or humanised. The antibody molecule is preferably a monoclonal antibody molecule. Examples of antibodies are the immunoglobulin isotypes, such as immunoglobulin G, and their isotypic subclasses, such as IgG1, IgG2, IgG3 and IgG4, as well as fragments thereof.
[0099] The term "antibody molecule", as used herein, thus includes antibody fragments, provided said fragments comprise a CDR-based antigen binding site for PD-L1 and a LAG-3 antigen binding site located in a constant domain, such as a CH1, CH2, or CH3 domain, preferably a CH3 domain, of the antibody molecule. Unless the context requires otherwise, the term "antibody molecule", as used herein, is thus equivalent to "antibody molecule or fragment thereof".
[0100] It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing the CDRs, or variable regions, and/or the constant domain sequences providing the LAG-3 antigen binding site, into a different immunoglobulin. Introduction of the CDRs of one immunoglobulin into another immunoglobulin is described for example in EP-A-184187, GB 2188638A or EP-A-239400. Similar techniques could be employed for the relevant constant domain sequences. Alternatively, a hybridoma or other cell producing an antibody molecule may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.
[0101] As antibodies can be modified in a number of ways, the term "antibody molecule" should be construed as covering antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023.
[0102] An example of an antibody fragment comprising both CDR sequences and CH3 domain is a minibody, which comprises an scFv joined to a CH3 domain (Hu et al. (1996), Cancer Res., 56(13):3055-61).
[0103] The antibody molecule of the present invention binds to PD-L1 and LAG-3. Binding in this context may refer to specific binding. The term "specific" may refer to the situation in which the antibody molecule will not show any significant binding to molecules other than its specific binding partner(s), here PD-L1 and LAG-3. The term "specific" is also applicable where the antibody molecule is specific for particular epitopes, such as epitopes on PD-L1 and LAG-3, that are carried by a number of antigens in which case the antibody molecule will be able to bind to the various antigens carrying the epitope.
[0104] LAG-3 shares 40% sequence identity with CD4, its most closely related protein. The present inventors tested the FS18-7-9 Fcab, which comprises the amino acid sequences set forth in SEQ ID NOs 1 to 3, for binding to CD4. The FS18-7-9 Fcab showed no binding to CD4, demonstrating that this molecule binds LAG-3 specifically. Thus, in a preferred embodiment, the LAG-3 binding site of an antibody molecule of the present invention does not bind, or does not show any significant binding, to CD4.
[0105] An antibody molecule of the invention preferably comprises a LAG-3 antigen binding site. The LAG-3 antigen binding site is located in a constant domain of the antibody molecule, such as a CH1, CH2, CH3 or CH4 domain. Preferably, the LAG-3 antigen binding site is located in the CH3 domain of the antibody molecule. The LAG-3 binding site preferably comprises the amino acid sequences WDEPWGED (SEQ ID NO: 1) and PYDRWVWPDE. (SEQ ID NO: 3). These sequences were present in all of the lead anti-LAG-3 Fcab clones identified by the present inventors following an extensive screening and characterisation program as described in the examples.
[0106] The amino acid sequences set forth in SEQ ID NOs 1 and 2 are preferably located in structural loops of the constant domain of the antibody molecule. The introduction of sequences into the structural loop regions of antibody constant domains to create new antigen-binding sites is described, for example, in WO2006/072620 and WO2009/132876.
[0107] The structural loops of antibody constant domains include the AB, CD and EF loops. In the CH3 domain, the AB, CD, and EF loops are located at residues 11-18, 43-78 and 92-101 of the CH3 domain, where the amino acid residue numbering is according to the ImMunoGeneTics (IMGT) numbering scheme. The amino acid sequence set forth in SEQ ID NO: 1 is preferably located in the AB loop of the constant domain. The amino acid sequence set forth in SEQ ID NO: 3 is preferably located in the EF loop of the constant domain. More preferably, the amino acid sequence set forth in SEQ ID NO: 1 is located at residues 11 to 18 of the CH3 domain; and/or the amino acid sequence set forth in SEQ ID NO: 3 is located at residues 92 to 101 of the CH3 domain, wherein the amino acid residue numbering is according to the IMGT numbering scheme.
[0108] In addition, the antibody molecule preferably comprises the amino acid sequence set forth in SEQ ID NO: 2, 8, 13, 18, 23, 28, 33, 38, 43, or 48, more preferably SEQ ID NO: 2, 28, or 38, yet more preferably SEQ ID NO: 2, in a structural loop of a constant domain of the antibody molecule. The structural loop is preferably the CD loop and the constant domain is preferably the CH3 domain. The amino acid sequence set forth in SEQ ID NO: 2, 8, 13, 18, 23, 28, 33, 38, 43, or 48 is preferably located at residues 43 to 78 of the CH3 domain, wherein the amino acid residue numbering is according to the IMGT numbering scheme.
[0109] An antibody molecule of the invention may further comprise a glutamic acid residue (E) at position 36 and/or a tyrosine residue (Y) at position 85.2 of the CH3 domain (as shown in FIG. 1A), wherein the amino acid residue numbering is according to the IMGT numbering scheme. In particular, an antibody molecule which comprises the CD structural loop region set forth in SEQ ID NO: 8 preferably further comprises a glutamic acid residue (E) at position 36 of the CH3 domain. Similarly, an antibody molecule which comprises the CD structural loop region set forth in SEQ ID NO: 18 preferably further comprises a tyrosine residue (Y) at position 85.2 of the CH3 domain.
[0110] In a preferred embodiment, the antibody molecule of the invention comprises a CH3 domain which comprises, has, or consists of the sequence set forth in SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50, preferably a CH3 domain with the sequence set forth in SEQ ID NO: 5, 30, or 40, more preferably, a CH3 domain with the sequence set forth in SEQ ID NO: 5.
[0111] The antibody molecule of the invention may comprise a CH3 domain which comprises, has, or consists of the sequence set forth in SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50, wherein the CH3 domain sequence further comprises a lysine residue (K) at the immediate C-terminus of the sequence shown in SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50. Thus, for example, the antibody molecule of the invention may comprise a CH3 domain which comprises, has, or consists of the sequence set forth in SEQ ID NO: 5 with a lysine residue at the C-terminus of the sequence shown in SEQ ID NO: 5. The sequence of such a CH3 domain would then be as follows:
TABLE-US-00002 (SEQ ID NO: 135) GQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQ KSLSLSPGK
[0112] In addition, the antibody molecule of the invention may comprise a CH2 domain of an immunoglobulin G molecule, such as a CH2 domain of an IgG1, IgG2, IgG3, or IgG4 molecule. Preferably the antibody molecule of the invention comprises a CH2 domain of an IgG1 molecule. The CH2 domain may have the sequence set forth in SEQ ID NO: 53.
[0113] The CH2 domain of the antibody molecule may comprise a mutation to reduce or abrogate binding of the CH2 domain to one or more Fc .gamma. receptors, such as Fc.gamma.RI, Fc.gamma.RIIa, Fc.gamma.RIIb, Fc.gamma.RIII and/or to complement. CH2 domains of human IgG domains normally bind to Fc .gamma. receptors and complement and the inventors postulate that reduced binding to Fc .gamma. receptors will reduce the antibody-dependent cell-mediated cytotoxicity (ADCC) and reduced binding to complement will reduce the complement-dependent cytotoxicity (CDC) activity of the antibody molecule. Mutations for reduce or abrogate binding of the CH2 domain to one or more Fc .gamma. receptors and complement are known and include the "LALA mutation" described in Bruhns, et al. (2009) and Xu et al. (2000). Thus, the antibody molecule may comprise a CH2 domain, wherein the CH2 domain comprises alanine residues at positions 4 and 5 of the CH2 domain, wherein the numbering is according to the IMGT numbering scheme. For example, the antibody molecule comprises an IgG1 CH2 domain which comprises, has, or consists of the sequence set forth in SEQ ID NO: 54.
[0114] The antibody molecule of the present invention comprises a CDR-based antigen binding site for PD-L1. The term "CDR-based antigen binding site" refers to the antigen-binding site of an antibody molecule variable region which is composed of six CDRs. The preparation of antibody molecules against PD-L1, and determination of the CDR sequences of such antibody molecules, is well within the capabilities of the skilled person and many suitable techniques are known in the art.
[0115] Preferably, the antibody molecule of the invention comprises the HCDR3 of antibody 84G09. The HCDR3 is known to play a role in determining the specificity of an antibody molecule (Segal et al., (1974), PNAS, 71:4298-4302; Amit et al., (1986), Science, 233:747-753; Chothia et al., (1987), J. Mol. Biol., 196:901-917; Chothia et al., (1989), Nature, 342:877-883; Caton et al., (1990), J. Immunol., 144:1965-1968; Sharon et al., (1990a), PNAS, 87:4814-4817; Sharon et al., (1990b), J. Immunol., 144:4863-4869; Kabat et al., (1991b), J. Immunol., 147:1709-1719).
[0116] The antibody molecule may further comprise the HCDR1, HCDR2, LCDR1, LCDR2 and/or LCDR3 of antibody 84G09. The skilled person would have no difficulty in determining the sequences of the CDRs from the VH and VL domain sequences of antibody 84G09 shown in SEQ ID NOs 92 and 93, respectively. The CDR sequences may, for example, be determined according to Kabat (Kabat, E. A. et al., (1991)) or the IMGT numbering scheme.
[0117] The sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of antibody 84G09, according to the IMGT numbering scheme, are set out in SEQ ID NOs 86, 87, 88, 89, 90, and 91, respectively.
[0118] The sequences of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of antibody 84G09, according to Kabat, are set out in SEQ ID NOs 136, 137, 138, 139, 140, and 141, respectively.
[0119] The antibody may also comprise the VH and/or VL domain of antibody 84G09. The VH and VL domain sequences of antibody 84G09 shown in SEQ ID NOs 92 and 93, respectively.
[0120] In a preferred embodiment, the antibody molecule of the invention comprises (i) a CDR-based antigen binding site for PD-L1 comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of antibody 84G09, and (ii) a LAG-3 antigen binding site located in a CH3 domain of the antibody molecule, wherein the LAG-3 binding site comprises the amino acid sequences set forth in SEQ ID NOs 1 and 3, and an amino acid sequence selected from the group consisting of: SEQ ID NOs 2, 8, 13, 18, 23, 28, 33, 38, 43, and 48.
[0121] More preferably, the antibody molecule of the invention comprises (i) a CDR-based antigen binding site for PD-L1 comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of antibody 84G09, and (ii) a LAG-3 antigen binding site located in a CH3 domain of the antibody molecule, wherein the LAG-3 binding site comprises the amino acid sequences set forth in SEQ ID NOs 1 and 3, and an amino acid sequence selected from the group consisting of: SEQ ID NOs 2, 28, and 38.
[0122] Yet more preferably, the antibody molecule of the invention comprises (i) a CDR-based antigen binding site for PD-L1 comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences of antibody 84G09, and (ii) a LAG-3 antigen binding site located in a CH3 domain of the antibody molecule, wherein the LAG-3 binding site comprises the amino acid sequences set forth in SEQ ID NOs 1, 2 and 3.
[0123] In a preferred embodiment, the antibody molecule of the invention comprises a VH domain and a VL domain which comprises, has, or consists of the sequence set forth in SEQ ID NOs 92 and 93, respectively, and a CH3 domain which comprises, has, or consists of the sequence set forth in SEQ ID NO: 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50, preferably a CH3 which comprises, has, or consists of the sequence set forth in SEQ ID NO: 5, 30, or 40, more preferably, a CH3 domain which comprises, has, or consists of the sequence set forth in SEQ ID NO: 5.
[0124] In a further preferred embodiment, the antibody molecule comprises a heavy chain which comprises, has, or consists of the sequence set forth in SEQ ID NOs: 94 to 113 and a light chain which comprises, has, or consists of the sequence set forth in SEQ ID NO: 116. More preferably, the antibody molecule comprises a heavy chain which comprises, has, or consists of the sequence set forth in SEQ ID NOs: 94, 95, 104, 105, 108, and 109 and a light chain which comprises, has, or consists of the sequence set forth in SEQ ID NO: 116. Most preferably, the antibody molecule comprises a heavy chain which comprises, has, or consists of the sequence set forth in SEQ ID NO: 94 or 95 and a light chain which comprises, has, or consists of the sequence set forth in SEQ ID NO: 116.
[0125] The antibody molecules of the present invention may also comprise variants of the structural loop, CH3 domain, CH2 domain, CH2 and CH3 domain, light chain or heavy chain sequences disclosed herein, provided the VL and VH domains of the light and heavy chain sequences, respectively, remain unchanged. Suitable variants can be obtained by means of methods of sequence alteration, or mutation, and screening. In a preferred embodiment, an antibody molecule comprising one or more variant sequences retains one or more of the functional characteristics of the parent antibody molecule, such as binding specificity and/or binding affinity for LAG-3 and PD-L1. For example, an antibody molecule comprising one or more variant sequences preferably binds to LAG-3 and/or PD-L1 with the same affinity, or a higher affinity, than the (parent) antibody molecule. The parent antibody molecule is an antibody molecule which does not comprise the amino acid substitution(s), deletion(s), and/or insertion(s) which have been incorporated into the variant antibody molecule.
[0126] For example, an antibody molecule of the invention may comprise a structural loop, CH3 domain, CH2 domain, CH2 and CH3 domain, light chain or heavy chain sequence which has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to a structural loop, CH3 domain, CH2 domain, CH2 and CH3 domain, light chain or heavy chain sequence disclosed herein, provided the VL and VH domains of the light and heavy chain sequences, respectively, remain unchanged.
[0127] In a preferred embodiment, the antibody molecule of the invention comprises a CH3 domain sequence which has at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the CH3 domain sequence set forth in SEQ ID NO: 4, 5, or 135.
[0128] In a further preferred embodiment, the antibody molecule of the invention comprises a CH3 and CH2 domain sequence, which has at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the CH2 and CH3 domain sequence set forth in SEQ ID NO: 6 or 7.
[0129] Sequence identity is commonly defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty=12 and gap extension penalty=4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195-197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may be used.
[0130] An antibody molecule of the invention may also comprise a structural loop, CH3 domain, CH2 domain, CH2 and CH3 domain, light chain or heavy chain sequence which has one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue), preferably 20 alterations or fewer, 15 alterations or fewer, 10 alterations or fewer, 5 alterations or fewer, 4 alterations or fewer, 3 alterations or fewer, 2 alterations or fewer, or 1 alteration compared with a structural loop, CH3 domain, CH2 domain, CH2 and CH3 domain, light chain or heavy chain sequence disclosed herein, provided the VL and VH domains of the light and heavy chain sequences, respectively, remain unchanged. In particular, alterations may be made in one or more framework regions of the antibody molecule outside the VH and VL domain sequences.
[0131] In a preferred embodiment, the antibody molecule of the invention may comprise a CH3 domain sequence with one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue), preferably 20 alterations or fewer, 15 alterations or fewer, 10 alterations or fewer, 5 alterations or fewer, 4 alterations or fewer, 3 alterations or fewer, 2 alterations or fewer, or 1 alteration compared with the CH3 domain sequence set forth in SEQ ID NO: 4, 5, or 135.
[0132] In a further preferred embodiment, the antibody molecule of the invention comprises a CH3 and CH2 domain sequence, with one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue), preferably 20 alterations or fewer, 15 alterations or fewer, 10 alterations or fewer, 5 alterations or fewer, 4 alterations or fewer, 3 alterations or fewer, 2 alterations or fewer, or 1 alteration compared with the CH2 and CH3 domain sequence set forth in SEQ ID NO: 6 or 7.
[0133] Also contemplated is an antibody molecule which competes with an antibody molecule of the invention for binding to LAG-3 and/or PD-L1, or which binds to the same epitope on LAG-3 and/or PD-L1 as an antibody molecule of the invention, wherein the antibody molecule comprises both a CDR-based antigen binding site for PD-L1 and a LAG-3 antigen binding site located in a CH3 domain of the antibody molecule. Methods for determining competition for an antigen by two antibodies are known in the art. For example, competition of binding to an antigen by two antibodies can be determined using BIAcore. Methods for mapping the epitope bound by an antibody are similarly known in the art.
[0134] The antibody molecule of the invention preferably binds to LAG-3 with an affinity (K.sub.D) of 1.times.10.sup.-9 M or an affinity which is greater. For example, the antibody molecule of the invention may bind to LAG-3 with an affinity (K.sub.D) of 8.times.10.sup.-10 M, or an affinity which is greater.
[0135] Fcabs have a smaller binding interface than monoclonal antibodies as the binding sites of Fcabs form a relatively compact antibody fragment with two binding sites situated in close proximity. In contrast, the Fab arms of a typical mAb are separated by a flexible hinge region. The two antigen binding sites of an Fcab are also spatially close to each other, as compared with those of a typical mAb. Based on this smaller binding interface and reduced flexibility of the two binding sites it was surprising that the anti-LAG-3 Fcabs were able to bind to and inhibit LAG-3 with similar affinity and potency as a monoclonal antibody benchmark.
[0136] The antibody molecule of the invention preferably binds to PD-L1 with an affinity (K.sub.D) of 1.times.10.sup.-9 M or an affinity which is greater.
[0137] The binding affinity of an antibody molecule to a cognate antigen, such as LAG-3 or PD-L1 can be determined by surface plasmon resonance (SPR), for example. The binding affinity of an antibody molecule to a cognate antigen, such as LAG-3 or PD-L1, expressed on a cell surface can be determined by flow cytometry.
[0138] The antibody molecule of the present invention is preferably capable of binding to LAG-3 and PD-L1 expressed on the surface of a cell. The cell is preferably a cancer cell.
[0139] The antibody molecule of the present invention is preferably capable of simultaneously binding to LAG-3 and PD-L1. In a preferred embodiment, the antibody molecule of the present invention is capable of simultaneously binding to LAG-3 and PD-L1, wherein the LAG-3 and PD-L1 are expressed on the surface of a single cell, or on the surface of two separate cells.
[0140] The antibody molecule of the invention may bind to human LAG-3, murine LAG-3, and/or cynomolgus monkey LAG-3. Preferably, the antibody molecule of the invention binds to human LAG-3. Most preferably, the antibody molecule of the invention binds to human LAG-3 and human PD-L1.
[0141] The antibody molecule of the invention comprises (i) a CDR-based antigen binding site for PD-L1; and (ii) a LAG-3 antigen binding site located in a constant domain of the antibody molecule. Antibody molecules which do not comprise a LAG-3 antigen binding site located in a constant domain, such as a CH3 domain, of the antibody molecule thus do not form part of the present invention. Similarly, a molecule which does not comprise a CDR-based antigen binding site for PD-L1 does not form part of the present invention.
[0142] The antibody molecule of the present invention may be conjugated to a therapeutic agent or detectable label. In this case, the antibody molecule may be referred to as a conjugate. For example, the antibody molecule may be conjugated to an immune system modulator, cytotoxic molecule, radioisotope, or detectable label. The immune system modulator or cytotoxic molecule may be a cytokine. The detectable label may be a radioisotope, e.g. a non-therapeutic radioisotope.
[0143] The antibody molecule may be conjugated to the therapeutic agent or detectable label, by means of a peptide bond or linker, i.e. within a fusion polypeptide comprising said therapeutic agent or detectable label and the antibody molecule or a polypeptide chain component thereof. Other means for conjugation include chemical conjugation, especially cross-linking using a bifunctional reagent (e.g. employing DOUBLE-REAGENTS.TM. Cross-linking Reagents Selection Guide, Pierce).
[0144] The antibody molecule and the therapeutic agent or detectable label may thus be connected to each other directly, for example through any suitable chemical bond or through a linker, for example a peptide linker.
[0145] The peptide linker may be a short (2-20, preferably 2-15, residue stretch of amino acids). Suitable examples of peptide linker sequences are known in the art. One or more different linkers may be used. The linker may be about 5 amino acids in length.
[0146] The chemical bond may be, for example, a covalent or ionic bond. Examples of covalent bonds include peptide bonds (amide bonds) and disulphide bonds. For example the antibody molecule and therapeutic or diagnostic agent may be covalently linked. For example by peptide bonds (amide bonds). Thus, the antibody molecule and therapeutic or diagnostic agent may be produced (secreted) as a single chain polypeptide.
[0147] The invention also provides isolated nucleic acids encoding the antibodies molecules of the invention. The skilled person would have no difficulty in preparing such nucleic acids using methods well-known in the art. An isolated nucleic acid may be used to express the antibody molecule of the invention, for example, by expression in a bacterial, yeast, insect or mammalian host cell. A preferred host cell is a mammalian cell such as a CHO, HEK or NSO cell. The nucleic acid will generally be provided in the form of a recombinant vector for expression.
[0148] The isolated nucleic acid may, for example, comprise the sequence set forth in SEQ ID NO: 142, 4, 9, 14, 19, 24, 29, 34, 39, 44, or 49, which encode the CH3 domains of FS18-7-9 (CHO codon optimised nucleotide sequence), FS18-7-9 (HEK293-expressed nucleotide sequence), FS18-7-32, FS18-7-33, FS18-7-36, FS18-7-58, FS18-7-62, FS18-7-65, FS18-7-78, FS18-7-88, and FS18-7-95, respectively.
[0149] In vitro host cells comprising such nucleic acids and vectors are part of the invention, as is their use for expressing the antibody molecules of the invention, which may subsequently be purified from cell culture and optionally formulated into a pharmaceutical composition. The present invention thus further provides a method of producing the antibody molecule of the invention, comprising culturing the recombinant host cell of the invention under conditions for production of the antibody molecule. Methods for culturing suitable host cells as mentioned above are well-known in the art. The method may further comprise isolating and/or purifying the antibody molecule. The method may also comprise formulating the antibody molecule into a pharmaceutical composition, optionally with a pharmaceutically acceptable excipient or other substance as described below.
[0150] PD-L1 is known to be expressed on many cancer cells, while expression of LAG-3 on cancer cells is more limited. Both are expressed on cells of the immune system. In particular, LAG-3 is known to be expressed on exhausted T cells within the tumour environment. In addition, the present inventors have shown that the use of an antibody molecule which binds to both LAG-3 and PD-L1 is effective in suppressing tumour growth in syngeneic mouse models of cancer, and that such antibody molecules are more effective than the administration of two binding molecules which bind LAG-3 and PD-L1, respectively.
[0151] Thus, an antibody molecule of the invention may be used in a method of treating cancer in a patient. The patient is preferably a human patient.
[0152] Cells of the cancer to be treated using the antibody molecule of the invention may express LAG-3, e.g. on their cell surface. In one embodiment, cells of the cancer to be treated may have been determined to express LAG-3, e.g. on their cell surface. For example, B cell lymphomas have been shown to express LAG-3 on their cell surface. Methods for determining the expression of an antigen on a cell surface are known in the art and include, for example, flow cytometry.
[0153] Example 4 below shows that the antibody molecules of the present invention can be used to treat tumours with high levels of LAG-3-expressing immune cells, such as LAG-3-expressing TILs, in mice. Thus, in addition, or alternatively, tumours of the cancer to be treated using the antibody molecule of the invention may comprise LAG-3 expressing immune cells. LAG-3 expressing immune cells, such as LAG-3 expressing TILs, are present between tumour cells in many cancers. In one embodiment, tumours of the cancer to be treated using the antibody molecule of the invention have been determined to contain LAG-3 expressing immune cells. Methods for determining the presence of LAG-3 expressing immune cells in a tumour or in the periphery of the tumour are known in the art.
[0154] Example 4 below also shows that the antibody molecules of the present invention can be used to treat tumours which express PD-L1 on their cell surface. Thus, in addition, or alternatively, cells of the cancer to be treated using the antibody molecule of the invention may express PD-L1, e.g. on their cell surface. In addition, or alternatively, tumours of the cancer to be treated may comprise immune cells, such as TILs, that express PD-L1. Cells of the cancer to be treated may have been determined to express PD-L1, e.g. on their cell surface. In addition, or alternatively, tumours of the cancer to be treated may have been determined to contain immune cells, such as TILs, that express PD-L1.
[0155] Cell surface expression of LAG-3 and PD-L1 is expected to allow the antibody molecule to bind to LAG-3 and PD-L1 expressed on the surface of the immune cell and/or the cancer cell. This is thought to result in directed therapy, bridging, and localisation of cancer cells and immune cells.
[0156] A cancer to be treated using an antibody molecule of the invention may be selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma (such as diffuse large B-cell lymphoma, indolent non-Hodgkin's lymphoma, mantle cell lymphoma, ovarian cancer, prostate cancer, colorectal cancer, fibrosarcoma, renal cell carcinoma, melanoma, pancreatic cancer, breast cancer, glioblastoma multiforme, lung cancer (such as non-small cell lung cancer), head and neck cancer (such as head and neck squamous cell carcinoma), stomach cancer (gastric cancer), bladder cancer, cervical cancer, uterine cancer, vulvar cancer, testicular cancer, penile cancer, leukemia (such as chronic lymphocytic leukemia, myeloid leukemia, acute lymphoblastoid leukaemia, or chronic lymphoblastoid leukaemia), multiple myeloma, squamous cell cancer, testicular cancer, esophageal cancer (such as adenocarcinoma of the gastroesophageal junction), Kaposi's sarcoma, and central nervous system (CNS) lymphoma, hepatocellular carcinoma, nasopharyngeal cancer, Merkel cell carcinoma, and mesothelioma. Tumours of these cancers are known, or expected, to express PD-L1 on their cell surface and/or contain immune cells, such as TILs, expressing PD-L1 and/or LAG-3.
[0157] Treatment of renal cell carcinoma, lung cancer (such as non-small cell lung cancer), nasopharyngeal cancer, colorectal cancer, melanoma, stomach cancer (gastric cancer), esophageal cancer (such as adenocarcinoma of the gastroesophageal junction), ovarian cancer, cervical cancer, bladder cancer, head and neck cancer (such as head and neck squamous cell carcinoma), leukemia (such as chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma (such as diffuse large B-cell lymphoma, indolent non-Hodgkin's lymphoma, mantle cell lymphoma), and multiple myeloma using anti-LAG-3 antibodies has been investigated in clinical trials and shown promising results. Thus, the cancer to be treated using the antibody molecules of the present invention may be a renal cell carcinoma, lung cancer (such as non-small cell lung cancer), nasopharyngeal cancer, colorectal cancer, melanoma, stomach cancer (gastric cancer), esophageal cancer (such as adenocarcinoma of the gastroesophageal junction), ovarian cancer, cervical cancer, bladder cancer, head and neck cancer (such as head and neck squamous cell carcinoma), leukemia (such as chronic lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma (such as diffuse large B-cell lymphoma, indolent non-Hodgkin's lymphoma, mantle cell lymphoma), or multiple myeloma.
[0158] Treatment of melanoma, colorectal cancer, breast cancer, bladder cancer, renal cell carcinoma, bladder cancer, gastric cancer, head and neck cancer (such as squamous cell carcinoma of the head and neck), mesothelioma, lung cancer (such as non-small-cell lung cancer), ovarian cancer, Merkel-cell carcinoma, pancreatic cancer, melanoma and hepatocellular carcinoma using anti-PD-L1 antibodies has also been investigated in clinical trials and shown promising results. Thus, the cancer to be treated using the antibody molecules of the present invention may be a melanoma, colorectal cancer, breast cancer, bladder cancer, renal cell carcinoma, bladder cancer, gastric cancer, head and neck cancer (such as squamous cell carcinoma of the head and neck), mesothelioma, lung cancer (such as non-small-cell lung cancer), ovarian cancer, Merkel-cell carcinoma, pancreatic cancer, melanoma, or hepatocellular carcinoma.
[0159] Preferred cancers for treatment using the antibody molecules of the present invention are lung cancer (such as non-small-cell lung cancer), bladder cancer, head and neck cancer (squamous cell carcinoma of the head and neck), diffuse large B cell lymphoma, gastric cancer, pancreatic cancer and hepatocellular carcinoma. Tumours of these cancers are known to comprise LAG-3 expressing immune cells and to express PD-L1 either on their cell surface or to comprise immune cells expressing PD-L1.
[0160] Where the application refers to a particular type of cancer, such as breast cancer, this refers to a malignant transformation of the relevant tissue, in this case a breast tissue. A cancer which originates from malignant transformation of a different tissue, e.g. ovarian tissue, may result in metastatic lesions in another location in the body, such as the breast, but is not thereby a breast cancer as referred to herein but an ovarian cancer.
[0161] The cancer may be a primary or secondary cancer. Thus, the antibody molecule of the present invention may be for use in a method of treating cancer in a patient, wherein the cancer is a primary tumour and/or a tumour metastasis.
[0162] The antibody molecules of the invention are designed to be used in methods of treatment of patients, preferably human patients. Antibody molecules will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the antibody molecule, such as a pharmaceutically acceptable excipient. For example, a pharmaceutical composition of the present invention, may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be by injection, e.g. intravenous or subcutaneous. The antibody molecule may be administered intravenously, or subcutaneously.
[0163] Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
[0164] For intravenous injection, or injection at the site of affliction, the antibody molecule, or pharmaceutical composition comprising the antibody molecule, is preferably in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be employed, as required. Many methods for the preparation of pharmaceutical formulations are known to those skilled in the art. See e.g. Robinson ed., Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York, 1978.
[0165] A composition comprising an antibody molecules according to the present invention may be administered alone or in combination with other treatments, concurrently or sequentially or as a combined preparation with another therapeutic agent or agents, dependent upon the condition to be treated. For example, an antibody molecule of the invention may be administered in combination with an existing therapeutic agent for the disease to be treated, e.g. a cancer as mentioned above. For example, an antibody molecule of the present invention may be administered to the patient in combination with a second anti-cancer therapy, such as chemotherapy, anti-tumour vaccination (also referred to as a cancer vaccination), radiotherapy, immunotherapy, an oncolytic virus, chimeric antigen receptor (CAR) T-cell therapy, or hormone therapy.
[0166] It is expected that the antibody molecule of the invention may act as an adjuvant in anti-cancer therapy, such as chemotherapy, anti-tumour vaccination, or radiotherapy. Without wishing to be bound by theory, it is thought that administration of the antibody molecule to the patient as part of chemotherapy, anti-tumour vaccination, or radiotherapy will trigger a greater immune response against the cancer associated antigens LAG-3 and PD-L1, than is achieved with chemotherapy, anti-tumour vaccination, or radiotherapy alone. For example, anti-LAG-3 therapies have shown good efficacy in treating viral based pathologies in mice (Blackburn S D, et al., 2009).
[0167] A method of treating cancer in a patient may thus comprise administering to the patient a therapeutically effective amount of an antibody molecule according to the present invention in combination with a chemotherapeutic agent, anti-tumour vaccine, radionuclide, immunotherapeutic agent, oncolytic viruses, CAR-T cells, or agent for hormone therapy. The chemotherapeutic agent, anti-tumour vaccine, radionuclide, immunotherapeutic agent, oncolytic viruses, CAR-T cells, or agent for hormone therapy is preferably a chemotherapeutic agent, anti-tumour vaccine, radionuclide, immunotherapeutic agent, oncolytic viruses, CAR-T cells, or agent for hormone therapy for the cancer in question, i.e. a chemotherapeutic agent, anti-tumour vaccine, radionuclide, immunotherapeutic agent, oncolytic viruses, CAR-T cells, or agent for hormone therapy which has been shown to be effective in the treatment of the cancer in question. The selection of a suitable chemotherapeutic agent, anti-tumour vaccine, radionuclide, immunotherapeutic agent, oncolytic viruses, CAR-T cells, or agent for hormone therapy which have been shown to be effective for the cancer in question is well within the capabilities of the skilled practitioner.
[0168] For example, where the method comprises administering to the patient a therapeutically effective amount of an antibody molecule according to the present invention in combination with a chemotherapeutic agent, the chemotherapeutic agent from the group consisting of: taxanes, cyctotoxic antibiotics, tyrosine kinase inhibitors, PARP inhibitors, B_RAF enzyme inhibitors, alkylating agents, platinum analogs, nucleoside analogs, thalidomide derivatives, antineoplastic chemotherapeutic agents and others. Taxanes include docetaxel, paclitaxel and nab-paclitaxel; cytotoxic antibiotics include actinomycin, bleomycin, anthracyclines, doxorubicin and valrubicin; tyrosine kinase inhibitors include erlotinib, gefitinib, axitinib, PLX3397, imatinib, cobemitinib and trametinib; PARP inhibitors include piraparib; B-Raf enzyme inhibitors include vemurafenib and dabrafenib; alkylating agents include dacarbazine, cyclophosphamide, temozolomide; platinum analogs include carboplatin, cisplatin and oxaliplatin; nucleoside analogs include gemcitabine and azacitidine; antineoplastics include fludarabine. Other chemotherapeutic agents suitable for use in the present invention include methotrexate, defactinib, entinostat, pemetrexed, capecitabine, eribulin, irinotecan, fluorouracil, and vinblastine.
