Patent application title: FC FUSION PROTEINS
Inventors:
Henning Walczak (Heidelberg, DE)
IPC8 Class: AA61K39395FI
USPC Class:
4241341
Class name: Immunoglobulin, antiserum, antibody, or antibody fragment, except conjugate or complex of the same with nonimmunoglobulin material structurally-modified antibody, immunoglobulin, or fragment thereof (e.g., chimeric, humanized, cdr-grafted, mutated, etc.) antibody, immunoglobulin, or fragment thereof fused via peptide linkage to nonimmunoglobulin protein, polypeptide, or fragment thereof (i.e., antibody or immunoglobulin fusion protein or polypeptide)
Publication date: 2011-12-15
Patent application number: 20110305697
Abstract:
The invention relates to fusion proteins comprising at least a first
domain and a second domain selected from a constant Fc immunoglobulin
domain; wherein there is at least one amino acid overlap between the
first domain and the second domain in the fusion region. In a preferred
embodiment of the invention, the first domain is a ligand-binding
receptor domain comprising the extra-cellular domain of a
membrane-anchored receptor or a ligand-binding fragment thereof.Claims:
1. A fusion protein comprising (i) a first domain comprising a
ligand-binding domain of a TRAIL receptor or a TNF receptor fused to (ii)
a second domain comprising a portion of a constant immunoglobulin domain,
wherein there is at least one amino acid overlap between the first domain
and the second domain in the fusion region.
2. The fusion protein according to claim 1, wherein the receptor is TRAIL-R1 and the fusion protein comprises the amino acid sequence of SEQ ID NO: 21, 22, 23, or 24.
3. The fusion protein according to claim 1, wherein the receptor is TRAIL-R2 and the fusion protein comprises the amino acid sequence of SEQ ID NO: 27, 28, 29, 30, 31, or 32.
4. The fusion protein according to claim 1, wherein the receptor is TRAIL-R2 and the fusion protein comprises the amino acid sequence of SEQ ID NO: 34, 35, 36, 37, or 38.
5. The fusion protein according to claim 1, wherein the receptor is TRAIL-R2 and the fusion protein comprises the amino acid sequence of SEQ ID NO: 41, 42, 43, or 44.
6. The fusion protein according to claim 1, wherein the receptor is TRAIL-R3 and the fusion protein comprises the amino acid sequence of SEQ ID NO: 47, 48, 49, 50, 51, or 52.
7. The fusion protein according to claim 1, wherein the receptor is TRAIL-R3 and the fusion protein comprises the amino acid sequence of SEQ ID NO: 54, 55, 56, 57, or 58.
8. The fusion protein according to claim 1, wherein the receptor is TRAIL-R4 and the fusion protein comprises the amino acid sequence of SEQ ID NO: 61, 62, 63, or 64.
9. The fusion protein according to claim 1, wherein the receptor is TNF-R1 and the fusion protein comprises the amino acid sequence of SEQ ID NO: 67, 68, 69, 70, 71, 72, or 73.
10. The fusion protein according to claim 1, wherein the receptor is TNF-R2 and the fusion protein comprises the amino acid sequence of SEQ ID NO: 76, 77, 78, 79, 80, 81, or 82.
11. The fusion protein of claim 1, wherein the second domain is an Fc fragment of a constant heavy immunoglobulin domain comprising the CH2 and CH3 domain.
12. The fusion protein of claim 1, further comprising an N-terminal signal sequence.
13. An isolated nucleic acid sequence encoding the fusion protein of claim 1.
14. The nucleic acid sequence of claim 13, which is operatively linked to an expression control sequence.
15. A vector comprising the nucleic acid sequence of claim 13.
16. An isolated cell transformed or transfected with the nucleic acid sequence of claim 13.
17. The cell of claim 16, which is a prokaryotic cell.
18. The cell of claim 16, which is a eukaryotic cell.
19. A pharmaceutical composition comprising the fusion protein of claim 1 and a pharmaceutically acceptable carrier.
20. A pharmaceutical composition comprising the nucleic acid sequence of claim 13 and a pharmaceutically acceptable carrier.
Description:
[0001] This application is a divisional of U.S. application Ser. No.
10/551,004, filed Apr. 12, 2007; which is National Stage of International
Application PCT/EP2004/003239, filed Mar. 26, 2004, published Oct. 7,
2004, under PCT Article 21(2) in English; which claims the priority of
European Patent Application No. 03006949.6, filed Mar. 26, 2003. The
content of the above-applications are incorporated herein by reference in
their entirety.
REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM
[0002] The Sequence Listing is concurrently submitted herewith the specification as an ASCII formatted text file via EFS-Web with a file name of Sequence Listing.txt with a creation date of Aug. 18, 2011, and a size of 84 kilobytes. The Sequence Listing filed via EFS-Web is part of the specification and is hereby incorporated in its entirety by reference herein.
DESCRIPTION
[0003] The invention relates to fusion proteins comprising at least a biologically active polypeptide domain and a second domain selected from a constant immunoglobulin domain.
[0004] Fusion proteins comprising an immunoglobulin heavy and/or light chain dimer or an immunoglobulin heavy and/or light chain tetramer, in which an amino acid sequence of a ligand-binding partner which is a receptor, a carrier protein, a hormone, a growth factor or an enzyme, is substituted for the variable region of at least one immunoglobulin chain, are described in EP-A-0 526 452. A fusion protein comprising the extra cellular domain of the death receptor CD95 (APO-1; Fas) fused to an immunoglobulin Fc fragment is described in WO 95/27735. N-terminally truncated derivatives of the APO-1 molecule optionally fused to immunoglobulin Fc fragments are disclosed in EP-A-0 965 637. A fusion protein consisting of soluble IL-15Rα and Fc fragments is disclosed in WO 98/36768. A fusion protein consisting of an antagonist IL-15 mutant and an Fc IgG2a fragment is disclosed by Kim et al. (J. Immunol. 160 (1998), 5742-5748). These documents are incorporated herein by reference.
[0005] Although it has been shown that fusion proteins as described above have high biological activity in vitro and in vivo, there are concerns with regard to the immunogenic potential of such molecules since there is a fusion region between two protein domains of different origin comprising a non-naturally occurring amino sequence which may elicit an undesired immune response in an organism to which the fusion protein is administered.
[0006] WO 02/066514 describes artificial fusion proteins having a reduced immunogenicity compared to the parent non-modified molecule when exposed to a species in vivo. These proteins essentially consist of an immunoglobulin molecule or a fragment thereof covalently fused via its C-terminus to the N-terminus of a biologically active non-immunoglobulin molecule, preferably a polypeptide or protein or a biologically active fragment thereof. The molecules have amino acid sequences which are altered in one or more amino acid residue positions but, in principle, have the same biological activity as compared with the non-altered molecules. The changes are made in regions of the molecules which are identified as T-cell epitopes, which contribute to an immune reaction in a living host. A disadvantage of this procedure, however, is that not all epitopes, particularly not B-cell epitopes, can be reliably eliminated. Furthermore, the introduction of non-naturally occurring amino acid sequences can lead to the generation of neo-epitopes.
[0007] Thus, it was an object of the present invention to provide fusion proteins with at least two domains of different origin having a reduced immunogenic potential.
[0008] Thus, the present invention relates to a fusion protein comprising
(i) at least one first domain comprising a biologically active polypeptide and (ii) a heterologous second domain comprising at least a portion of a constant immunoglobulin domain, wherein there is at least one amino acid overlap between the first domain and the second domain in the fusion region.
[0009] The fusion protein may be a monomeric protein or a multimeric protein, e.g. a dimeric or tetrameric protein, which may be formed by multimerisation via the constant immunoglobulin domain.
[0010] According to the present invention, the design of a fusion protein comprises i) the selection of at least one first domain and a second domain which is heterologous to the first domain and ii) the selection of at least one terminal amino acid which is common to the first and the second domain, e.g. the last amino acid(s) of the first domain is (are) selected such that they are identical with the first amino acid(s) of the second domain. Preferably, the overlap has a length of one, two or three amino acids. Thus, a fusion protein is obtained which is free from a non-naturally occurring transition between the last amino acid of one domain and the first amino acid of another domain.
[0011] In an embodiment of the invention, the first domain(s) is (are) located at the N-terminus of the fusion protein, whereas the second domain is located at the C-terminus. Thus, in this embodiment, at least one carboxy terminal amino acid of a first domain overlaps with at least one amino terminal acid of the second domain.
[0012] In a further embodiment the second domain is located at the N-terminus of the fusion protein and the first domain(s) is (are) located at the C-terminus. Thus, in this embodiment, at least one carboxy terminal amino acid of the second domain overlaps with at least one amino terminal acid of a first domain.
[0013] In cases where the fusion protein comprises more than one, e.g. two or three, first domains, these domains are preferably located sequentially at the N-terminus or the C-terminus of the fusion protein and the second domain at the C-terminus or at the N-terminus, respectively. It should be noted that the first domains in such proteins may be the same or different. Transitions between individual first domains are preferably designed such that there is also at least one amino acid overlap (and thus not a non-naturally occurring transition between the last amino acid of one domain and the first amino acid of the other domain) between the individual first domains. Fusion proteins comprising multiple first domains are disclosed in WO 00/18932 which is incorporated herein by reference.