[0169] Vaccination strategies for the treatment of cancers has been both implemented in the clinic and discussed in detail within scientific literature (such as Rosenberg, S. 2000 Development of Cancer Vaccines). This mainly involves strategies to prompt the immune system to respond to various cellular markers expressed by autologous or allogenic cancer cells by using those cells as a vaccination method, both with or without granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF provokes a strong response in antigen presentation and works particularly well when employed with said strategies.
[0170] Administration may be in a "therapeutically effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. Thus "treatment" of a specified disease refers to amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular patient being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the type of antibody molecule, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and may depend on the severity of the symptoms and/or progression of a disease being treated. Appropriate doses of antibody molecules are well known in the art (Ledermann et al. (1991) Int. J. Cancer 47: 659-664; and Bagshawe et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). Specific dosages indicated herein, or in the Physician's Desk Reference (2003) as appropriate for an antibody molecule being administered, may be used. A therapeutically effective amount or suitable dose of an antibody molecule can be determined by comparing it's in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the size and location of the area to be treated, and the precise nature of the antibody molecule. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician. Treatment may be given before, and/or after surgery, and may be administered or applied directly at the anatomical site of surgical treatment.
[0171] Further aspects and embodiments of the invention will be apparent to those skilled in the art given the present disclosure including the following experimental exemplification.
[0172] All documents mentioned in this specification are incorporated herein by reference in their entirety.
[0173] "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
[0174] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.
[0175] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above.
EXAMPLES
Example 1--Selection and characterisation of Fcab molecules
[0176] 1.1 Naive Selection and Affinity Maturation of Anti-Human LAG-3 Fcabs
[0177] 1.1.1 Naive Selection
[0178] Naive phage libraries displaying the CH3 domain of human IgG1 (IMGT numbering 1.4-130) with randomisation within the AB (residues 14-18) and EF (residues 92-101) loops were used for selection with recombinant Fc-tagged human LAG-3 (LAG-3 Fc) antigen (R&D systems, 2319-L3-050). The libraries were selected in three rounds using antigen captured on Protein A (Life Technologies, 10002D) or Protein G (Life Technologies, 10004D) beads. The outputs were screened by ELISA and positive binders sub-cloned and expressed as soluble Fcabs (containing a truncated hinge) in Pichia pastoris using EasySelect Pichia Expression Kit (Life Technologies, K1740-01). The Fcabs were then screened for binding to recombinant human LAG-3 Fc on the Biacore 3000 (GE Healthcare). Briefly, LAG-3 Fc (R&D systems, 2319-L3-050) was coupled at a density of 7200 RU to a CM5 chip (GE Healthcare, BR-100012) using amine coupling (GE Healthcare, BR-1000-50). Fcabs were diluted in HBS-P (GE Healthcare, BR100368) buffer and injected at 250 nM, 500 nM and 1000 nM for 3 min and then allowed to dissociate in buffer for 5 min. Reference subtracted data (LAG-3 Fc flow cell 2-blank flow cell) was analyzed using BIAevaluation 3.2 software to identify binding. Fcabs were then tested for binding to HEK cell-expressed human LAG-3 (LAG-3 cloned into pcDNA5FRT vector [Life Technologies, V6010-20] [See section 1.4.5 for methodology]). Briefly, HEK 293 cells overexpressing human LAG-3 grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 .mu.g/ml Hygromycin B (Melford Laboratories Ltd, Z2475), 15 .mu.g/ml Blasticidin (Melford Laboratories Ltd, B1105) and 1 .mu.g/ml Doxycyclin (Sigma, D9891) were detached from tissue culture flasks using cell dissociation buffer (Life Technologies, 13151-014) and seeded in V-bottom 96-well plates at 2.times.10.sup.5 cells/well. Fcabs were incubated with the cells at 5 .mu.M in a 100 .mu.l volume for 1 h at 4.degree. C. The plates were washed the secondary antibody (Anti-human Fc-488, Jackson ImmunoResearch, 109-546-098) was diluted 1:1000 in PBS and 100 .mu.l was added to the cells and incubated for 30 min at 4.degree. C. The plates were washed and the cells were resuspended in 100 .mu.l PBS containing 1 .mu.g/ml DAPI (Biotium, 40043). The plate was read on a BD FACSCanto II cytometer (BD Biosciences) and the data analysed using FlowJoX. The Fcabs were then expressed in mammalian cells by transformation using lipofectamine (Life Technologies, 11668-019) into Flp-In T-Rex 293 cells (Life Technologies, R780-07). The LAG-3 binding Fcabs were tested for inhibition of binding of human MHC class II on A375 cells (ATCC, CRL-1619) to recombinant LAG-3 Fc (using the methodology in example 1.6). 54 unique Fcab sequences were identified from three rounds of phage selection, and 12 of these Fcabs were determined to bind to LAG-3 Fc by BIAcore analysis and/or bind to LAG-3 expressing HEK cells. Three of the selected Fcabs were also able to inhibit the interaction of LAG-3 with MHC class II and were selected for affinity maturation. The three Fcabs were termed FS18-3, FS18-7 and FS18-21.
[0179] 1.1.2 Affinity Maturation
[0180] First Affinity maturation
[0181] Six phage display affinity maturation libraries were constructed by randomising five residues in the AB loop (residues 14-18) and either five (residues 92-94 and 97-98) or eight (residues 92-94 and 97-101) residues in the EF loop of each of the three Fcabs identified using the naive selection process described above.
[0182] The affinity maturation libraries were selected using recombinant human LAG-3 Fc (R&D systems, 2319-L3-050) and HEK cells expressing human LAG-3 (as described above). The outputs were screened by phage ELISA, the positive binders were subcloned and expressed as soluble Fcabs (containing a truncated hinge) in HEK Expi293 cells (Fcabs cloned into pTT5 vector [National Research Council of Canada] transfected using ExpiFectamine 293 Transfection kit [Life Technologies, A14524] into Expi293F cells [Life technologies, A14527]). The HEK expressed soluble Fcabs were then screened for binding to cell expressed human LAG-3, binding to cell expressed cynomolgus LAG-3 (methodology as example 1.4.3), and the ability to block MHC class II binding to recombinant LAG-3 Fc (methodology as in example 1.6). The blocking Fcabs were further tested to determine whether they were able to reverse LAG-3 induced inhibition of IL-2 secretion in a T cell activation assay (methodology as in example 2.1). 61 unique anti-LAG-3 Fcabs were identified from the six affinity maturation libraries using these screening methods. Affinity matured Fcabs from the FS18-7 lineage were shown to have the highest level of cross-reactivity with cynomolgus monkey LAG-3. The three Fcabs from this lineage with the strongest binding to cynomolgus monkey LAG-3 Fc and the highest activity in the T cell activation assay (termed FS18-7-7, FS18-7-9, and FS18-7-11) were selected for further affinity maturation. These three Fcabs were also shown to block the interaction of LAG-3 Fc with cell expressed MHC class II.
[0183] Second Affinity Maturation
[0184] A pool of the three Fcabs (FS18-7-7, FS18-7-9, and FS18-7-11) from the first affinity maturation was used to create further affinity maturation libraries. The CD loop was hard randomized using randomized primers from ELLA Biotech. A portion of amino acid positions in the CD loop (residues 45.1-78) was randomized using an equimolar distribution of amino acids excluding cysteine. Error prone PCR was also carried out across the entire CH3 domain sequence to introduce additional mutations that might enhance binding.
[0185] The affinity maturation libraries were generated in phage and selections performed against biotinylated recombinant LAG-3 avi-Fc (BPS Bioscience, 71147) and HEK hLAG-3 cells and screened for binding to recombinant LAG-3 Fc (R&D systems, 2319-L3-050) by phage ELISA. 86 unique Fcabs (containing a truncated hinge) were expressed in HEK293F cells. Selected Fcabs were also screened for activity in a T cell activation assay as described above. The nine Fcabs identified during the second affinity maturation with the highest activity in the T cell activation assay (FS18-7-32; FS18-7-33; FS18-7-36; FS18-7-58; FS18-7-62; FS18-7-65; FS18-7-78; FS18-7-88; and FS18-7-95), as well as the parental Fcab clone, FS18-7-9, were then further characterised as described below. A sequence alignment of these nine Fcabs against the parental Fcab clone, FS18-7-9, is shown in FIG. 1A. FIG. 1B details the percentage sequence identity of each of the nine Fcab clones to the parental Fcab clone, FS18-7-9. Fcabs originating from affinity maturation of the two other parental Fcab clones, FS18-7-7 and FS18-7-11, were not as promising candidates as those originating from affinity maturation of FS18-7-9 and were therefore not pursued further.
[0186] 1.2 Selection of Surrogate Fcab Specific for Mouse LAG-3
[0187] Fcab FS18-7, which was selected using the naive selection protocol described above, was used to generate phage libraries to select against mouse LAG-3. Two rounds of affinity maturation were performed, and Fcab clones FS18-7-108-29 and FS18-7-108-35, which showed high-affinity, specific binding to mouse LAG-3 were selected following affinity maturation. The ability of FS18-7-108-29 and FS18-7-108-35 to inhibit mouse LAG-3 in a T cell activation assay was confirmed. Epitope mapping using the Octet (Forteo Bio) showed that the anti-mouse LAG-3 Fcabs compete with the anti-human LAG-3 Fcabs (selected following the second affinity maturation as described above) for binding to human LAG-3. There are between 4 and 8 residue differences between the anti-human LAG-3 and anti-mouse LAG-3 Fcabs. It is therefore expected that the anti-mouse LAG-3 Fcabs represent suitable surrogates for the binding and function of the anti-human LAG-3 Fcabs in mice.
[0188] 1.3 Construction and Expression of Mock mAb.sup.2
[0189] "mock" mAb.sup.2 comprising the lead anti-human LAG-3 and anti-mouse LAG-3 Fcabs identified in 1.1 and 1.2 above were prepared in order to allow the characterisation of these Fcabs in mAb.sup.2 format. These mock mAb.sup.2 were prepared from the anti-LAG-3 Fcabs and the variable regions of anti-FITC antibody 4420 (see SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO: 85 for details) (Bedzyk, W. D., et al. 1989 and Bedzyk, W. D., et al. 1989). The mock mAb.sup.2 were prepared both with (SEQ ID NO: 63, 65, 67, 69, 71, 73, 75, 77, 79, and 81) and without (SEQ ID NO: 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82) the LALA mutation in the CH2 domain of the heavy chain (see section 1.5 below for details) and further comprised the light chain of the anti-FITC mAb 4420 (SEQ ID NO: 85). The mock mAb.sup.2 were produced by transient expression in HEK293-6E cells and purified using mAb Select SuRe protein A columns.
[0190] 1.4 Binding Affinity of Fcabs to LAG-3
[0191] 1.4.1 Binding Affinity of Fcabs to Human LAG-3 as Determined by Surface Plasmon Resonance (SPR)
[0192] A BIAcore T200 (GE Healthcare) was used to measure the affinity of the anti-human LAG-3 Fcabs in the mock mAb.sup.2 format for human LAG-3. Flow cell 4 of a CM5 sensor chip (GE Healthcare, BR1005-30) was immobilised with human LAG-3-Fc (R&D Systems, 2319-L3-050), and flow cell 3 was immobilised with buffer for reference using the amine coupling kit (GE Healthcare, BR-1000-50). LAG-3-Fc was diluted to 5 .mu.g/ml in sodium acetate pH5 (ForteoBio, 18-1069) and injected at a flow rate of 10 .mu.l/min for 12 seconds followed by deactivation of the surface by injection of ethanolamine for 420 sec. The Immobilisation level was 158 RU. The mock mAb.sup.2 (or control anti-human LAG-3 mAb, 25F7) were diluted in HBS-P buffer (GE Healthcare, BR-1003-68) in a 2-fold dilution series from 4 .mu.g/ml. The control mAb/mock mAb.sup.2 were injected with an association time of 240 seconds at 30 .mu.l/min, and a dissociation time 300 seconds at 30 .mu.l/min. The surface was regenerated using 25 mM NaOH for 30 seconds at 100 .mu.l/min. The data was double reference subtracted and analysed using the BIAevaluation 3.2 software to calculate kinetic constants. The Fcabs in mock mAb.sup.2 format had affinities for human LAG-3 in the range of 0.8-1.1 nM (Table 1), which is similar to the affinity of the benchmark anti-human LAG-3 mAb 25F7. This was surprising because Fcabs have a smaller binding interface than monoclonal antibodies as the binding sites of Fcabs form a relatively compact antibody fragment with two binding sites situated in close proximity. In contrast, the Fab arms of a typical mAb are separated by a flexible hinge region. Based on this smaller binding interface and the associated reduced flexibility of the two binding sites in the Fc region, it was unexpected that the anti-LAG-3 Fcabs were able to bind to and inhibit LAG-3 with similar affinity and potency as the benchmark antibody 25F7.
TABLE-US-00003 TABLE 1 Binding affinity of LAG-3 specific Fcabs in mock mAb.sup.2 format to human LAG-3 Anti-human LAG-3 Fcab in mock mAb.sup.2 format and benchmark anti-human LAG-3 mAb, 25F7 K.sub.D (M) FS18-7-9 8.3 .times. 10.sup.-10 FS18-7-62 9.5 .times. 10.sup.-10 FS18-7-78 8.4 .times. 10.sup.-10 FS18-7-32 8.6 .times. 10.sup.-10 FS18-7-36 8.9 .times. 10.sup.-10 FS18-7-65 1.1 .times. 10.sup.-9 25F7 3.2 .times. 10.sup.-10
[0193] 1.4.2 Binding Affinity of Surrogate Fcab Specific for Mouse LAG-3 to Mouse LAG-3 as Determined by SPR
[0194] A Biacore 3000 (GE Healthcare) was used to measure the affinity of the surrogate Fcabs specific for mouse LAG-3 to mouse LAG-3. Amine coupling (amine coupling kit, GE Healthcare, BR-1000-50) was used to coat mLAG-3 Fc (R&D Systems, 3328-L3-050) diluted in 10 mM sodium acetate pH 5.0 (ForteBio, 18-1069) directly to a CM5 chip (GE Healthcare, BR-1000-12). Flow cell 1 was coated with Mouse Fc (SinoBiological, 51094-MNAH), and flow cell 2 was coated with mLAG-3 Fc at 950 RU. Fcabs were diluted in HBS-P buffer (GE Healthcare, BR-1003-68) and injected at various concentrations (fourfold dilutions from 100 nM) for 3 min at 20 .mu.l/min and then allowed to dissociate in buffer for 12 min. The chip was regenerated by injection of 10 mM glycine pH 2.5 for 30 s at 30 .mu.l/min. Data was double reference subtracted and analyzed using BIAevaluation 3.2 software to calculate kinetic constants. The tested surrogate Fcabs bound to mouse LAG-3 with single digit nanomolar affinity as set out in Table 2.
TABLE-US-00004 TABLE 2 Binding affinity (K.sub.D) of surrogate LAG-3 specific Fcabs to mouse LAG-3 Surrogate Fcabs specific for mouse LAG-3 Affinity K.sub.D (nM) FS18-7-108-29 1.5 FS18-7-108-35 2.1
[0195] 1.4.3 Binding Affinity of Fcabs to Human LAG-3 Expressed on Cells as Determined by Flow Cytometry
[0196] Production of cell lines over-expressing LAG-3 Lentiviral transduction methodology was used to generate DO11.10 cells (National Jewish Health) over-expressing human, cynomolgus or mouse LAG-3 using the Lenti-X HTX Packaging System (Clontech, Cat. No 631249). Lenti-X expression vector (pLVX) (Clontech, Cat. No 631253), containing the mouse LAG-3 cDNA (SEQ ID NO: 96), human LAG-3 cDNA (SEQ ID NO: 95) or cynomolgus LAG-3 cDNA (SEQ ID NO: 97), was co-transfected with a Lenti-X HTX Packaging Mix into the Lenti-X 293T Cell Line (Clontech, Cat. No 632180) to generate virus. The DO11.10 cell line was transduced using the lentiviral vectors produced with the Lenti-X HTX Packaging System.
[0197] The affinity of the anti-human LAG-3 Fcabs in mock mAb.sup.2 format to cells expressing human LAG-3 (DO11.10 cell line transfected with human LAG-3) was measured using flow cytometry. mAb.sup.2 and control mAb dilutions (2.times.final concentration) were prepared in triplicate in 1.times.DPBS (Gibco, 14190-094). DO11.10:LAG-3 cell suspensions were prepared in PBS+2% BSA (Sigma, A7906) and seeded at 4.times.10.sup.-6 cell/ml with 50 .mu.l/well in V-bottomed 96-well plates (Costar, 3897). 50 .mu.l of the mAb.sup.2 or control mAb (anti human LAG-3 mAb, 25F7) dilutions were added to the wells containing cells (final volume 100 .mu.l) and incubated at 4.degree. C. for 1 hour. The plates were washed and 100 .mu.l/well of secondary antibody (anti-human Fc-488 antibody, Jackson ImmunoResearch, 109-546-098) diluted 1:1000 in PBS+2% BSA was then added and incubated for 30 mins at 4.degree. C. in the dark. The plates were washed and resuspended in 100 .mu.l of PBS containing DAPI (Biotium, 40043) at 1 mg/ml. The plates were read using Canto II flow cytometer (BD Bioscience). Dead cells were excluded and the fluorescence in the FITC channel (488 nm/530/30) was measured. The data was fitted using log (agonist) vs response in GraphPad Prism Software. All tested Fcabs in mock mAb.sup.2 format and the benchmark anti-human LAG-3 mAb, 25F7, bound human LAG-3 with similar affinity (EC.sub.50), in the range of 1.2-2.1 nM as set out in Table 3.
TABLE-US-00005 TABLE 3 Binding affinity of anti-human LAG-3 Fcabs in mock mAb.sup.2 format to DO11.10 cells expressing human LAG-3 as determined by flow cytometry Anti-human LAG-3 Fcab in mock mAb.sup.2 format and benchmark EC.sub.50 anti-human LAG-3 mAb, 25F7 (nM) FS18-7-9 1.2 FS18-7-32 1.6 FS18-7-33 1.5 FS18-7-36 1.5 FS18-7-62 2.1 FS18-7-65 1.6 FS18-7-78 1.7 25F7 2.1
[0198] 1.4.4 Binding Affinity of Fcabs to Cynomolgus LAG-3 Expressed on Cells as Determined by Flow Cytometry
[0199] The affinity of the anti-human LAG-3 Fcabs in mock mAb.sup.2 format to cells expressing cynomolgus LAG-3 (DO11.10 cell line transfected with cynomolgus LAG-3) was measured using flow cytometry. mAb.sup.2 and control mAb dilutions (2.times. final concentration) were prepared in triplicate in 1.times.DPBS (Gibco, 14190-094). DO11.10:LAG-3 cell suspensions were prepared in PBS+2% BSA (Sigma, A7906) and seeded at 4.times.10.sup.-6 cell/ml with 50 .mu.l/well in V-bottomed 96-well plates (Costar, 3897). 50 .mu.l of the mAb.sup.2 or control mAb (anti human LAG-3 mAb, 25F7) dilutions were added to the wells containing cells (final volume 100 .mu.l) and incubated at 4.degree. C. for 1 hour. The plates were washed and 100 .mu.l/well of secondary antibody (anti-human Fc-488 antibody, Jackson ImmunoResearch, 109-546-098) diluted 1:1000 in PBS+2% BSA was then added and incubated for 30 mins at 4.degree. C. in the dark. The plates were washed and resuspended in 100 .mu.l of PBS containing DAPI (Biotium, 40043) at 1 mg/ml. The plates were read using Canto II flow cytometer (BD Bioscience). The dead cells were excluded and the fluorescence in the FITC channel (488 nm/530/30) was measured. The data was fit using log (agonist) vs response in GraphPad Prism Software. The tested Fcabs in mock mAb.sup.2 format bound to cynomolgus LAG-3 with 0.5-0.6 nM affinity indicating that toxicology studies in cynomolgus monkeys would be expected to be predictive of effects seen in humans (see Table 4). The benchmark anti-human LAG-3 mAb, 25F7, binds cynomolgus LAG-3 with a 15-fold poorer affinity (EC.sub.50) (Table 4).
TABLE-US-00006 TABLE 4 Binding affinity of anti-LAG-3 Fcabs to DO11.10 cells expressing cynomolgus LAG-3 by flow cytometry Anti-human LAG-3 Fcab in mock mAb.sup.2 format and benchmark anti-human LAG-3 mAb, 25F7 EC.sub.50 (nM) FS18-7-9 0.6 FS18-7-62 0.5 FS18-7-78 0.5 25F7 9.0
[0200] 1.4.5 Binding Affinity of Surrogate Anti-Mouse LAG-3 Fcabs and Anti-Human LAG-3 Fcab to Mouse LAG-3 Expressed on Cells as Determined by Flow Cytometry
[0201] Production of HEK Cells Over-Expressing mLAG-3
[0202] The mouse LAG-3 sequence (SEQ ID NO: 96) was subdloned into pcDNA5FRT vector (Life Technologies, V6010-20) using KpnI (NEB, R0142) and NotI (NEB, R0146) restriction digestion. The vector was then transformed into Flp-In T-REx 293 HEK cell line (Life Technologies, R780-07) using Lipofectamine 2000 (Life Technologies, 11668-019). Transformed Flp-In T-REx 293 cells were grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 .mu.g/ml Hygromycin B (Melford Laboratories Ltd, Z2475), 15 .mu.g/ml Blasticidin (Melford Laboratories Ltd, B1105) for 3-4 weeks until colonies of stably transformed cells were apparent. These colonies were amplified in the presence of 1 .mu.g/ml Doxycyclin (Sigma, D9891) and tested for mouse LAG-3 expression using PE conjugated anti-mouse LAG-3 (clone C9B7W, BD Biosciences, 552380).
[0203] The affinity of the surrogate anti-mouse LAG-3 Fcabs (containing the truncated hinge; SEQ ID NO: 58) to cell-expressed mouse LAG-3 was determined using flow cytometry. HEK cells expressing mLAG-3 grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 .mu.g/ml Hygromycin B (Melford Laboratories Ltd, Z2475), 15 .mu.g/ml Blasticidin (Melford Laboratories Ltd, B1105) and 1 .mu.g/ml Doxycyclin (Sigma, D9891) were detached from tissue culture flasks using cell dissociation buffer (Life Technologies, 13151-014) and seeded in V-bottom 96-well plates (Costar, 3897) at 2.times.10.sup.5 cells/well. The plates were centrifuged at 1500 rpm for 3 min at 4.degree. C. to pellet the cells. A dilution series of the Fcabs (or control mAb) were incubated with the cells in a 100 .mu.l volume for 1 h at 4.degree. C. The plates were washed and secondary antibody (Anti-human Fc-488, Jackson ImmunoResearch, 109-546-098 for Fcabs or Anti-Rat IgG (H+L), Alexa Fluor 488 Conjugate, ThermoFisher, A-11006 for C9B7W) was diluted 1:1000 in PBS and 100 .mu.l was added to the cells for 30 min at 4.degree. C. (plates were kept in the dark). The plates were then washed and the cells resuspended in 100 .mu.l PBS containing 1 .mu.g/ml DAPI (Biotium, 40043). The plates were read using Canto II flow cytometer (BD Bioscience). Dead cells were excluded and the fluorescence in the FITC channel (488 nm/530/30) was measured. The data was fit using log (agonist) vs response in GraphPad Prism Software. The tested Fcabs bound to mouse LAG-3 with similar affinity (see Table 5). The benchmark LAG-3 mAb, C9B7W (2B Scientific, BE0174-50MG), binds mouse LAG-3 with 17-fold poorer affinity (EC.sub.50) than the Fcabs (Table 5).
TABLE-US-00007 TABLE 5 Binding affinity of surrogate anti-mouse LAG-3 Fcabs to HEK cells expressing mouse LAG-3 by flow cytometry Anti-mouse LAG-3 Fcabs and benchmark anti-mouse LAG-3 mAb, EC.sub.50 C9B7W (nM) FS18-7-108-29 4.5 FS18-7-108-35 4.5 C9B7W 79
[0204] The affinity of the anti-human LAG-3 Fcab FS18-7-9 in mock mAb.sup.2 format to cell-expressed mouse LAG-3 was determined using flow cytometry. HEK cells expressing mLAG-3 grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 .mu.g/ml Hygromycin B (Melford Laboratories Ltd, Z2475), 15 .mu.g/ml Blasticidin (Melford Laboratories Ltd, B1105) and 1 .mu.g/ml Doxycyclin (Sigma, D9891) were detached from tissue culture flasks using cell dissociation buffer (Life Technologies, 13151-014). Cells were collected by centrifuged at 1500 rpm for 3 min at 4.degree. C. to pellet the cells and then resuspended in 1.times.DPBS then seeded in V-bottom 96-well plates (Costar, 3897) at 1.2.times.10.sup.5 cells/well in 30 .mu.l. A 1:1 volume of a dilution series of the mAb.sup.2 (or control mAb) was added and incubated with the cells for 1 h at 4.degree. C. The plates were washed and secondary antibody (Anti-human Fc-488, Jackson ImmunoResearch, 109-546-098) was diluted 1:1000 in PBS and 60 .mu.l was added to the cells for 30 min at 4.degree. C. (plates were kept in the dark). The plates were then washed and the cells resuspended in 60 .mu.l PBS containing 1 .mu.g/ml DAPI (Biotium, 40043). The plates were read using Canto II flow cytometer (BD Bioscience). Dead cells were excluded and the fluorescence in the FITC channel (488 nm/530/30) was measured. The data was fitted using log (agonist) vs response in GraphPad Prism Software. The anti-human LAG-3 Fcab FS18-7-9 in mock mAb.sup.2 format bound to mouse LAG-3 with an EC.sub.50 of 19 nM compared to an EC.sub.50 of 2.6 nM for the surrogate anti-mouse LAG-3 Fcab FS18-7-9-108 (Table 6). The human mAb, 25F7 does not show any detectable binding to mouse LAG-3, indicating that the human LAG-3 Fcab, FS18-7-9, has a different binding epitope on LAG-3 than that of 25F7.
TABLE-US-00008 TABLE 6 Binding affinity of human anti-LAG-3 Fcab FS18-7-9 to HEK cells expressing mouse LAG-3 by flow cytometry Anti-human LAG-3 Fcab, anti-mouse LAG-3 Fcab and benchmark anti-human LAG-3 mAb, 25F7 EC.sub.50 (nM) FS18-7-108-29 2.6 FS18-7-9 19 25F7 No binding
[0205] 1.5 Binding Affinity of Fcabs to Fc Receptors
[0206] The introduction of the LALA mutation in the CH2 domain of human IgG1 is known to reduce Fc .gamma. receptor binding (Bruhns, P., et al. (2009); and Xu, D. et al. (2000)). BIAcore was used to confirm that the LALA mutation had reduced the binding affinity of the Fcabs (in mock mAb.sup.2 format) to Fc.gamma. receptors. The human Fc.gamma.R binding assay was performed on a Biacore T200 instrument (GE Healthcare) using the Fcabs in the mock mAb.sup.2 format. Human Fc.gamma.Rs (R&D Systems, 1257-FC, 1330-CD, 1875-CD, 4325-FC) were immobilized using amine coupling (amine coupling kit, GE Healthcare, BR-1000-50) onto a Series S CM5 chip (GE Healthcare, BR-1005-30) to a surface density of 370 RU for Fc.gamma.RI, 264 RU for Fc.gamma.RIII (high affinity human Fc.gamma.Rs) and 500 RU for Fc.gamma.RIIa and Fc.gamma.RIIb (low affinity human Fc.gamma.Rs). For each immobilized chip a flow cell was left blank for background subtraction. Fc.gamma.R were immobilized using a concentration of 5 .mu.g/ml in sodium acetate pH5 (ForteBio, 18-1069) and injected at a flow rate of 10 .mu.l/min in 15 second cycles until the required immobilization level was reached.
[0207] For the high affinity Fc.gamma.RI and Fc.gamma.RIII, 200 .mu.g/ml of mAbs or mock mAb.sup.2 were flowed across the chip for 3 min at a flow rate of 30 .mu.l/min and the dissociation was followed for 5 min. Running buffer was HBS-P (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% v/v Surfactant P20, GE Healthcare, BR-1003-68). For the low affinity Fc.gamma.RIIa and Fc.gamma.RIIb the concentration of mock mAb.sup.2 was increased to 500 .mu.g/ml.
[0208] The positive control was a wild type IgG1 isotype mAb, which was compared to controls LALA IgG1 mAb and monoclonal IgG2 and IgG4 isotype mAbs to irrelevant targets. The flow cells were regenerated by injecting 10 mM sodium hydroxide (VWR, 28244.262) at a flow rate of 100 .mu.l/min for 30 seconds. The data analysis was performed with BiaEvaluation software version 3.2 RC1 by double referencing against the blank flow cell (without immobilized Fc.gamma.R) and subtracting a buffer cycle from test mAb.sup.2. The results are shown in Table 7.
TABLE-US-00009 TABLE 7 Binding response of anti-human LAG-3 Fcabs in mock mAb.sup.2 format (comprising LALA mutation as detailed above) to human Fc.gamma. receptors by SPR Binding response at end of association (RU) mAb/mock mAb.sup.2 Fc.gamma.RI Fc.gamma.RIII Fc.gamma.RIIa Fc.gamma.RIIb FS18-7-9 1.4 6.6 -9.8 -8.5 FS18-7-62 -0.9 0.7 -10 -8.5 FS18-7-78 -0.3 4.0 -10.7 -9.2 mock mAb LALA 2 8.0 -12.7 -9.6 IgG2 0 1.9 9.7 7.4 IgG4 9 3.1 4.3 15.1 mock mAb IgG1 26 44 13 17.7
[0209] All mock mAb.sup.2 tested (all comprising the LALA mutation as set out above) showed significantly reduced binding to the tested Fc.gamma. receptors compared to the control antibody (mock mAb IgG1) without the LALA mutation, indicating that the LALA mutation has reduced Fc.gamma. receptor binding by these mock mAb.sup.2 and therefore is expected to reduce ADCC activity of the mAb.sup.2.
[0210] 1.6 Blocking of MHC Class II Binding to LAG-3
[0211] The ability of the Fcabs (containing the truncated hinge; SEQ ID NO: 58) to block the interaction between recombinant human or mouse LAG-3 Fc and human MHC Class II was studied by measuring binding of LAG-3 Fc to A375 cells, a melanoma cell line that expresses human MHC Class II. A375 (ATCC, CRL-1619) cells grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-106) were detached from cell culture flasks using cell dissociation buffer (Life Technologies, 13151-014) and seeded in V-bottom 96-well plates (Costar, 3897) at 2.times.10.sup.5 cells/well. The plates were centrifuged at 1500 rpm for 3 min at 4.degree. C. to pellet the cells. The relevant concentrations of Fcab or control mAb were incubated with 1 .mu.g/ml LAG-3 Fc (human LAG-3-Fc R&D Systems, 2319-L3-050 or mouse LAG-3 Fc R&D Systems, 3328-L3-050) in 100 .mu.l DMEM containing 10% FBS for 1 h at 4.degree. C. LAG-3/Fcab mix was added to the A375 cells and incubated for 1 h at 4.degree. C. Cells were washed. Secondary antibody (Alexa Fluor 488 conjugated goat anti-human Fc F(ab').sub.2, Jackson Immunoresearch, 109-546-098 or Goat anti-mouse IgG (H+L) 488 conjugate, Life Technologies, A-1101) was diluted 1:1000 in PBS and 100 .mu.l was added to the cells for 30 min at 4.degree. C. (plates were kept in the dark). Cells were washed once in PBS and resuspended in 100 .mu.l PBS+1 .mu.g/ml DAPI (Biotium, 40043). The plates were read on a BD FACSCanto II cytometer (BD Biosciences) and the data analysed using FlowJo software.
[0212] Both anti-mouse LAG-3 Fcabs were able to inhibit the interaction of human MHC class II with mouse LAG-3, whereas the control anti-mouse LAG-3 mAb (C9B7W, 2B Scientific, BE0174-50MG) was not (see Table 8).
TABLE-US-00010 TABLE 8 Surrogate anti-mouse LAG-3 Fcabs inhibit binding of mouse LAG-3 to MHC class II Surrogate anti-mouse LAG-3 Fcabs and control anti-mouse LAG-3 mAb C9B7W IC.sub.50 (nM) F518-7-108-29 0.6 F518-7-108-35 0.7 C9B7W No blocking
[0213] The anti-human LAG-3 Fcabs tested were also able to inhibit the interaction of human MHC class II with human LAG-3 with a similar potency as the control anti-human LAG-3 mAb (25F7).