[0014] The first domain of the fusion protein comprises a biologically active polypeptide, i.e. a polypeptide which is capable of interacting with, e.g. binding to, a binding partner, e.g. another polypeptide, in its natural environment in a cell or an organism and which is preferably capable of exhibiting a pharmacological activity. The first domain is preferably a non-immunoglobulin polypeptide. The first domain may be a naturally occurring polypeptide or a variant thereof having desired, e.g. increased or reduced, biological activity or a fragment of a naturally occurring polypeptide or a variant thereof. The first domain is preferably selected from the ligand-binding domain of a receptor and a receptor-binding domain of a ligand. The terms "ligand" and "receptor" are understood in this context such that ligands are defined as proteins known to function to bind specifically to receptor molecules. The term "receptor" includes soluble or membrane-anchored receptor proteins having a hydrophobic transmembrane region or a phospholipid anchor. Further, the term "receptor" encompasses carrier proteins as well as hormones, cellular adhesive proteins, lectins, growth factors, enzymes, etc.
[0015] In a preferred embodiment of the invention the first domain is a ligand-binding receptor domain comprising the extra-cellular domain of a membrane-anchored receptor or a ligand-binding fragment thereof. The receptor is preferably selected from death receptors, growth factor receptors and cytokine receptors. More preferably, the receptor is selected from CD95 (APO-1; Fas), TRAIL receptors, TNF receptors, VEGF receptors, an interleukin receptor such as IL-15Rα. Most preferably the receptor is CD95, a TRAIL receptor, e.g. the TRAIL receptor-1, the TRAIL receptor-2, the TRAIL receptor-3 or the TRAIL receptor-4 or a TNF receptor, e.g. the TNF receptor-1 or the TNF receptor-2.
[0016] In a further embodiment, the first domain is a receptor-binding ligand domain. The ligand is preferably selected from death ligands such as the CD95 ligand, TRAIL, TNF, e.g. TNF-α or TNF-β, growth factors, e.g. VEGF and cytokines, such as interferons or interleukins, e.g. IL-15 or variants thereof.
[0017] In a still further embodiment, the fusion protein comprises multiple first domains which may be the same or different. A preferred example of such a multiple fusion protein is a VEGF Trap fusion protein comprising the second extracellular domain of the VEGF receptor 1 (Flt-1) with the third domain of the VEGF receptor 2 (KDR/FIK-1) and an IgG constant region.
[0018] The first domain protein is preferably a mammalian protein, more preferably a human protein. For therapeutic purposes in particular, the use of human proteins is preferred.
[0019] The second domain of the fusion protein comprises at least a portion of a constant immunoglobulin domain, e.g. a constant heavy immunoglobulin domain or a constant light immunoglobulin domain. Preferably, the second domain comprises at least a portion of a constant heavy immunoglobulin domain. The constant heavy immunoglobulin domain is preferably an Fc fragment comprising the CH2 and CH3 domain and, optionally, at least a part of the hinge region. The immunoglobulin domain may be an IgG, IgM, IgD or IgE immunoglobulin domain or a modified immunoglobulin domain derived therefrom. Preferably, the second domain comprises at least a portion of a constant IgG immunoglobulin domain. The IgG immunoglobulin domain may be selected from IgG1, IgG2, IgG3 of IgG4 domains or from modified domains such as are described in U.S. Pat. No. 5,925,734. The immunoglobulin domain may exhibit effector functions, particularly effector functions selected from ADCC and/or CDC. In some embodiments, however, modified immunoglobulin domains having modified, e.g. at least partially deleted, effector functions may be used.
[0020] Designing the fusion protein of the present invention comprises a selection of the terminal amino acid(s) of the first domain and of the second domain in order to create an at least one amino acid overlap between both domains. In order to achieve this goal it is usually necessary to delete one or several amino acids from a first and/or second domain and/or to add one or several amino acids from the naturally occurring adjacent domain to the first and/or second domain. For example, it may be necessary to provide a first domain having a deletion of preferably up to 10 and, more preferably, up to 6 amino acids, e.g. 1, 2, 3, 4, 5 or 6 amino acids from naturally occurring domain boundaries. On the other hand, it may be required to add preferably up to 10 and, more preferably, up to 6 amino acids, e.g. 1, 2, 3, 4, 5 or 6 amino acids from a naturally occurring adjacent domain to the first and/or second domain. When deleting and/or adding amino acids, however, one has to take care that the biological activity of the first domain and/or the second domain is not detrimentally affected.
[0021] The fusion protein of the invention may comprise an N-terminal signal sequence which allows secretion from a host cell after recombinant expression. The signal sequence may be a signal sequence which is homologous to the first domain of the fusion protein. Alternatively, the signal sequence may also be a heterologous signal sequence, e.g. the Igκ or the Igλ signal peptide sequence. In a different embodiment, the fusion protein is free from an N-terminal sequence, thus representing the mature form of the fusion protein.
[0022] The overlap between the first and the second domain or between two first domains has a length of preferably 1, 2 or 3 amino acids. More preferably the overlap has a length of one amino acid. Examples of overlapping amino acids are S, E, K, H, T, P and D.
[0023] The present invention is explained in detail below with regard to several specific preferred embodiments. It should be noted, however, that further fusion proteins of the invention may be manufactured by analogous means.
[0024] In a first preferred embodiment the first domain is the extracellular domain of human CD95. The extracellular domain of the fusion protein preferably comprises the amino acid sequence up to amino acid 170, 171, 172 or 173 of human CD95. Preferably, the extracellular domain of CD95 is fused with a human IgG Fc fragment, e.g. a human IgG1 Fc fragment. The amino acid sequence of the human CD95 molecule is shown in FIG. 1. The amino acid sequence of the human IgG1 chain constant domain is shown in FIG. 2. Especially preferred is the fusion protein comprising the amino acid sequence as shown in FIGS. 3A and 3B, wherein the overlapping amino acid sequence is S.
[0025] In a further especially preferred embodiment the first domain is the extracellular domain of a human TRAIL receptor, e.g. the human TRAIL receptor-1, the human TRAIL receptor-2, the human TRAIL receptor-3 and the human TRAIL receptor-4. The extracellular domain preferably comprises the amino acid sequence up to amino acid 232, 233, 234, 235, 236, 237, 238, 239 (TRAILR-1), 204, 205, 206, 207, 208, 209, 210 (TRAILR-2 long), 185, 186, 187, 188, 189, 190, 191 (TRAILR-2 long--without repeat), 179, 180, 181, 182, 183, 184 (TRAILR-2 short), 228, 229, 230, 231, 232, 233, 234, 235, 236, (TRAILR-3), 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 (TRAILR-3 without repeat) and 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 (TRAILR-4). Especially preferred is the human TRAIL receptor-2. The extracellular human TRAIL receptor domain may be fused with a human IgG-1 Fc fragment. The amino acid sequences of human TRAIL receptors are shown in FIG. 4 (TRAILR-1), FIG. 6 (TRAILR-2 long), FIG. 9 (TRAILR-2 short), FIG. 11 (TRAIL-3) and FIG. 14 (TRAILR-4). Specific examples of preferred fusion proteins comprise amino acid sequences as shown in FIGS. 5, 7, 8, 10, 12, 13 and 15.
[0026] In still a further preferred embodiment the fusion protein comprises a first domain which is the extracellular domain of a human TNF receptor, e.g. a human TNF receptor-1 or a human TNF receptor-2. The extracellular domain preferably comprises the amino acid sequence up to amino acid 203, 204, 205, 206, 207, 208, 209, 210, 211 (TNF-R1) or 248, 249, 250, 251, 252, 253, 254, 255, 256, 257 (TNF-R2). The extracellular domain of the human TNF receptor may be fused to a human IgG-1 Fc fragment. The amino acid sequence of human TNF receptors are shown in FIGS. 16 (TNF-R1) and 18 (TNF-R2). Specific examples of preferred fusion protein comprise amino acid sequences as shown in FIGS. 17 and 19.
[0027] A further aspect of the present invention relates to a nucleic acid molecule encoding a fusion protein as described above. The nucleic acid molecule may be a DNA molecule, e.g. a double-stranded or single-stranded DNA molecule, or an RNA molecule. The nucleic acid molecule may encode the fusion protein or a precursor thereof, e.g. a pro- or pre-proform of the fusion protein which may comprise a signal sequence or other heterologous amino acid portions for secretion or purification which are preferably located at the N- and/or C-terminus of the fusion protein. The heterologous amino acid portions may be linked to the first and/or second domain via a protease cleavage site, e.g. a Factor Xa, thrombin or IgA protease cleavage site.
[0028] The nucleic acid molecule may be operatively linked to an expression control sequence, e.g. an expression control sequence which allows expression of the nucleic acid molecule in a desired host cell. The nucleic acid molecule may be located on a vector, e.g. a plasmid, a bacteriophage, a viral vector, a chromosal integration vector, etc. Examples of suitable expression control sequences and vectors are described for example by Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, and Ausubel et al. (1989), Current Protocols in Molecular Biology, John Wiley & Sons.
[0029] Various expression vector/host cell systems may be used to express the nucleic acid sequences encoding the fusion proteins of the present invention. Suitable host cells include, but are not limited to, prokaryotic cells such as bacteria, e.g. E. coli, eukaryotic host cells such as yeast cells, insect cells, plant cells or animal cells, preferably mammalian cells and, more preferably, human cells.
[0030] Further, the invention relates to a non-human organism transformed or transfected with a nucleic acid molecule as described above. Such transgenic organisms may be generated by known methods of genetic transfer including homologous recombination.
[0031] A further aspect of the present invention relates to a pharmaceutical composition comprising as an active agent at least one fusion protein or a nucleic acid molecule coding thereof as described above. In an especially preferred embodiment, the first domain is a soluble death receptor, e.g. the extracellular domain of a death receptor as described above for use in the prophylaxis and/or treatment of disorders associated with apoptosis. Most preferably, the first domain is the extracellular CD95 domain.
[0032] In this embodiment of the invention the composition may be used in the prophylaxis and/or treatment of disorders selected from autoimmune disorders, AIDS, heart disorders, e.g. myocardial infarction, graft-versus-host-disorders, transplant rejection, brain damage, e.g. stroke, spinal cord injuries, e.g. paraplegia, sepsis, hepatitis, disorders associated with inflammation, ischemic reperfusion injury and renal disorders. These disorders and further disorders which may be treated by administration of death receptor fusion proteins, particularly CD95 fusion proteins, are described in WO 95/27735, WO 99/50413, WO 01/41803, EP-A-0 965 637 and EP-A-0 992 243 which are herein incorporated by reference.
[0033] The fusion protein is administered to a subject in need thereof, particularly a human patient, in a sufficient dose for the treatment of the specific conditions by suitable means. For example, the fusion protein may be formulated as a pharmaceutical composition together with pharmaceutically acceptable carriers, diluents and/or adjuvants. Therapeutic efficacy and toxicity may be determined according to standard protocols. The pharmaceutical composition may be administered systemically, e.g. intraperitoneally, intramuscularly or intravenously or locally, e.g. intranasally, subcutaneously or intrathecally. Preferred is intravenous administration.
[0034] Especially preferred is a death ligand inhibitor, e.g. a soluble extracellular CD95 or TRAIL receptor domain fused to an Fc fragment.
[0035] The dose of the fusion protein administered will of course be dependent on the subject to be treated, on the subject's weight, the type and severity of the injury, the manner of administration and the judgement of the prescribing physician. For the administration of CD95 or TRAIL-R fusion proteins, a daily dose of 0.001 to 100 mg/kg is suitable.
[0036] Moreover, the invention relates to a method for manufacturing a fusion protein comprising [0037] (i) at least one first domain comprising a biologically active protein fused to [0038] (ii) a second domain comprising at least a portion of a constant immunoglobulin domain with reduced immunogenic potential, wherein the first domain is fused to the second domain with at least one amino acid overlap.
[0039] Still a further aspect of the present relates to a fusion protein comprising: [0040] (i) at least one first domain comprising a biologically active polypeptide fused to [0041] (ii) a heterologous second domain which is capable of oligomerising the fusion protein wherein there is at least one amino acid overlap between the first and the second domain in the fusion region.
[0042] Fusion proteins comprising heterologous second domains which are capable or oligomerising the fusion proteins in the absence of third proteins are described in WO 01/49866 and in WO 02/090553, for example, which are incorporated herein by reference. The presence of at least one amino acid overlap, e.g. one, two or three amino acids overlap, between the first and the second domain in the fusion proteins leads--as explained above--to fusion proteins with reduced immunogenic potential.
[0043] The first domain in this oligomerising fusion protein is defined as above. Preferably, the first domain is an extracellular domain of a membrane-anchored receptor, or a ligand-binding fragment thereof. Especially preferred is that the receptor is selected from CD95, a TRAIL receptor, particularly the TRAIL receptor-2 and a TNF receptor, particularly the TNF receptor-2. Alternatively, the first domain may be a receptor-binding ligand domain, wherein the ligand is preferably selected from CD95 ligand, TRAIL and TNF. Specific examples of preferred first domains are as described above.
[0044] The second domain of the fusion protein comprises an oligomerising portion of a protein. Preferably, the second domain is capable of di- tri- tetra- or pentamerising the fusion protein. In this context, particular reference is made to the disclosure of WO 01/49866 and WO 02/090553, which are herein incorporated by reference. Preferred examples of second domains are C1q, MBP (Mannose Binding Protein), SP-A (Lung Surfactant Protein-A), SP-D (Lung Surfactant Protein-D), BC (Bovine Serum Conglutinine), CL43 (Bovine Collectine-43), ACRP-30 (a protein from the C1q family) and COMP (Cartilage Oligomeric Matrix Protein) or the collagen domain of EDA or a functionally active derivative thereof. Especially preferred are portions of ACRP-30, particularly of the human ACRP-30 protein, e.g. amino acids 18 to 108, or 18 to 110 or of COMP.
[0045] As described above, the first domain(s) of the fusion protein may be located at the N- or C-terminus and the second domain at the C- or N-terminus. Further, both the first and the second domains are preferably from the same species, more preferably of human origin. Furthermore, the features relating to preferred embodiments of the fusion proteins based on immunoglobulins also apply to the oligomerising fusion proteins.
[0046] The reduced immunogenic potential of the fusion protein results from the lack of non-naturally occurring transitions between the first and the second domain in the fusion proteins, which in turn leads to a decreased potential for the formation of neo-epitopes resulting from the fusion between two heterologous polypeptides.
[0047] The present invention is illustrated further by the following Figures and Examples.
FIGURE LEGEND
[0048] FIG. 1: the amino acid sequence of the human CD95 (APO-1; Fas) protein (SEQ ID NO: 13);
[0049] FIG. 2: the amino acid sequence of the human IgG-1 chain C-region (SEQ ID NO: 14);
[0050] FIGS. 3A and 3B: a preferred example of a CD95-Fc IgG1 fusion protein with an overlapping amino acid (SEQ ID NOs: 15-18);
[0051] FIG. 4: the amino acid sequence of the human TRAIL receptor-1 (SEQ ID NO: 19);
[0052] FIG. 5: preferred examples of TRAIL-R1Fc IgG1 fusion proteins with overlapping amino acids (SEQ ID NO: 20-24);
[0053] FIG. 6: the amino acid sequence of human TRAIL receptor-2 (long form) (SEQ ID NO: 25);
[0054] FIG. 7: preferred examples of TRAIL-R2 (long) Fc IgG1 fusion proteins with overlapping amino acids, including a repeat sequence (SEQ ID NO: 26-32);
[0055] FIG. 8: preferred examples of TRAIL-R2 (long form) Fc fusion proteins with overlapping amino acids (without repeat sequence) (SEQ ID NO: 33-38);
[0056] FIG. 9: the amino acid sequence of human TRAIL-R2 (short form) (SEQ ID NO: 39);
[0057] FIG. 10: preferred examples of TRAIL-R2 (short) Fc IgG1 fusion proteins with overlapping amino acids (SEQ ID NO: 40-44);
[0058] FIG. 11: the amino acid sequence of human TRAIL receptor R3 (SEQ ID NO: 45);
[0059] FIG. 12: preferred examples of TRAIL-R3Fc IgG1 fusion proteins with overlapping amino acids (repeats included) (SEQ ID NO: 46-52);
[0060] FIG. 13: preferred examples of TRAIL-R3Fc IgG1 fusion proteins with overlapping amino acids (repeats not included) (SEQ ID NO: 53-58);
[0061] FIG. 14: the amino acid sequence of human TRAIL receptor-4 (SEQ ID NO: 59);
[0062] FIG. 15: preferred examples of TRAIL-R4Fc IgG1 fusion proteins with overlapping amino acids (SEQ ID NO: 60-64);
[0063] FIG. 16: the amino acid sequence of human tumor necrosis factor receptor-1 (SEQ ID NO: 65);
[0064] FIG. 17: preferred examples of TNF-R1Fc IgG1 fusion proteins with overlapping amino acids (SEQ ID NO: 66-73);
[0065] FIG. 18: the amino acid sequence of human tumor necrosis factor receptor-2 (SEQ ID NO: 74);
[0066] FIG. 19: preferred examples of TNF-R2Fc IgG1 fusion proteins with overlapping amino acids (SEQ ID NO: 75-82).
EXAMPLES
Example 1
Fusion Protein Consisting of the Human CD95 Extracellular Domain and the Human IgG1 Fc Domain with Overlapping Amino Acids
Human CD95 Extracellular Domain
[0067] Bases 221-736 of Human CD95 (Genbank Acc. No. X63717). Utilized Sequence from Oehm, A., "Purification and Molecular Cloning of the APO-1 Cell Surface Antigen, a Member of the Tumour Necrosis Factor/Nerve Growth Factor Receptor Superfamily," Journal of Biological Chemistry Vol. 267, No. 15, pp. 10709-10715, 1992. cDNA was created from total RNA isolated from Peripheral Blood Lymphocytes (PBL) from donor blood by RT-PCR using Oligo dT primer. PCRs were used to amplify the cDNA of the extracellular domain of CD95 by including a restriction Hind III Site and a Kozak Sequence at the 5' of the Extracellular domain and at the 3' a Bgl II site (termination of the extracellular domain).