TABLE-US-00011 TABLE 9 Anti-human LAG-3 Fcabs inhibit binding of human LAG-3 to MHC class II Anti-human LAG-3 Fcabs and control anti-human LAG-3 mAb 25F7 IC.sub.50 (nM) FS18-7-108-33 2.6 FS18-7-108-78 2.4 25F7 3.6
Example 2: Preparation and Characterisation of mAb and mAb.sup.2 Molecules
[0214] 2.1 Preparation of mAb 84G09
[0215] 2.1.1 DNA Construct Generation
[0216] DNA inserts encoding variable heavy and light chain regions of 84G09 were codon optimised for mammalian expression and synthesised by DNA2.0 (Menlo Park, Calif., USA). The inserts supplied in pJ-Amp-high host vector were sub-cloned into expression vectors pFS-hHC2.1-G1 m17(z) LALA (IgG heavy chain containing LALA mutation) or pFS-hHC2.1-G1m17(z) (IgG heavy chain without LALA mutation) and pFShK1.0 (IgG kappa light chain) via EcoRI and NheI restriction digest.
[0217] Fidelity of cloning was verified by colony PCR and subsequent nucleotide sequencing analysis by a third party (GATC Biotech).
[0218] 2.1.2 Cell Maintenance
[0219] HEK293-6E cells (NRCC), were sub-cultured in pre-warmed F17 medium (Invitrogen, A13835-01) supplemented with 4 mM of GlutaMAX-1 (Invitrogen, 35050-038), 0.1% of Pluronic F-68 (Invitrogen A13835-01) and 25 .mu.g/ml of geneticin (Invitrogen, 10131-027). Cells were incubated at 37.degree. C., 140 rpm, 5% CO.sub.2 and subcultured at 0.3.times.10.sup.6 cells/ml on a three then four day regime.
[0220] 2.1.3 Transient Transfection
[0221] HEK293-6E cells were transfected transiently using PEIpro at 1 mg/ml (Polyplus, PPLU115). 24 hours prior transfection cells were seeded at 0.8.times.10.sup.6 cells/ml in culture medium. For each 200 ml of cell culture, a DNA mixture was prepared by mixing 10 ml of warmed Opti-MEMI (Invitrogen, 11058-021), 100 .mu.g endotoxin-free DNA encoding the heavy chain and 100 .mu.g endotoxin-free DNA encoding the light chain. PEI mixture was prepared by mixing 10 ml of warmed Opti-MEMI and 200 .mu.l of PEIpro and vortexing. The DNA mixture was quickly added to the vortexed PEI mixture, mixed by vortex pulsing 3 times for 1 s, incubated 3 min at room temperature and added drop by drop to the cells. 48 hours after transfection 20 ml of F17 plus supplements with 0.5% Tryptone N1 (TekniScience Inc., 19553) was added to each flask. 6 days after transfection cells were harvested by centrifugation at 4500 rpm for 40 min. Supernatant was then filtered with 0.22 .mu.m polyethersulfone filter unit (Millipore, SCGPU01 RE, SCGPU02RE, SCGPU05RE, SCGPU11RE) and stored at +4.degree. C. until purification.
[0222] 2.1.4 Protein A Chromatography
[0223] Clarified supernatants were purified using pre-packed 5 ml HiTrap MabSelect SuRe columns (GE Healthcare, 11-0034-95) on an .ANG.KTAexplorer or .ANG.KTAxpress. Briefly, the columns were equilibrated with 50 mM Tris-HCl, 250 mM NaCl at pH 7.0, unbound material washed with the same buffer at 5 ml/min. The products were eluted with 10 mM sodium formate pH 3.0 at 5 ml/min. Eluted samples were immediately buffer exchange into PBS pH 7.4 using PD-10 columns (GE Healthcare, 17-0851-01) pre-equilibrated with PBS pH 7.4 according to the manufacturer recommendations.
[0224] 2.1.5 Product Concentration Measurement by Spectrometry
[0225] Absorbance at 280 nm of each purified product was measured using LabChip DS (PerkinElmer, 133089) with DropPlate 96 D+ (PerkinElmer, CLS135136). The product concentration was calculated using extinction coefficient (A280 of 1 mg/ml) calculated using VectorNTI Advance v11.5.4 software (Thermofisher Scientific, A13784)
[0226] 2.1.6 Product Concentration
[0227] When necessary, purified fractions were concentrated using Amicon Ultra-4 Centrifugal Filter Unit 30K (Millipore, UFC803024). After equilibration of the Ultracel regenerated cellulose membrane with PBS pH 7.4 by centrifugation 10 min at 3000 rpm, samples were loaded to the 4 ml unit and centrifuged at 3000 rpm until desired protein concentration was reached.
[0228] 2.1.6 Filter Sterilisation
[0229] Final samples were filtered using pre-wet Millex-GV PVDF syringe filters (Millipore, SLGV013SL).
[0230] 2.2 Preparation of Human LAG-3/PD-L1 mAb.sup.2
[0231] The heavy chains of the mAb.sup.2 molecules FS18-7-9/84G09 (SEQ ID NOs 94 and 95), FS18-7-32/84G09 (SEQ ID NOs 96 and 97), FS18-7-33/84G09 (SEQ ID NOs 98 and 99), FS18-7-36/84G09 (SEQ ID NOs 100 and 101), FS18-7-58/84G09 (SEQ ID NOs 102 and 103), FS18-7-62/84G09 (SEQ ID NOs 104 and 105), FS18-7-65/84G09 (SEQ ID NOs 106 and 107), FS18-7-78/84G09 (SEQ ID NOs 108 and 109), FS18-7-88/84G09 (SEQ ID NOs 110 and 111) and FS18-7-95/84G09 (SEQ ID NOs 112 and 113) were prepared by replacing the CH3 domains of the monoclonal antibodies 84G09 (with and without the LALA mutation) with the CH3 domains of the human LAG-3 specific Fcabs FS18-7-9, FS18-7-32, FS18-7-33, FS18-7-36, FS18-7-58, FS18-7-62, FS18-7-65, FS18-7-78, FS18-7-88 and FS18-7-95 within XhoI and BamH/sites present in the sequence of the unmodified CH3 domain of human IgG1. The heavy chain of the mAb.sup.2 were co-transfected with the light chain of 84G09 (SEQ ID NO: 116) as described for mAb 84G09 in section 2.1 above. The mAb.sup.2 were then expressed and purified as described for mAb 84G09 in section 2.1 above.
[0232] 2.3 Preparation of Human LAG-3/Mock mAb.sup.2
[0233] The anti-FITC mAb (with and without LALA mutation) was prepared as described for mAb 84G09 in section 2.1 above using the heavy chains (SEQ ID NOs 83 and 84; with and without LALA mutation) and light chain (SEQ ID NO: 85) of mAb 4420.
[0234] The heavy chains of the mAb.sup.2 molecules FS18-7-9/4420 (SEQ ID NOs 63 and 64), FS18-7-32/4420 (SEQ ID NOs 65 and 66), FS18-7-33/4420 (SEQ ID NOs 67 and 68, FS18-7-36/4420 (SEQ ID NOs 69 and 70), FS18-7-58/4420 (SEQ ID NOs 71 and 72), FS18-7-62/4420 (SEQ ID NOs 73 and 74), FS18-7-65/4420 (SEQ ID NOs 75 and 76), FS18-7-78/4420 (SEQ ID NOs 77 and 78), FS18-7-88/4420 (SEQ ID NOs 79 and 80) and FS18-7-95/4420 (SEQ ID NOs 81 and 82) were prepared by replacing the CH3 domains of the monoclonal antibodies 4420 (with and without the LALA mutation) with the CH3 domains of the human LAG-3 specific Fcabs FS18-7-9, FS18-7-32, FS18-7-33, FS18-7-36, FS18-7-58, FS18-7-62, FS18-7-65, FS18-7-78, FS18-7-88 and FS18-7-95 within XhoI and BamH/sites present in the sequence of the unmodified CH3 domain of human IgG1. The heavy chains of the mAb.sup.2 were co-transfected with the light chain of mAb 4420 as described for mAb 84G09 in section 2.1 above. The proteins were then expressed and purified as described for mAb 84G09 in section 2.1 above.
[0235] 2.4 Preparation of Mouse LAG-3/PD-L1 mAb.sup.2
[0236] The mouse anti-PD-L1 mAb (with and without LALA mutation) was prepared as described for mAb 84G09 in section 2.1 above using the heavy chain (SEQ ID NOs 122 and 123) and light chain (SEQ ID NO: 119) of mAb S1.
[0237] The heavy chain of the mAb.sup.2 molecules FS18-7-108-29/S1 (SEQ ID NOs 117 and 118) and FS18-7-108-35/S1 (SEQ ID NOs 120 and 121) were prepared by replacing the CH3 domains of the monoclonal antibodies S1 (with and without the LALA mutation) with the CH3 domains of the mouse LAG-3 specific Fcabs FS18-7-108-29 and FS18-7-108-35 within XhoI and BamH/sites present in the sequence of the unmodified CH3 domain of human IgG1. The heavy chain of the mAb.sup.2 were co-transfected with the light chain of S1 as described for mAb 84G09 in section 2.1 above. The proteins were then expressed and purified as described for mAb 84G09 in section 2.1 above.
[0238] 2.5 Binding Affinity and Kinetics of mAb.sup.2 for Human LAG-3 and Human PD-L1
[0239] Protein L (Thermo, 21189) was immobilized on flow cells 1 and 2 of a Series S CM5 chip (GE Healthcare, BR-1005-30) by amine coupling (GE Healthcare, BR-1000-50) to a surface density of 2000 RU by following the manufacturer's instructions for the BIAcore T200 instrument. For LAG-3 binding, the mAb.sup.2 samples (all containing the LALA mutation) were captured on flow cell 2 only and human LAG-3Fc (R&D Systems, 2319-L3) at 4 concentrations in a two-fold dilution series starting at 0.5 nM were flowed across both flow cell 1 and 2 at a flow rate of 30 .mu.l/min. The association time was 3 min and the dissociation time was 6 min. Running buffer was HBS-P (GE Healthcare, BR-1003-68). Both flow cells were regenerated by injecting 10 mM sodium hydroxide (NaOH) at a flow rate of 100 .mu.l/min for 20 seconds. The data were analysed by double referencing against the blank flow cell.
[0240] For PD-L1 binding, four concentrations in a two-fold dilution series of PD-L1 Fc (R&D Systems, 156-B7), starting at 40 nM, were flowed across mAb.sup.2 captured on the same Protein L chip. All others conditions were the same as for LAG-3 binding (see above).
[0241] The binding kinetics were fit with a 1:1 Langmuir model to generate binding association (k.sub.a) and dissociation (k.sub.d) rates. Equilibrium binding constants (K.sub.D) were calculated by dividing the dissociation rate by the association rate for each sample. Data analysis was performed with BiaEvaluation software version 3.2. The results are shown in Tables 10 and 11.
TABLE-US-00012 TABLE 10 Binding affinity and kinetics of mAb.sup.2 to human LAG-3 as determined by SPR mAb.sup.2 K.sub.D (pM) k.sub.a (1/Ms) .times. 10.sup.6 k.sub.d (1/s) .times. 10.sup.-4 84G09LALA No binding -- -- FS18-7-09/84G09LALA 56 6.3 3.5 FS18-7-32/84G09LALA 49 5.6 2.8 FS18-7-33/84G09LALA 43 4.2 1.8 FS18-7-36/84G09LALA 38 4.4 1.7 FS18-7-62/84G09LALA 36 5.4 2.0 FS18-7-65/84G09 LALA 39 5.9 2.3 FS18-7-78/84G09 LALA 29 4.8 1.4
TABLE-US-00013 TABLE 11 Binding affinity and kinetics of mAb.sup.2 to human PD-L1 as determined by SPR mAb.sup.2 K.sub.D (nM) k.sub.a (1/Ms) .times. 10.sup.5 k.sub.d (1/s) .times. 10.sup.-4 84G09LALA 1.2 3.8 4.6 FS18-7-09/84G09LALA 1.0 3.8 3.9 FS18-7-32/84G09LALA 1.0 3.4 3.2 FS18-7-33/84G09LALA 1.1 3.7 4.2 FS18-7-36/84G09LALA 3.5 1.6 5.6 FS18-7-62/84G09LALA 1.3 1.7 2.2 FS18-7-65/84G09 LALA 1.1 1.3 1.4 FS18-7-78/84G09 LALA 1.0 1.0 1.0
[0242] The binding affinities for human PD-L1 and human LAG-3 were comparable for all the mAb.sup.2 tested. The mAb.sup.2 binding affinities for human PD-L1 were comparable to 84G09, indicating that introduction of the LAG-3 binding site into the CH3 domain did not affect PD-L1 binding.
[0243] 2.6 Simultaneous Binding of mAb.sup.2 to Human LAG-3 and Human PD-L1
[0244] The ability of mAb.sup.2 (FS18-7-09/84G09, FS18-7-32/84G09, FS18-7-33/84G09, FS18-7-36/84G09, FS18-7-62/84G09, FS18-7-65/84G09, and FS18-7-78/84G09 all with the LALA mutation) to bind simultaneously to LAG-3 and PD-L1 was tested by SPR. Human PD-L1 Fc (R&D Systems, 156-B7) was immobilized on flow cell 2 of a Series S CM5 chip (GE Healthcare, BR-1005-30) to a surface density of 150 RU by following the manufacturer's instructions. Flow cell 1 was activated and deactivated without any protein immobilised for background subtraction. For each sample, 10 .mu.g/ml of mAb.sup.2 was flowed across flow cells 1 and 2, at a flow rate of 10 .mu.l/min for 3 min. Subsequently, 40 nM of LAG-3Fc (R&D Systems, 2319-L3) was flowed across both flow cell 1 and 2 at a flow rate of 10 .mu.l/min for 3 min. For each binding step dissociation was followed for 3 min. Sensor chip was regenerated after each cycle with a 15 s injection of 25 mM NaOH at a flow rate of 100 .mu.l/min.
[0245] All mAb.sup.2 tested were capable of simultaneously binding to LAG-3 and PD-L1. The parental anti-PD-L1 mAb, 84G09, only binds to PD-L1.
[0246] 2.7. Simultaneous Binding of Surrogate mAb.sup.2 to Murine LAG-3 and Murine PD-L1
[0247] The ability of the two surrogate mouse mAb.sup.2 (FS18-7-108-29/S1 and FS18-7-108-35/S1, both containing the LALA mutation) to bind simultaneously to murine LAG-3 and murine PD-L1 was tested by SPR on a BIAcore 3000 (GE Healthcare). Murine PD-L1 Fc (R&D Systems, 1019-B7-100) was immobilized on flow cell 4 of a CM5 chip to a surface density of 830 RU according to the manufacturer's instructions. Flow cell 3 was immobilised with 820 RU of human-Fc (R&D system, 110-HG) for background subtraction. For each sample, 50 nM of mAb.sup.2 was flowed across flow cells 1 and 2, at a flow rate of 20 .mu.l/min for 150 sec. Subsequently, 50 nM of murine LAG-3Fc (R&D Systems, 3328-L3-050) was flowed across both flow cell 3 and 4 at a flow rate of 20 .mu.l/min for 150 sec. For each binding step dissociation was followed for 3 min. Sensor chip was regenerated after each cycle with 2.times.10 .mu.l of 50 mM NaOH. Both mAb.sup.2 tested were capable of simultaneously binding to murine LAG-3 and murine PD-L1 and are therefore suitable surrogates for the human LAG-3/PD-L1 mAb.sup.2.
[0248] 2.8 Binding to Human Fc.gamma. Receptors by mAb.sup.2 Comprising LALA Mutation
[0249] Human Fc.gamma. receptors were immobilized on a CM5 chip to a surface density of approximately 200 RU for Fc.gamma. RI (R&D Systems, 1257-FC) and Fc.gamma. RIIIa (R&D Systems, 4325-FC) and approximately 500 RU for Fc.gamma. RIIa (R&D Systems, 1330-CD) and Fc.gamma. RIIb/c (R&D Systems, 1875-CD), according to the manufacturer's instruction for the BIAcore3000 instrument. For Fc.gamma. RI and Fc.gamma. RIIIa, 100 .mu.g/ml of mAbs or mAb.sup.2 were flowed across the chip for 3 min at a flow rate of 10 .mu.l/min and the dissociation was followed for 5 min. Running buffer was PBS (Lonza, BE17-516F)+0.05% (v/v) P20 surfactant (GE Healthcare, BR-1000-54). The positive control was wild-type IgG1 4420-mAb. Monoclonal IgG2 and IgG4 mAbs to irrelevant targets (20H4 and MOR7490) and mouse IgG1 (Sigma, P5305) were included as reference points. No regeneration was required due to the fast dissociation rates of the binding complexes. For Fc.gamma. RIIa and Fc.gamma. RIIb/c testing the concentration of mAb.sup.2 was increased to 500 .mu.g/ml to compensate for the weaker binding to these two receptors. The results are shown in Table 12.
TABLE-US-00014 TABLE 12 Binding response of anti-LAG-3/PD-L1 mAb.sup.2 to human Fc.gamma. receptors as determined by SPR Binding response at end of association (RU) mAb/mAb.sup.2 Fc.gamma.RI Fc.gamma.RIII Fc.gamma.RIIa Fc.gamma.RIIb/c FS18-7-9/84G09 LALA <1 <5 <1 <2 FS18-7-33/84G09 LALA <1 <5 <1 <2 FS18-7-62/84G09 LALA <1 <5 <1 <2 FS18-7-78/84G09 LALA <1 <5 <1 <2 84G09 mAb LALA <1 <5 <1 <2 IgG2 <1 <5 9 <2 IgG4 8 <5 8 9 4420 mAb IgG1 30 28 29 18 Mouse IgG1 <1 <5 7 <2
[0250] As expected, the LALA variant introduced into the mAb or mAb.sup.2 reduces the ability of these molecules to bind human Fc.gamma. receptors.
[0251] 2.9 Binding of mAb.sup.2 to Cells Expressing Human and Cynomolgus LAG-3
[0252] The human LAG-3 sequence (SEQ ID NO: 126) or cynomolgus LAG-3 sequence (SEQ ID NO: 128) were subcloned into pcDNA5FRT vector (Life Technologies, V6010-20) using KpnI (NEB, R0142) and NotI (NEB, R0146) restriction digestion. The vector was then transformed into Flp-In T-REx 293 HEK cell line (Life Technologies, R780-07) using Lipofectamine 2000 (Life Technologies, 11668-019). Transformed Flp-In T-REx 293 cells were grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 .mu.g/ml Hygromycin B (Melford Laboratories Ltd, Z2475), 15 .mu.g/ml Blasticidin (Melford Laboratories Ltd, B1105) for 3-4 weeks until colonies of stably transformed cells were apparent. These colonies were amplified in the presence of 1 .mu.g/ml Doxycyclin (Sigma, D9891) and tested for LAG-3 expression was confirmed by flow cytometry.
[0253] The affinity of the mAb.sup.2 (all containing the LALA mutation) for cell expressed human or cynomolgus LAG-3 was determined using flow cytometry. HEK cells expressing human or cynomolgus LAG-3 grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 .mu.g/ml Hygromycin B (Melford Laboratories Ltd, Z2475), 15 .mu.g/ml Blasticidin (Melford Laboratories Ltd, B1105) and 1 .mu.g/ml Doxycyclin (Sigma, D9891) were detached from tissue culture flasks using cell dissociation buffer (Life Technologies, 13151-014) and seeded in V-bottom 96-well plates (Costar, 3897) at 2.times.10.sup.5 cells/well. The plates were centrifuged at 1500 rpm for 3 min at 4.degree. C. to pellet the cells. A dilution series of the mAb.sup.2 (or control mAb) were incubated with the cells in a 100 .mu.l volume for 1 h at 4.degree. C. The plates were washed and secondary antibody (Anti-human Fc-488, Jackson ImmunoResearch, 109-546-098) was diluted 1:1000 in PBS and 100 .mu.l was added to the cells for 30 min at 4.degree. C. (plates were kept in the dark). The plates were washed and then cells were resuspended in 100 .mu.l PBS containing 1 .mu.g/ml DAPI (Biotium, 40043). The plates were read using Canto II flow cytometer (BD Bioscience). Dead cells were excluded and the fluorescence in the FITC channel (488 nm/530/30) was measured. The data was fit using log (agonist) vs response in GraphPad Prism Software. The plates were read on a BD FACSCanto II cytometer (BD Biosciences) and the data analysed using FlowJo. The results are shown in Table 13.
TABLE-US-00015 TABLE 13 Binding affinity of anti-LAG-3/PD-L1 mAb.sup.2 to HEK cells expressing human or cynomolgus LAG-3 as determined by flow cytometry human LAG-3 cyno LAG-3 EC.sub.50 EC.sub.50 (nM) 95% Conf. Int. (nM) 95% Conf. Int. FS18-7-09/84G09LALA 3.179 2.311 to 4.372 29.9 22.16 to 40.35 FS18-7-62/84G09LALA 4.079 2.736 to 6.083 26.37 15.80 to 44.02 FS18-7-78/84G09LALA 2.526 1.474 to 4.329 24.75 19.31 to 31.73 25F7 4.192 2.791 to 6.297 156.5 107.3 to 228.2
[0254] The results confirm mAb.sup.2 binding to human and cynomologus LAG-3 expressed on HEK cells. Regarding the calculated EC.sub.50 values, tested mAb.sup.2 show better or equal binding to the human LAG-3 and at least two times better binding to the cynomologus LAG-3 when compared with control anti-LAG-3 antibody 25F7. No cross-reactivity with other proteins expressed on the surface of the HEK cell line was observed.
[0255] 2.10 Binding of mAb.sup.2 to Cells Expressing Human and Cynomolgus PD-L1
[0256] The human PD-L1 sequence (SEQ ID NO: 129) or cynomolgus PD-L1 sequence (SEQ ID NO: 131) were subcloned into pcDNA5FRT vector (Life Technologies, V6010-20) using KpnI (NEB, R0142) and NotI (NEB, R0146) restriction digestion. The vector was then transformed into Flp-In T-REx 293 HEK cell line (Life Technologies, R780-07) using Lipofectamine 2000 (Life Technologies, 11668-019). Transformed Flp-In T-REx 293 cells were grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 .mu.g/ml Hygromycin B (Melford Laboratories Ltd, Z2475), 15 .mu.g/ml Blasticidin (Melford Laboratories Ltd, B1105) for 3-4 weeks until colonies of stably transformed cells were apparent. These colonies were amplified in the presence of 1 .mu.g/ml Doxycyclin (Sigma, D9891) and LAG-3 expression was confirmed by flow cytometry.
[0257] The affinity of the mAb.sup.2 (all containing the LALA mutation) binding to cell expressed human or cynomolgus PD-L1 or to parental (untransformed cells) was determined using flow cytometry. HEK cells expressing human or cynomolgus PD-L1 grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 .mu.g/ml Hygromycin B (Melford Laboratories Ltd, Z2475), 15 .mu.g/ml Blasticidin (Melford Laboratories Ltd, B1105) and 1 .mu.g/ml Doxycyclin (Sigma, D9891) were detached from tissue culture flasks using cell dissociation buffer (Life Technologies, 13151-014) and seeded in V-bottom 96-well plates (Costar, 3897) at 2.times.10.sup.5 cells/well. The plates were centrifuged at 1500 rpm for 3 min at 4.degree. C. to pellet the cells. A dilution series of the mAb.sup.2 (or control mAb) was incubated with the cells in a 100 .mu.l volume for 1 h at 4.degree. C. The plates were washed and secondary antibody (Anti-human Fc-488, Jackson ImmunoResearch, 109-546-098) was diluted 1:1000 in PBS and 100 .mu.l was added to the cells for 30 min at 4.degree. C. (plates were kept in the dark). The plates were washed then cells were resuspended in 100 .mu.l PBS containing 1 .mu.g/ml DAPI (Biotium, 40043). The plates were read using Canto II flow cytometer (BD Bioscience). Dead cells were excluded and the fluorescence in the FITC channel (488 nm/530/30) was measured. The data was fit using log (agonist) vs response in GraphPad Prism Software. The plates were read on a BD FACSCanto II cytometer (BD Biosciences) and the data analysed using FlowJo. The results are shown in Table 14.
TABLE-US-00016 TABLE 14 Binding affinity of anti-LAG-3/PD-L1 mAb.sup.2 to HEK cells expressing human or cynomolgus PD-L1 as measured by flow cytometry human PD-L1 HEK cynomolgus PD-L1 EC.sub.50 EC.sub.50 (nM) 95% Conf. Int. (nM) 95% Conf. Int. 84G09 3.107 2.230 to 4.331 1.641 1.244 to 2.164 FS18-7-09/84G09 3.19 2.208 to 4.610 1.674 1.393 to 2.012 FS18-7-09/84G09 3.51 2.570 to 4.794 1.814 1.503 to 2.189 FS18-7-33/84G09 3.448 2.436 to 4.881 1.895 1.566 to 2.292 FS18-7-32/84G09 3.816 2.749 to 5.298 1.984 1.644 to 2.393 FS18-7-58/84G09 3.775 2.957 to 4.820 1.861 1.541 to 2.247 FS18-7-33/84G09 3.225 2.214 to 4.700 1.733 1.397 to 2.150 FS18-7-65/84G09 3.999 2.782 to 5.750 1.845 1.455 to 2.339 FS18-7-36/84G09 3.907 2.844 to 5.367 1.999 1.580 to 2.528 FS18-7-88/84G09 3.492 2.541 to 4.800 1.814 1.464 to 2.247 FS18-7-58/84G09 3.907 2.998 to 5.093 2.033 1.622 to 2.550 84G09 (parental) 3.051 2.181 to 4.267 1.959 1.515 to 2.533
[0258] All of the tested LAG-3/PD-L1 mAb.sup.2 bound to human PD-L1 and cyno PD-L1 with an EC.sub.50 close to that of the 84G09 mAb, demonstrating that the PD-L1 binding affinity was not affected by the introduction of the LAG-3 binding site into the CH3 domain of the mAb.sup.2.
[0259] 2.11 Binding of Surrogate Mouse mAb.sup.2 to Cells Expressing Mouse LAG-3 or Mouse PD-L1
[0260] The murine LAG-3 sequence (SEQ ID NO: 127) or murine PD-L1 sequence (SEQ ID NO: 130) were subcloned into pcDNA5FRT vector (Life Technologies, V6010-20) using KpnI (NEB, R0142) and NotI (NEB, R0146) restriction digestion. The vector was then transformed into Flp-In T-REx 293 HEK cell line (Life Technologies, R780-07) using Lipofectamine 2000 (Life Technologies, 11668-019). Transformed Flp-In T-REx 293 cells were grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 .mu.g/ml Hygromycin B (Melford Laboratories Ltd, Z2475), 15 .mu.g/ml Blasticidin (Melford Laboratories Ltd, B1105) for 3-4 weeks until colonies of stably transformed cells were apparent. These colonies were amplified in the presence of 1 .mu.g/ml Doxycyclin (Sigma, D9891) and LAG-3 or PD-L1 expression was confirmed by flow cytometry.
[0261] The affinity of the mAb.sup.2 (all containing the LALA mutations) binding to cell expressed murine LAG-3 or murine PD-L1 was determined using flow cytometry. HEK cells expressing murine LAG-3 or murine PD-L1 grown in DMEM (Life Technologies, 61965-026) containing 10% FBS (Life Technologies, 10270-1-6), 100 .mu.g/ml Hygromycin B (Melford Laboratories Ltd, Z2475), 15 .mu.g/ml Blasticidin (Melford Laboratories Ltd, B1105) and 1 .mu.g/ml Doxycyclin (Sigma, D9891) were detached from tissue culture flasks using cell dissociation buffer (Life Technologies, 13151-014) and seeded in V-bottom 96-well plates (Costar, 3897) at 2.times.10.sup.5 cells/well. The plates were centrifuged at 1500 rpm for 3 min at 4.degree. C. to pellet the cells. A dilution series of the mAb.sup.2 (or control mAb) were incubated with the cells in a 60 .mu.l volume for 1 h at 4.degree. C. The plates were washed and secondary antibody (Anti-human Fc-488, Jackson ImmunoResearch, 109-546-098 for mAb.sup.2 or Anti-Rat IgG (H+L), Alexa Fluor 488, ThermoFisher, A-11006 for anti-LAG-3 control, C9B7W) was diluted 1:1000 in PBS and 50 .mu.l was added to the cells for 30 min at 4.degree. C. (plates were kept in the dark). The plates were washed then cells were resuspended in 50 .mu.l FACS Cell Fix (BD Bioscience, 340181) for 15 minutes, then washed and resuspended in 100 .mu.l PBS containing 1 .mu.g/ml DAPI (Biotium, 40043). The plates were read using Canto II flow cytometer (BD Bioscience). Dead cells were excluded and the fluorescence in the FITC channel (488 nm/530/30) was measured. The data was fit using log (agonist) vs response in GraphPad Prism Software. The plates were read on a BD FACSCanto II cytometer (BD Biosciences) and the data analysed using FlowJo. The results are shown in Table 15.
TABLE-US-00017 TABLE 15 Binding affinity of surrogate anti-mouse LAG-3/PD-L1 mAb.sup.2 to HEK cells expressing murine LAG-3 or murine PD-L1 by flow cytometry murine LAG-3 HEK murine PD-L1 HEK EC.sub.50 EC.sub.50 (nM) 95% Conf. Int. (nM) 95% Conf. Int. C9B7W 27 12.27 to 58.04 N/A -- FS18-7-108-29/S1 2.27 1.064 to 4.857 11.9 7.627 to 18.68 FS18-7-108-29/4420 2.29 1.256 to 4.164 N/A -- FS18-7-108-35/S1 3.84 2.126 to 6.926 12.3 8.873 to 17.16 FS18-7-108-35/4420 2.38 0.7819 to 7.239 N/A --
[0262] The surrogate mAb.sup.2 were able to bind to cell-expressed murine LAG-3 and to cell expressed murine PD-L1. The binding affinity of the surrogate mAb.sup.2 to cell expressed murine LAG-3 is approximately the same as the affinity of the anti-human LAG-3/PD-L1 mAb.sup.2 to human LAG-3 (2.3-3.8 nM compared to 2.5-4.2 nM) and the binding affinity of the surrogate mAb.sup.2 to cell-expressed murine PD-L1 is within 3-fold the affinity of the anti-human LAG-3/PD-L1 mAb.sup.2 to human PD-L1 (11.9-12.3 nM compared to 3.1-4.0 nM), demonstrating that these mAb.sup.2 are suitable surrogates for the anti-human LAG-3/PD-L1 mAb.sup.2 for use in vivo studies in mice.
Example 3: Activity of mAb.sup.2 Molecules in T Cell Activation Assays and an SEB Assay
[0263] 3.1 T Cell Activation Assay
[0264] An IL-2 release assay based on the DO11.10 OVA T-lymphocyte and LK35.2 B-lymphocyte hybridoma cell lines was used for functional screening of the mAb.sup.2. IL-2 release is a marker of T cell activation. T cells, expressing endogenous murine PD-1, were transfected with either empty vector (pLVX) or human LAG-3 construct. B-cells were transfected with empty vector (pLVX) or human PD-L1 construct.
[0265] Three combinations of these four cell lines were used side by side for testing T cell activation by the mAb.sup.2:
[0266] DO11.10 pLVX+LK35.2 hPD-L1 for anti-PD-L1 activity;
[0267] DO11.10 hLAG-3+LK35.2 pLVX for anti-LAG-3 activity;
[0268] DO11.10 hLAG-3+LK35.2 hPD-L1 for simultaneous anti-LAG-3/anti-PD-L1 activity.
[0269] All mAb.sup.2 (all containing the LALA mutation) were tested twice in this T cell activation assay. Cross-reactivity with cynomolgus LAG-3 and PD-L1 was tested in a functional T cell activation assay using cells which overexpress cynomolgus targets (cPD-L1 and cLAG-3).
[0270] Production of T Cell Lines Over-Expressing LAG-3
[0271] Lentiviral transduction methodology was used to generate DO11.10 cells (National Jewish Health) overexpressing human, cynomolgus or mouse LAG-3 using the Lenti-X HTX Packaging System (Cat. No 631249). Lenti-X expression vector (pLVX) (Cat. No 631253), containing the mouse LAG-3 cDNA (SEQ ID NO: 127), human LAG-3 cDNA (SEQ ID NO: 126) or cynomolgus LAG-3 cDNA (SEQ ID NO: 128), was co-transfected with a Lenti-X HTX Packaging Mix into the Lenti-X 293T Cell Line (Cat. No 632180) to generate virus. The DO11.10 cell line was transduced using the lentiviral vectors produced with the Lenti-X HTX Packaging System.