[0068] PCR primers for the amplification of CD95 cDNA with Taq polymerase:
TABLE-US-00001 Sense huCD95-Hind III: (SEQ ID NO: 1) TATA AAGCTT GCC ACC ATG CTG GGC ATC TG Antisense huCD95-Bgl II: (SEQ ID NO: 2) TATA AGATCT GGA TCC TTC CTC TTT GC
Human IgG1 Fc Domain
[0069] Sequence: 2050-2745 bp. Sequence used from, Ellison, J., "The nucleotide sequence of human immunoglobulin C gene", Nucleic Acid Research, Volume 10 Number 13, 1982. cDNA was created from total RNA isolated from Peripheral Blood Lymphocytes (PBL) from donor blood by RT-PCR using Oligo dT primer. A PCR was used to amplify the cDNA of human IgG1 Fc (partial hinge-CH3) by including a restriction Bgl II site at the 5' of the primer and at the 3' primer after the stop codon, an Xho I site.
[0070] PCR primers for the amplification of IgG1 Fc cDNA with Taq polymerase:
TABLE-US-00002 Sense hulgG1Fc-BglII: (SEQ ID NO: 3) TATA AGATCT TGT GAC AAA ACT CAC ACA TG Antisense hulgG1Fc-XhoI: (SEQ ID NO: 4) TATA CTCGAG TCA TTT ACC CGG AGA CAG GG
Cloning Procedure:
[0071] Following amplification the IgG1 Fc PCR product was digested with Bgl II and Xho I. The CD95 PCR product was digested with Hind III and Bgl II and pcDNA3.1 (with CMV promoter) with Hind III and Xho I. The products were purified via gel extraction (Qiagen Kit).
[0072] The hulgG1 Fc and CD95 fragments were ligated with T4 ligase into pcDNA3.1. After transfection of One Shot Top 10 chemically competent cells (E. coli) from Invitrogen Ordering # C4040-10 and amplification, a plasmid preparation was performed with Qiagen Plasmid Prep Kit.
[0073] A three point ligation was performed by digesting pcDNA3.1 with HindIII and XhoI, CD95 EC with HindIII and Bgl II, and hulgG1 Fc with BglII and XhoI. The presence of the CD95-hulgG1 Fc insert in pcDNA3.1 was verified by sequencing and restriction enzyme analysis. The vector containing insert was digested with HindIII and XbaI and the insert was ligated into pcDNA3.1 containing the EF-1 promoter.
[0074] The Kozak sequence of the original CD95-Fc construct was changed from GCCACCATGC to GCCGCCACCATGG by amplification of the whole CD95-Fc product with the primers SEQ ID 5 and SEQ ID 6.
Primers for Changing the Kozak Sequence from GCCACCATGC to GCCGCCACCATGG:
TABLE-US-00003 ShuCD95EC_altKozak (SEQ ID NO. 5) TATA AAGCTT GCC GCC ACC ATG GTG GGC ATC AS698 hulgG1Fc-Xho1 (SEQ ID NO: 6) TATA CTCGAG TCA TTT ACC CGG AGA CAG GG
Cloning Procedure:
[0075] The PCR product was cloned in pcDNA3.1N5 His Topo vector from Invitrogen (Ordering # K4800-01), digested with Hind III and Xba I as well as pcDNA3.1 containing the pEF promoter and ligated with T4 Ligase.
Expression and Isolation
[0076] The construct encoding the final product was transfected into cell lines suitable for protein expression. Transfection can be performed by any standard method know to those skilled in the art. Examples include electroporation, liposomal mediated transfer, calcium phosphate transfection. Cell lines suitable for the expression include 293T cells, COS-1, COS-7 and CHO cells. Other cell lines may be used.
[0077] In this example, 293T cells were transiently transfected by the calcium phosphate method. Alternatively, CHO cells were transfected utilizing FuGene6 and stable clones were selected.
[0078] The desired protein can be purified from the cell culture medium by chromatographic methods. Methods include but are not limited to affinity chromatography on protein-G or protein-A columns, ion-exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatograpy or a combination of these methods.
[0079] In the example the supernatant was purified on IgG columns (Amersham Pharmacia) according to the manufacturers instructions, leading to a highly purified product in a single step.
Example 2
[0080] Fusion protein consisting of the TRAIL receptor-2 and the human IgG1 Fc domain with overlapping amino acids
Human IgG1 Fc Domain:
[0081] Sequence used from, Ellison, J., "The nucleotide sequence of human immunoglobulin C gene", Nucleic Acid Research, Volume 10 Number 13, 1982. cDNA was created from total RNA isolated from Peripheral Blood Lymphocytes (PBL) from donor blood by RT-PCR using Oligo dT primer. A PCR was used to amplify the cDNA of human IgG1 Fc (partial hinge-CH3) with an overlapping sequence to TRAILR2 at the 5' end and at the 3' end after the stop codon an EcoRI site.
TABLE-US-00004 I. Primer: Sense_hulgG1 (SEQ ID NO: 7) cca ggg act cct gcc TCT TGT GAC AAA ACT CAC ACA TG (Capital letters => part of hulgG1) II. Primer: Antisense_ERIhulgG1 (SEQ ID NO: 8) TATA gaa ttc tca ttt acc cgg aga cag gg
TRAILR2:
[0082] Utilized Sequence from Walczak H., "TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL" The EMBO Journal Vol. 16, No. 17, pp. 5386-5397, 1997. (Accession number DDBJ/EMBL/GenBank: AF016849) cDNA was created from total RNA isolated from Peripheral Blood Lymphocytes (PBL) from donor blood by RT-PCR using an Oligo dT primer. A PCR was used to amplify the cDNA of TRAILR2 domain by including a restriction site Hind III and a Kozak Sequence at the 5' end and at the 3' end an overlapping sequence to human IgG1.
TABLE-US-00005 III. Primer: Sense_HIII_TRAILR2 (SEQ ID NO: 9) TATA aag ctt gcc gcc acc atg gaa caa cgg gga cag aac IV. Primer: Antisense_TRAILR2 (SEQ ID NO: 10) gtg agt ttt gtc aca aga GGC AGG AGT CCC TGG (Capital letters => part huTRAIL-R2, in reverse)
Cloning Procedure:
[0083] Following the amplification a gel extraction was performed to isolate the modified inserts. Then a third PCR utilizing both fragments was performed. Due to the overlap of both fragments and the primers at the end, this PCR joins in one product. Afterwards the product was digested with Hind III and EcoR I and ligated in a suitable expression vector, e.g. pcDNA3.1 (Invitrogen).
TABLE-US-00006 III. Primer: Sense_HIII_TRAILR2 (SEQ ID NO: 11) TATA aag ctt gcc gcc acc atg gaa caa cgg gga cag aac II. Primer: Antisense_ERIhulgG1 (SEQ ID NO: 12) TATA gaa ttc tca ttt acc cgg aga cag gg
Expression
[0084] The construct was cloned and expressed in suitable host cells as described in Example 1.
Example 3
Use of a CD95-Fc Construct for the Regeneration and Functional Recovery after Spinal Cord Injury
[0085] The CD95-Fc construct with overlapping amino acids as described in Example 1 was used for the treatment of spinal cord-injury in a mouse model as described by Demjen et al., Nat. Med. (Mar. 7, 2004). It was found that administration of the construct promotes regeneration and functional recovery after spinal cord injury.
Example 4
Use of CD95-Fc Construct for the Attenuation of Brain Damage in Stroke
[0086] The CD95-Fc construct with overlapping amino acids was investigated for its influence on primary ischemic death and secondary inflammatory injury in a mouse model as described by Martin-Villalba et al. (Cell Death Differ. 8 (2001), 679-686). It was found that administration of the CD95-Fc construct resulted in a significant decrease in both infarct volumes and mortality.
Sequence CWU
1
82130DNAArtificial SequenceDescription of Artificial Sequence PCR primer
for the amplification of CD95 cDNA 1tataaagctt gccaccatgc tgggcatctg
30227DNAArtificial
SequenceDescription of Artificial Sequence PCR primer for the
amplification of CD95 cDNA 2tataagatct ggatccttcc tctttgc
27330DNAArtificial SequenceDescription of
Artificial Sequence PCR primer for the amplification of IgG1 Fc
cDNA 3tataagatct tgtgacaaaa ctcacacatg
30430DNAArtificial SequenceDescription of Artificial Sequence PCR
primer for the amplification of IgG1 Fc cDNA 4tatactcgag tcatttaccc
ggagacaggg 30531DNAArtificial
SequenceDescription of Artificial Sequence PCR primer for the
changing the Kozak Sequence from GCCACCATGC to GCCGCCACCATGG
5tataaagctt gccgccacca tggtgggcat c
31630DNAArtificial SequenceDescription of Artificial Sequence PCR primer
for the changing the Kozak Sequence from GCCACCATGC to
GCCGCCACCATGG 6tatactcgag tcatttaccc ggagacaggg
30738DNAArtificial SequenceDescription of Artificial Sequence
primer for amplifying cDNA of human IgG1 Fc (partial hinge CH3)
7ccagggactc ctgcctcttg tgacaaaact cacacatg
38830DNAArtificial SequenceDescription of Artificial Sequence primer
for amplifying cDNA of human IgG1 Fc (partial hinge CH3) 8tatagaattc
tcatttaccc ggagacaggg
30940DNAArtificial SequenceDescription of Artificial Sequence primer
used to amplify the cDNA of TRAILR2 domain 9tataaagctt gccgccacca
tggaacaacg gggacagaac 401033DNAArtificial
SequenceDescription of Artificial Sequence primer used to amplify
the cDNA of TRAILR2 domain 10gtgagttttg tcacaagagg caggagtccc tgg
331140DNAArtificial SequenceDescription of
Artificial Sequence primer for PCR used to utlilize fragments for
cloning purposes 11tataaagctt gccgccacca tggaacaacg gggacagaac
401230DNAArtificial SequenceDescription of Artificial
Sequence primer for PCR used to utlilize fragments for cloning
purposes 12tatagaattc tcatttaccc ggagacaggg
3013335PRThumanCD95 >sp/P25445/TNR6_HUMAN Tumor necrosis
factor receptor superfamily 6 precursor (FASL-receptor)
(Apoptosis-mediating surface antigen FAS) (Apo-1 antigen) (CD95) -
Homo sapiens (Human) 13Met Leu Gly Ile Trp Thr Leu Leu Pro Leu Val Leu
Thr Ser Val Ala1 5 10
15Arg Leu Ser Ser Lys Ser Val Asn Ala Gln Val Thr Asp Ile Asn Ser
20 25 30Lys Gly Leu Glu Leu Arg Lys
Thr Val Thr Thr Val Glu Thr Gln Asn 35 40
45Leu Glu Gly Leu His His Asp Gly Gln Phe Cys His Lys Pro Cys
Pro 50 55 60Pro Gly Glu Arg Lys Ala
Arg Asp Cys Thr Val Asn Gly Asp Glu Pro65 70
75 80Asp Cys Val Pro Cys Gln Glu Gly Lys Glu Tyr
Thr Asp Lys Ala His 85 90
95Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly
100 105 110Leu Glu Val Glu Ile Asn
Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg 115 120
125Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His
Cys Asp 130 135 140Pro Cys Thr Lys Cys
Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr145 150
155 160Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser
Arg Ser Asn Leu Gly Trp 165 170
175Leu Cys Leu Leu Leu Leu Pro Ile Pro Leu Ile Val Trp Val Lys Arg
180 185 190Lys Glu Val Gln Lys
Thr Cys Arg Lys His Arg Lys Glu Asn Gln Gly 195
200 205Ser His Glu Ser Pro Thr Leu Asn Pro Glu Thr Val
Ala Ile Asn Leu 210 215 220Ser Asp Val
Asp Leu Ser Lys Tyr Ile Thr Thr Ile Ala Gly Val Met225
230 235 240Thr Leu Ser Gln Val Lys Gly
Phe Val Arg Lys Asn Gly Val Asn Glu 245
250 255Ala Lys Ile Asp Glu Ile Lys Asn Asp Asn Val Gln
Asp Thr Ala Glu 260 265 270Gln
Lys Val Gln Leu Leu Arg Asn Trp His Gln Leu His Gly Lys Lys 275
280 285Glu Ala Tyr Asp Thr Leu Ile Lys Asp
Leu Lys Lys Ala Asn Leu Cys 290 295
300Thr Leu Ala Glu Lys Ile Gln Thr Ile Ile Leu Lys Asp Ile Thr Ser305
310 315 320Asp Ser Glu Asn
Ser Asn Phe Arg Asn Glu Ile Gln Ser Leu Val 325
330 33514330PRThumanIgG1 > sp/P01857/GC1_HUMAN Ig
gamma-1 chain C region - Homo sapiens (Human) 14Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5
10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65
70 75 80Tyr Ile Cys Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp145 150 155 160Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185
190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210
215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260
265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290
295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
33015400PRTArtificial SequenceMUTAGEN(1)..(400)CD95-Fc fusion
protein (AA 1-172 CD95 and AA 102-330 IgG1) 15Met Leu Gly Ile Trp
Thr Leu Leu Pro Leu Val Leu Thr Ser Val Ala1 5
10 15Arg Leu Ser Ser Lys Ser Val Asn Ala Gln Val
Thr Asp Ile Asn Ser 20 25
30Lys Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gln Asn
35 40 45Leu Glu Gly Leu His His Asp Gly
Gln Phe Cys His Lys Pro Cys Pro 50 55
60Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro65
70 75 80Asp Cys Val Pro Cys
Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His 85
90 95Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys
Asp Glu Gly His Gly 100 105
110Leu Glu Val Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg
115 120 125Cys Lys Pro Asn Phe Phe Cys
Asn Ser Thr Val Cys Glu His Cys Asp 130 135
140Pro Cys Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu
Thr145 150 155 160Ser Asn
Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Cys Asp Lys Thr
165 170 175His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser 180 185
190Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg 195 200 205Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 210
215 220Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala225 230 235
240Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
245 250 255Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 260
265 270Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 275 280 285Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 290
295 300Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys305 310 315
320Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
325 330 335Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 340
345 350Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser 355 360 365Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 370
375 380Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys385 390 395
4001643PRThumanCD95 extracellular domain (AA 131-173) 16Pro Asn
Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp Pro Cys1 5
10 15Thr Lys Cys Glu His Gly Ile Ile
Lys Glu Cys Thr Leu Thr Ser Asn 20 25
30Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Asn 35
401722PRThumanhuIgG1 (AA 99-120) 17Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala1 5 10
15Pro Glu Leu Leu Gly Gly 201860PRTArtificial
SequenceCD95-Fc fusion protein of CD95 extracellular domain (AA
131-173) and huIgG1 (AA99-120) with an overlapping amino acid (CD95
AA 172 and huIgG1 AA 102) 18Pro Asn Phe Phe Cys Asn Ser Thr Val Cys
Glu His Cys Asp Pro Cys1 5 10
15Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr Ser Asn
20 25 30Thr Lys Cys Lys Glu Glu
Gly Ser Arg Ser Cys Asp Lys Thr His Thr 35 40
45Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 50
55 6019468PRThumanTRAIL-R1
>sp/000220/T10A_HUMAN Tumor necrosis factor receptor superfamily
member 10A precursor (Death receptor 4) (TNF-related
apoptosis-including ligand receptor 1) (TRAIL receptor-1) (TRAIL-R1)
19Met Ala Pro Pro Pro Ala Arg Val His Leu Gly Ala Phe Leu Ala Val1
5 10 15Thr Pro Asn Pro Gly Ser
Ala Ala Ser Gly Thr Glu Ala Ala Ala Ala 20 25
30Thr Pro Ser Lys Val Trp Gly Ser Ser Ala Gly Arg Ile
Glu Pro Arg 35 40 45Gly Gly Gly
Arg Gly Ala Leu Pro Thr Ser Met Gly Gln His Gly Pro 50
55 60Ser Ala Arg Ala Arg Ala Gly Arg Ala Pro Gly Pro
Arg Pro Ala Arg65 70 75