[0272] Production of Antigen Presenting Cells Over-Expressing PD-L1
[0273] Lentiviral transduction methodology was used to generate LK35.2 B cell lymphoma (ATCC, HB-98) overexpressing human, cynomolgus or mouse PD-L1 using the Lenti-X HTX Packaging System (Cat. No 631249). Lenti-X expression vector (pLVX) (Cat. No 631253), containing the mouse PD-L1 cDNA (SEQ ID NO: 130), human PD-L1 cDNA (SEQ ID NO: 129 or cynomolgus PD-L1 cDNA (SEQ ID NO: 131), was co-transfected with a Lenti-X HTX Packaging Mix into the Lenti-X 293T Cell Line (Cat. No 632180) to generate virus. The LK35.2 cell line was transduced using the lentiviral vectors produced with the Lenti-X HTX Packaging System.
[0274] Media and Peptide
[0275] Cell culture medium: DMEM (Gibco, 61965-026) 10% FBS (Gibco, 10270-106), 1 mM Sodium Pyruvate (Gibco, 11360-070), 1 .mu.g/ml puromycin (Gibco, A11138-03) Experimental medium: complete DO11.10 culture medium without puromycin. OVA peptide (MW=1773.9 Da): H-ISQAVHAAHAEINEAGR-OH (Pepscan)
[0276] Cells:
[0277] DO11.10 hLAG-3: DO11.10 T cell hybridoma transduced with a lentiviral vector to overexpress human LAG-3;
[0278] DO11.10 pLVX: DO11.10 T cell hybridoma transduced with an empty lentiviral vector;
[0279] DO11.10 cLAG-3: DO11.10 T cell hybridoma transduced with a lentiviral vector to overexpress cynomolgus LAG-3.
[0280] LK 35.2 hPD-L1: B cell hybridoma transduced with a lentiviral vector containing hPD-L1 to overexpress human PD-1;
[0281] LK 35.2 PLVX: B cell hybridoma transduced with an empty lentiviral (pLVX) vector;
[0282] LK 35.2 cPD-L1: B cell hybridoma transduced with a lentiviral vector to overexpress cynomolgus PD-1.
[0283] DO11.10 cells (either DO11.10 pLVX cells or DO11.10 hLAG-3 cells) at 0.3.times.10.sup.6 cells/ml were mixed at a 1:1 ratio with antibodies at 3.times. final concentration. Antibodies and DO11.10 cells were incubated at 37.degree. C., 5% CO.sub.2 for 1 hour. LK 35.2 cells (both pLVX and PD-L1-pLVX) were incubated at 3.times.10.sup.5 cells/ml experimental media with the OVA peptide at 1.5 .mu.M for 30 min. LK 35.2 cells+OVA were added to DO11.10 cells/treatment mix at a 1:2 ratio in the following combinations:
[0284] Human Functional Screen
[0285] DO11.10 pLVX+LK35.2 hPD-L1,
[0286] DO11.10 hLAG-3+LK35.2 pLVX,
[0287] DO11.10 hLAG-3+LK35.2 hPD-L1;
[0288] Cynomolgus Cross Reactivity Screen
[0289] DO11.10 pLVX+LK35.2 cPD-L1,
[0290] DO11.10 cLAG-3+LK35.2 pLVX,
[0291] DO11.10 cLAG-3+LK35.2 cPD-L1;
[0292] Cells were incubated at 37.degree. C., 5% CO.sub.2 for 24 hours. Supernatants were collected and assayed with mouse IL-2 ELISA kit (eBioscience, 88-7024-88 or R&D systems, SM2000) following the manufacturer's instructions. Plates were read at 450 nm using the plate reader with the Gen5 Software, BioTek. Absorbance values of 570 nm were subtracted from those of 450 nm (Correction). The standard curve for calculation of cytokine concentration was based on four parameter logistic curve fit (Gen5 Software, BioTek). The concentration of mIL-2 was plotted vs the log concentration of Fcab or mAb and the resulting curves were fitted using the log (agonist) vs response equation in GraphPad Prism. Table 16 shows the EC.sub.50 values and the maximal IL-2 release of the mAb.sup.2 and control mAbs, calculated as a percentage of the control (84G09+25F7). FIGS. 2A-2F show representative plots of IL-2 release by the panel of cell lines treated with FS18-7-9/84G09, FS18-7-62/84G09, or FS18-7-78/84G09, and controls. In the T cell activation assay with both targets present (DO11.10 hLAG-3+LK35.2 hPD-L1), IL-2 release was only induced when both LAG-3 and PD-L1 were inhibited, for example when the mAb.sup.2 or a combination of LAG-3 and PD-L1 antibodies was used. Therefore the mAb.sup.2 has benefit over a single agent alone. In the assays with just LAG-3 (DO11.10 hLAG-3+LK35.2 pvlx) or PD-L1 (DO11.10 pvlx+LK35.2 PD-L1) the mAb.sup.2 showed similar activity as the single agents in inhibiting these targets, demonstrating that the mAb.sup.2 is the only single molecule which was able to lead to T cell activation in the presence of LAG-3, PD-L1 or LAG-3+PD-L1.
TABLE-US-00018 TABLE 16 EC.sub.50 and maximal IL2 release (as calculated by percentage of IL2 release compared to control - 84G09 + 25F7) for nine mAb.sup.2 AVERAGE SD Maximal IL2 release AVERAGE calculated as % ctrl mAb.sup.2/control Abs EC.sub.50 SD (84G09 + 25F7) FS18-7-09/84G09LALA 0.75 0.20 65.89 24.04 F518-7-33/84G09LALA 0.82 0.35 63.95 36.56 F518-7-78/84G09LALA 0.82 0.38 64.39 39.28 F518-7-62/84G09LALA 0.65 0.02 58.97 18.13 F518-7-65/84G09LALA 0.81 0.20 70.25 2.22 F518-7-95/84G09LALA 1.10 -- 79.77 -- F518-7-32/84G09LALA 1.19 0.00 82.94 14.67 FS18-7-36/84G09LALA 1.05 0.32 71.56 8.83 F518-7-58/84G09LALA 1.16 0.21 79.40 0.10 84G09LALA + 25F7 1.32 0.25 100.00
[0293] One of the mAb.sup.2 (FS18-7-9/84G09 containing the LALA mutation) was tested for cynomolgus functional cross reactivity in the DO11.1-/LK35.2 T cell activation assay. FIGS. 3A-3C show the results in the panel of three cell based assays. FS18-7-9/84G09 was able to induce T cell activation in the presence of both cLAG-3+cPD-L1 and cLAG-3 or cPD-L1 alone, indicating that the mAb.sup.2 are functionally cynomolgus cross-reactive and therefore suitable for use in primate based safety studies.
[0294] 3.2 Staphylococcal Enterotoxin B Assay
[0295] Three mAb.sup.2 (all containing the LALA mutation) were tested in the human-PBMC based Staphylococcal Enterotoxin B assay (SEB assay). Staphylococcal Enterotoxin B is a superantigen, and binds to MHC class II molecules on antigen presenting cells (APCs) and the v.beta. chain of the T cell receptor (TCR), causing non-specific activation of T cells and cytokine release. There is no requirement for antigen to be present to see T cell activation. The SEB assay uses stimulated human cells (PBMCs) with physiological levels of checkpoint inhibitors, and can be used to confirm that T cell activation is enhanced by the mAb.sup.2 in a human system. Three mAb.sup.2 were tested in the SEB system with cells coming from four different donors.
[0296] Generation of Expanded T Cells
[0297] PMBCs were isolated from leukocyte cones by Ficoll gradient seperation. CD4+ T cells were isolated using human CD4+ T Cell Isolation Kit (Miltenyi Biotec Ltd, 130-096-533) according to the manufacturer's instruction. Human T-Activator CD3/CD28 Dynabeads (Life technologies, 11131 D) were resuspended by vortexing. Beads were transferred to a sterile 15 ml tube and 10 ml RPMI (Life Technologies, 61870044) with 10% FBS (Life Technologies, 10270106) and 1.times. Penicillin Streptomycin (Life Technologies, 15140122) was added to wash Dynabeads. The supernatant was discarded. The required amount of CD4+ T cells at 1.0.times.10.sup.6 cells/ml in RPMI with 10% FBS and 1.times. Penicillin Streptomycin Solution and 50 IU/ml recombinant human IL2 (Peprotech, 200-02-50 ug) with 3:1 bead to cell ratio were transferred to T75 flask (Greiner Bio-one, 690195) and incubated at 37.degree. C.+5% CO.sub.2. After 3 days the cells were gently resuspended and counted. The cell density was maintained between 0.8-1.times.10.sup.6 cells/ml by adding fresh media (RPMI-10% FBS+Penicillin Streptomycin Solution 1.times.+50 IU/ml rhuIL2) as needed. On day 7 or 8, the CD3/28 beads were removed and CD4+ T cells were rested overnight at 1.times.10.sup.6 cells/ml fresh media RPMI-10% FBS+Penicillin Streptomycin Solution 1.times. with reduced 10 IU/ml rhuIL2. The cells were stored frozen until required.
[0298] Generation of MoiDCs
[0299] Untouched monocytes were isolated from human PBMCs using human Pan Monocyte Isolation Kit, (Miltenyi Biotec Ltd, 130-096-537) following the manufacturer's instructions. Monocytes were differentiated to iDCs using human Mo-DC Differentiation Medium (Miltenyi Biotec Ltd, 130-094-812) following the manufacturer's instructions.
[0300] SEB Assay
[0301] Expanded T cells were thawed one day before the experiment, washed with AIM medium (Gibco, 12055-091) and incubated at 37.degree. C., 5% CO.sub.2 overnight at 1.times.10.sup.6 cells/ml in AIM medium. 2 .mu.M concentration of each antibody/mixture was prepared in DPBS (Gibco, 14190-169) and diluted 1:10 in medium (30 .mu.l+270 .mu.l) to obtain 200 nM. In a 96 well plate, serial dilutions were carried out at 1:10 (30 .mu.l+270 .mu.l experimental medium; 2.times. final conc.). MoiDCs were thawed, washed with AIM medium and mixed with T cells from the same donor at a 1:10 ratio (5 ml of iDCs at 2.times.10.sup.5 cells/ml were combined with 5 ml of T cells at 2.times.10.sup.6 cells/ml). 20 .mu.l of SEB (Sigma, S4881) at 0.1 .mu.g/ml was added to 10 ml of the cells. In a round bottom 96 well plate, 100 .mu.l of the cell/SEB mixture was added to 100 .mu.l of the antibody dilution, giving a ratio of 10.sup.4 iDC cells to 10.sup.5 T cells with 0.1 ng/ml SEB in 200 .mu.l of AIM media per well with final antibody concentrations of 100, 10, 1, 0.1, 0.01, 0.001 nM. Cells were incubated at 37.degree. C., 5% CO2 for 3 days. Supernatants were collected and assayed immediately with human IFN.gamma. ELISA kit (R&D Systems, PDIF50) or frozen down at -20.degree. C. for further analysis. The assay was performed according to the kit manufacturer's instructions using supernatants diluted 1:30 with PBA (DPBS, 2% BSA (Sigma, A7906-100G)). The concentration of human IFN.gamma. was plotted vs the log concentration of mAb.sup.2 or mAb and the resulting curves were fitted using the log (agonist) vs response equation in GraphPad Prism software. Table 17 shows the EC.sub.50 values and the span of the IFN.gamma. release in the SEB assay with cells from four different cell donors (Donors A to D). FIG. 4 shows a representative plot of the SEB assay with a single donor. In all six assays the LAG-3/PD-L1 mAb.sup.2 and the combination of the LAG-3/FITC mAb.sup.2+84G09LALA showed greater activation than the 84G09LALA mAb alone, whereas when the LAG-3/FITC mAb.sup.2 or 4420 mAb were assayed they did not show significant activation. The results of the SEB assay therefore confirm the results of the DO11.10/LK35.2 assay in a more physiological system.
TABLE-US-00019 TABLE 17 EC.sub.50 values calculated for six mAb.sup.2 based on six SEB assays with 4 different cell donors. EC.sub.50 (nM) 95% Conf. Int. SPAN 95% Conf. Int. Assay 1 Donor A FS18-7-09/84G09LALA 0.1551 0.08521 to 0.2824 12280 10802 to 13758 FS18-7-62/84G09LALA 0.1424 0.08665 to 0.2340 12120 10897 to 13342 FS18-7-78/84G09LALA 0.2774 0.1220 to 0.6310 14156 11970 to 16341 84G09 LALA 0.1884 0.08960 to 0.3962 10077 8612 to 11542 FS18-7-09/4420LALA -- -- 1010 384.8 to 1635 FS18-7-62/4420LALA -- -- 416.1 -443.1 to 1275 FS18-7-78/4420LALA -- -- 411 -1103 to 1925 84G09 LALA + FS18-7- 0.2455 0.1030 to 0.5850 11836 9888 to 13785 09/4420LAL 84G09 LALA + FS18-7- 0.1601 0.05399 to 0.4750 11448 8958 to 13937 62/4420LAL 84G09 LALA + FS18-7- 0.1774 0.06863 to 0.4587 12250 9955 to 14544 78/4420LAL 4420 LALA -- -- 218.6 -35.91 to 473.1 Assay 1 Donor B FS18-7-09/84G09LALA 0.07316 0.02255 to 0.2374 11825 8919 to 14730 FS18-7-62/84G09LALA 0.03236 0.007146 to 0.1465 12170 8419 to 15921 FS18-7-78/84G09LALA 0.09718 0.03128 to 0.3019 15198 11594 to 18802 84G09 LALA 0.08562 0.03403 to 0.2154 12343 9957 to 14730 FS18-7-09/4420LALA -- -- 1337 -195.8 to 2870 FS18-7-62/4420LALA -- -- 2508 -2118 to 7133 FS18-7-78/4420LALA -- -- 1425 -58.52 to 2908 84G09 LALA + FS18-7- 0.2861 0.1005 to 0.8143 16037 12890 to 19184 09/4420LAL 84G09 LALA + FS18-7- 0.1431 0.02857 to 0.7172 13907 9358 to 18455 62/4420LAL 84G09 LALA + FS18-7- 0.3775 0.08929 to 1.596 14418 10502 to 18334 78/4420LAL 4420 LALA -- -- 218.6 1028 to 3558 Assay 2 Donor A FS18-7-09/84G09LALA 0.2761 0.1654 to 0.4611 20727 18729 to 22724 FS18-7-33/84G09LALA 0.1934 0.07990 to 0.4681 171 03 14156 to 20049 FS18-7-78/84G09LALA 0.2384 0.1596 to 0.3560 19822 18311 to 21333 84G09 LALA 0.3788 0.1166 to 1.230 11745 9138 to 14352 FS18-7-09/4420LALA -- -- 1991 -1927 to 5909 FS18-7-33/4420LALA -- -- ~1.053e+006 (Very wide) FS18-7-78/4420LALA -- -- ~72657 (Very wide) 84G09 LALA + FS18-7- 0.5202 0.2624 to 1.031 23346 20230 to 26462 09/4420LALA 84G09 LALA + FS18-7- 0.2881 0.1529 to 0.5428 22529 19853 to 25205 33/4420LALA 84G09 LALA + FS18-7- 0.4335 0.2089 to 0.8996 20955 18038 to 23873 78/4420LALA Assay 2 Donor B FS18-7-09/84G09LALA 0.139 0.04157 to 0.4646 21572 16269 to 26874 FS18-7-33/84G09LALA 0.07278 0.01615 to 0.3280 19554 13408 to 25699 FS18-7-78/84G09LALA 0.1356 0.03552 to 0.5178 22319 16212 to 28426 84G09 LALA 0.1959 0.03365 to 1.140 12077 7938 to 16216 FS18-7-09/4420 LALA -- -- 1272 -963.4 to 3507 FS18-7-33/4420 LALA -- -- 2272 -190.8 to 4735 FS18-7-78/4420LALA -- -- 1960 -1287 to 5207 84G09 LALA + FS18-7- 0.2889 0.07772 to 1.074 23661 17835 to 29488 09/4420LALA 84G09 LALA + FS18-7- 0.3 0.06936 to 1.298 26145 18971 to 33320 33/4420LALA 84G09 LALA + FS18-7- 0.271 0.03980 to 1.845 25489 16292 to 34686 78/4420LALA Assay 1 Donor C FS18-7-09/84G09LALA 0.06739 0.01933 to 0.2349 2987 15107 to 27710 84G09 LALA 0.08347 0.0331 to 0.2105 1509 11046 to 17414 84G09 LALA + FS18-7- 0.1618 0.06635 to 0.3944 1744 13346 to 20704 09/4420LALA Assay 1 Donor D FS18-7-09/84G09LALA 0.04373 0.01797 to 0.1064 1850 15995 to 23799 84G09 LALA 0.07043 0.03483 to 0.1424 1045 10996 to 15405 84G09 LALA + FS18-7- 0.1351 0.08359 to 0.2182 968 15318 to 19403 09/4420LALA
Example 4: In Vivo Activity of mAb.sup.2 Molecules in Murine Tumour Models
[0302] 4.1 Activity of mAb.sup.2 Molecules in a MC38 Non-Established Tumour Model
[0303] The MC38 syngeneic tumour model was used in this experiment as MC38 tumours are known to express PD-L1 on their cell surface and to be highly immunogenic resulting in increased LAG-3 expression on immune cells in the tumour and tumour periphery.
[0304] The surrogate mouse mAb.sup.2 FS18-7-108-29/S1 containing the LALA mutation (SEQ ID NO: 117 and 119) referred to as FS18-29/S1 was tested for in vivo activity using a MC38 syngeneic mouse tumour growth model. The ability of the mAb.sup.2 to inhibit tumour growth was compared to that of the LAG-3/mock mAb.sup.2, FS18-7-108-29/4420 containing the LALA mutation (SEQ ID NO: 132 and 85) referred to as FS18-29/4420, the benchmark anti-LAG-3 mAb C9B7W (2B scientific; Catalogue Number BE0174-50MG), the benchmark anti-PD-L1 mAb S1 containing the LALA mutation (SEQ ID NO: 122 and 119) and to a combination of mAbs C9B7W and S1.
[0305] C57BL/6 female mice (Charles River) aged 8-10 weeks and weighing 20-25 g each were rested for one week prior to the study start. All animals were micro-chipped and given a unique identifier. Each cohort had 10 mice. The MC38 colon carcinoma cell line (S. Rosenberg, NIH) was initially expanded, stored, and then pre-screened by IDEXX Bioresearch for pathogens using the IMPACT I protocol and shown to be pathogen free. MC38 cells were thawed from -150.degree. C. storage and added to 20 ml DMEM (Gibco, 61965-026) with 10% FCS (Gibco, 10270-106) in a T175 tissue culture flask. Mice were anaesthetised using isoflurane (Abbott Laboratories) and each animal received 2.times.10.sup.6 cells injected subcutaneously in the left flank. 7-8 days following tumour cell inoculation, mice which did not have tumours at this point were removed from the study.
[0306] All of the mAb.sup.2 molecules and the control antibodies were analysed within 24 hours prior to injection by SEC-HPLC profiling and checked for impurities. Antibodies were prepared at a final concentration of 10 mg/kg in PBS and combined with a second antibody in the combination studies. The mAb.sup.2 molecules and the control antibodies were administered to the mice by intraperitoneal (IP) injection on days 8, 11, and 14 following tumour inoculation. Accurate measurements of tumours were taken, any drug dosing due on the day in question was performed, and the mice subjected to close observation for the remainder of the trial. Tumour volume measurements were taken with callipers to determine the longest axis and the shortest axis of the tumour. The following formula was used to calculate the tumour volume:
L.times.(S.sup.2)/2
[0307] Where L=longest axis; S=shortest axis
[0308] The trial was halted at day 20 when the tumour burden was considered close to restrictions. All mice were humanely sacrificed and the tumours were excised and weighed.
[0309] The results are shown in FIGS. 5-6. LAG-3/PD-L1 mAb.sup.2 (FS18-29/S1) treated mice had end tumours with significantly smaller weights than those treated with a combination of benchmark mAbs, C9B7W and S1. Specifically, statistical analysis of end tumour weights was performed using a two tailed Student's t-test within the GraphPad Prism software package demonstrating a statistical significant difference between administration of FS18-29/S1 and administration of the combination of benchmark antibodies C9B7W and S1 (p=0.0125), indicating that LAG-3 and PD-L1 inhibition provided by the same molecule resulted in a synergistic effect on end tumour weights compared with LAG-3 and PD-L1 inhibition provided by separate molecules.
[0310] The surrogate mAb.sup.2 FS18-29/S1 also had a marked effect on tumour growth preventing the establishment in 6 of 8 growing MC38 tumours, and slowing the growth of the remaining 2. Administration of the benchmark anti-LAG-3 and PD-L1 antibodies in combination slowed tumour growth in 7 animals with no animals being tumour free.
[0311] FS18-29/4420 alone had no marked effect on tumour growth, indicating that for maximal efficacy, the mAb.sup.2 requires the anti-PD-L1 Fab. The benchmark anti-mouse LAG-3 antibody alone had little or no effect on resulting tumour growth while the benchmark anti-mouse PD-L1 prevented establishment of 1 of 7 tumours in this cohort, and had some overall effect of slowing tumour growth.
[0312] Syngeneic mouse models are accepted as appropriate murine systems for testing the anti-tumour effect of inhibiting therapeutic targets and have been used extensively to validate development of human therapeutics.
[0313] 4.2 Activity of mAb2 Molecules in a MC38 Established Tumour Model
[0314] The surrogate mouse mAb.sup.2 FS18-7-108-29/S1 containing the LALA mutation, (SEQ ID NO: 117 and 119) referred to as FS18-29/S1, was tested for in vivo activity in a MC38 syngeneic mouse tumour growth model. The ability of the mAb.sup.2 to inhibit tumour growth was compared to that of the LAG-3/mock mAb.sup.2, FS18-7-108-29/4420 containing the LALA mutation (SEQ ID NO: 132 and 85), referred to as FS18-29/4420, the benchmark LAG-3 mAb C9B7W, the benchmark PD-L1 mAb S1 containing the LALA mutation (SEQ ID NO: 122 and 119) and to a combination of C9B7W and S1.
[0315] C57BL/6 female mice (Charles River) aged 8-10 weeks and weighing 20-25 g each were rested for one week prior to the study start. All animals were micro-chipped and given a unique identifier. Each cohort had 10 mice. The MC38 colon carcinoma cell line (S. Rosenberg, NIH) was initially expanded, stored, and then pre-screened by IDEXX Bioresearch for pathogens using the IMPACT I protocol and shown to be pathogen free.
[0316] MC38 cells (approximately 3-5.times.10.sup.6) was thawed from -150.degree. C. storage and added to 20 ml DMEM (Gibco, 61965-026) with 10% FCS (Gibco, 10270-106) in a T175 tissue culture flask. Mice were anaesthetised using isofluorane (Abbot Laboratories) and 2.times.10.sup.6 cells in 100 .mu.l were injected subcutaneously into the left flank of each mouse. 7-8 days following tumour cell inoculation, mice were routinely monitored for health and tumour growth appropriate for initiation of the study. When the majority of mice exhibited tumours of 5-10 mm diameter, they were sorted and randomised back into study cohorts. Any mice which did not have appropriately sized tumours at this point were removed from the study.
[0317] All of the mAb.sup.2 molecules and the control antibodies were analysed within 24 hours prior to injection by SEC-HPLC profiling and checked for impurities. Antibodies were prepared at a final concentration of 10 mg/kg in PBS and combined with the second antibody for the combination studies. The mAb.sup.2 molecules and the control antibodies were administered to the mice on days 15, 18, and 21 following tumour inoculation by IP injection. Animals were health screened under anaesthesia three times a week in a blinded fashion, during which time accurate measurements of tumours were taken. Tumour volume measurements were taken with callipers to determine the longest axis and the shortest axis of the tumour. The formula used to calculate the tumour volume was as set out in section 4.1 above.
[0318] The trial was halted at day 24 when the tumour burden was considered close to restrictions. All mice were humanely sacrificed and the tumours were excised and weighed. The results are shown in FIGS. 7-8.
[0319] Effective control or suppression of tumour growth in syngeneic tumour models in mice is best accomplished through therapeutic intervention at early time points (starting tumour volumes of less than 40 mm.sup.3). The later the intervention is administered, the more difficult it is to observe positive effects with respect to tumour growth, though perhaps this is more akin to the situation in the human clinical setting.
[0320] FS18-29/S1 had a positive effect in both suppressing tumour growth and preventing the establishment of MC38 colon carcinoma in immune competent mice when given at early time points in C57BL/6 mice. When administered at later time points (starting tumour volumes of 50-125 mm.sup.3), FS18-29/S1 was just as effective in suppressing tumour growth as the combination of benchmark antibodies. FS18-29/4420 alone had no noticeable impact on tumour growth and S1 and C9B7W both had a mild effect on resulting tumour growth.
[0321] 4.3 Activity of mAb.sup.2 Molecules in a CT26 Non-Established Tumour Model
[0322] The CT26 syngeneic tumour model was used in this experiment as CT26 tumours are known to express PD-L1 on their cell surface and to be highly immunogenic resulting in increased LAG-3 expression on immune cells in the tumour and periphery.
[0323] The surrogate mouse mAb.sup.2 FS18-7-108-29/S1 containing the LALA mutation (SEQ ID NO: 117 and 119), referred to as FS18-29/S1, and FS18-7-108-35/S1 containing the LALA mutation (SEQ ID NO: 120 and 119), referred to as FS18-35/S1, were tested for in vivo activity in a CT26 syngeneic mouse tumour growth model. The ability of the mAb.sup.2 to inhibit tumour growth was compared to that of the LAG-3/mock mAb.sup.2, FS18-7-108-29/4420 containing the LALA mutation (SEQ ID NO: 132 and 85), referred to as FS18-29/4420, and FS18-7-108-35/4420 containing the LALA mutation (SEQ ID NO: 133 and 85), referred to as FS18-35/4420, the benchmark LAG-3 mAb C9B7W, the benchmark PD-L1 mAb S1 containing the LALA mutation (SEQ ID NO: 122 and 119) and to a combination of C9B7W and S1.
[0324] BALB/c female mice (Charles River) aged 8-10 weeks and weighing 20-25 g each were rested for one week prior to the study start. All animals were micro-chipped and given a unique identifier. Each cohort had 10 mice. The CT26 colon carcinoma cell line (ATCC, CRL-2638) was initially expanded, stored, and then pre-screened by IDEXX Bioresearch for pathogens using the IMPACT I protocol and shown to be pathogen free. CT26 cells (approximately 3-5.times.10.sup.6) were thawed from -150.degree. C. storage and added to 20 ml DMEM (Gibco, 61965-026) with 10% FCS (Gibco, 10270-106) in a T175 tissue culture flask. 7-8 days following tumour cell inoculation, mice were routinely monitored for health and tumour growth appropriate for initiation of the study. When the majority of mice exhibited tumours with a diameter of 3-5 mm.sup.3, they were sorted and randomised back into study cohorts. Any mice which did not have tumours at this point were removed from the study.
[0325] All of the mAb.sup.2 molecules and the control antibodies were analysed within 24 hours of injection by SEC-HPLC profiling and checked for impurities. Antibodies were prepared at a final concentration of 10 mg/kg in PBS and combined with a second antibody in the combination studies. The mAb.sup.2 molecules and the control antibodies were administered to the mice on days 8, 11, and 14 following tumour inoculation. Animals were health screened during which time accurate measurements of tumours were taken. Tumour volume measurements were taken with callipers to determine the longest axis and the shortest axis of the tumour. The formula used to calculate the tumour volume was as set out in section 4.1 above: The trial was halted at day 20 when the tumour burden was considered close to restrictions.
[0326] All mice were humanely sacrificed and the tumours were excised and weighed. The results are shown in FIGS. 9-10.
[0327] Statistical analysis of the end tumour weights was performed using a two tailed Student's t-test within the GraphPad Prism software package. Statistical analysis of the tumour growth curves was determined using compare Growth Curves function from the Statistical Modeling package, statmod (Elso et al., 2004 and Baldwin et al., 2007), available from the R Project for Statistical Computing.
[0328] There was a demonstrated statistical significant difference between FS18-35/S1 mAb.sup.2 and IgG control (normal growth) in suppressing tumour growth. Such a statistically significant difference was not observed with either the combination of benchmark antibodies, or FS18-29/S1 mAb.sup.2 versus the IgG control group, or versus any other cohort in this trial.
[0329] The CT26 tumour model is an aggressive, fast growing tumour model, one that is inherently prone to mice developing intestinal metastasis, and as a result has a very limited therapeutic window.
[0330] Surprisingly the combination of benchmark LAG-3 and PD-L1 antibodies did not significantly suppress tumour growth compared to the IgG control cohort. However, the FS18-35/S1 treated cohort did reveal a significant suppression of growth compared to IgG control. FS18-29/S1, while it did suppress tumour growth as well compared to IgG control, it was not statistically significant. This trial shows a second tumour model in which the mouse reactive LAG-3/PD-L1 mAb.sup.2 has demonstrated a positive effect in slowing tumour growth at least to the same degree as administration of a combination of benchmark monoclonal antibodies.
[0331] 4.4 Effect of LALA Mutation on Tumour Growth Inhibition in the MC38 Non-Established Tumour Model
[0332] Two mAb.sup.2 (FS18-7-108-29/S1 LALA and FS18-7-108-29/S1) were tested to examine potential differences in anti-tumour activity of these mAb.sup.2 with and without the LALA mutation in the Fc region. The surrogate mouse mAb.sup.2 FS18-7-108-29/S1 referred to as FS18-29/S1 with (SEQ ID NO: 117 and 119) and without LALA mutation (SEQ ID NO: 118 and 119) were tested for in vivo activity using a MC38 syngeneic mouse tumour growth model. The ability of the mAb.sup.2 to inhibit tumour growth was compared to that of the LAG-3/mock mAb.sup.2, FS18-7-108-29/4420 with (SEQ ID NO: 132 and 85) and without LALA mutation (SEQ ID NO: 134 and 85) referred to as FS18-29/4420LALA and FS18-29/4420 and a combination of the LAG-3/mock mAb.sup.2 with and without LALA mutation with the benchmark PD-L1 mAb S1 with (SEQ ID NO: 122 and 119) and without the LALA mutation (SEQ ID NO: 123 and 119).
[0333] C57BL/6 female mice (Charles River) aged 8-10 weeks and weighing 20-25 g each were rested for one week prior to the study start. All animals were micro-chipped and given a unique identifier. Each cohort had 10 mice. The MC38 colon carcinoma cell line (S. Rosenberg, NIH) was initially expanded, stored, and then pre-screened by IDEXX Bioresearch for pathogens using the IMPACT I protocol and shown to be pathogen free. MC38 cells (approximately 3-5.times.10.sup.6) were thawed from -150.degree. C. storage and added to 20 ml DMEM (Gibco, 61965-026) with 10% FCS (Gibco, 10270-106) in a T175 tissue culture flask. Mice were anaesthetised using isofluorane (Abbot Laboratories) and 2.times.10.sup.6 cells in 100 .mu.l were injected subcutaneously into the left flank of each mouse. Mice were allowed to recover under observation and the inoculation date noted as Day 0. 7-8 days following tumour cell inoculation, mice were routinely monitored for health and tumour growth appropriate for initiation of the study. Any mice which did not have tumours at this point were removed from the study.
[0334] All of the mAb.sup.2 molecules and the control antibodies were analysed within 24 hours prior to injection by SEC-HPLC profiling and checked for impurities. Antibodies were prepared at a final concentration of 10 mg/kg in PBS and combined with a second antibody in the combination studies. The mAb.sup.2 molecules and the control antibodies were administered to the mice on days 8, 11, and 14 following tumour inoculation by IP injection. Animals were health screened under anaesthesia three times a week in a blinded fashion, during which time accurate measurements of tumours were taken. Tumour volume measurements were taken with callipers to determine the longest axis and the shortest axis of the tumour. The formula used to calculate the tumour volume was as set out in section 4.1.
[0335] All mice were humanely sacrificed and the tumours were excised and weighed. The results are shown in FIGS. 11-12.
[0336] This trial confirms that the presence or absence of the LALA mutation, which abrogates ADCC activity, has no statistically significant effect on tumour growth in the MC38 colon carcinoma model, however those mAb.sup.2 which included the LALA mutation tended to result in increased tumour growth suppression. Nevertheless, the rationale for including the mutation to potentially inhibit ADCC activity against T cells expressing LAG-3 or PD-L1 is justified as the LALA mutation has no detrimental effect on the anti-tumour activity of LAG-3/PD-L1 mAb.sup.2. There was some evidence to suggest that inclusion of the LALA mutation was only critical for the PD-L1 antibody.
[0337] This trial also examined whether the LAG-3/PD-L1 mAb.sup.2 may have increased efficacy over administration of the individual antibodies (LAG-3 LALA+PD-L1 LALA). In this case there was no significant difference between these two cohorts. Both groups suppressed growth in the MC38 colon carcinoma model.
[0338] 4.5 Conclusion
[0339] Overall, it is clear from the above results that there is a synergistic effect on tumour growth suppression when a mAb.sup.2 molecule comprising binding sites for both LAG-3 and PD-L1 is administered to mice in the mouse models tested. Based on these results, it is expected that the antibody molecules of the invention will show a superior effect in the treatment of cancer in human patients, in particular in supressing tumour growth, than administration of two separate molecules which bind LAG-3 and PD-L1, respectively.
Example 5: Effect of mAb2 Treatment on T Cell LAG-3 Expression
[0340] The mechanism by which the surrogate mouse mAb.sup.2 FS18-7-108-29/S1 containing the LALA mutation, (SEQ ID NOs: 117 and 119) referred to as FS18-29/S1 led to decreased tumour burden was tested in a MC38 syngeneic mouse tumour growth model expressing ovalbumin (MC38.OVA). The effect of FS18-29/S1 was compared to that of the LAG-3/mock mAb.sup.2, FS18-7-108-29/4420 containing the LALA mutation (SEQ ID NOs: 132 and 85), referred to as FS18-29/4420, the benchmark PD-L1 mAb S1 containing the LALA mutation (SEQ ID NOs: 122 and 119) and to a combination of FS18-29/4420 and S1.
[0341] On the day of implant, cultured MC38.OVA cells were harvested during log phase growth (Confluency .about.75%) and resuspended in PBS at a concentration of 1.times.10.sup.7 cells/mL. Tumours were initiated by first anesthetizing each animal with isoflurane, then subcutaneously implanting 1.times.10.sup.6 MC38.OVA cells (0.1 mL suspension) into the left flank of each test animal. Eleven days after tumour cell implantation animals were randomised, using a deterministic randomisation method, into five groups with individual tumour volumes of 32 to 62.5 mm.sup.3. Animals were dosed at 10 mg/kg antibody or mAb.sup.2 on day 12, 14 and 16 after tumour inoculation, and tumours collected from three animals/group at days 19 and 23 after tumour inoculation. GentleMACS.TM. Dissociator was used to dissociate the tumour with cells subsequently sieved through a 70 m cell strainer to obtain a single cell suspension. 1.times.10.sup.6 cells/well on a 96-well plate were resuspended in FACS buffer with 1:3000 viability stain and Fc block (anti-CD16/32 antibody). Cells for FACS analysis were stained using a Master Mix that included labelled antibodies against CD43, CD8a, CD4, FoxP3, and LAG-3. For the FoxP3 intracellular staining cells were fixed and permeabilized prior to staining with the FoxP3 antibody. Samples were run on the Canto flow cytometer with a compensation matrix and a minimum of 500,000 events counted.
[0342] In this experiment, TILs were examined for LAG-3 expression after the third dose of antibody/mAb.sup.2 had been administered, when a separation in the growth of the tumour between control and mAb.sup.2 treatments is seen but before there is a large difference between tumour sizes which might skew results. At this time point, LAG-3 expression on TILs was found to be markedly decreased in animals treated with the mAb.sup.2 FS18-29/S1 or with the combination of FS18-29/4420 and S1. Specifically, as shown in FIG. 13, LAG-3 expression on CD8, CD4 and FoxP3 tumour infiltrating lymphocytes (TILs) was decreased after treatment with FS18-29/S1 at day 19 and 23 after tumour inoculation, which corresponds to 3 and 7 days after the last antibody/mAb.sup.2 dosing, respectively. The decrease in LAG-3 expression was more pronounced at day 23 but was still evident at day 19. Animals given a combination of FS18-29/4420 and S1 also show a decrease in LAG-3 expression on TILs but the effect was delayed until day 23, while treatment with FS18-29/4420 or S1 administered individually had little to no effect on LAG-3 expression on TILs.
[0343] These results show that dual inhibition of LAG-3 and PD-L1 is required for a decrease in LAG-3 expression by TILs, as this phenomenon was not seen in animals treated with single agents against LAG-3 or PD-L1. Without wishing to be bound by theory, it is thought that dual anti-LAG-3 and anti-PD-L1 treatment leads to a decrease in LAG-3 expression on TILS, thereby reducing the inhibitory effect of LAG-3 and allowing the TILs to overcome exhaustion. Once the TILs become activated, they are able to recognise neo-antigens expressed by the tumour and mount a response against it. This is therefore thought to be the mechanism by which treatment with anti-LAG3/PD-L1 mAb.sup.2 results in a reduction in the tumour burden.
Example 6: Antibody Dependent Cellular Cytotoxicity and Complement Dependent Cytotoxicity Activity of mAb.sup.2
[0344] IgG1 antibodies usually exhibit effector functions via conserved interaction sites within the constant region of the molecule. These include Antibody Dependent Cellular Cytotoxicity (ADCC), mediated by binding to Fc.gamma.R expressed on Monocytes/Macrophages, dendritic cells, NK cells, neutrophils and other granulocytes, and Complement Dependent Cytotoxicity (CDC), mediated by induction of the complements cascade initiated by binding to C1q complement component. Since LAG-3 is predominantly expressed on activated T cells and PD-L1 is expressed on these but also on tumour cells at high levels, the ability of mAb.sup.2 FS18-7-9/84G09 (SEQ ID NOs 94 and 116) to induce ADCC and CDC was investigated.
[0345] Specifically, it was tested whether FS18-7-9/84G09 treatment of LAG-3 or PD-L1 expressing cells, followed by incubation with either NK cells or complement, induces lysis of the respective target cells. In addition, since FS18-7-9/84G09 is a bispecific antibody, it was also tested whether target engagement on one of the specificities affects effector function towards cells expressing the target for the other specificity.
[0346] Understanding the effector functions of mAb.sup.2 FS18-7-9/84G09 is useful for a number of reasons, including determining whether a mutation which reduces effector functions, such as the LALA mutation, should be included in the molecule to protect LAG-3 expressing effector T cells engaged in tumour killing from FS18-7-9/84G09-mediated ADCC and/or CDC.
[0347] 6.1 Study Design
[0348] Raji cells recombinantly expressing LAG-3 or PD-L1 were used for all assays, using their endogenous expression of CD20 as a control for targeting with a generic version of Rituximab to demonstrate functional activity of the added complement and NK cell preparations independent of the recombinant expression of the target proteins. Target expression was confirmed prior to these experiments.
[0349] To determine the basic CDC activity towards LAG-3 or PD-L1 expressing cells, this activity was measured using LDH release, measured by conversion of a substrate into a fluorescent dye (CytoTox-ONE.TM. by Promega). To measure which target cells in a cell mixture comprising both LAG-3 expressing cells and PD-L1 expressing cells were being lysed, differential CDC was measured by flow cytometry of differentially fluorescently labelled target cells after incubation with the mAb.sup.2/antibody, detecting dead cells using a fluorescent dye excluded from live cells.
[0350] In order to determine ADCC activity towards LAG-3 or PD-L1 expressing cells, this activity was measured using NK cells isolated from frozen PBMC and LDH release measured colourimetrically (CytoTox 96 by Promega). For all these studies, Rituximab in various isotypes and Fc configurations was used as a control. No reliable method for measuring ADCC differentially is known, so differential ADCC activity was not measured.
[0351] In all experiments, the PD-L1 specific mAb (84G09) and the LAG-3 specific mAb (25F7) were used as controls. An IgG isotype control (4420) which was either used as negative control or to generate the CDC background activity was also used. The LALA versions of the respective antibodies and mAb.sup.2 (excluding 25F7) were also compared to the IgG1 wild-type versions in the CDC and ADCC assays to determine the effect of this mutation on these effector functions.
[0352] 6.2 Materials and Methods
[0353] 6.2.1 CDC Assays
[0354] All antibodies/mAb.sup.2, including Rituximab, were diluted in 10 point 1 in 2 serial dilutions. Control wells containing the IgG (4420 LALA) at the highest concentration used were also prepared. Cell suspensions of Raji cells recombinantly expressing LAG-3 or PD-L1 respectively, were prepared in serum free medium for the LDH release assay and added to an equal volume of the prepared antibodies/mAb.sup.2.
[0355] For the Flow Cytometry based CDC assay, cell suspensions of 5.times.10.sup.7 cells were prepared and resuspended in either 0.5 .mu.M CellTracker deep red (CellTracker.TM. Deep Red, Thermo Fisher, #C34565) or 5 .mu.M for CellTracker Green (CellTracker.TM. Green CMFDA (5-chloromethylfluorescein diacetate, Thermo Fisher, #C7025) in serum free medium. After a 30 min incubation at 37.degree. C., cells were washed in serum free medium and either added to the prepared antibody/mAb.sup.2-containing wells directly, or combined with the other differentially stained cell line at equal volumes and then added to the antibody/mAb.sup.2-containing wells as described above. For both assays, after a 30 min incubation under cell culture conditions, wells were topped up with an equal volume of baby rabbit complement, 10% in serum free medium (Baby Rabbit Complement, TEBU-bio, #CL3441). Plates were incubated for 4 hrs in cell culture conditions. For the LDH release CDC assay, 100% lysis controls were generated by adding Triton X 100 to half of the 4420 LALA treated wells and the Cytotox assay was performed according to manufacturer's instructions (CytotoxOne, Promega, G7891). After obtaining the reads, the signal from the 100% lysis controls was set to 100% and the signals from the sample wells were calculated as a percentage of that level.
[0356] For the Flow Cytometry based CDC assay, at the end of the incubation period, the dead cell dye (SYTOX.RTM. Blue Dead Cell Stain, Thermo Fisher, #S34857) was diluted 1 in 500 in PBS and wells were topped up with an equal volume. Flow Cytometry was performed on a Cytoflex flow cytometer gating on the intact cell populations based on FSC and SSC and detecting the percentage of Sytox positive cells (channel PB450) of both the CellTracker.TM. Deep Red positive and the CellTracker.TM. Green CMFDA positive cell populations.
[0357] 6.3.2 ADCC Assay
[0358] ADCC was measured as described previously (Broussas, Matthieu; Broyer, Lucile; and Goetsch, Liliane. (2013) Evaluation of Antibody-Dependent Cell Cytotoxicity Using Lactate Dehydrogenase (LDH) Measurement in Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols, Methods in Molecular Biology. New York: Springer Science+Business Media. Volume 988, pp 305-317). Briefly, target cells were pre-incubated with the antibodies before adding primary NK cells isolated from human PBMCs (NK cell isolation kit, Miltenyi Biotec, 130-092-657) at a ratio of 20 to 1 for 4 hrs. The cytotoxicity assay was performed following manufacturer's instructions (CytoTox 96 Non-Radioactive Cytotoxicity Assay, Promega, G1780). % lysis was calculated based on 100% target cell lysis, taking into account spontaneous lysis of effector and target cells.
[0359] 6.3 Results and Conclusions
[0360] 6.3.1 CDC Assay
[0361] PD-L1 expressing Raji cells were targeted by the anti-CD20 antibody Rituximab, which resulted in a maximum lysis of <60% when measuring CDC by generic LDH release. Anti-PD-L1 antibody 84G09 (which comprises the F(ab).sub.2 portion of mAb.sup.2 FS18-7-9/84G09), and FS18-7-9/84G09 in IgG1 format, showed higher maximum lysis and also higher lysis potency, with estimated half maximal doses of about half of that required by Rituximab in IgG1 format. This shows that the introduction of the LAG-3 binding site into the 84G09 antibody did not change its PD-L1 targeting activity with respect to potency or max response, as both were very similar when 84G09 and FS18-7-9/84G09 were compared. The introduction of the LALA mutation resulted in a reduced max response for Rituximab, 84G09 and FS18-7-9/84G09, however, the potency was only reduced for 84G09 and FS18-7-9/84G09. As expected, the anti-LAG-3 antibody 25F7 had no effect on cell viability of the PD-L1 expressing Raji cells, as these cells did not express human LAG-3. These results are shown in FIG. 14A.
[0362] LAG-3 expressing Raji cells were targeted for CDC by the anti-CD20 antibody Rituximab, however, the LAG-3 antibody 25F7 showed even better potency with an estimated half maximal dose of about half of that required for Rituximab. None of the other antibodies showed any CDC activity against LAG-3 expressing Raji cells, including FS18-7-9/84G09. The introduction of the LALA mutation had very limited effect on the CDC activity of Rituximab (FIG. 14B).
[0363] 6.3.2 Differential CDC Assay
[0364] A differential CDC assay, employing flow cytometry, was developed by the inventors to distinguish which target expressing cells were lysed when treated with FS18-7-9/84G09 or control antibodies. This assay was used to confirm the results from the basic LDH release CDC assay described above. Compared to the IgG isotype control antibody (4420), which had no effect on the percentage of live cells, Rituximab mediated a reduction of live cells and an increase of dead cells, both of PD-L1 and LAG-3 expressing cells. However, FS18-7-9/84G09 had no effect on LAG-3 expressing cells, but very efficiently lysed PD-L1 expressing cells. Similarly, the mixture of the LAG-3 specific antibody 25F7 and the PD-L1 antibody 84G09 also showed a dose-dependent decrease in live cells and a reciprocal increase in dead cells, however, the maximal lysis of LAG-3 expressing cells was only just over 50% of cells, but was reached already at a concentration around 1 nM, the lowest dose to achieve maximal lysis of all antibodies tested. This confirms the previous finding that the LAG-3 binding site in the CH3 domain of FS18-7-9/84G09 does not induce CDC mediated lysis of LAG-3 expressing target cells. In addition, this experiment shows that the presence of LAG-3 expressing cells has no effect on the CDC activity of FS18-7-9/84G09 towards PD-L1 expressing cells. The results are shown in FIG. 15.
[0365] 6.3.3 ADCC Assay
[0366] PD-L1 expressing Raji cells were targeted for ADCC by the anti-CD20 antibody Rituximab, FS18-7-9/84G09, and 84G09 with very similar efficacy and potency, resulting in a maximum lysis of around 40% of cells. Rituximab and 84G09 containing the LALA mutation showed no ADCC-mediated lysis and FS18-7-9/84G09 containing the LALA mutation showed no or very low ADCC-meditated lysis of PD-L1 expressing target cells. The LAG-3 specific antibody 25F7 and the isotype control 4420 with and without the LALA mutation showed no activity in this assay.
[0367] These results show that the introduction of the LAG-3 binding site into antibody 84G09 did not change its PD-L1 targeted ADCC activity in potency or max response, since both were very similar to the PD-L1 specific antibody 84G09. The introduction of the LALA mutation resulted in abrogation of ADCC activity (FIG. 16A).
[0368] LAG-3 expressing Raji cells were targeted by Rituximab and 25F7 for ADCC-mediated lysis, resulting in maximum lysis of about 40%. FS18-7-9/84G09 also mediated lysis of LAG-3 expressing cells by ADCC, be it with a much lower potency and efficacy, only reaching just under 20% lysis at 2.5 nM concentration. The introduction of the LALA mutation abrogated all ADCC activity of Rituximab and FS18-7-9/84G09. 84G09 and the isotype control 4420 with and without the LALA mutation showed no ADCC activity in this assay as expected, as these antibodies do not bind LAG-3 (FIG. 16B).
[0369] Sequence listing
TABLE-US-00020 Amino acid sequences of Fcab FS18-7-9 loop regions FS18-7-9 AB loop-WDEPWGED (SEQ ID NO: 1) FS18-7-9 CD loop-SNGQPENNY (SEQ ID NO: 2) FS18-7-9 EF loop-PYDRWVWPDE (SEQ ID NO: 3) Nucleotide sequence of Fcab FS18-7-9 CH3 domain (SEQ ID NO: 4) GGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCTGGGATGAGCCGTGGGGTGAA GACGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC TTCTTCCTCTACAGCAAGCTCACCGTGCCGTATGATAGGTGGGTTTGGCCGGATGAGTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT CHO codon optimised nucleotide sequence of Fcab FS18-7-9 CH3 domain (SEQ ID NO: 142) GGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCTCCATCCTGGGATGAGCCCTGGGGCGA GGATGTGTCTCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGGAATGGGAG AGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACTCCGACGGCTCA TTCTTCCTGTACAGCAAGCTGACAGTGCCCTACGACAGATGGGTGTGGCCCGACGAGTTCTCCT GCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGAGCCCCG GC Amino acid sequence of Fcab FS18-7-9 CH3 domain (SEQ ID NO: 5) GQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-9 CH3 domain comprising C-terminal lysine (SEQ ID NO: 135) GQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPGK Amino acid sequence of Fcab FS18-7-9 CH2 and CH3 domains, comprising LALA mutation (underlined) (SEQ ID NO: 6) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGED VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVM HEALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-9 CH2 and CH3 domains without LALA mutation (SEQ ID NO: 7) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHE ALHNHYTQKSLSLSPG Amino acid sequences of Fcab FS18-7-32 loop regions FS18-7-32 AB loop-WDEPWGED (SEQ ID NO: 1) FS18-7-32 CD loop-SNGQPENNY (SEQ ID NO: 8) FS18-7-32 EF loop-PYDRWVWPDE (SEQ ID NO: 3) Nucleotide sequence of Fcab FS18-7-32 CH3 domain (SEQ ID NO: 9) GGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCTGGGATGAGCCGTGGGGTGAA GACGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGAAATCGCCGTGGAGTGGGAG AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC TTCTTCCTCTACAGCAAGCTCACCGTGCCGTATGATAGGTGGGTTTGGCCGGATGAGTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT Amino acid sequence of Fcab FS18-7-32 CH3 domain (SEQ ID NO: 10) GQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSEIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-32 CH2 and CH3 domains, comprising LALA mutation (underlined) (SEQ ID NO: 11) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGED VSLTCLVKGFYPSEIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVM HEALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-32 CH2 and CH3 domains without LALA mutation (SEQ ID NO: 12) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVS LTCLVKGFYPSEIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHE ALHNHYTQKSLSLSPG Amino acid sequences of Fcab FS18-7-33 loop regions FS18-7-33 AB loop-WDEPWGED (SEQ ID NO: 1) FS18-7-33 CD loop-SNGQPEDNY (SEQ ID NO: 13) FS18-7-33 EF loop-PYDRWVWPDE (SEQ ID NO: 3) Nucleotide sequence of Fcab FS18-7-33 CH3 domain (SEQ ID NO: 14) GGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCTGGGATGAGCCGTGGGGTGAA GACGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGCAGCCGGAGGACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC TTCTTCCTCTACAGCAAGCTCACCGTGCCGTATGATAGGTGGGTTTGGCCGGATGAGTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT Amino acid sequence of Fcab FS18-7-33 CH3 domain (SEQ ID NO: 15) GQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGQPEDNYKTTPPVLDSDGSFFL YSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-33 CH2 and CH3 domains, comprising LALA mutation (underlined) (SEQ ID NO: 16) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGED VSLTCLVKGFYPSDIAVEWESNGQPEDNYKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVM HEALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-33 CH2 and CH3 domains without LALA mutation (SEQ ID NO: 17) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVS LTCLVKGFYPSDIAVEWESNGQPEDNYKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHE ALHNHYTQKSLSLSPG Amino acid sequences of Fcab FS18-7-36 loop regions FS18-7-36 AB loop-WDEPWGED (SEQ ID NO: 1) FS18-7-36 CD loop-SNGQPENNY (SEQ ID NO: 18) FS18-7-36 EF loop-PYDRWVWPDE (SEQ ID NO: 3) Nucleotide sequence of Fcab FS18-7-36 CH3 domain (SEQ ID NO: 19) GGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCTGGGATGAGCCGTGGGGTGAA GACGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC TACTTCCTCTACAGCAAGCTCACCGTGCCGTATGATAGGTGGGTTTGGCCGGATGAGTTCTCAT GCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGG T Amino acid sequence of Fcab FS18-7-36 CH3 domain (SEQ ID NO: 20) GQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSYFL YSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of CH2 + CH3 of Fcab FS18-7-36 CH2 and CH3 domains, comprising LALA mutation (underlined) (SEQ ID NO: 21) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGED VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSYFLYSKLTVPYDRWVWPDEFSCSVM HEALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-36 CH2 and CH3 domains without LALA mutation (SEQ ID NO: 22) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSYFLYSKLTVPYDRWVWPDEFSCSVMHE ALHNHYTQKSLSLSPG Amino acid sequences of Fcab FS18-7-58 loop regions FS18-7-58 AB loop-WDEPWGED (SEQ ID NO: 1) FS18-7-58 CD loop-SNGYPEIEF (SEQ ID NO: 23) FS18-7-58 EF loop-PYDRWVWPDE (SEQ ID NO: 3) Nucleotide sequence of Fcab FS18-7-58 CH3 domain (SEQ ID NO: 24) GGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCTGGGATGAGCCGTGGGGTGAA GACGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGTATCCAGAAATCGAATTCAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT TCTTCCTCTACAGCAAGCTCACCGTGCCTTATGATAGGTGGGTTTGGCCGGATGAGTTCTCATGC TCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT Amino acid sequence of Fcab FS18-7-58 CH3 domain (SEQ ID NO: 25) GQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYPElEFKTTPPVLDSDGSFFLY SKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-58 CH2 and CH3 domains, comprising LALA mutation (underlined) (SEQ ID NO: 26) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGED VSLTCLVKGFYPSDIAVEWESNGYPEIEFKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMH EALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-58 CH2 and CH3 domains without LALA mutation (SEQ ID NO: 27) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVS LTCLVKGFYPSDIAVEWESNGYPEIEFKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEA LHNHYTQKSLSLSPG Amino acid sequences of Fcab FS18-7-62 loop regions FS18-7-62 AB loop-WDEPWGED (SEQ ID NO: 1) FS18-7-62 CD loop-SNGIPEWNY (SEQ ID NO: 28) FS18-7-62 EF loop-PYDRWVWPDE (SEQ ID NO: 3) Nucleotide sequence of Fcab FS18-7-62 CH3 domain (SEQ ID NO: 29) GGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCTGGGATGAGCCGTGGGGTGAA GACGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGATCCCAGAATGGAACTATAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT TCTTCCTCTACAGCAAGCTCACCGTGCCGTATGATAGGTGGGTTTGGCCGGATGAGTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT Amino acid sequence of Fcab FS18-7-62 CH3 domain (SEQ ID NO: 30) GQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGIPEWNYKTTPPVLDSDGSFFL YSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-62 CH2 and CH3 domains, comprising LALA mutation (underlined) (SEQ ID NO: 31) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGED VSLTCLVKGFYPSDIAVEWESNGIPEWNYKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMH EALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-62 CH2 and CH3 domains without LALA mutation (SEQ ID NO: 32) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVS LTCLVKGFYPSDIAVEWESNGIPEWNYKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEA LHNHYTQKSLSLSPG Amino acid sequences of Fcab FS18-7-65 loop regions FS18-7-65 AB loop-WDEPWGED (SEQ ID NO: 1) FS18-7-65 CD loop-SNGYAEYNY (SEQ ID NO: 33) FS18-7-65 EF loop-PYDRWVWPDE (SEQ ID NO: 3) Nucleotide sequence of Fcab FS18-7-65 CH3 domain (SEQ ID NO: 34) GGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCTGGGATGAGCCGTGGGGTGAA GACGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGTATGCAGAATATAACTATAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT TCTTCCTCTACAGCAAGCTCACCGTGCCGTATGATAGGTGGGTTTGGCCGGATGAGTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT Amino acid sequence of Fcab FS18-7-65 CH3 domain (SEQ ID NO: 35) GQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYAEYNYKTTPPVLDSDGSFFL YSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-65 CH2 and CH3 domains, comprising LALA mutation (underlined) (SEQ ID NO: 36) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGED VSLTCLVKGFYPSDIAVEWESNGYAEYNYKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVM HEALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-65 CH2 and CH3 domains without LALA mutation (SEQ ID NO: 37) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVS LTCLVKGFYPSDIAVEWESNGYAEYNYKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHE ALHNHYTQKSLSLSPG Amino acid sequences of Fcab FS18-7-78 loop regions FS18-7-78 AB loop-WDEPWGED (SEQ ID NO: 1) FS18-7-78 CD loop-SNGYKEENY (SEQ ID NO: 38) FS18-7-78 EF loop-PYDRWVWPDE (SEQ ID NO: 3) Nucleotide sequence of Fcab FS18-7-78 CH3 domain (SEQ ID NO: 39) GGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCTGGGATGAGCCGTGGGGTGAA GACGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGTATAAAGAAGAAAACTATAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT TCTTCCTCTACAGCAAGCTCACCGTGCCGTATGATAGGTGGGTTTGGCCGGATGAGTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT Amino acid sequence of Fcab FS18-7-78 CH3 domain (SEQ ID NO: 40) GQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYKEENYKTTPPVLDSDGSFFL YSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-78 CH2 and CH3 domains, comprising LALA mutation (underlined) (SEQ ID NO: 41) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGED VSLTCLVKGFYPSDIAVEWESNGYKEENYKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVM HEALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-78 CH2 and CH3 domains without LALA mutation (SEQ ID NO: 42) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVS LTCLVKGFYPSDIAVEWESNGYKEENYKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHE ALHNHYTQKSLSLSPG Amino acid sequences of Fcab FS18-7-88 loop regions FS18-7-88 AB loop-WDEPWGED (SEQ ID NO: 1) FS18-7-88 CD loop-SNGVPELNV (SEQ ID NO: 43) FS18-7-88 EF loop-PYDRWVWPDE (SEQ ID NO: 3) Nucleotide sequence of Fcab FS18-7-88 CH3 domain (SEQ ID NO: 44) GGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCTGGGATGAGCCGTGGGGTGAA GACGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGGTTCCAGAACTGAACGTTAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT TCTTCCTCTACAGCAAGCTCACCGTGCCGTATGATAGGTGGGTTTGGCCGGATGAGTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT Amino acid sequence of Fcab FS18-7-88 CH3 domain (SEQ ID NO: 45) GQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGVPELNVKTTPPVLDSDGSFFL YSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-88 CH2 and CH3 domains, comprising LALA mutation (underlined) (SEQ ID NO: 46) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGED VSLTCLVKGFYPSDIAVEWESNGVPELNVKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMH EALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-88 CH2 and CH3 domains without LALA mutation (SEQ ID NO: 47) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVS LTCLVKGFYPSDIAVEWESNGVPELNVKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEA LHNHYTQKSLSLSPG Amino acid sequences of Fcab FS18-7-95 loop regions FS18-7-95 AB loop-WDEPWGED (SEQ ID NO: 1) FS18-7-95 CD loop-SNGYQEDNY (SEQ ID NO: 48) FS18-7-95 EF loop-PYDRWVWPDE (SEQ ID NO: 3) Nucleotide sequence of Fcab FS18-7-95 CH3 domain (SEQ ID NO: 49) GGCCAGCCTCGAGAACCACAGGTGTACACCCTGCCCCCATCCTGGGATGAGCCGTGGGGTGAA GACGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG AGCAATGGGTATCAGGAAGATAACTATAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT TCTTCCTCTACAGCAAGCTCACCGTGCCGTATGATAGGTGGGTTTGGCCGGATGAGTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGT Amino acid sequence of Fcab FS18-7-95 CH3 domain (SEQ ID NO: 50) GQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYQEDNYKTTPPVLDSDGSFFL YSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-95 CH2 and CH3 domains, comprising LALA mutation (underlined) (SEQ ID NO: 51) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGED VSLTCLVKGFYPSDIAVEWESNGYQEDNYKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVM HEALHNHYTQKSLSLSPG Amino acid sequence of Fcab FS18-7-95 CH2 and CH3 domains without LALA mutation (SEQ ID NO: 52) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVS LTCLVKGFYPSDIAVEWESNGYQEDNYKTTPPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHE ALHNHYTQKSLSLSPG Amino acid sequence of the wild-type human IgG1 CH2 domain (SEQ ID NO: 53) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK Amino acid sequence of the human IgG1 CH2 domain comprising the "LALA mutation" (underlined) (SEQ ID NO: 54) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK Amino acid sequence of "wild-type" Fcab CH2 and CH3 domains without LALA mutation (SEQ ID NO: 55). APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG Amino acid sequence of "wild-type" Fcab CH2 and CH3 domains, comprising LALA mutation (underlined) (SEQ ID NO: 56) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPG Amino acid sequence of the human IgG1 hinge region (SEQ ID NO: 57) EPKSCDKTHTCPPCP Amino acid sequence of the human IgG1 truncated hinge region (SEQ ID NO: 58) TCPPCP Amino acid sequence anti-mouse LAG-3 Fcab FS18-7-108-29, comprising LALA mutation (underlined) (SEQ ID NO: 59) The CH3 domain is shown in italics. The AB, CD and EF loops of the CH3 domain are shown in bold and underlined. TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS VSLTCLVKGFYPSDIVVEWE KTTPPVLDSDGSFFLYSKLTV FS CSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-mouse LAG-3 Fcab FS18-7-108-29 without LALA mutation (SEQ ID NO: 60) The CH3 domain is shown in italics. The AB, CD and EF loops of the CH3 domain are shown in bold and underlined. TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS VSLTCLVKGFYPSDIVVEWE KTTPPVLDSDGSFFLYSKLTVPFERWMWPDEFS CSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-mouse LAG-3 Fcab FS18-7-108-35, comprising LALA mutation (underlined) (SEQ ID NO: 61) The CH3 domain is shown in italics. The AB, CD and EF loop regions are shown in bold and underlined. TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS VSLTCLVKGFYPSDISVEWE KTTPPVLDSDGSFFLYSKLTVPFERWMWPDEFS CSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-mouse LAG-3 Fcab FS18-7-108-35 without LALA mutation (SEQ ID NO: 62) The CH3 domain is shown in italics. The AB, CD and EF loop regions are shown in bold and underlined. TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS VSLTCLVKGFYPSDISVEWE KTTPPVLDSDGSFFLYSKLTVPFERWMWPDEFS CSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7-9/4420 comprising LALA mutation (SEQ ID NO: 63) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7-9/4420 without LALA mutation (SEQ ID NO: 64) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7-32/4420 comprising LALA mutation (SEQ ID NO: 65) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSEIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7- 32/without LALA mutation (SEQ ID NO: 66) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSEIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7-33/4420 comprising LALA mutation (SEQ ID NO: 67) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGQPEDNYKTTPP VLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7-33/4420 without LALA mutation (SEQ ID NO: 68) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGQPEDNYKTTPPVL DSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7-36/4420 comprising LALA mutation (SEQ ID NO: 69) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSYFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7-36/4420 without LALA mutation (SEQ ID NO: 70) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSYFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7-58/4420 comprising LALA mutation (SEQ ID NO: 71) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYPEIEFKTTPPV LDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7-58/4420 without LALA mutation (SEQ ID NO: 72) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYPEIEFKTTPPVLD SDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7-62/4420 comprising LALA mutation (SEQ ID NO: 73) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGIPEWNYKTTPP VLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7-62/4420 without LALA mutation (SEQ ID NO: 74) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGIPEWNYKTTPPVL DSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7-65/4420 comprising LALA mutation (SEQ ID NO: 75) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYAEYNYKTTPP VLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7-65/4420 without LALA mutation (SEQ ID NO: 76) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYAEYNYKTTPPVL DSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7-78/4420 comprising LALA mutation (SEQ ID NO: 77) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYKEENYKTTPP VLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7-78/4420 without LALA mutation (SEQ ID NO: 78) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYKEENYKTTPPVL DSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7-88/4420 comprising LALA mutation (SEQ ID NO: 79) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGVPELNVKTTPP VLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7-88/4420 without LALA mutation (SEQ ID NO: 80) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGVPELNVKTTPPVL DSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb2 FS18-7-95/4420 comprising LALA mutation (SEQ ID NO: 81) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYQEDNYKTTPP VLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-human LAG-3/FITC mAb.sup.2 FS18-7-95/4420 without LALA mutation (SEQ ID NO: 82) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYQEDNYKTTPPVL DSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-FITC mAb 4420 comprising LALA mutation (SEQ ID NO: 83) Position of the CDRs are underlined. Position of LALA mutation is in bold. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-FITC mAb 4420 without LALA mutation (SEQ ID NO: 84) Position of the CDRs are underlined. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSDS VKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-FITC mAb 4420 light chain (SEQ ID NO: 85) Position of the CDRs are underlined. DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKVSNRFSGVPDRF SGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC Amino acid sequences of the CDRs of anti-PD-L1 antibody 84G09 (according to IMGT) HCDR1 GFTFDDYA (SEQ ID NO: 86) HCDR2 ISWKSNII (SEQ ID NO: 87)
HCDR3 ARDITGSGSYGWFDP (SEQ ID NO: 88) LCDR1 QSISSY (SEQ ID NO: 89) LCDR2 VAS (SEQ ID NO: 90) LCDR3 QQSYSNPIT (SEQ ID NO: 91) Amino acid sequences of the CDRs of anti-PD-L1 antibody 84G09 (according to Kabat) HCDR1 DYAMH (SEQ ID NO: 136) HCDR2 GISWKSNIIGYADSVKG (SEQ ID NO: 137) HCDR3 DITGSGSYGWFDP (SEQ ID NO: 138) LCDR1 RASQSISSYLN (SEQ ID NO: 139) LCDR2 VASSLQS (SEQ ID NO: 140) LCDR3 QQSYSNPIT (SEQ ID NO: 141) Amino acid sequence of the anti-PD-L1 antibody 84G09 VH domain (SEQ ID NO: 92) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSS Amino acid sequence of the anti-PD-L1 antibody 84G09 VL domain (SEQ ID NO: 93) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKPLIYVASSLQSGVPSSFSGSGS GTDFTLTISSLQPEDFATYYCQQSYSNPITFGQGTRLEIK Amino acid sequence of the anti-human LAG-3/PD-L1 mAb.sup.2 FS18-7-9/84G09 with LALA mutation heavy chain (SEQ ID NO: 94) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb.sup.2 FS18-7-9/84G09 heavy chain (SEQ ID NO: 95) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb2 FS18-7-32/84G09 with LALA mutation heavy chain (SEQ ID NO: 96) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSEIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb.sup.2 FS18-7-32/84G09 heavy chain (SEQ ID NO: 97) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSEIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb.sup.2 FS18-7-33/84G09 with LALA mutation heavy chain (SEQ ID NO: 98) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGQPEDNYKTT PPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb.sup.2 FS18-7-33/84G09 heavy chain (SEQ ID NO: 99) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGQPEDNYKTTP PVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb.sup.2 FS18-7-36/84G09 with LALA mutation heavy chain (SEQ ID NO: 100) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSYFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb.sup.2 FS18-7-36/84G09 heavy chain (SEQ ID NO: 101) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSYFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb.sup.2 FS18-7-58/84G09 with LALA mutation heavy chain (SEQ ID NO: 102) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYPEIEFKTTP PVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb.sup.2 FS18-7-58/84G09 heavy chain (SEQ ID NO: 103) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYPEIEFKTTPP VLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb.sup.2 FS18-7-62/84G09 with LALA mutation heavy chain (SEQ ID NO: 104) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGIPEWNYKTT PPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb.sup.2 FS18-7-62/84G09 heavy chain (SEQ ID NO: 105) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGIPEWNYKTTP PVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb.sup.2 FS18-7-65/84G09 with LALA mutation heavy chain (SEQ ID NO: 106) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYAEYNYKTT PPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb.sup.2 FS18-7-65/84G09 heavy chain (SEQ ID NO: 107) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYAEYNYKTTP PVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb.sup.2 FS18-7-78/84G09 with LALA mutation heavy chain (SEQ ID NO: 108) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYKEENYKTT PPVLDSDGSFFLYSKLTV FSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb.sup.2 FS18-7-78/84G09 heavy chain (SEQ ID NO: 109) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYKEENYKTTP PVLDSDGSFFLYSKLTV FSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb.sup.2 FS18-7-88/84G09 with LALA mutation heavy chain (SEQ ID NO: 110) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGVPELNVKTT PPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb.sup.2 FS18-7-88/84G09 heavy chain (SEQ ID NO: 111) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGVPELNVKTTP PVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb.sup.2 FS18-7-95/84G09 with LALA mutation heavy chain (SEQ ID NO: 112) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYQEDNYKTT PPVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human LAG-3/PD-L1 mAb2 FS18-7-95/84G09 heavy chain (SEQ ID NO: 113) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIAVEWESNGYQEDNYKTTP PVLDSDGSFFLYSKLTVPYDRWVWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human PD-L1 mAb 84G09 with LALA heavy chain (SEQ ID NO: 114) Position of the CDRs are underlined. Position of LALA mutation is in bold. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human PD-L1 mAb 84G09 heavy chain (SEQ ID NO: 115) Position of the CDRs are underlined. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQTPGKGLEWVSGISWKSNIIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCARDITGSGSYGWFDPWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-human PD-L1 mAb 84G09 light chain (SEQ ID NO: 116) Position of the CDRs are underlined. DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKPLIYVASSLQSGVPSSFSGSGS GTDFTLTISSLQPEDFATYYCQQSYSNPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC Amino acid sequence of the anti-mouse LAG-3/PD-L1 mAb.sup.2 FS18-7-108-29/S1 with LALA mutation heavy chain (SEQ ID NO: 117) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVK GRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSAASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIVVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVPFERWMWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-mouse LAG-3/PD-L1 mAb.sup.2 FS18-7-108-29/S1 heavy chain (SEQ ID NO: 118) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVK GRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSAASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIVVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVPFERWMWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-mouse PD-L1 mAb S1 light chain (SEQ ID NO: 119) Position of the CDRs are underlined. DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQYLFTPPTFGQGTKVEIKRTVAAPSVF1FPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC Amino acid sequence of the anti-mouse LAG-3/PD-L1 mAb.sup.2 FS18-7-108-35/S1 with LALA mutation heavy chain (SEQ ID NO: 120) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVK GRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSAASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVPFERWMWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the anti-mouse LAG-3/PD-L1 mAb.sup.2 FS18-7-108-35/S1 heavy chain (SEQ ID NO: 121) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVK GRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSAASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVPFERWMWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the control PD-L1 mAb S1 with LALA mutation heavy chain (SEQ ID NO: 122) Position of the CDRs are underlined. Position of LALA mutation is in bold. EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVK GRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSAASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the control PD-L1 mAb S1 heavy chain (SEQ ID NO: 123) Position of the CDRs are underlined. Position of LALA mutation is in bold. EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVK GRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSAASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the control anti-human LAG-3 mAb 25F7 heavy chain (SEQ ID NO: 124) Position of the CDRs are underlined. QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSR VTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the control anti-human LAG-3 mAb 25F7 light chain (SEQ ID NO: 125) Position of the CDRs are underlined. EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSG TDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC Amino acid sequence of human LAG-3 (SEQ ID NO: 126) MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQ PDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGD FSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRP ASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLE PPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAG TYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPW LEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLILGVLSLLLLV TGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPEQL Amino acid sequence of mouse LAG-3 (SEQ ID NO: 127) MREDLLLGFLLLGLLWEAPVVSSGPGKELPVVWAQEGAPVHLPCSLKSPNLDPNFLRRGGVIWQHQ PDSGQPTPIPALDLHQGMPSPRQPAPGRYTVLSVAPGGLRSGRQPLHPHVQLEERGLQRGDFSLWL RPALRTDAGEYHATVRLPNRALSCSLRLRVGQASMIASPSGVLKLSDWVLLNCSFSRPDRPVSVHW FQGQNRVPVYNSPRHFLAETFLLLPQVSPLDSGTWGCVLTYRDGFNVSITYNLKVLGLEPVAPLTVYA AEGSRVELPCHLPPGVGTPSLLIAKWTPPGGGPELPVAGKSGNFTLHLEAVGLAQAGTYTCSIHLQG QQLNATVTLAVITVTPKSFGLPGSRGKLLCEVTPASGKERFVWRPLNNLSRSCPGPVLEIQEARLLAE RWQCQLYEGQRLLGATVYAAESSSGAHSARRISGDLKGGHLVLVLILGALSLFLLVAGAFGFHWWRK QLLLRRFSALEHGIQPFPAQRKIEELERELETEMGQEPEPEPEPQLEPEPRQL Amino acid sequence of cynomolqus LAG-3 (SEQ ID NO: 128) MWEAQFLGLLFLQPLWVAPVKPPQPGAEISVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQ PDSGPPAAAPGHPPVPGHRPAAPYSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRG DFSLWLRPARRADAGEYRATVHLRDRALSCRLRLRVGQASMTASPPGSLRTSDWVILNCSFSRPDR PASVHWFRSRGQGRVPVQGSPHHHLAESFLFLPHVGPMDSGLWGCILTYRDGFNVSIMYNLTVLGL EPATPLTVYAGAGSRVELPCRLPPAVGTQSFLTAKWAPPGGGPDLLVAGDNGDFTLRLEDVSQAQA GTYICHIRLQGQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPASGQEHFVWSPLNTPSQRSFSGP WLEAQEAQLLSQPWQCQLHQGERLLGAAVYFTELSSPGAQRSGRAPGALRAGHLPLFLILGVLFLLL LVTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPELEPEPELERELGPEPEPGPEPEP EQL Amino acid sequence of human PD-L1 (SEQ ID NO: 129) MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFV HGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPY NKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTT TNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKK CGIQDTNSKKQSDTHLEET Amino acid sequence of murine PD-L1 (SEQ ID NO: 130) MRIFAGIIFTACCHLLRAFTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKEDEQVIQFV AGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYR KINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATA NDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTHWVLLGSILLFLIVVSTVLLFLRKQVRMLDVE KCGVEDTSSKNRNDTQFEET Amino acid sequence of cynomoldus PD-L1 (SEQ ID NO: 131) MRIFAVFIFTIYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLTSLIVYWEMEDKNIIQFVH GEEDLKVQHSNYRQRAQLLKDQLSLGNAALRITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYN KINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLLNVTSTLRINTTA NEIFYCIFRRLDPEENHTAELVIPELPLALPPNERTHLVILGAIFLLLGVALTFIFYLRKGRMMDMKKCG IRVTNSKKQRDTQLEET