80Glu Ala Ser Pro Arg Leu Arg Val His Lys Thr Phe Lys Phe Val Val
85 90 95Val Gly Val Leu Leu Gln
Val Val Pro Ser Ser Ala Ala Thr Ile Lys 100
105 110Leu His Asp Gln Ser Ile Gly Thr Gln Gln Trp Glu
His Ser Pro Leu 115 120 125Gly Glu
Leu Cys Pro Pro Gly Ser His Arg Ser Glu His Pro Gly Ala 130
135 140Cys Asn Arg Cys Thr Glu Gly Val Gly Tyr Thr
Asn Ala Ser Asn Asn145 150 155
160Leu Phe Ala Cys Leu Pro Cys Thr Ala Cys Lys Ser Asp Glu Glu Glu
165 170 175Arg Ser Pro Cys
Thr Thr Thr Arg Asn Thr Ala Cys Gln Cys Lys Pro 180
185 190Gly Thr Phe Arg Asn Asp Asn Ser Ala Glu Met
Cys Arg Lys Cys Ser 195 200 205Arg
Gly Cys Pro Arg Gly Met Val Lys Val Lys Asp Cys Thr Pro Trp 210
215 220Ser Asp Ile Glu Cys Val His Lys Glu Ser
Gly Asn Gly His Asn Ile225 230 235
240Trp Val Ile Leu Val Val Thr Leu Val Val Pro Leu Leu Leu Val
Ala 245 250 255Val Leu Ile
Val Cys Cys Cys Ile Gly Ser Gly Cys Gly Gly Asp Pro 260
265 270Lys Cys Met Asp Arg Val Cys Phe Trp Arg
Leu Gly Leu Leu Arg Gly 275 280
285Pro Gly Ala Glu Asp Asn Ala His Asn Glu Ile Leu Ser Asn Ala Asp 290
295 300Ser Leu Ser Thr Phe Val Ser Glu
Gln Gln Met Glu Ser Gln Glu Pro305 310
315 320Ala Asp Leu Thr Gly Val Thr Val Gln Ser Pro Gly
Glu Ala Gln Cys 325 330
335Leu Leu Gly Pro Ala Glu Ala Glu Gly Ser Gln Arg Arg Arg Leu Leu
340 345 350Val Pro Ala Asn Gly Ala
Asp Pro Thr Glu Thr Leu Met Leu Phe Phe 355 360
365Asp Lys Phe Ala Asn Ile Val Pro Phe Asp Ser Trp Asp Gln
Leu Met 370 375 380Arg Gln Leu Asp Leu
Thr Lys Asn Glu Ile Asp Val Val Arg Ala Gly385 390
395 400Thr Ala Gly Pro Gly Asp Ala Leu Tyr Ala
Met Leu Met Lys Trp Val 405 410
415Asn Lys Thr Gly Arg Asn Ala Ser Ile His Thr Leu Leu Asp Ala Leu
420 425 430Glu Arg Met Glu Glu
Arg His Ala Lys Glu Lys Ile Gln Asp Leu Leu 435
440 445Val Asp Ser Gly Lys Phe Ile Tyr Leu Glu Asp Gly
Thr Gly Ser Ala 450 455 460Val Ser Leu
Glu4652039PRThumanTrail R1 extracellular domain (AA 201-239) 20Ala Glu
Met Cys Arg Lys Cys Ser Arg Gly Cys Pro Arg Gly Met Val1 5
10 15Lys Val Lys Asp Cys Thr Pro Trp
Ser Asp Ile Glu Cys Val His Lys 20 25
30Glu Ser Gly Asn Gly His Asn 352154PRTArtificial
SequenceTrail-R1-Fc fusion protein of Trail R1 extracellular domain
(AA 201-239) and huIgG1 (AA99-120) with an overlapping amino acid
(TRAILR1 AA 233 and huIgG1 AA 99) 21Ala Glu Met Cys Arg Lys Cys Ser
Arg Gly Cys Pro Arg Gly Met Val1 5 10
15Lys Val Lys Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Val
His Lys 20 25 30Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 35
40 45Pro Glu Leu Leu Gly Gly 502251PRTArtificial
SequenceTrail-R1-Fc fusion protein of Trail R1 extracellular domain
(AA 201-239) and huIgG1 (AA 99-120) with an overlapping amino acid
(TRAILR1 AA 232 and huIgG1 AA 101) 22Ala Glu Met Cys Arg Lys Cys Ser
Arg Gly Cys Pro Arg Gly Met Val1 5 10
15Lys Val Lys Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Val
His Lys 20 25 30Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 35
40 45Leu Gly Gly 502352PRTArtificial
SequenceTrail-R1-Fc fusion protein of Trail R1 extracellular domain
(AA 201-239) and huIgG1 (AA99-120) with an overlapping amino acid
(TRAILR1 AA 234 and huIgG1 AA 102) 23Ala Glu Met Cys Arg Lys Cys Ser
Arg Gly Cys Pro Arg Gly Met Val1 5 10
15Lys Val Lys Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Val
His Lys 20 25 30Glu Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 35
40 45Leu Leu Gly Gly 502451PRTArtificial
SequenceTrail-R1-Fc fusion protein of Trail R1 extracellular domain
(AA 201-239) and huIgG1 (AA99-120) with an overlapping amino acid
(TRAILR1 AA 238 and huIgG1 AA 107) 24Ala Glu Met Cys Arg Lys Cys Ser
Arg Gly Cys Pro Arg Gly Met Val1 5 10
15Lys Val Lys Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Val
His Lys 20 25 30Glu Ser Gly
Asn Gly His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 35
40 45Leu Gly Gly 5025440PRThumanTrail-R2
>sp/014763/T10B_HUMAN Tumor necrosis factor receptor superfamily
member 10B precursor (Death receptor 5) (TNF-related
apoptosis-including ligand receptor 2) (TRAIL receptor-2) (TRAIL-R2)
25Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala Arg Lys1
5 10 15Arg His Gly Pro Gly Pro
Arg Glu Ala Arg Gly Ala Arg Pro Gly Pro 20 25
30Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val
Leu Leu Leu 35 40 45Val Ser Ala
Glu Ser Ala Leu Ile Thr Gln Gln Asp Leu Ala Pro Gln 50
55 60Gln Arg Ala Ala Pro Gln Gln Lys Arg Ser Ser Pro
Ser Glu Gly Leu65 70 75
80Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser
85 90 95Cys Lys Tyr Gly Gln Asp
Tyr Ser Thr His Trp Asn Asp Leu Leu Phe 100
105 110Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val
Glu Leu Ser Pro 115 120 125Cys Thr
Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe 130
135 140Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys
Cys Arg Thr Gly Cys145 150 155
160Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile
165 170 175Glu Cys Val His
Lys Glu Ser Gly Thr Lys His Ser Gly Glu Ala Pro 180
185 190Ala Val Glu Glu Thr Val Thr Ser Ser Pro Gly
Thr Pro Ala Ser Pro 195 200 205Cys
Ser Leu Ser Gly Ile Ile Ile Gly Val Thr Val Ala Ala Val Val 210
215 220Leu Ile Val Ala Val Phe Val Cys Lys Ser
Leu Leu Trp Lys Lys Val225 230 235
240Leu Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly Asp Pro
Glu 245 250 255Arg Val Asp
Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp Asn Val Leu 260
265 270Asn Glu Ile Val Ser Ile Leu Gln Pro Thr
Gln Val Pro Glu Gln Glu 275 280
285Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gly Val Asn Met Leu Ser 290
295 300Pro Gly Glu Ser Glu His Leu Leu
Glu Pro Ala Glu Ala Glu Arg Ser305 310
315 320Gln Arg Arg Arg Leu Leu Val Pro Ala Asn Glu Gly
Asp Pro Thr Glu 325 330
335Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala Asp Leu Val Pro Phe Asp
340 345 350Ser Trp Glu Pro Leu Met
Arg Lys Leu Gly Leu Met Asp Asn Glu Ile 355 360
365Lys Val Ala Lys Ala Glu Ala Ala Gly His Arg Asp Thr Leu
Tyr Thr 370 375 380Met Leu Ile Lys Trp
Val Asn Lys Thr Gly Arg Asp Ala Ser Val His385 390
395 400Thr Leu Leu Asp Ala Leu Glu Thr Leu Gly
Glu Arg Leu Ala Lys Gln 405 410
415Lys Ile Glu Asp His Leu Leu Ser Ser Gly Lys Phe Met Tyr Leu Glu
420 425 430Gly Asn Ala Asp Ser
Ala Met Ser 435 4402640PRThumanTrail R2 (long)
extracellular domain (AA 171-210), "repeat" included 26Thr Pro Trp
Ser Asp Ile Glu Cys Val His Lys Glu Ser Gly Thr Lys1 5
10 15His Ser Gly Glu Ala Pro Ala Val Glu
Glu Thr Val Thr Ser Ser Pro 20 25
30Gly Thr Pro Ala Ser Pro Cys Ser 35
402758PRTArtificial SequenceTrail-R2(long)-Fc fusion protein of Trail R1
extracellular domain (AA 171-210)Trail R2 (long) extracellular
domain (AA 171-210), "repeat" included) and huIgG1 (AA99-120) with
an overlapping amino acid 27Thr Pro Trp Ser Asp Ile Glu Cys Val His
Lys Glu Ser Gly Thr Lys1 5 10
15His Ser Gly Glu Ala Pro Ala Val Glu Glu Thr Val Thr Ser Ser Pro
20 25 30Gly Thr Pro Ala Ser Pro
Cys Ser Cys Asp Lys Thr His Thr Cys Pro 35 40
45Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 50
552855PRTArtificial SequenceTrail-R2(long)-Fc fusion protein of Trail
R2 extracellular domain (AA 171-210; "repeat" included) and
huIgG1 (AA99-120) with an overlapping amino acid (TRAIL-R2(long) AA
207 and huIgG1 AA 102) 28Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys
Glu Ser Gly Thr Lys1 5 10
15His Ser Gly Glu Ala Pro Ala Val Glu Glu Thr Val Thr Ser Ser Pro
20 25 30Gly Thr Pro Ala Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro 35 40
45Ala Pro Glu Leu Leu Gly Gly 50
552958PRTArtificial SequenceTrail-R2(long)-Fc fusion protein of Trail R2
extracellular domain (AA 171-210; "repeat" included) and huIgG1
(AA99-120) with an overlapping amino acid (TRAIL-R2(long) AA 208 and
huIgG1 AA 100) 29Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu Ser