Amino acid sequence of the heavy chain of anti-mouse LAG-3/FITC mAb2 FS18-7-108- 29/4420 comprising LALA mutation (SEQ ID NO: 132) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSD SVKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIVVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVPFERWMWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-mouse LAG-3/FITC mAb2 FS18-7-108- 35/4420 comprising LALA mutation (SEQ ID NO: 133) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. Position of LALA mutation is in bold. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSD SVKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDISVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVPFERWMWPDEFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of the heavy chain of anti-mouse LAG-3/FITC mAb2 FS18-7-108- 29/4420 (SEQ ID NO: 134) Position of the CDRs are underlined, and the AB, CD, and EF loop sequences are in bold and underlined. EVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYNYETYYSD SVKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTSVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSWDEPWGEDVSLTCLVKGFYPSDIVVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVPFERWMWPDEFSCSVMHEALHNHYTQKSLSLSPG
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[0388] Sega E I, Leveson-Gower D B, Florek M, Schneidawind D, Luong R H, Negrin R S. Role of lymphocyte activation gene-3 (Lag-3) in conventional and regulatory T cell function in allogeneic transplantation. PLoS One. 9(1), e86551 (2014).
[0389] Wolchok J et al; Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med. 369(2), 122-33 (2013).
[0390] Woo S R, Turnis M E, Goldberg M V, Bankoti J, Selby M, Nirschl C J, Bettini M L, Gravano D M, Vogel P, Liu C L, Tangsombatvisit S, Grosso J F, Netto G, Smeltzer M P, Chaux A, Utz P J, Workman C J, Pardoll D M, Korman A J, Drake C G, Vignali D A. Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T cell function to promote tumoral immune escape. Cancer Res. 72(4), 917-2 (2012).
[0391] Workman C J, Vignali D A. Negative regulation of T cell homeostasis by lymphocyte activation gene-3 (CD223). J Immunol. 2005 Jan. 15; 174(2):688-95.
[0392] Workman C J, Vignali D A. The CD4-related molecule, LAG-3 (CD223), regulates the expansion of activated T cells. Eur J Immunol. 2003 April; 33(4):970-9.
Sequence CWU
1
1
14218PRTArtificial SequenceFS18-7-9 AB loop 1Trp Asp Glu Pro Trp Gly Glu
Asp1 529PRTArtificial SequenceFS18-7-9 CD loop 2Ser Asn Gly
Gln Pro Glu Asn Asn Tyr1 5310PRTArtificial SequenceFS18-7-9
EF loop 3Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu1 5
104318DNAArtificial SequenceNucleotide sequence of Fcab
FS18-7-9 CH3 domain 4ggccagcctc gagaaccaca ggtgtacacc ctgcccccat
cctgggatga gccgtggggt 60gaagacgtca gcctgacctg cctggtcaaa ggcttctatc
ccagcgaaat cgccgtggag 120tgggagagca atgggcagcc ggagaacaac tacaagacca
cgcctcccgt gctggactcc 180gacggctcct tcttcctcta cagcaagctc accgtgccgt
atgataggtg ggtttggccg 240gatgagttct catgctccgt gatgcatgag gctctgcaca
accactacac acagaagagc 300ctctccctgt ctccgggt
3185106PRTArtificial SequenceAmino acid sequence
of Fcab FS18-7-9 CH3 domain 5Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Trp Asp1 5 10
15Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe
20 25 30Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40
45Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe 50 55 60Phe Leu Tyr Ser Lys Leu
Thr Val Pro Tyr Asp Arg Trp Val Trp Pro65 70
75 80Asp Glu Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr 85 90
95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 100
1056216PRTArtificial SequenceAmino acid sequence of Fcab FS18-7-9 CH2 and
CH3 domains, comprising LALA mutation 6Ala Pro Glu Ala Ala Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10
15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val 20 25 30Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35
40 45Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu 50 55 60Gln
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65
70 75 80Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln 100 105 110Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro 115
120 125Trp Gly Glu Asp Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro 130 135
140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn145
150 155 160Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165
170 175Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg
Trp Val Trp Pro Asp Glu 180 185
190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
195 200 205Lys Ser Leu Ser Leu Ser Pro
Gly 210 2157216PRTArtificial SequenceAmino acid
sequence of Fcab FS18-7-9 CH2 and CH3 domains without LALA mutation
7Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1
5 10 15Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25
30Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr 35 40 45Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50
55 60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His65 70 75
80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100
105 110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Trp Asp Glu Pro 115 120 125Trp Gly
Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130
135 140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn145 150 155
160Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175Tyr Ser Lys Leu
Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu 180
185 190Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln 195 200 205Lys
Ser Leu Ser Leu Ser Pro Gly 210 21589PRTArtificial
SequenceFS18-7-32 CD loop 8Ser Asn Gly Gln Pro Glu Asn Asn Tyr1
59318DNAArtificial SequenceNucleotide sequence of Fcab FS18-7-32 CH3
domain 9ggccagcctc gagaaccaca ggtgtacacc ctgcccccat cctgggatga
gccgtggggt 60gaagacgtca gcctgacctg cctggtcaaa ggcttctatc ccagcgaaat
cgccgtggag 120tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt
gctggactcc 180gacggctcct tcttcctcta cagcaagctc accgtgccgt atgataggtg
ggtttggccg 240gatgagttct catgctccgt gatgcatgag gctctgcaca accactacac
acagaagagc 300ctctccctgt ctccgggt
31810106PRTArtificial SequenceAmino acid sequence of Fcab
FS18-7-32 CH3 domain 10Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Trp Asp1 5 10
15Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe
20 25 30Tyr Pro Ser Glu Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40
45Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe 50 55 60Phe Leu Tyr Ser Lys Leu
Thr Val Pro Tyr Asp Arg Trp Val Trp Pro65 70
75 80Asp Glu Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr 85 90
95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 100
10511216PRTArtificial SequenceAmino acid sequence of Fcab FS18-7-32 CH2
and CH3 domains, comprising LALA mutation 11Ala Pro Glu Ala Ala Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5
10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 20 25 30Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35
40 45Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu 50 55
60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65
70 75 80Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln 100 105
110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro
115 120 125Trp Gly Glu Asp Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135
140Ser Glu Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn145 150 155 160Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175Tyr Ser Lys Leu Thr Val Pro
Tyr Asp Arg Trp Val Trp Pro Asp Glu 180 185
190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 195 200 205Lys Ser Leu Ser
Leu Ser Pro Gly 210 21512216PRTArtificial
SequenceAmino acid sequence of Fcab FS18-7-32 CH2 and CH3 domains
without LALA mutation 12Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys1 5 10
15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu 50 55 60Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His65 70
75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro 115 120
125Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro 130 135 140Ser Glu Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn145 150
155 160Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu 165 170
175Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu
180 185 190Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 195
200 205Lys Ser Leu Ser Leu Ser Pro Gly 210
215139PRTArtificial SequenceFS18-7-33 CD loop 13Ser Asn Gly Gln Pro
Glu Asp Asn Tyr1 514318DNAArtificial SequenceNucleotide
sequence of Fcab FS18-7-33 CH3 domain 14ggccagcctc gagaaccaca
ggtgtacacc ctgcccccat cctgggatga gccgtggggt 60gaagacgtca gcctgacctg
cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 120tgggagagca atgggcagcc
ggaggacaac tacaagacca cgcctcccgt gctggactcc 180gacggctcct tcttcctcta
cagcaagctc accgtgccgt atgataggtg ggtttggccg 240gatgagttct catgctccgt
gatgcatgag gctctgcaca accactacac acagaagagc 300ctctccctgt ctccgggt
31815106PRTArtificial
SequenceAmino acid sequence of Fcab FS18-7-33 CH3 domain 15Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp1 5
10 15Glu Pro Trp Gly Glu Asp Val Ser
Leu Thr Cys Leu Val Lys Gly Phe 20 25
30Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu 35 40 45Asp Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55
60Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val
Trp Pro65 70 75 80Asp
Glu Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly 100 10516216PRTArtificial
SequenceAmino acid sequence of Fcab FS18-7-33 CH2 and CH3 domains,
comprising LALA mutation 16Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys1 5 10
15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu 50 55 60Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His65 70
75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro 115 120
125Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro 130 135 140Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asp Asn145 150
155 160Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu 165 170
175Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu
180 185 190Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 195
200 205Lys Ser Leu Ser Leu Ser Pro Gly 210
21517216PRTArtificial SequenceAmino acid sequence of Fcab FS18-7-33
CH2 and CH3 domains without LALA mutation 17Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5
10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 20 25 30Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35
40 45Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu 50 55
60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65
70 75 80Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln 100 105
110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro
115 120 125Trp Gly Glu Asp Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135
140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asp
Asn145 150 155 160Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175Tyr Ser Lys Leu Thr Val Pro
Tyr Asp Arg Trp Val Trp Pro Asp Glu 180 185
190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 195 200 205Lys Ser Leu Ser
Leu Ser Pro Gly 210 215189PRTArtificial
SequenceFS18-7-36 CD loop 18Ser Asn Gly Gln Pro Glu Asn Asn Tyr1
519318DNAArtificial SequenceNucleotide sequence of Fcab FS18-7-36
CH3 domain 19ggccagcctc gagaaccaca ggtgtacacc ctgcccccat cctgggatga
gccgtggggt 60gaagacgtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat
cgccgtggag 120tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt
gctggactcc 180gacggctcct acttcctcta cagcaagctc accgtgccgt atgataggtg
ggtttggccg 240gatgagttct catgctccgt gatgcatgag gctctgcaca accactacac
acagaagagc 300ctctccctgt ctccgggt
31820106PRTArtificial SequenceAmino acid sequence of Fcab
FS18-7-36 CH3 domain 20Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Trp Asp1 5 10
15Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe
20 25 30Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40
45Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Tyr 50 55 60Phe Leu Tyr Ser Lys Leu
Thr Val Pro Tyr Asp Arg Trp Val Trp Pro65 70
75 80Asp Glu Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr 85 90
95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 100
10521216PRTArtificial SequenceAmino acid sequence of CH2+CH3 of Fcab
FS18-7-36 CH2 and CH3 domains, comprising LALA mutation 21Ala Pro Glu Ala
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5
10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val 20 25
30Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
35 40 45Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu 50 55
60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65
70 75 80Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln 100 105
110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro
115 120 125Trp Gly Glu Asp Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135
140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn145 150 155 160Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu
165 170 175Tyr Ser Lys Leu Thr Val Pro
Tyr Asp Arg Trp Val Trp Pro Asp Glu 180 185
190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 195 200 205Lys Ser Leu Ser
Leu Ser Pro Gly 210 21522216PRTArtificial
SequenceAmino acid sequence of Fcab FS18-7-36 CH2 and CH3 domains
without LALA mutation 22Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys1 5 10
15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu 50 55 60Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His65 70
75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro 115 120
125Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro 130 135 140Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn145 150
155 160Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Tyr Phe Leu 165 170
175Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu
180 185 190Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 195
200 205Lys Ser Leu Ser Leu Ser Pro Gly 210
215239PRTArtificial SequenceFS18-7-58 CD loop 23Ser Asn Gly Tyr Pro
Glu Ile Glu Phe1 524318DNAArtificial SequenceNucleotide
sequence of Fcab FS18-7-58 CH3 domain 24ggccagcctc gagaaccaca
ggtgtacacc ctgcccccat cctgggatga gccgtggggt 60gaagacgtca gcctgacctg
cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 120tgggagagca atgggtatcc
agaaatcgaa ttcaagacca cgcctcccgt gctggactcc 180gacggctcct tcttcctcta
cagcaagctc accgtgcctt atgataggtg ggtttggccg 240gatgagttct catgctccgt
gatgcatgag gctctgcaca accactacac acagaagagc 300ctctccctgt ctccgggt
31825106PRTArtificial
SequenceAmino acid sequence of Fcab FS18-7-58 CH3 domain 25Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp1 5
10 15Glu Pro Trp Gly Glu Asp Val Ser
Leu Thr Cys Leu Val Lys Gly Phe 20 25
30Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Tyr Pro
Glu 35 40 45Ile Glu Phe Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55
60Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val
Trp Pro65 70 75 80Asp
Glu Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly 100 10526216PRTArtificial
SequenceAmino acid sequence of Fcab FS18-7-58 CH2 and CH3 domains,
comprising LALA mutation 26Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys1 5 10
15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu 50 55 60Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His65 70
75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro 115 120
125Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro 130 135 140Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Tyr Pro Glu Ile Glu145 150
155 160Phe Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu 165 170
175Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu
180 185 190Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 195
200 205Lys Ser Leu Ser Leu Ser Pro Gly 210
21527216PRTArtificial SequenceAmino acid sequence of Fcab FS18-7-58
CH2 and CH3 domains without LALA mutation 27Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5
10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 20 25 30Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35
40 45Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu 50 55
60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65
70 75 80Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln 100 105
110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro
115 120 125Trp Gly Glu Asp Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135
140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Tyr Pro Glu Ile
Glu145 150 155 160Phe Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175Tyr Ser Lys Leu Thr Val Pro
Tyr Asp Arg Trp Val Trp Pro Asp Glu 180 185
190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 195 200 205Lys Ser Leu Ser
Leu Ser Pro Gly 210 215289PRTArtificial
SequenceFS18-7-62 CD loop 28Ser Asn Gly Ile Pro Glu Trp Asn Tyr1
529318DNAArtificial SequenceNucleotide sequence of Fcab FS18-7-62
CH3 domain 29ggccagcctc gagaaccaca ggtgtacacc ctgcccccat cctgggatga
gccgtggggt 60gaagacgtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat
cgccgtggag 120tgggagagca atgggatccc agaatggaac tataagacca cgcctcccgt
gctggactcc 180gacggctcct tcttcctcta cagcaagctc accgtgccgt atgataggtg
ggtttggccg 240gatgagttct catgctccgt gatgcatgag gctctgcaca accactacac
acagaagagc 300ctctccctgt ctccgggt
31830106PRTArtificial SequenceAmino acid sequence of Fcab
FS18-7-62 CH3 domain 30Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Trp Asp1 5 10
15Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe
20 25 30Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Ile Pro Glu 35 40
45Trp Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe 50 55 60Phe Leu Tyr Ser Lys Leu
Thr Val Pro Tyr Asp Arg Trp Val Trp Pro65 70
75 80Asp Glu Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr 85 90
95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 100
10531216PRTArtificial SequenceAmino acid sequence of Fcab FS18-7-62 CH2
and CH3 domains, comprising LALA mutation 31Ala Pro Glu Ala Ala Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5
10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 20 25 30Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35
40 45Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu 50 55
60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65
70 75 80Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln 100 105
110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro
115 120 125Trp Gly Glu Asp Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135
140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Ile Pro Glu Trp
Asn145 150 155 160Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175Tyr Ser Lys Leu Thr Val Pro
Tyr Asp Arg Trp Val Trp Pro Asp Glu 180 185
190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 195 200 205Lys Ser Leu Ser
Leu Ser Pro Gly 210 21532216PRTArtificial
SequenceAmino acid sequence of Fcab FS18-7-62 CH2 and CH3 domains
without LALA mutation 32Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys1 5 10
15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu 50 55 60Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His65 70
75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro 115 120
125Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro 130 135 140Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Ile Pro Glu Trp Asn145 150
155 160Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu 165 170
175Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu
180 185 190Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 195
200 205Lys Ser Leu Ser Leu Ser Pro Gly 210
215339PRTArtificial SequenceFS18-7-65 CD loop 33Ser Asn Gly Tyr Ala
Glu Tyr Asn Tyr1 534318DNAArtificial SequenceNucleotide
sequence of Fcab FS18-7-65 CH3 domain 34ggccagcctc gagaaccaca
ggtgtacacc ctgcccccat cctgggatga gccgtggggt 60gaagacgtca gcctgacctg
cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 120tgggagagca atgggtatgc
agaatataac tataagacca cgcctcccgt gctggactcc 180gacggctcct tcttcctcta
cagcaagctc accgtgccgt atgataggtg ggtttggccg 240gatgagttct catgctccgt
gatgcatgag gctctgcaca accactacac acagaagagc 300ctctccctgt ctccgggt
31835106PRTArtificial
SequenceAmino acid sequence of Fcab FS18-7-65 CH3 domain 35Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp1 5
10 15Glu Pro Trp Gly Glu Asp Val Ser
Leu Thr Cys Leu Val Lys Gly Phe 20 25
30Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Tyr Ala
Glu 35 40 45Tyr Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55
60Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val
Trp Pro65 70 75 80Asp
Glu Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly 100 10536216PRTArtificial
SequenceAmino acid sequence of Fcab FS18-7-65 CH2 and CH3 domains,
comprising LALA mutation 36Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys1 5 10
15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu 50 55 60Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His65 70
75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro 115 120
125Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro 130 135 140Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Tyr Ala Glu Tyr Asn145 150
155 160Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu 165 170
175Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu
180 185 190Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 195
200 205Lys Ser Leu Ser Leu Ser Pro Gly 210
21537216PRTArtificial SequenceAmino acid sequence of Fcab FS18-7-65
CH2 and CH3 domains without LALA mutation 37Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5
10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 20 25 30Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35
40 45Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu 50 55
60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65
70 75 80Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln 100 105
110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro
115 120 125Trp Gly Glu Asp Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135
140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Tyr Ala Glu Tyr
Asn145 150 155 160Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175Tyr Ser Lys Leu Thr Val Pro
Tyr Asp Arg Trp Val Trp Pro Asp Glu 180 185
190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 195 200 205Lys Ser Leu Ser
Leu Ser Pro Gly 210 215389PRTArtificial
SequenceFS18-7-78 CD loop 38Ser Asn Gly Tyr Lys Glu Glu Asn Tyr1
539318DNAArtificial SequenceNucleotide sequence of Fcab FS18-7-78
CH3 domain 39ggccagcctc gagaaccaca ggtgtacacc ctgcccccat cctgggatga
gccgtggggt 60gaagacgtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat
cgccgtggag 120tgggagagca atgggtataa agaagaaaac tataagacca cgcctcccgt
gctggactcc 180gacggctcct tcttcctcta cagcaagctc accgtgccgt atgataggtg
ggtttggccg 240gatgagttct catgctccgt gatgcatgag gctctgcaca accactacac
acagaagagc 300ctctccctgt ctccgggt
31840106PRTArtificial SequenceAmino acid sequence of Fcab
FS18-7-78 CH3 domain 40Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Trp Asp1 5 10
15Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe
20 25 30Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Tyr Lys Glu 35 40
45Glu Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe 50 55 60Phe Leu Tyr Ser Lys Leu
Thr Val Pro Tyr Asp Arg Trp Val Trp Pro65 70
75 80Asp Glu Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr 85 90
95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 100
10541216PRTArtificial SequenceAmino acid sequence of Fcab FS18-7-78 CH2
and CH3 domains, comprising LALA mutation 41Ala Pro Glu Ala Ala Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5
10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 20 25 30Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35
40 45Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu 50 55
60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65
70 75 80Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln 100 105
110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro
115 120 125Trp Gly Glu Asp Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135
140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Tyr Lys Glu Glu
Asn145 150 155 160Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175Tyr Ser Lys Leu Thr Val Pro
Tyr Asp Arg Trp Val Trp Pro Asp Glu 180 185
190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 195 200 205Lys Ser Leu Ser
Leu Ser Pro Gly 210 21542216PRTArtificial
SequenceAmino acid sequence of Fcab FS18-7-78 CH2 and CH3 domains
without LALA mutation 42Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys1 5 10
15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu 50 55 60Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His65 70
75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro 115 120
125Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro 130 135 140Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Tyr Lys Glu Glu Asn145 150
155 160Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu 165 170
175Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu
180 185 190Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 195
200 205Lys Ser Leu Ser Leu Ser Pro Gly 210
215439PRTArtificial SequenceFS18-7-88 CD loop 43Ser Asn Gly Val Pro
Glu Leu Asn Val1 544318DNAArtificial SequenceNucleotide
sequence of Fcab FS18-7-88 CH3 domain 44ggccagcctc gagaaccaca
ggtgtacacc ctgcccccat cctgggatga gccgtggggt 60gaagacgtca gcctgacctg
cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 120tgggagagca atggggttcc
agaactgaac gttaagacca cgcctcccgt gctggactcc 180gacggctcct tcttcctcta
cagcaagctc accgtgccgt atgataggtg ggtttggccg 240gatgagttct catgctccgt
gatgcatgag gctctgcaca accactacac acagaagagc 300ctctccctgt ctccgggt
31845106PRTArtificial
SequenceAmino acid sequence of Fcab FS18-7-88 CH3 domain 45Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp1 5
10 15Glu Pro Trp Gly Glu Asp Val Ser
Leu Thr Cys Leu Val Lys Gly Phe 20 25
30Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Val Pro
Glu 35 40 45Leu Asn Val Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55
60Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val
Trp Pro65 70 75 80Asp
Glu Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly 100 10546216PRTArtificial
SequenceAmino acid sequence of Fcab FS18-7-88 CH2 and CH3 domains,
comprising LALA mutation 46Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys1 5 10
15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu 50 55 60Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His65 70
75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro 115 120
125Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro 130 135 140Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Val Pro Glu Leu Asn145 150
155 160Val Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu 165 170
175Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu
180 185 190Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 195
200 205Lys Ser Leu Ser Leu Ser Pro Gly 210
21547216PRTArtificial SequenceAmino acid sequence of Fcab FS18-7-88
CH2 and CH3 domains without LALA mutation 47Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5
10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 20 25 30Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35
40 45Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu 50 55
60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65
70 75 80Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln 100 105
110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro
115 120 125Trp Gly Glu Asp Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135
140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Val Pro Glu Leu
Asn145 150 155 160Val Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175Tyr Ser Lys Leu Thr Val Pro
Tyr Asp Arg Trp Val Trp Pro Asp Glu 180 185
190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 195 200 205Lys Ser Leu Ser
Leu Ser Pro Gly 210 215489PRTArtificial
SequenceFS18-7-95 CD loop 48Ser Asn Gly Tyr Gln Glu Asp Asn Tyr1
549318DNAArtificial SequenceNucleotide sequence of Fcab FS18-7-95
CH3 domain 49ggccagcctc gagaaccaca ggtgtacacc ctgcccccat cctgggatga
gccgtggggt 60gaagacgtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat
cgccgtggag 120tgggagagca atgggtatca ggaagataac tataagacca cgcctcccgt
gctggactcc 180gacggctcct tcttcctcta cagcaagctc accgtgccgt atgataggtg
ggtttggccg 240gatgagttct catgctccgt gatgcatgag gctctgcaca accactacac
acagaagagc 300ctctccctgt ctccgggt
31850106PRTArtificial SequenceAmino acid sequence of Fcab
FS18-7-95 CH3 domain 50Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Trp Asp1 5 10
15Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe
20 25 30Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Tyr Gln Glu 35 40
45Asp Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe 50 55 60Phe Leu Tyr Ser Lys Leu
Thr Val Pro Tyr Asp Arg Trp Val Trp Pro65 70
75 80Asp Glu Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr 85 90
95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 100
10551216PRTArtificial SequenceAmino acid sequence of Fcab FS18-7-95 CH2
and CH3 domains, comprising LALA mutation 51Ala Pro Glu Ala Ala Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5
10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 20 25 30Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35
40 45Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu 50 55
60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65
70 75 80Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln 100 105
110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro
115 120 125Trp Gly Glu Asp Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135
140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Tyr Gln Glu Asp
Asn145 150 155 160Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175Tyr Ser Lys Leu Thr Val Pro
Tyr Asp Arg Trp Val Trp Pro Asp Glu 180 185
190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 195 200 205Lys Ser Leu Ser
Leu Ser Pro Gly 210 21552216PRTArtificial
SequenceAmino acid sequence of Fcab FS18-7-95 CH2 and CH3 domains
without LALA mutation 52Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys1 5 10
15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu 50 55 60Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His65 70
75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
100 105 110Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro 115 120
125Trp Gly Glu Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro 130 135 140Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Tyr Gln Glu Asp Asn145 150
155 160Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu 165 170
175Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro Asp Glu
180 185 190Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln 195
200 205Lys Ser Leu Ser Leu Ser Pro Gly 210
21553110PRTArtificial SequenceAmino acid sequence of the wild-type
human IgG1 CH2 domain 53Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys1 5 10
15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
20 25 30Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
Glu 50 55 60Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His65 70
75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys 85 90
95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100
105 11054110PRTArtificial SequenceAmino
acid sequence of the human IgG1 CH2 domain comprising the "LALA
mutation" 54Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys1 5 10 15Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20
25 30Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50
55 60Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His65 70 75
80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys 85 90 95Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100
105 11055216PRTArtificial SequenceAmino acid
sequence of "wild-type" Fcab CH2 and CH3 domains without LALA
mutation 55Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys1 5 10 15Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20
25 30Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr 35 40
45Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50
55 60Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His65 70 75
80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys 85 90 95Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100
105 110Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu 115 120
125Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
130 135 140Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn145 150
155 160Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu 165 170
175Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
180 185 190Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln 195 200
205Lys Ser Leu Ser Leu Ser Pro Gly 210
21556216PRTArtificial SequenceAmino acid sequence of "wild-type" Fcab CH2
and CH3 domains, comprising LALA mutation 56Ala Pro Glu Ala Ala Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5
10 15Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val 20 25 30Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35
40 45Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu 50 55
60Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65
70 75 80Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85
90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln 100 105
110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
115 120 125Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135
140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn145 150 155 160Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
165 170 175Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185
190Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln 195 200 205Lys Ser Leu Ser
Leu Ser Pro Gly 210 2155715PRTArtificial SequenceAmino
acid sequence of the human IgG1 hinge region 57Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys Pro1 5
10 15586PRTArtificial SequenceAmino acid sequence of
the human IgG1 truncated hinge region 58Thr Cys Pro Pro Cys Pro1
559222PRTArtificial SequenceAmino acid sequence anti-mouse
LAG-3 Fcab FS18-7-108-29, comprising LALA mutation 59Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val1 5
10 15Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr 20 25
30Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
35 40 45Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys 50 55
60Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser65
70 75 80Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 85
90 95Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile 100 105
110Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
115 120 125Pro Ser Trp Asp Glu Pro Trp
Gly Glu Asp Val Ser Leu Thr Cys Leu 130 135
140Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu Trp Glu Ser
Asn145 150 155 160Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
165 170 175Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Pro Phe Glu Arg 180 185
190Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu
Ala Leu 195 200 205His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 210 215
22060222PRTArtificial SequenceAmino acid sequence of the
anti-mouse LAG-3 Fcab FS18-7-108-29 without LALA mutation 60Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val1 5
10 15Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr 20 25
30Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu 35 40 45Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 50 55
60Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser65 70 75 80Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
85 90 95Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile 100 105
110Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 115 120 125Pro Ser Trp Asp
Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu 130
135 140Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu
Trp Glu Ser Asn145 150 155
160Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
165 170 175Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Pro Phe Glu Arg 180
185 190Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val Met
His Glu Ala Leu 195 200 205His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 210
215 22061222PRTArtificial SequenceAmino acid sequence of
the anti-mouse LAG-3 Fcab FS18-7-108-35, comprising LALA mutation
61Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val1
5 10 15Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 20 25
30Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu 35 40 45Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 50
55 60Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser65 70 75
80Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
85 90 95Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 100
105 110Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro 115 120 125Pro Ser
Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu 130
135 140Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val
Glu Trp Glu Ser Asn145 150 155
160Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
165 170 175Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Pro Phe Glu Arg 180
185 190Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val
Met His Glu Ala Leu 195 200 205His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 210
215 22062222PRTArtificial SequenceAmino acid sequence of
the anti-mouse LAG-3 Fcab FS18-7-108-35 without LALA mutation 62Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val1
5 10 15Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 20 25
30Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu 35 40 45Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 50 55
60Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser65 70 75
80Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
85 90 95Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 100
105 110Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro 115 120 125Pro Ser
Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys Leu 130
135 140Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val
Glu Trp Glu Ser Asn145 150 155
160Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
165 170 175Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Pro Phe Glu Arg 180
185 190Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val
Met His Glu Ala Leu 195 200 205His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 210
215 22063447PRTArtificial SequenceAmino acid sequence of
the heavy chain of anti-human LAG-3/FITC mAb2 FS18-7-9/4420
comprising LALA mutation 63Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu
Val Gln Pro Gly Arg1 5 10
15Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30Trp Met Asn Trp Val Arg Gln
Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40
45Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser
Asp 50 55 60Ser Val Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65 70
75 80Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu
Asp Met Gly Ile Tyr 85 90
95Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr
100 105 110Ser Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120
125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly 130 135 140Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150
155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195
200 205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr 210 215 220His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser225
230 235 240Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 245
250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro 260 265 270Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275
280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val 290 295
300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305
310 315 320Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325
330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu 340 345
350Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys
355 360 365Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375
380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp385 390 395 400Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp
405 410 415Arg Trp Val Trp Pro Asp Glu
Phe Ser Cys Ser Val Met His Glu Ala 420 425
430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly 435 440 44564447PRTArtificial
SequenceAmino acid sequence of the heavy chain of anti-human
LAG-3/FITC mAb2 FS18-7-9/4420 without LALA mutation 64Glu Val Lys Leu Asp
Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe
Thr Phe Ser Asp Tyr 20 25
30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
35 40 45Ala Gln Ile Arg Asn Lys Pro Tyr
Asn Tyr Glu Thr Tyr Tyr Ser Asp 50 55
60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65
70 75 80Val Tyr Leu Gln Met
Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr 85
90 95Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr
Trp Gly Gln Gly Thr 100 105
110Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135
140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn145 150 155 160Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340
345 350Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp
Val Ser Leu Thr Cys 355 360 365Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370
375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr
Asp 405 410 415Arg Trp Val
Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala 420
425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 440
44565447PRTArtificial SequenceAmino acid sequence of the heavy chain of
anti-human LAG-3/FITC mAb2 FS18-7-32/4420 comprising LALA mutation
65Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15Pro Met Lys Leu Ser Cys
Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu
Glu Trp Val 35 40 45Ala Gln Ile
Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50
55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Ser Ser65 70 75
80Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr
85 90 95Tyr Cys Thr Gly Ser Tyr
Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130
135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn145 150 155
160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180
185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser 195 200 205Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Ala Ala Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala 275 280
285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr305 310
315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330
335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350Pro Pro Ser Trp Asp Glu
Pro Trp Gly Glu Asp Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Glu Ile Ala Val Glu Trp
Glu Ser 370 375 380Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390
395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Pro Tyr Asp 405 410
415Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
44566447PRTArtificial SequenceAmino acid sequence of the
heavy chain of anti-human LAG-3/FITC mAb2 FS18-7-32/without LALA
mutation 66Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly
Arg1 5 10 15Pro Met Lys
Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20
25 30Trp Met Asn Trp Val Arg Gln Ser Pro Glu
Lys Gly Leu Glu Trp Val 35 40
45Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50
55 60Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ser Lys Ser Ser65 70 75
80Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly
Ile Tyr 85 90 95Tyr Cys
Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110Ser Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 115 120
125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn145 150
155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr 210 215 220His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser225 230
235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg 245 250
255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275
280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305
310 315 320Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr 325
330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu 340 345 350Pro
Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys 355
360 365Leu Val Lys Gly Phe Tyr Pro Ser Glu
Ile Ala Val Glu Trp Glu Ser 370 375
380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385
390 395 400Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp 405
410 415Arg Trp Val Trp Pro Asp Glu Phe Ser Cys
Ser Val Met His Glu Ala 420 425
430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 44567447PRTArtificial
SequenceAmino acid sequence of the heavy chain of anti-human
LAG-3/FITC mAb2 FS18-7-33/4420 comprising LALA mutation 67Glu Val Lys Leu
Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Pro Met Lys Leu Ser Cys Val Ala Ser Gly
Phe Thr Phe Ser Asp Tyr 20 25
30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
35 40 45Ala Gln Ile Arg Asn Lys Pro Tyr
Asn Tyr Glu Thr Tyr Tyr Ser Asp 50 55
60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65
70 75 80Val Tyr Leu Gln Met
Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr 85
90 95Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr
Trp Gly Gln Gly Thr 100 105
110Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135
140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn145 150 155 160Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340
345 350Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp
Val Ser Leu Thr Cys 355 360 365Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370
375 380Asn Gly Gln Pro Glu Asp Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr
Asp 405 410 415Arg Trp Val
Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala 420
425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 440
44568447PRTArtificial SequenceAmino acid sequence of the heavy chain of
anti-human LAG-3/FITC mAb2 FS18-7-33/4420 without LALA mutation 68Glu
Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15Pro Met Lys Leu Ser Cys Val
Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu
Trp Val 35 40 45Ala Gln Ile Arg
Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50 55
60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Ser Ser65 70 75
80Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr
85 90 95Tyr Cys Thr Gly Ser Tyr
Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130
135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn145 150 155
160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180
185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser 195 200 205Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala 275 280
285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr305 310
315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330
335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350Pro Pro Ser Trp Asp Glu
Pro Trp Gly Glu Asp Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser 370 375 380Asn Gly Gln Pro Glu
Asp Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390
395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Pro Tyr Asp 405 410
415Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
44569447PRTArtificial SequenceAmino acid sequence of the
heavy chain of anti-human LAG-3/FITC mAb2 FS18-7-36/4420 comprising
LALA mutation 69Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Pro Met
Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20
25 30Trp Met Asn Trp Val Arg Gln Ser Pro
Glu Lys Gly Leu Glu Trp Val 35 40
45Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50
55 60Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ser Lys Ser Ser65 70 75
80Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly
Ile Tyr 85 90 95Tyr Cys
Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110Ser Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 115 120
125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn145 150
155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr 210 215 220His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser225 230
235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg 245 250
255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275
280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305
310 315 320Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr 325
330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu 340 345 350Pro
Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys 355
360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375
380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385
390 395 400Ser Asp Gly Ser
Tyr Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp 405
410 415Arg Trp Val Trp Pro Asp Glu Phe Ser Cys
Ser Val Met His Glu Ala 420 425
430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 44570447PRTArtificial
SequenceAmino acid sequence of the heavy chain of anti-human
LAG-3/FITC mAb2 FS18-7-36/4420 without LALA mutation 70Glu Val Lys Leu
Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Pro Met Lys Leu Ser Cys Val Ala Ser Gly
Phe Thr Phe Ser Asp Tyr 20 25
30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
35 40 45Ala Gln Ile Arg Asn Lys Pro Tyr
Asn Tyr Glu Thr Tyr Tyr Ser Asp 50 55
60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65
70 75 80Val Tyr Leu Gln Met
Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr 85
90 95Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr
Trp Gly Gln Gly Thr 100 105
110Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135
140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn145 150 155 160Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340
345 350Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp
Val Ser Leu Thr Cys 355 360 365Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370
375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr
Asp 405 410 415Arg Trp Val
Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala 420
425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 440
44571447PRTArtificial SequenceAmino acid sequence of the heavy chain of
anti-human LAG-3/FITC mAb2 FS18-7-58/4420 comprising LALA mutation
71Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15Pro Met Lys Leu Ser Cys
Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu
Glu Trp Val 35 40 45Ala Gln Ile
Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50
55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Ser Ser65 70 75
80Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr
85 90 95Tyr Cys Thr Gly Ser Tyr
Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130
135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn145 150 155
160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180
185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser 195 200 205Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Ala Ala Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala 275 280
285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr305 310
315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330
335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350Pro Pro Ser Trp Asp Glu
Pro Trp Gly Glu Asp Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser 370 375 380Asn Gly Tyr Pro Glu
Ile Glu Phe Lys Thr Thr Pro Pro Val Leu Asp385 390
395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Pro Tyr Asp 405 410
415Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
44572447PRTArtificial SequenceAmino acid sequence of the
heavy chain of anti-human LAG-3/FITC mAb2 FS18-7-58/4420 without
LALA mutation 72Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Pro Met
Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20
25 30Trp Met Asn Trp Val Arg Gln Ser Pro
Glu Lys Gly Leu Glu Trp Val 35 40
45Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50
55 60Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ser Lys Ser Ser65 70 75
80Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly
Ile Tyr 85 90 95Tyr Cys
Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110Ser Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 115 120
125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn145 150
155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr 210 215 220His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser225 230
235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg 245 250
255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275
280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305
310 315 320Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr 325
330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu 340 345 350Pro
Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys 355
360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375
380Asn Gly Tyr Pro Glu Ile Glu Phe Lys Thr Thr Pro Pro Val Leu Asp385
390 395 400Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp 405
410 415Arg Trp Val Trp Pro Asp Glu Phe Ser Cys
Ser Val Met His Glu Ala 420 425
430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 44573447PRTArtificial
SequenceAmino acid sequence of the heavy chain of anti-human
LAG-3/FITC mAb2 FS18-7-62/4420 comprising LALA mutation 73Glu Val Lys Leu
Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Pro Met Lys Leu Ser Cys Val Ala Ser Gly
Phe Thr Phe Ser Asp Tyr 20 25
30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
35 40 45Ala Gln Ile Arg Asn Lys Pro Tyr
Asn Tyr Glu Thr Tyr Tyr Ser Asp 50 55
60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65
70 75 80Val Tyr Leu Gln Met
Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr 85
90 95Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr
Trp Gly Gln Gly Thr 100 105
110Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135
140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn145 150 155 160Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340
345 350Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp
Val Ser Leu Thr Cys 355 360 365Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370
375 380Asn Gly Ile Pro Glu Trp Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr
Asp 405 410 415Arg Trp Val
Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala 420
425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 440
44574447PRTArtificial SequenceAmino acid sequence of the heavy chain of
anti-human LAG-3/FITC mAb2 FS18-7-62/4420 without LALA mutation 74Glu
Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15Pro Met Lys Leu Ser Cys Val
Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu
Trp Val 35 40 45Ala Gln Ile Arg
Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50 55
60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Ser Ser65 70 75
80Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr
85 90 95Tyr Cys Thr Gly Ser Tyr
Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130
135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn145 150 155
160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180
185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser 195 200 205Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala 275 280
285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr305 310
315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330
335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350Pro Pro Ser Trp Asp Glu
Pro Trp Gly Glu Asp Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser 370 375 380Asn Gly Ile Pro Glu
Trp Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390
395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Pro Tyr Asp 405 410
415Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
44575447PRTArtificial SequenceAmino acid sequence of the
heavy chain of anti-human LAG-3/FITC mAb2 FS18-7-65/4420 comprising
LALA mutation 75Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Pro Met
Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20
25 30Trp Met Asn Trp Val Arg Gln Ser Pro
Glu Lys Gly Leu Glu Trp Val 35 40
45Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50
55 60Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ser Lys Ser Ser65 70 75
80Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly
Ile Tyr 85 90 95Tyr Cys
Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110Ser Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 115 120
125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn145 150
155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr 210 215 220His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser225 230
235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg 245 250
255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275
280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305
310 315 320Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr 325
330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu 340 345 350Pro
Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys 355
360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375
380Asn Gly Tyr Ala Glu Tyr Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385
390 395 400Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp 405
410 415Arg Trp Val Trp Pro Asp Glu Phe Ser Cys
Ser Val Met His Glu Ala 420 425
430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 44576447PRTArtificial
SequenceAmino acid sequence of the heavy chain of anti-human
LAG-3/FITC mAb2 FS18-7-65/4420 without LALA mutation 76Glu Val Lys Leu
Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Pro Met Lys Leu Ser Cys Val Ala Ser Gly
Phe Thr Phe Ser Asp Tyr 20 25
30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
35 40 45Ala Gln Ile Arg Asn Lys Pro Tyr
Asn Tyr Glu Thr Tyr Tyr Ser Asp 50 55
60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65
70 75 80Val Tyr Leu Gln Met
Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr 85
90 95Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr
Trp Gly Gln Gly Thr 100 105
110Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135
140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn145 150 155 160Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340
345 350Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp
Val Ser Leu Thr Cys 355 360 365Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370
375 380Asn Gly Tyr Ala Glu Tyr Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr
Asp 405 410 415Arg Trp Val
Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala 420
425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 440
44577447PRTArtificial SequenceAmino acid sequence of the heavy chain of
anti-human LAG-3/FITC mAb2 FS18-7-78/4420 comprising LALA mutation
77Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15Pro Met Lys Leu Ser Cys
Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu
Glu Trp Val 35 40 45Ala Gln Ile
Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50
55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Ser Ser65 70 75
80Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr
85 90 95Tyr Cys Thr Gly Ser Tyr
Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130
135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn145 150 155
160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180
185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser 195 200 205Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Ala Ala Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala 275 280
285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr305 310
315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330
335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350Pro Pro Ser Trp Asp Glu
Pro Trp Gly Glu Asp Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser 370 375 380Asn Gly Tyr Lys Glu
Glu Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390
395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Pro Tyr Asp 405 410
415Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
44578447PRTArtificial SequenceAmino acid sequence of the
heavy chain of anti-human LAG-3/FITC mAb2 FS18-7-78/4420 without
LALA mutation 78Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Pro Met
Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20
25 30Trp Met Asn Trp Val Arg Gln Ser Pro
Glu Lys Gly Leu Glu Trp Val 35 40
45Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50
55 60Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ser Lys Ser Ser65 70 75
80Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly
Ile Tyr 85 90 95Tyr Cys
Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110Ser Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 115 120
125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn145 150
155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr 210 215 220His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser225 230
235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg 245 250
255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275
280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305
310 315 320Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr 325
330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu 340 345 350Pro
Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys 355
360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375
380Asn Gly Tyr Lys Glu Glu Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385
390 395 400Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp 405
410 415Arg Trp Val Trp Pro Asp Glu Phe Ser Cys
Ser Val Met His Glu Ala 420 425
430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 44579447PRTArtificial
SequenceAmino acid sequence of the heavy chain of anti-human
LAG-3/FITC mAb2 FS18-7-88/4420 comprising LALA mutation 79Glu Val Lys Leu
Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Pro Met Lys Leu Ser Cys Val Ala Ser Gly
Phe Thr Phe Ser Asp Tyr 20 25
30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
35 40 45Ala Gln Ile Arg Asn Lys Pro Tyr
Asn Tyr Glu Thr Tyr Tyr Ser Asp 50 55
60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65
70 75 80Val Tyr Leu Gln Met
Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr 85
90 95Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr
Trp Gly Gln Gly Thr 100 105
110Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135
140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn145 150 155 160Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340
345 350Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp
Val Ser Leu Thr Cys 355 360 365Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370
375 380Asn Gly Val Pro Glu Leu Asn Val Lys Thr
Thr Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr
Asp 405 410 415Arg Trp Val
Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala 420
425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 440
44580447PRTArtificial SequenceAmino acid sequence of the heavy chain of
anti-human LAG-3/FITC mAb2 FS18-7-88/4420 without LALA mutation 80Glu
Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15Pro Met Lys Leu Ser Cys Val
Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu
Trp Val 35 40 45Ala Gln Ile Arg
Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50 55
60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Ser Ser65 70 75
80Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr
85 90 95Tyr Cys Thr Gly Ser Tyr
Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130
135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn145 150 155
160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180
185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser 195 200 205Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala 275 280
285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr305 310
315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330
335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350Pro Pro Ser Trp Asp Glu
Pro Trp Gly Glu Asp Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser 370 375 380Asn Gly Val Pro Glu
Leu Asn Val Lys Thr Thr Pro Pro Val Leu Asp385 390
395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Pro Tyr Asp 405 410
415Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
44581447PRTArtificial SequenceAmino acid sequence of the
heavy chain of anti-human LAG-3/FITC mAb2 FS18-7-95/4420 comprising
LALA mutation 81Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Pro Met
Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20
25 30Trp Met Asn Trp Val Arg Gln Ser Pro
Glu Lys Gly Leu Glu Trp Val 35 40
45Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50
55 60Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ser Lys Ser Ser65 70 75
80Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly
Ile Tyr 85 90 95Tyr Cys
Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110Ser Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 115 120
125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn145 150
155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr 210 215 220His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser225 230
235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg 245 250
255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275
280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305
310 315 320Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr 325
330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu 340 345 350Pro
Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys 355
360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375
380Asn Gly Tyr Gln Glu Asp Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385
390 395 400Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp 405
410 415Arg Trp Val Trp Pro Asp Glu Phe Ser Cys
Ser Val Met His Glu Ala 420 425
430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 44582447PRTArtificial
SequenceAmino acid sequence of the heavy chain of anti-human
LAG-3/FITC mAb2 FS18-7-95/4420 without LALA mutation 82Glu Val Lys Leu
Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Pro Met Lys Leu Ser Cys Val Ala Ser Gly
Phe Thr Phe Ser Asp Tyr 20 25
30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val
35 40 45Ala Gln Ile Arg Asn Lys Pro Tyr
Asn Tyr Glu Thr Tyr Tyr Ser Asp 50 55
60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65
70 75 80Val Tyr Leu Gln Met
Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr 85
90 95Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr
Trp Gly Gln Gly Thr 100 105
110Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135
140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn145 150 155 160Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340
345 350Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp
Val Ser Leu Thr Cys 355 360 365Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370
375 380Asn Gly Tyr Gln Glu Asp Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr
Asp 405 410 415Arg Trp Val
Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala 420
425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 440
44583447PRTArtificial SequenceAmino acid sequence of the heavy chain of
anti-FITC mAb 4420 comprising LALA mutation 83Glu Val Lys Leu Asp Glu
Thr Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr
Phe Ser Asp Tyr 20 25 30Trp
Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35
40 45Ala Gln Ile Arg Asn Lys Pro Tyr Asn
Tyr Glu Thr Tyr Tyr Ser Asp 50 55
60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65
70 75 80Val Tyr Leu Gln Met
Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr 85
90 95Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr
Trp Gly Gln Gly Thr 100 105
110Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135
140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn145 150 155 160Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340
345 350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys 355 360 365Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370
375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser 405 410 415Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420
425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 440
44584447PRTArtificial SequenceAmino acid sequence of the heavy chain of
anti-FITC mAb 4420 without LALA mutation 84Glu Val Lys Leu Asp Glu Thr
Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10
15Pro Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe
Ser Asp Tyr 20 25 30Trp Met
Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val 35
40 45Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr
Glu Thr Tyr Tyr Ser Asp 50 55 60Ser
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser65
70 75 80Val Tyr Leu Gln Met Asn
Asn Leu Arg Val Glu Asp Met Gly Ile Tyr 85
90 95Tyr Cys Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp
Gly Gln Gly Thr 100 105 110Ser
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115
120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly 130 135
140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145
150 155 160Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165
170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser Ser 180 185
190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215
220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser225 230 235 240Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro 260 265
270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala 275 280 285Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340
345 350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys 355 360 365Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370
375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420
425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly 435 440
44585219PRTArtificial SequenceAmino acid sequence of the anti-FITC mAb
4420 light chain 85Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Ser Leu Gly1 5 10
15Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30Asn Gly Asn Thr Tyr Leu Arg
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45Pro Lys Val Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val
Pro 50 55 60Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70
75 80Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr
Phe Cys Ser Gln Ser 85 90
95Thr His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110Arg Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120
125Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe 130 135 140Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln145 150
155 160Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser 165 170
175Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
180 185 190Lys His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195
200 205Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210
215868PRTArtificial SequenceAmino acid sequences of the
CDRs of anti-PD-L1 antibody 84G09 (according to IMGT) HCDR1 86Gly
Phe Thr Phe Asp Asp Tyr Ala1 5878PRTArtificial
SequenceAmino acid sequences of the CDRs of anti-PD-L1 antibody
84G09 (according to IMGT) HCDR2 87Ile Ser Trp Lys Ser Asn Ile Ile1
58815PRTArtificial SequenceAmino acid sequences of the CDRs of
anti-PD-L1 antibody 84G09 (according to IMGT) HCDR3 88Ala Arg Asp
Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro1 5
10 15896PRTArtificial SequenceAmino acid
sequences of the CDRs of anti-PD-L1 antibody 84G09 (according to
IMGT) LCDR1 89Gln Ser Ile Ser Ser Tyr1 5903PRTArtificial
SequenceAmino acid sequences of the CDRs of anti-PD-L1 antibody
84G09 (according to IMGT) LCDR2 90Val Ala Ser1919PRTArtificial
SequenceAmino acid sequences of the CDRs of anti-PD-L1 antibody
84G09 (according to IMGT) LCDR3 91Gln Gln Ser Tyr Ser Asn Pro Ile Thr1
592122PRTArtificial SequenceAmino acid sequence of the
anti-PD-L1 antibody 84G09 VH domain 92Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Asp Asp Tyr 20 25 30Ala Met
His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile
Gly Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85
90 95Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp
Phe Asp Pro Trp 100 105 110Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115
12093107PRTArtificial SequenceAmino acid sequence of the anti-PD-L1
antibody 84G09 VL domain 93Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile 35 40
45Tyr Val Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Ser Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70
75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Tyr Ser Asn Pro Ile 85 90
95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100
10594451PRTArtificial SequenceAmino acid sequence of the
anti-human LAG-3/PD-L1 mAb2 FS18-7-9/84G09 with LALA mutation heavy
chain 94Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20
25 30Ala Met His Trp Val Arg Gln Thr Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser
Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr
Cys 85 90 95Ala Arg Asp
Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130
135 140Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser 210 215 220Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala225 230
235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275
280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305
310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325
330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 340 345 350Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val 355
360 365Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385
390 395 400Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405
410 415Val Pro Tyr Asp Arg Trp Val Trp Pro Asp
Glu Phe Ser Cys Ser Val 420 425
430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445Ser Pro Gly
45095451PRTArtificial SequenceAmino acid sequence of the anti-human
LAG-3/PD-L1 mAb2 FS18-7-9/84G09 heavy chain 95Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Asp Asp Tyr 20 25 30Ala Met
His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile
Gly Tyr Ala Asp Ser Val 50 55 60Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85
90 95Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp
Phe Asp Pro Trp 100 105 110Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115
120 125Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145
150 155 160Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165
170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr 180 185
190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205His Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser 210 215
220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu225 230 235 240Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser 260 265
270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu 275 280 285Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290
295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340
345 350Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp
Gly Glu Asp Val 355 360 365Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370
375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro385 390 395
400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
405 410 415Val Pro Tyr Asp
Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val 420
425 430Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu 435 440 445Ser
Pro Gly 45096451PRTArtificial SequenceAmino acid sequence of the
anti-human LAG-3/PD-L1 mAb2 FS18-7-32/84G09 with LALA mutation heavy
chain 96Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20
25 30Ala Met His Trp Val Arg Gln Thr Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser
Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr
Cys 85 90 95Ala Arg Asp
Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 130
135 140Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser 210 215 220Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala225 230
235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275
280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305
310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325
330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 340 345 350Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val 355
360 365Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Glu Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385
390 395 400Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405
410 415Val Pro Tyr Asp Arg Trp Val Trp Pro Asp
Glu Phe Ser Cys Ser Val 420 425
430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445Ser Pro Gly
45097451PRTArtificial SequenceAmino acid sequence of the anti-human
LAG-3/PD-L1 mAb2 FS18-7-32/84G09 heavy chain 97Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Asp Asp Tyr 20 25 30Ala
Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Gly Ile Ser Trp Lys Ser Asn Ile
Ile Gly Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85
90 95Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly
Trp Phe Asp Pro Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 195 200 205His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210
215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260
265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290
295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340
345 350Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro
Trp Gly Glu Asp Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Glu Ile Ala Val 370
375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro385 390 395
400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr 405 410 415Val Pro Tyr
Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val 420
425 430Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly 45098451PRTArtificial SequenceAmino acid sequence of
the anti-human LAG-3/PD-L1 mAb2 FS18-7-33/84G09 with LALA mutation
heavy chain 98Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Arg1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20
25 30Ala Met His Trp Val Arg Gln Thr Pro Gly
Lys Gly Leu Glu Trp Val 35 40
45Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr
Tyr Cys 85 90 95Ala Arg
Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser 210 215 220Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala225 230
235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275
280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305
310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325
330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 340 345 350Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val 355
360 365Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asp Asn Tyr Lys Thr Thr Pro385
390 395 400Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405
410 415Val Pro Tyr Asp Arg Trp Val Trp Pro Asp
Glu Phe Ser Cys Ser Val 420 425
430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445Ser Pro Gly
45099451PRTArtificial SequenceAmino acid sequence of the anti-human
LAG-3/PD-L1 mAb2 FS18-7-33/84G09 heavy chain 99Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Asp Asp Tyr 20 25 30Ala
Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Gly Ile Ser Trp Lys Ser Asn Ile
Ile Gly Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85
90 95Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly
Trp Phe Asp Pro Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 195 200 205His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210
215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260
265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290
295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340
345 350Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro
Trp Gly Glu Asp Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370
375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asp
Asn Tyr Lys Thr Thr Pro385 390 395
400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr 405 410 415Val Pro Tyr
Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val 420
425 430Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly 450100451PRTArtificial SequenceAmino acid sequence of
the anti-human LAG-3/PD-L1 mAb2 FS18-7-36/84G09 with LALA mutation
heavy chain 100Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20
25 30Ala Met His Trp Val Arg Gln Thr Pro
Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr
Tyr Cys 85 90 95Ala Arg
Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser 210 215 220Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala225 230
235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275
280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305
310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325
330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 340 345 350Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val 355
360 365Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro385
390 395 400Pro Val Leu Asp
Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Thr 405
410 415Val Pro Tyr Asp Arg Trp Val Trp Pro Asp
Glu Phe Ser Cys Ser Val 420 425
430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445Ser Pro Gly
450101451PRTArtificial SequenceAmino acid sequence of the anti-human
LAG-3/PD-L1 mAb2 FS18-7-36/84G09 heavy chain 101Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Asp Asp Tyr 20 25 30Ala
Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Gly Ile Ser Trp Lys Ser Asn Ile
Ile Gly Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85
90 95Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly
Trp Phe Asp Pro Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 195 200 205His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210
215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260
265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290
295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340
345 350Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro
Trp Gly Glu Asp Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370
375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro385 390 395
400Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu
Thr 405 410 415Val Pro Tyr
Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val 420
425 430Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly 450102451PRTArtificial SequenceAmino acid sequence of
the anti-human LAG-3/PD-L1 mAb2 FS18-7-58/84G09 with LALA mutation
heavy chain 102Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20
25 30Ala Met His Trp Val Arg Gln Thr Pro
Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr
Tyr Cys 85 90 95Ala Arg
Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser 210 215 220Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala225 230
235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275
280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305
310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325
330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 340 345 350Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val 355
360 365Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Tyr Pro Glu Ile Glu Phe Lys Thr Thr Pro385
390 395 400Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405
410 415Val Pro Tyr Asp Arg Trp Val Trp Pro Asp
Glu Phe Ser Cys Ser Val 420 425
430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445Ser Pro Gly
450103451PRTArtificial SequenceAmino acid sequence of the anti-human
LAG-3/PD-L1 mAb2 FS18-7-58/84G09 heavy chain 103Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Asp Asp Tyr 20 25 30Ala
Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Gly Ile Ser Trp Lys Ser Asn Ile
Ile Gly Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85
90 95Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly
Trp Phe Asp Pro Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 195 200 205His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210
215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260
265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290
295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340
345 350Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro
Trp Gly Glu Asp Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370
375 380Glu Trp Glu Ser Asn Gly Tyr Pro Glu Ile
Glu Phe Lys Thr Thr Pro385 390 395
400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr 405 410 415Val Pro Tyr
Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val 420
425 430Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly 450104451PRTArtificial SequenceAmino acid sequence of
the anti-human LAG-3/PD-L1 mAb2 FS18-7-62/84G09 with LALA mutation
heavy chain 104Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20
25 30Ala Met His Trp Val Arg Gln Thr Pro
Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr
Tyr Cys 85 90 95Ala Arg
Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser 210 215 220Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala225 230
235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275
280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305
310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325
330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 340 345 350Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val 355
360 365Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Ile Pro Glu Trp Asn Tyr Lys Thr Thr Pro385
390 395 400Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405
410 415Val Pro Tyr Asp Arg Trp Val Trp Pro Asp
Glu Phe Ser Cys Ser Val 420 425
430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445Ser Pro Gly
450105451PRTArtificial SequenceAmino acid sequence of the anti-human
LAG-3/PD-L1 mAb2 FS18-7-62/84G09 heavy chain 105Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Asp Asp Tyr 20 25 30Ala
Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Gly Ile Ser Trp Lys Ser Asn Ile
Ile Gly Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85
90 95Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly
Trp Phe Asp Pro Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 195 200 205His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210
215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260