Gly Thr Lys1 5 10 15His
Ser Gly Glu Ala Pro Ala Val Glu Glu Thr Val Thr Ser Ser Pro 20
25 30Gly Thr Pro Ala Ser Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro 35 40
45Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 50
553055PRTArtificial SequenceTrail-R2(long)-Fc fusion protein of Trail R2
extracellular domain (AA 171-210; "repeat" included) and huIgG1
(AA99-120) with an overlapping amino acid (TRAIL-R2(long) AA 205
and huIgG1 AA 100) 30Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu
Ser Gly Thr Lys1 5 10
15His Ser Gly Glu Ala Pro Ala Val Glu Glu Thr Val Thr Ser Ser Pro
20 25 30Gly Thr Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro 35 40
45Ala Pro Glu Leu Leu Gly Gly 50
553156PRTArtificial SequenceTrail-R2(long)-Fc fusion protein of Trail R1
extracellular domain (AA 171-210; "repeat" included) and huIgG1
(AA99-120) with an overlapping amino acid (TRAIL-R2(long) AA 209 and
huIgG1 AA 103) 31Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu Ser
Gly Thr Lys1 5 10 15His
Ser Gly Glu Ala Pro Ala Val Glu Glu Thr Val Thr Ser Ser Pro 20
25 30Gly Thr Pro Ala Ser Pro Cys Asp
Lys Thr His Thr Cys Pro Pro Cys 35 40
45Pro Ala Pro Glu Leu Leu Gly Gly 50
553248PRTArtificial SequenceTrail-R2(long)-Fc fusion protein of Trail R2
extracellular domain (AA 171-210; "repeat" included) and huIgG1
(AA99-120) with an overlapping amino acid (TRAIL-R2(long) AA 204 and
huIgG1 AA 106) 32Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu Ser
Gly Thr Lys1 5 10 15His
Ser Gly Glu Ala Pro Ala Val Glu Glu Thr Val Thr Ser Ser Pro 20
25 30Gly Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly 35 40
453321PRThumanTrail R2 (long) extracellular domain (AA 171-191;
"repeat" not included) 33Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu
Ser Gly Thr Lys1 5 10
15His Ser Gly Glu Ala 203441PRTArtificial
SequenceTrail-R2(long)-Fc fusion protein of Trail R2 (long)
extracellular domain (AA 171-191; "repeat" not included) and huIgG1
(AA99-120) with an overlapping amino acid (TRAIL-R2(long) AA190 and
huIgG1 AA99) 34Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu Ser
Gly Thr Lys1 5 10 15His
Ser Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 20
25 30Cys Pro Ala Pro Glu Leu Leu Gly
Gly 35 403535PRTArtificial
SequenceTrail-R2(long)-Fc fusion protein of Trail R2 (long)
extracellular domain (AA171-191; "repeat" not included) and huIgG1
(AA99-120) with an overlapping amino acid (TRAIL-R2(long) AA186 and
huIgG1 AA101) 35Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu Ser
Gly Thr Lys1 5 10 15Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 20
25 30Leu Gly Gly
353636PRTArtificial SequenceTrail-R2(long)-Fc fusion protein of Trail R2
(long) extracellular domain (AA171-191; "repeat" not included)
and huIgG1 (AA99-120) with an overlapping amino acid (TRAIL-R2(long)
AA188 and huIgG1 AA102) 36Thr Pro Trp Ser Asp Ile Glu Cys Val His
Lys Glu Ser Gly Thr Lys1 5 10
15His Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
20 25 30Leu Leu Gly Gly
353729PRTArtificial SequenceTrail-R2(long)-Fc fusion protein of Trail R2
(long) extracellular domain (AA171-191; "repeat" not included)
and huIgG1 (AA99-120) with an overlapping amino acid (TRAIL-R2(long)
AA185 and huIgG1 AA106) 37Thr Pro Trp Ser Asp Ile Glu Cys Val His
Lys Glu Ser Gly Thr His1 5 10
15Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 20
253830PRTArtificial SequenceTrail-R2(long)-Fc fusion
protein of Trail R2 (long) extracellular domain (AA171-191; "repeat"
not included) and huIgG1 (AA99-120) with an overlapping amino
acid (TRAIL-R2(long) AA187 and huIgG1 AA107) 38Thr Pro Trp Ser Asp
Ile Glu Cys Val His Lys Glu Ser Gly Thr Lys1 5
10 15His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly 20 25
3039411PRThumanTrail-R2 (short) >sp/014763/T10B_HUMAN Tumor
necrosis factor receptor superfamily 10B precursor (Death receptor
5) (TNF-related apoptosis-inducing ligand receptor 2) (TRAIL
receptor-2) (TRAIL-R2) 39Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser
Gly Ala Arg Lys1 5 10
15Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg Pro Gly Pro
20 25 30Arg Val Pro Lys Thr Leu Val
Leu Val Val Ala Ala Val Leu Leu Leu 35 40
45Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln Asp Leu Ala Pro
Gln 50 55 60Gln Arg Ala Ala Pro Gln
Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu65 70
75 80Cys Pro Pro Gly His His Ile Ser Glu Asp Gly
Arg Asp Cys Ile Ser 85 90
95Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe
100 105 110Cys Leu Arg Cys Thr Arg
Cys Asp Ser Gly Glu Val Glu Leu Ser Pro 115 120
125Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly
Thr Phe 130 135 140Arg Glu Glu Asp Ser
Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys145 150
155 160Pro Arg Gly Met Val Lys Val Gly Asp Cys
Thr Pro Trp Ser Asp Ile 165 170
175Glu Cys Val His Lys Glu Ser Gly Ile Ile Ile Gly Val Thr Val Ala
180 185 190Ala Val Val Leu Ile
Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp 195
200 205Lys Lys Val Leu Pro Tyr Leu Lys Gly Ile Cys Ser
Gly Gly Gly Gly 210 215 220Asp Pro Glu
Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp225
230 235 240Asn Val Leu Asn Glu Ile Val
Ser Ile Leu Gln Pro Thr Gln Val Pro 245
250 255Glu Gln Glu Met Glu Val Gln Glu Pro Ala Glu Pro
Thr Gly Val Asn 260 265 270Met
Leu Ser Pro Gly Glu Ser Glu His Leu Leu Glu Pro Ala Glu Ala 275
280 285Glu Arg Ser Gln Arg Arg Arg Leu Leu
Val Pro Ala Asn Glu Gly Asp 290 295
300Pro Thr Glu Thr Leu Arg Gln Cys Phe Asp Asp Phe Ala Asp Leu Val305
310 315 320Pro Phe Asp Ser
Trp Glu Pro Leu Met Arg Lys Leu Gly Leu Met Asp 325
330 335Asn Glu Ile Lys Val Ala Lys Ala Glu Ala
Ala Gly His Arg Asp Thr 340 345
350Leu Tyr Thr Met Leu Ile Lys Trp Val Asn Lys Thr Gly Arg Asp Ala
355 360 365Ser Val His Thr Leu Leu Asp
Ala Leu Glu Thr Leu Gly Glu Arg Leu 370 375
380Ala Lys Gln Lys Ile Glu Asp His Leu Leu Ser Ser Gly Lys Phe
Met385 390 395 400Tyr Leu
Glu Gly Asn Ala Asp Ser Ala Met Ser 405
4104034PRThumanTrail-R2 (short) extracellular domain (AA 151 - AA
184) 40Glu Met Cys Arg Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val Lys1
5 10 15Val Gly Asp Cys Thr
Pro Trp Ser Asp Ile Glu Cys Val His Lys Glu 20
25 30Ser Gly4153PRTArtificial
SequenceTrail-R2(short)-Fc fusion protein of Trail R2 (short)
extracellular domain (AA 151-184) and huIgG1 (AA 99-120) with an
overlapping amino acid (TRAIL-R2(short) AA 182 and huIgG1 AA 99)
41Glu Met Cys Arg Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val Lys1
5 10 15Val Gly Asp Cys Thr Pro
Trp Ser Asp Ile Glu Cys Val His Lys Glu 20 25
30Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro 35 40 45Glu Leu Leu
Gly Gly 504250PRTArtificial SequenceTrail-R2(short)-Fc fusion protein
of Trail R2 (short) extracellular domain (AA 151-184) and
huIgG1 (AA 99-120) with an overlapping amino acid (TRAIL-R2(short)
AA 181 and huIgG1 AA 101) 42Glu Met Cys Arg Lys Cys Arg Thr Gly Cys Pro
Arg Gly Met Val Lys1 5 10
15Val Gly Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Val His Lys Ser
20 25 30Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu 35 40
45Gly Gly 504351PRTArtificial SequenceTrail-R2(short)-Fc
fusion protein of Trail R2 (short) extracellular domain (AA 151-184)
and huIgG1 (AA 99-120) with an overlapping amino acid
(TRAIL-R2(short) AA 183 and huIgG1 AA 102) 43Glu Met Cys Arg Lys Cys Arg
Thr Gly Cys Pro Arg Gly Met Val Lys1 5 10
15Val Gly Asp Cys Thr Pro Trp Ser Asp Ile Glu Cys Val
His Lys Glu 20 25 30Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 35
40 45Leu Gly Gly 504443PRTArtificial
SequenceTrail-R2(short)-Fc fusion protein of Trail R2 (short)
extracellular domain (AA 151-184) and huIgG1 (AA 99-120) with an