265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290
295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340
345 350Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro
Trp Gly Glu Asp Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370
375 380Glu Trp Glu Ser Asn Gly Ile Pro Glu Trp
Asn Tyr Lys Thr Thr Pro385 390 395
400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr 405 410 415Val Pro Tyr
Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val 420
425 430Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly 450106451PRTArtificial SequenceAmino acid sequence of
the anti-human LAG-3/PD-L1 mAb2 FS18-7-65/84G09 with LALA mutation
heavy chain 106Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20
25 30Ala Met His Trp Val Arg Gln Thr Pro
Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr
Tyr Cys 85 90 95Ala Arg
Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser 210 215 220Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala225 230
235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275
280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305
310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325
330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 340 345 350Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val 355
360 365Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Tyr Ala Glu Tyr Asn Tyr Lys Thr Thr Pro385
390 395 400Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405
410 415Val Pro Tyr Asp Arg Trp Val Trp Pro Asp
Glu Phe Ser Cys Ser Val 420 425
430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445Ser Pro Gly
450107451PRTArtificial SequenceAmino acid sequence of the anti-human
LAG-3/PD-L1 mAb2 FS18-7-65/84G09 heavy chain 107Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Asp Asp Tyr 20 25 30Ala
Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Gly Ile Ser Trp Lys Ser Asn Ile
Ile Gly Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85
90 95Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly
Trp Phe Asp Pro Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 195 200 205His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210
215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260
265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290
295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340
345 350Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro
Trp Gly Glu Asp Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370
375 380Glu Trp Glu Ser Asn Gly Tyr Ala Glu Tyr
Asn Tyr Lys Thr Thr Pro385 390 395
400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr 405 410 415Val Pro Tyr
Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val 420
425 430Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly 450108451PRTArtificial SequenceAmino acid sequence of
the anti-human LAG-3/PD-L1 mAb2 FS18-7-78/84G09 with LALA mutation
heavy chain 108Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20
25 30Ala Met His Trp Val Arg Gln Thr Pro
Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr
Tyr Cys 85 90 95Ala Arg
Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser 210 215 220Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala225 230
235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275
280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305
310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325
330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 340 345 350Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val 355
360 365Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Tyr Lys Glu Glu Asn Tyr Lys Thr Thr Pro385
390 395 400Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405
410 415Val Pro Tyr Asp Arg Trp Val Trp Pro Asp
Glu Phe Ser Cys Ser Val 420 425
430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445Ser Pro Gly
450109451PRTArtificial SequenceAmino acid sequence of the anti-human
LAG-3/PD-L1 mAb2 FS18-7-78/84G09 heavy chain 109Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Asp Asp Tyr 20 25 30Ala
Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Gly Ile Ser Trp Lys Ser Asn Ile
Ile Gly Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85
90 95Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly
Trp Phe Asp Pro Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 195 200 205His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210
215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260
265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290
295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340
345 350Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro
Trp Gly Glu Asp Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370
375 380Glu Trp Glu Ser Asn Gly Tyr Lys Glu Glu
Asn Tyr Lys Thr Thr Pro385 390 395
400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr 405 410 415Val Pro Tyr
Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val 420
425 430Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly 450110451PRTArtificial SequenceAmino acid sequence of
the anti-human LAG-3/PD-L1 mAb2 FS18-7-88/84G09 with LALA mutation
heavy chain 110Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20
25 30Ala Met His Trp Val Arg Gln Thr Pro
Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr
Tyr Cys 85 90 95Ala Arg
Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser 210 215 220Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala225 230
235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275
280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305
310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325
330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 340 345 350Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val 355
360 365Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Val Pro Glu Leu Asn Val Lys Thr Thr Pro385
390 395 400Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405
410 415Val Pro Tyr Asp Arg Trp Val Trp Pro Asp
Glu Phe Ser Cys Ser Val 420 425
430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445Ser Pro Gly
450111451PRTArtificial SequenceAmino acid sequence of the anti-human
LAG-3/PD-L1 mAb2 FS18-7-88/84G09 heavy chain 111Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Asp Asp Tyr 20 25 30Ala
Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Gly Ile Ser Trp Lys Ser Asn Ile
Ile Gly Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85
90 95Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly
Trp Phe Asp Pro Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 195 200 205His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210
215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260
265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290
295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340
345 350Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro
Trp Gly Glu Asp Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370
375 380Glu Trp Glu Ser Asn Gly Val Pro Glu Leu
Asn Val Lys Thr Thr Pro385 390 395
400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr 405 410 415Val Pro Tyr
Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val 420
425 430Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly 450112451PRTArtificial SequenceAmino acid sequence of
the anti-human LAG-3/PD-L1 mAb2 FS18-7-95/84G09 with LALA mutation
heavy chain 112Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Arg1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20
25 30Ala Met His Trp Val Arg Gln Thr Pro
Gly Lys Gly Leu Glu Trp Val 35 40
45Ser Gly Ile Ser Trp Lys Ser Asn Ile Ile Gly Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr
Tyr Cys 85 90 95Ala Arg
Asp Ile Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro Trp 100
105 110Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro 115 120
125Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150
155 160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro 165 170
175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser 210 215 220Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala225 230
235 240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu 245 250
255Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275
280 285Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr 290 295 300Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn305
310 315 320Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro 325
330 335Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln 340 345 350Val
Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val 355
360 365Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 370 375
380Glu Trp Glu Ser Asn Gly Tyr Gln Glu Asp Asn Tyr Lys Thr Thr Pro385
390 395 400Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405
410 415Val Pro Tyr Asp Arg Trp Val Trp Pro Asp
Glu Phe Ser Cys Ser Val 420 425
430Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445Ser Pro Gly
450113451PRTArtificial SequenceAmino acid sequence of the anti-human
LAG-3/PD-L1 mAb2 FS18-7-95/84G09 heavy chain 113Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Asp Asp Tyr 20 25 30Ala
Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35
40 45Ser Gly Ile Ser Trp Lys Ser Asn Ile
Ile Gly Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85
90 95Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly
Trp Phe Asp Pro Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 195 200 205His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210
215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260
265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290
295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340
345 350Val Tyr Thr Leu Pro Pro Ser Trp Asp Glu Pro
Trp Gly Glu Asp Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370
375 380Glu Trp Glu Ser Asn Gly Tyr Gln Glu Asp
Asn Tyr Lys Thr Thr Pro385 390 395
400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr 405 410 415Val Pro Tyr
Asp Arg Trp Val Trp Pro Asp Glu Phe Ser Cys Ser Val 420
425 430Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly 450114451PRTArtificial SequenceAmino acid sequence of
the anti-human PD-L1 mAb 84G09 with LALA heavy chain 114Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Asp Asp Tyr 20 25
30Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Gly Ile Ser Trp Lys Ser
Asn Ile Ile Gly Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85
90 95Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr
Gly Trp Phe Asp Pro Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 195 200 205His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210
215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Ala Ala225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260
265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290
295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340
345 350Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370
375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro385 390 395
400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420
425 430Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly 450115451PRTArtificial SequenceAmino acid sequence of
the anti-human PD-L1 mAb 84G09 heavy chain 115Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Asp Asp Tyr 20 25
30Ala Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Gly Ile Ser Trp Lys Ser Asn
Ile Ile Gly Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65
70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85
90 95Ala Arg Asp Ile Thr Gly Ser Gly Ser Tyr Gly
Trp Phe Asp Pro Trp 100 105
110Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr 130 135
140Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr145 150 155 160Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 195 200 205His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 210
215 220Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu225 230 235
240Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260
265 270His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu 275 280 285Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290
295 300Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn305 310 315
320Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340
345 350Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val 355 360 365Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370
375 380Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro385 390 395
400Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr 405 410 415Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420
425 430Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu 435 440
445Ser Pro Gly 450116214PRTArtificial SequenceAmino acid sequence of
the anti-human PD-L1 mAb 84G09 light chain 116Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5
10 15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Ser Ile Ser Ser Tyr 20 25
30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile
35 40 45Tyr Val Ala Ser Ser Leu Gln Ser
Gly Val Pro Ser Ser Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ser Tyr Ser Asn Pro Ile 85
90 95Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln145 150 155 160Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205Phe Asn Arg Gly
Glu Cys 210117447PRTArtificial SequenceAmino acid sequence of the
anti-mouse LAG-3/PD-L1 mAb2 FS18-7-108-29/S1 with LALA mutation
heavy chain 117Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser 20
25 30Trp Ile His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50
55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp
Thr Ser Lys Asn Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95Ala Arg
Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100
105 110Leu Val Thr Val Ser Ala Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 115 120
125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn145 150
155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr 210 215 220His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser225 230
235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg 245 250
255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275
280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305
310 315 320Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr 325
330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu 340 345 350Pro
Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys 355
360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Val Val Glu Trp Glu Ser 370 375
380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385
390 395 400Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Phe Glu 405
410 415Arg Trp Met Trp Pro Asp Glu Phe Ser Cys
Ser Val Met His Glu Ala 420 425
430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445118447PRTArtificial
SequenceAmino acid sequence of the anti-mouse LAG-3/PD-L1 mAb2
FS18-7-108-29/S1 heavy chain 118Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser
20 25 30Trp Ile His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110Leu Val Thr Val Ser
Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115
120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly 130 135 140Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145
150 155 160Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu Gln 165
170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser 180 185 190Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195
200 205Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr 210 215
220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser225
230 235 240Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245
250 255Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro 260 265
270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295
300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr305 310 315 320Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345
350Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu
Thr Cys 355 360 365Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Val Val Glu Trp Glu Ser 370
375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Phe Glu
405 410 415Arg Trp Met Trp Pro
Asp Glu Phe Ser Cys Ser Val Met His Glu Ala 420
425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly 435 440
445119214PRTArtificial SequenceAmino acid sequence of the anti-mouse
PD-L1 mAb S1 light chain 119Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala
20 25 30Val Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70
75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr Leu Phe Thr Pro Pro 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115
120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145
150 155 160Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165
170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr 180 185 190Ala
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195
200 205Phe Asn Arg Gly Glu Cys
210120447PRTArtificial SequenceAmino acid sequence of the anti-mouse
LAG-3/PD-L1 mAb2 FS18-7-108-35/S1 with LALA mutation heavy chain 120Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asp Ser 20 25
30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Ala Trp Ile Ser
Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
Thr Ala Tyr65 70 75
80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg His Trp Pro
Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100
105 110Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130
135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn145 150 155
160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180
185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser 195 200 205Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Ala Ala Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala 275 280
285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr305 310
315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330
335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350Pro Pro Ser Trp Asp Glu
Pro Trp Gly Glu Asp Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp
Glu Ser 370 375 380Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390
395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Pro Phe Glu 405 410
415Arg Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445121447PRTArtificial SequenceAmino acid sequence of the
anti-mouse LAG-3/PD-L1 mAb2 FS18-7-108-35/S1 heavy chain 121Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asp Ser 20 25
30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Trp Ile Ser Pro
Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr
Ala Tyr65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg His Trp Pro Gly
Gly Phe Asp Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro 115 120 125Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130
135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn145 150 155
160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180
185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser 195 200 205Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala 275 280 285Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr 325 330 335Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340
345 350Pro Pro Ser Trp Asp Glu Pro Trp Gly Glu
Asp Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Ser 370
375 380Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp385 390
395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Pro Phe Glu 405 410
415Arg Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445122447PRTArtificial SequenceAmino acid sequence of the
control PD-L1 mAb S1 with LALA mutation heavy chain 122Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5
10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Asp Ser 20 25
30Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Trp Ile Ser Pro Tyr Gly
Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65
70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95Ala Arg Arg His Trp Pro Gly Gly Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135
140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn145 150 155 160Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340
345 350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys 355 360 365Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370
375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser 405 410 415Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420
425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly 435 440
445123447PRTArtificial SequenceAmino acid sequence of the control PD-L1
mAb S1 heavy chain 123Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser
20 25 30Trp Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70
75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90
95Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110Leu Val Thr Val Ser Ala
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120
125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly 130 135 140Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150
155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195
200 205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr 210 215 220His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser225
230 235 240Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 245
250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro 260 265 270Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275
280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val 290 295
300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305
310 315 320Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325
330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu 340 345
350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375
380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp385 390 395 400Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala 420 425
430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly 435 440 445124449PRTArtificial
SequenceAmino acid sequence of the control anti-human LAG-3 mAb 25F7
heavy chain 124Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro
Ser Glu1 5 10 15Thr Leu
Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr 20
25 30Tyr Trp Asn Trp Ile Arg Gln Pro Pro
Gly Lys Gly Leu Glu Trp Ile 35 40
45Gly Glu Ile Asn His Arg Gly Ser Thr Asn Ser Asn Pro Ser Leu Lys 50
55 60Ser Arg Val Thr Leu Ser Leu Asp Thr
Ser Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95Phe Gly
Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gln 100
105 110Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val 115 120
125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser145 150
155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val 165 170
175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp 210 215 220Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly225 230
235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile 245 250
255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275
280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305
310 315 320Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu 325
330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr 340 345 350Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355
360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp 370 375
380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385
390 395 400Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405
410 415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His 420 425
430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445Gly125214PRTArtificial
SequenceAmino acid sequence of the control anti-human LAG-3 mAb 25F7
light chain 125Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly1 5 10 15Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20
25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu Ile 35 40
45Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50
55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Glu Pro65 70 75
80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp
Pro Leu 85 90 95Thr Phe
Gly Gln Gly Thr Asn Leu Glu Ile Lys Arg Thr Val Ala Ala 100
105 110Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150
155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210126525PRTArtificial
SequenceAmino acid sequence of human LAG-3 126Met Trp Glu Ala Gln Phe Leu
Gly Leu Leu Phe Leu Gln Pro Leu Trp1 5 10
15Val Ala Pro Val Lys Pro Leu Gln Pro Gly Ala Glu Val
Pro Val Val 20 25 30Trp Ala
Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser Pro Thr Ile 35
40 45Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg
Ala Gly Val Thr Trp Gln 50 55 60His
Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro Gly His Pro Leu65
70 75 80Ala Pro Gly Pro His Pro
Ala Ala Pro Ser Ser Trp Gly Pro Arg Pro 85
90 95Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly
Leu Arg Ser Gly 100 105 110Arg
Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu Arg Gly Arg Gln 115
120 125Arg Gly Asp Phe Ser Leu Trp Leu Arg
Pro Ala Arg Arg Ala Asp Ala 130 135
140Gly Glu Tyr Arg Ala Ala Val His Leu Arg Asp Arg Ala Leu Ser Cys145
150 155 160Arg Leu Arg Leu
Arg Leu Gly Gln Ala Ser Met Thr Ala Ser Pro Pro 165
170 175Gly Ser Leu Arg Ala Ser Asp Trp Val Ile
Leu Asn Cys Ser Phe Ser 180 185
190Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg Asn Arg Gly Gln
195 200 205Gly Arg Val Pro Val Arg Glu
Ser Pro His His His Leu Ala Glu Ser 210 215
220Phe Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser Gly Pro Trp
Gly225 230 235 240Cys Ile
Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile Met Tyr Asn
245 250 255Leu Thr Val Leu Gly Leu Glu
Pro Pro Thr Pro Leu Thr Val Tyr Ala 260 265
270Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu Pro Ala
Gly Val 275 280 285Gly Thr Arg Ser
Phe Leu Thr Ala Lys Trp Thr Pro Pro Gly Gly Gly 290
295 300Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp Phe
Thr Leu Arg Leu305 310 315
320Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Thr Cys His Ile His
325 330 335Leu Gln Glu Gln Gln
Leu Asn Ala Thr Val Thr Leu Ala Ile Ile Thr 340
345 350Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu
Gly Lys Leu Leu 355 360 365Cys Glu
Val Thr Pro Val Ser Gly Gln Glu Arg Phe Val Trp Ser Ser 370
375 380Leu Asp Thr Pro Ser Gln Arg Ser Phe Ser Gly
Pro Trp Leu Glu Ala385 390 395
400Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys Gln Leu Tyr Gln
405 410 415Gly Glu Arg Leu
Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser 420
425 430Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly
Ala Leu Pro Ala Gly 435 440 445His
Leu Leu Leu Phe Leu Ile Leu Gly Val Leu Ser Leu Leu Leu Leu 450
455 460Val Thr Gly Ala Phe Gly Phe His Leu Trp
Arg Arg Gln Trp Arg Pro465 470 475
480Arg Arg Phe Ser Ala Leu Glu Gln Gly Ile His Pro Pro Gln Ala
Gln 485 490 495Ser Lys Ile
Glu Glu Leu Glu Gln Glu Pro Glu Pro Glu Pro Glu Pro 500
505 510Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro
Glu Gln Leu 515 520
525127521PRTArtificial SequenceAmino acid sequence of mouse LAG-3 127Met
Arg Glu Asp Leu Leu Leu Gly Phe Leu Leu Leu Gly Leu Leu Trp1
5 10 15Glu Ala Pro Val Val Ser Ser
Gly Pro Gly Lys Glu Leu Pro Val Val 20 25
30Trp Ala Gln Glu Gly Ala Pro Val His Leu Pro Cys Ser Leu
Lys Ser 35 40 45Pro Asn Leu Asp
Pro Asn Phe Leu Arg Arg Gly Gly Val Ile Trp Gln 50 55
60His Gln Pro Asp Ser Gly Gln Pro Thr Pro Ile Pro Ala
Leu Asp Leu65 70 75
80His Gln Gly Met Pro Ser Pro Arg Gln Pro Ala Pro Gly Arg Tyr Thr
85 90 95Val Leu Ser Val Ala Pro
Gly Gly Leu Arg Ser Gly Arg Gln Pro Leu 100
105 110His Pro His Val Gln Leu Glu Glu Arg Gly Leu Gln
Arg Gly Asp Phe 115 120 125Ser Leu
Trp Leu Arg Pro Ala Leu Arg Thr Asp Ala Gly Glu Tyr His 130
135 140Ala Thr Val Arg Leu Pro Asn Arg Ala Leu Ser
Cys Ser Leu Arg Leu145 150 155
160Arg Val Gly Gln Ala Ser Met Ile Ala Ser Pro Ser Gly Val Leu Lys
165 170 175Leu Ser Asp Trp
Val Leu Leu Asn Cys Ser Phe Ser Arg Pro Asp Arg 180
185 190Pro Val Ser Val His Trp Phe Gln Gly Gln Asn
Arg Val Pro Val Tyr 195 200 205Asn
Ser Pro Arg His Phe Leu Ala Glu Thr Phe Leu Leu Leu Pro Gln 210
215 220Val Ser Pro Leu Asp Ser Gly Thr Trp Gly
Cys Val Leu Thr Tyr Arg225 230 235
240Asp Gly Phe Asn Val Ser Ile Thr Tyr Asn Leu Lys Val Leu Gly
Leu 245 250 255Glu Pro Val
Ala Pro Leu Thr Val Tyr Ala Ala Glu Gly Ser Arg Val 260
265 270Glu Leu Pro Cys His Leu Pro Pro Gly Val
Gly Thr Pro Ser Leu Leu 275 280
285Ile Ala Lys Trp Thr Pro Pro Gly Gly Gly Pro Glu Leu Pro Val Ala 290
295 300Gly Lys Ser Gly Asn Phe Thr Leu
His Leu Glu Ala Val Gly Leu Ala305 310
315 320Gln Ala Gly Thr Tyr Thr Cys Ser Ile His Leu Gln
Gly Gln Gln Leu 325 330
335Asn Ala Thr Val Thr Leu Ala Val Ile Thr Val Thr Pro Lys Ser Phe
340 345 350Gly Leu Pro Gly Ser Arg
Gly Lys Leu Leu Cys Glu Val Thr Pro Ala 355 360
365Ser Gly Lys Glu Arg Phe Val Trp Arg Pro Leu Asn Asn Leu
Ser Arg 370 375 380Ser Cys Pro Gly Pro
Val Leu Glu Ile Gln Glu Ala Arg Leu Leu Ala385 390
395 400Glu Arg Trp Gln Cys Gln Leu Tyr Glu Gly
Gln Arg Leu Leu Gly Ala 405 410
415Thr Val Tyr Ala Ala Glu Ser Ser Ser Gly Ala His Ser Ala Arg Arg
420 425 430Ile Ser Gly Asp Leu
Lys Gly Gly His Leu Val Leu Val Leu Ile Leu 435
440 445Gly Ala Leu Ser Leu Phe Leu Leu Val Ala Gly Ala
Phe Gly Phe His 450 455 460Trp Trp Arg
Lys Gln Leu Leu Leu Arg Arg Phe Ser Ala Leu Glu His465
470 475 480Gly Ile Gln Pro Phe Pro Ala
Gln Arg Lys Ile Glu Glu Leu Glu Arg 485
490 495Glu Leu Glu Thr Glu Met Gly Gln Glu Pro Glu Pro
Glu Pro Glu Pro 500 505 510Gln
Leu Glu Pro Glu Pro Arg Gln Leu 515
520128533PRTArtificial SequenceAmino acid sequence of cynomolgus LAG-3
128Met Trp Glu Ala Gln Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu Trp1
5 10 15Val Ala Pro Val Lys Pro
Pro Gln Pro Gly Ala Glu Ile Ser Val Val 20 25
30Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser
Pro Thr Ile 35 40 45Pro Leu Gln
Asp Leu Ser Leu Leu Arg Arg Ala Gly Val Thr Trp Gln 50
55 60His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro
Gly His Pro Pro65 70 75
80Val Pro Gly His Arg Pro Ala Ala Pro Tyr Ser Trp Gly Pro Arg Pro
85 90 95Arg Arg Tyr Thr Val Leu
Ser Val Gly Pro Gly Gly Leu Arg Ser Gly 100
105 110Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu
Arg Gly Arg Gln 115 120 125Arg Gly
Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg Arg Ala Asp Ala 130
135 140Gly Glu Tyr Arg Ala Thr Val His Leu Arg Asp
Arg Ala Leu Ser Cys145 150 155
160Arg Leu Arg Leu Arg Val Gly Gln Ala Ser Met Thr Ala Ser Pro Pro
165 170 175Gly Ser Leu Arg
Thr Ser Asp Trp Val Ile Leu Asn Cys Ser Phe Ser 180
185 190Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe
Arg Ser Arg Gly Gln 195 200 205Gly
Arg Val Pro Val Gln Gly Ser Pro His His His Leu Ala Glu Ser 210
215 220Phe Leu Phe Leu Pro His Val Gly Pro Met
Asp Ser Gly Leu Trp Gly225 230 235
240Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser Ile Met Tyr
Asn 245 250 255Leu Thr Val
Leu Gly Leu Glu Pro Ala Thr Pro Leu Thr Val Tyr Ala 260
265 270Gly Ala Gly Ser Arg Val Glu Leu Pro Cys
Arg Leu Pro Pro Ala Val 275 280
285Gly Thr Gln Ser Phe Leu Thr Ala Lys Trp Ala Pro Pro Gly Gly Gly 290
295 300Pro Asp Leu Leu Val Ala Gly Asp
Asn Gly Asp Phe Thr Leu Arg Leu305 310
315 320Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Ile
Cys His Ile Arg 325 330
335Leu Gln Gly Gln Gln Leu Asn Ala Thr Val Thr Leu Ala Ile Ile Thr
340 345 350Val Thr Pro Lys Ser Phe
Gly Ser Pro Gly Ser Leu Gly Lys Leu Leu 355 360
365Cys Glu Val Thr Pro Ala Ser Gly Gln Glu His Phe Val Trp
Ser Pro 370 375 380Leu Asn Thr Pro Ser
Gln Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala385 390
395 400Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp
Gln Cys Gln Leu His Gln 405 410
415Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser
420 425 430Pro Gly Ala Gln Arg
Ser Gly Arg Ala Pro Gly Ala Leu Arg Ala Gly 435
440 445His Leu Pro Leu Phe Leu Ile Leu Gly Val Leu Phe
Leu Leu Leu Leu 450 455 460Val Thr Gly
Ala Phe Gly Phe His Leu Trp Arg Arg Gln Trp Arg Pro465
470 475 480Arg Arg Phe Ser Ala Leu Glu
Gln Gly Ile His Pro Pro Gln Ala Gln 485
490 495Ser Lys Ile Glu Glu Leu Glu Gln Glu Pro Glu Leu
Glu Pro Glu Pro 500 505 510Glu
Leu Glu Arg Glu Leu Gly Pro Glu Pro Glu Pro Gly Pro Glu Pro 515
520 525Glu Pro Glu Gln Leu
530129290PRTArtificial SequenceAmino acid sequence of human PD-L1 129Met
Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu1
5 10 15Asn Ala Phe Thr Val Thr Val
Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25
30Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys
Gln Leu 35 40 45Asp Leu Ala Ala
Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55
60Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln
His Ser Ser65 70 75
80Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn
85 90 95Ala Ala Leu Gln Ile Thr
Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100
105 110Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys
Arg Ile Thr Val 115 120 125Lys Val
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val 130
135 140Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys
Gln Ala Glu Gly Tyr145 150 155
160Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser
165 170 175Gly Lys Thr Thr
Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180
185 190Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr
Asn Glu Ile Phe Tyr 195 200 205Cys
Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210
215 220Val Ile Pro Glu Leu Pro Leu Ala His Pro
Pro Asn Glu Arg Thr His225 230 235
240Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu
Thr 245 250 255Phe Ile Phe
Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys 260
265 270Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln
Ser Asp Thr His Leu Glu 275 280
285Glu Thr 290130290PRTArtificial SequenceAmino acid sequence of
murine PD-L1 130Met Arg Ile Phe Ala Gly Ile Ile Phe Thr Ala Cys Cys His
Leu Leu1 5 10 15Arg Ala
Phe Thr Ile Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr 20
25 30Gly Ser Asn Val Thr Met Glu Cys Arg
Phe Pro Val Glu Arg Glu Leu 35 40
45Asp Leu Leu Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val 50
55 60Ile Gln Phe Val Ala Gly Glu Glu Asp
Leu Lys Pro Gln His Ser Asn65 70 75
80Phe Arg Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys
Gly Asn 85 90 95Ala Ala
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100
105 110Cys Cys Ile Ile Ser Tyr Gly Gly Ala
Asp Tyr Lys Arg Ile Thr Leu 115 120
125Lys Val Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp
130 135 140Pro Ala Thr Ser Glu His Glu
Leu Ile Cys Gln Ala Glu Gly Tyr Pro145 150
155 160Glu Ala Glu Val Ile Trp Thr Asn Ser Asp His Gln
Pro Val Ser Gly 165 170
175Lys Arg Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val
180 185 190Thr Ser Ser Leu Arg Val
Asn Ala Thr Ala Asn Asp Val Phe Tyr Cys 195 200
205Thr Phe Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala Glu
Leu Ile 210 215 220Ile Pro Glu Leu Pro
Ala Thr His Pro Pro Gln Asn Arg Thr His Trp225 230
235 240Val Leu Leu Gly Ser Ile Leu Leu Phe Leu
Ile Val Val Ser Thr Val 245 250
255Leu Leu Phe Leu Arg Lys Gln Val Arg Met Leu Asp Val Glu Lys Cys
260 265 270Gly Val Glu Asp Thr
Ser Ser Lys Asn Arg Asn Asp Thr Gln Phe Glu 275
280 285Glu Thr 290131290PRTArtificial SequenceAmino
acid sequence of cynomolgus PD-L1 131Met Arg Ile Phe Ala Val Phe Ile Phe
Thr Ile Tyr Trp His Leu Leu1 5 10
15Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu
Tyr 20 25 30Gly Ser Asn Met
Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35
40 45Asp Leu Thr Ser Leu Ile Val Tyr Trp Glu Met Glu
Asp Lys Asn Ile 50 55 60Ile Gln Phe
Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Asn65 70
75 80Tyr Arg Gln Arg Ala Gln Leu Leu
Lys Asp Gln Leu Ser Leu Gly Asn 85 90
95Ala Ala Leu Arg Ile Thr Asp Val Lys Leu Gln Asp Ala Gly
Val Tyr 100 105 110Arg Cys Met
Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val 115
120 125Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln
Arg Ile Leu Val Val 130 135 140Asp Pro
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr145
150 155 160Pro Lys Ala Glu Val Ile Trp
Thr Ser Ser Asp His Gln Val Leu Ser 165
170 175Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu
Lys Leu Leu Asn 180 185 190Val
Thr Ser Thr Leu Arg Ile Asn Thr Thr Ala Asn Glu Ile Phe Tyr 195
200 205Cys Ile Phe Arg Arg Leu Asp Pro Glu
Glu Asn His Thr Ala Glu Leu 210 215
220Val Ile Pro Glu Leu Pro Leu Ala Leu Pro Pro Asn Glu Arg Thr His225
230 235 240Leu Val Ile Leu
Gly Ala Ile Phe Leu Leu Leu Gly Val Ala Leu Thr 245
250 255Phe Ile Phe Tyr Leu Arg Lys Gly Arg Met
Met Asp Met Lys Lys Cys 260 265
270Gly Ile Arg Val Thr Asn Ser Lys Lys Gln Arg Asp Thr Gln Leu Glu
275 280 285Glu Thr
290132447PRTArtificial SequenceAmino acid sequence of the heavy chain of
anti-mouse LAG-3/FITC mAb2 FS18-7-108-29/4420 comprising LALA
mutation 132Glu Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly
Arg1 5 10 15Pro Met Lys
Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20
25 30Trp Met Asn Trp Val Arg Gln Ser Pro Glu
Lys Gly Leu Glu Trp Val 35 40
45Ala Gln Ile Arg Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50
55 60Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ser Lys Ser Ser65 70 75
80Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly
Ile Tyr 85 90 95Tyr Cys
Thr Gly Ser Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110Ser Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro 115 120
125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn145 150
155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200
205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr 210 215 220His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser225 230
235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg 245 250
255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275
280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305
310 315 320Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr 325
330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu 340 345 350Pro
Pro Ser Trp Asp Glu Pro Trp Gly Glu Asp Val Ser Leu Thr Cys 355
360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Val Val Glu Trp Glu Ser 370 375
380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385
390 395 400Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Phe Glu 405
410 415Arg Trp Met Trp Pro Asp Glu Phe Ser Cys
Ser Val Met His Glu Ala 420 425
430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445133447PRTArtificial
SequenceAmino acid sequence of the heavy chain of anti-mouse
LAG-3/FITC mAb2 FS18-7-108-35/4420 comprising LALA mutation 133Glu
Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15Pro Met Lys Leu Ser Cys Val
Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu
Trp Val 35 40 45Ala Gln Ile Arg
Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50 55
60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Ser Ser65 70 75
80Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr
85 90 95Tyr Cys Thr Gly Ser Tyr
Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130
135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn145 150 155
160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180
185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser 195 200 205Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Ala Ala Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala 275 280
285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr305 310
315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330
335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350Pro Pro Ser Trp Asp Glu
Pro Trp Gly Glu Asp Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp
Glu Ser 370 375 380Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390
395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Pro Phe Glu 405 410
415Arg Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445134447PRTArtificial SequenceAmino acid sequence of the
heavy chain of anti-mouse LAG-3/FITC mAb2 FS18-7-108-29/4420 134Glu
Val Lys Leu Asp Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15Pro Met Lys Leu Ser Cys Val
Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25
30Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu
Trp Val 35 40 45Ala Gln Ile Arg
Asn Lys Pro Tyr Asn Tyr Glu Thr Tyr Tyr Ser Asp 50 55
60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
Lys Ser Ser65 70 75
80Val Tyr Leu Gln Met Asn Asn Leu Arg Val Glu Asp Met Gly Ile Tyr
85 90 95Tyr Cys Thr Gly Ser Tyr
Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr 100
105 110Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro 115 120 125Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130
135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn145 150 155
160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180
185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro Ser 195 200 205Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210
215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260
265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala 275 280
285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290
295 300Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr305 310
315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330
335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350Pro Pro Ser Trp Asp Glu
Pro Trp Gly Glu Asp Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu Trp
Glu Ser 370 375 380Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390
395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Pro Phe Glu 405 410
415Arg Trp Met Trp Pro Asp Glu Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445135107PRTArtificial SequenceAmino acid sequence of Fcab
FS18-7-9 CH3 domain comprising C-terminal lysine 135Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Trp Asp1 5
10 15Glu Pro Trp Gly Glu Asp Val Ser Leu Thr
Cys Leu Val Lys Gly Phe 20 25
30Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
35 40 45Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe 50 55
60Phe Leu Tyr Ser Lys Leu Thr Val Pro Tyr Asp Arg Trp Val Trp Pro65
70 75 80Asp Glu Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr 85
90 95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
100 1051365PRTArtificial SequenceAmino acid
sequences of the CDRs of anti-PD-L1 antibody 84G09 (according to
Kabat) HCDR1 136Asp Tyr Ala Met His1 513717PRTArtificial
SequenceAmino acid sequences of the CDRs of anti-PD-L1 antibody
84G09 (according to Kabat) HCDR2 137Gly Ile Ser Trp Lys Ser Asn Ile Ile
Gly Tyr Ala Asp Ser Val Lys1 5 10
15Gly13813PRTArtificial SequenceAmino acid sequences of the CDRs
of anti-PD-L1 antibody 84G09 (according to Kabat) HCDR3 138Asp Ile
Thr Gly Ser Gly Ser Tyr Gly Trp Phe Asp Pro1 5
1013911PRTArtificial SequenceAmino acid sequences of the CDRs of
anti-PD-L1 antibody 84G09 (according to Kabat) LCDR1 139Arg Ala Ser
Gln Ser Ile Ser Ser Tyr Leu Asn1 5
101407PRTArtificial SequenceAmino acid sequences of the CDRs of
anti-PD-L1 antibody 84G09 (according to Kabat) LCDR2 140Val Ala Ser
Ser Leu Gln Ser1 51419PRTArtificial SequenceAmino acid
sequences of the CDRs of anti-PD-L1 antibody 84G09 (according to
Kabat) LCDR3 141Gln Gln Ser Tyr Ser Asn Pro Ile Thr1
5142318DNAArtificial SequenceCHO codon optimised nucleotide sequence of
Fcab FS18-7-9 CH3 domain 142ggccagcccc gggaacccca ggtgtacaca
ctgcctccat cctgggatga gccctggggc 60gaggatgtgt ctctgacctg tctcgtgaaa
ggcttctacc cctccgatat cgccgtggaa 120tgggagagca acggccagcc cgagaacaac
tacaagacca ccccccctgt gctggactcc 180gacggctcat tcttcctgta cagcaagctg
acagtgccct acgacagatg ggtgtggccc 240gacgagttct cctgctccgt gatgcacgag
gccctgcaca accactacac ccagaagtcc 300ctgtccctga gccccggc
318
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