overlapping amino acid (TRAIL-R2(short) AA 180 and huIgG1 AA 107)
44Glu Met Cys Arg Lys Cys Arg Thr Gly Cys Pro Arg Gly Met Val Lys1
5 10 15Val Gly Asp Cys Thr Pro
Trp Ser Asp Ile Glu Cys Val His Thr Cys 20 25
30Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 35
4045259PRTArtificial
SequenceTrail-R3>sp/014798/T10C_HUMAN Tumor necrosis factor
receptor superfamily member 10C precursor;Decoy receptor
1;DcR1;Decoy TRAIL receptor without death domain;TNF-related
apoptosis inducing ligand r3 45Met Ala Arg Ile Pro Lys Thr Leu Lys Phe
Val Val Val Ile Val Ala1 5 10
15Val Leu Leu Pro Val Leu Ala Tyr Ser Ala Thr Thr Ala Arg Gln Glu
20 25 30Glu Val Pro Gln Gln Thr
Val Ala Pro Gln Gln Gln Arg His Ser Phe 35 40
45Lys Gly Glu Glu Cys Pro Ala Gly Ser His Arg Ser Glu His
Thr Gly 50 55 60Ala Cys Asn Pro Cys
Thr Glu Gly Val Asp Tyr Thr Asn Ala Ser Asn65 70
75 80Asn Glu Pro Ser Cys Phe Pro Cys Thr Val
Cys Lys Ser Asp Gln Lys 85 90
95His Lys Ser Ser Cys Thr Met Thr Arg Asp Thr Val Cys Gln Cys Lys
100 105 110Glu Gly Thr Phe Arg
Asn Glu Asn Ser Pro Glu Met Cys Arg Lys Cys 115
120 125Ser Arg Cys Pro Ser Gly Glu Val Gln Val Ser Asn
Cys Thr Ser Trp 130 135 140Asp Asp Ile
Gln Cys Val Glu Glu Phe Gly Ala Asn Ala Thr Val Glu145
150 155 160Thr Pro Ala Ala Glu Glu Thr
Met Asn Thr Ser Pro Gly Thr Pro Ala 165
170 175Pro Ala Ala Glu Glu Thr Met Asn Thr Ser Pro Gly
Thr Pro Ala Pro 180 185 190Ala
Ala Glu Glu Thr Met Thr Thr Ser Pro Gly Thr Pro Ala Pro Ala 195
200 205Ala Glu Glu Thr Met Thr Thr Ser Pro
Gly Thr Pro Ala Pro Ala Ala 210 215
220Glu Glu Thr Met Thr Thr Ser Pro Gly Thr Pro Ala Ser Ser His Tyr225
230 235 240Leu Ser Cys Thr
Ile Val Gly Ile Ile Val Leu Ile Val Leu Leu Ile 245
250 255Val Phe Val4636PRThumanTrail-R3
extracellular domain (AA 201-236; "repeats" included) 46Ser Pro Gly Thr
Pro Ala Pro Ala Ala Glu Glu Thr Met Thr Thr Ser1 5
10 15Pro Gly Thr Pro Ala Pro Ala Ala Glu Glu
Thr Met Thr Thr Ser Pro 20 25
30Gly Thr Pro Ala 354755PRTArtificial SequenceTrail-R3-Fc fusion
protein of Trail-R3 extracellular domain (AA 201-236; "repeats"
included) and huIgG1 (AA 99-120) with an overlapping amino acid
(TRAIL-R3 AA 235 and huIgG1 AA 100) 47Ser Pro Gly Thr Pro Ala Pro
Ala Ala Glu Glu Thr Met Thr Thr Ser1 5 10
15Pro Gly Thr Pro Ala Pro Ala Ala Glu Glu Thr Met Thr
Thr Ser Pro 20 25 30Gly Thr
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 35
40 45Ala Pro Glu Leu Leu Gly Gly 50
554852PRTArtificial SequenceTrail-R3-Fc fusion protein of
Trail-R3 extracellular domain (AA 201-236; "repeats" included)
and huIgG1 (AA 99-120) with an overlapping amino acid (TRAIL-R3 AA
232and huIgG1 AA 100) 48Ser Pro Gly Thr Pro Ala Pro Ala Ala Glu Glu Thr
Met Thr Thr Ser1 5 10
15Pro Gly Thr Pro Ala Pro Ala Ala Glu Glu Thr Met Thr Thr Ser Pro
20 25 30Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu 35 40
45Leu Leu Gly Gly 504949PRTArtificial SequenceTrail-R3-Fc
fusion protein of Trail-R3 extracellular domain (AA 201-236;
"repeats" included) and huIgG1 (AA 99-120) with an overlapping
amino acid (TRAIL-R3 AA 231 and huIgG1 AA 102) 49Ser Pro Gly Thr Pro
Ala Pro Ala Ala Glu Glu Thr Met Thr Thr Ser1 5
10 15Pro Gly Thr Pro Ala Pro Ala Ala Glu Glu Thr
Met Thr Thr Ser Cys 20 25
30Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
35 40 45Gly5048PRTArtificial
SequenceTrail-R3-Fc fusion protein of Trail-R3 extracellular domain
(AA 201-236; "repeats" included) and huIgG1 (AA 99-120) with an
overlapping amino acid (TRAIL-R3 AA 234 and huIgG1 AA 106) 50Ser Pro
Gly Thr Pro Ala Pro Ala Ala Glu Glu Thr Met Thr Thr Ser1 5
10 15Pro Gly Thr Pro Ala Pro Ala Ala
Glu Glu Thr Met Thr Thr Ser Pro 20 25
30Gly Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly 35 40 455144PRTArtificial
SequenceTrail-R3-Fc fusion protein of Trail-R3 extracellular domain
(AA 201-236; "repeats" included) and huIgG1 (AA 99-120) with an
overlapping amino acid (TRAIL-R3 AA 230 and huIgG1 AA 106) 51Ser Pro
Gly Thr Pro Ala Pro Ala Ala Glu Glu Thr Met Thr Thr Ser1 5
10 15Pro Gly Thr Pro Ala Pro Ala Ala
Glu Glu Thr Met Thr Thr His Thr 20 25
30Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 35
405243PRTArtificial SequenceTrail-R3-Fc fusion protein of
Trail-R3 extracellular domain (AA 201-236; "repeats" included)
and huIgG1 (AA 99-120) with an overlapping amino acid (TRAIL-R3 AA
229 and huIgG1 AA 106) 52Ser Pro Gly Thr Pro Ala Pro Ala Ala Glu Glu
Thr Met Thr Thr Ser1 5 10
15Pro Gly Thr Pro Ala Pro Ala Ala Glu Glu Thr Met Thr His Thr Cys
20 25 30Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly 35 405341PRThumanTrail-R3
extracellular domain (AA 121-161, "repeats" not included) 53Ser Pro Glu
Met Cys Arg Lys Cys Ser Arg Cys Pro Ser Gly Glu Val1 5
10 15Gln Val Ser Asn Cys Thr Ser Trp Asp
Asp Ile Gln Cys Val Glu Glu 20 25
30Phe Gly Ala Asn Ala Thr Val Glu Thr 35
405461PRTArtificial SequenceTrail-R3-Fc fusion protein of Trail-R3
extracellular domain (AA 121-161; "repeats"not included) and huIgG1
(AA 99-120) with an overlapping amino acid (TRAIL-R3 AA 160 and
huIgG1 AA 99) 54Ser Pro Glu Met Cys Arg Lys Cys Ser Arg Cys Pro Ser Gly
Glu Val1 5 10 15Gln Val
Ser Asn Cys Thr Ser Trp Asp Asp Ile Gln Cys Val Glu Glu 20
25 30Phe Gly Ala Asn Ala Thr Val Glu Pro
Lys Ser Cys Asp Lys Thr His 35 40
45Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 50
55 605553PRTArtificial SequenceTrail-R3-Fc fusion
protein of Trail-R3 extracellular domain (AA 121-161; "repeats"not
included) and huIgG1 (AA 99-120) with an overlapping amino acid
(TRAIL-R3 AA 152 and huIgG1 AA 99) 55Ser Pro Glu Met Cys Arg Lys
Cys Ser Arg Cys Pro Ser Gly Glu Val1 5 10
15Gln Val Ser Asn Cys Thr Ser Trp Asp Asp Ile Gln Cys
Val Glu Glu 20 25 30Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 35
40 45Glu Leu Leu Gly Gly 505652PRTArtificial
SequenceTrail-R3-Fc fusion protein of Trail-R3 extracellular domain
(AA 121-161; "repeats"not included) and huIgG1 (AA 99-120) with an
overlapping amino acid (TRAIL-R3 AA 151 and huIgG1 AA 99) 56Ser
Pro Glu Met Cys Arg Lys Cys Ser Arg Cys Pro Ser Gly Glu Val1
5 10 15Gln Val Ser Asn Cys Thr Ser
Trp Asp Asp Ile Gln Cys Val Glu Pro 20 25
30Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu 35 40 45Leu Leu Gly Gly
505755PRTArtificial SequenceTrail-R3-Fc fusion protein of Trail-R3
extracellular domain (AA 121-161; "repeats"not included) and huIgG1
(AA 99-120) with an overlapping amino acid (TRAIL-R3 AA 161 and
huIgG1 AA 106) 57Ser Pro Glu Met Cys Arg Lys Cys Ser Arg Cys Pro Ser Gly
Glu Val1 5 10 15Gln Val
Ser Asn Cys Thr Ser Trp Asp Asp Ile Gln Cys Val Glu Glu 20
25 30Phe Gly Ala Asn Ala Thr Val Glu Thr
His Thr Cys Pro Pro Cys Pro 35 40
45Ala Pro Glu Leu Leu Gly Gly 50 555852PRTArtificial
SequenceTrail-R3-Fc fusion protein of Trail-R3 extracellular domain
(AA 121-161; "repeats"not included) and huIgG1 (AA 99-120) with an
overlapping amino acid (TRAIL-R3 AA 158 and huIgG1 AA 106) 58Ser
Pro Glu Met Cys Arg Lys Cys Ser Arg Cys Pro Ser Gly Glu Val1
5 10 15Gln Val Ser Asn Cys Thr Ser
Trp Asp Asp Ile Gln Cys Val Glu Glu 20 25
30Phe Gly Ala Asn Ala Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu 35 40 45Leu Leu Gly Gly
5059386PRThumanTrail-R4>sp/Q9UBN6/T10D_HUMAN Tumor necrosis
factor receptor superfamily member 10D precursor;Decoy receptor 2;
DcR2; TNF-related apoptosis-inducing ligand receptor 4) 59Met Gly
Leu Trp Gly Gln Ser Val Pro Thr Ala Ser Ser Ala Arg Ala1 5
10 15Gly Arg Tyr Pro Gly Ala Arg Thr
Ala Ser Gly Thr Arg Pro Trp Leu 20 25
30Leu Asp Pro Lys Ile Leu Lys Phe Val Val Phe Ile Val Ala Val
Leu 35 40 45Leu Pro Val Arg Val
Asp Ser Ala Thr Ile Pro Arg Gln Asp Glu Val 50 55
60Pro Gln Gln Thr Val Ala Pro Gln Gln Gln Arg Arg Ser Leu
Lys Glu65 70 75 80Glu
Glu Cys Pro Ala Gly Ser His Arg Ser Glu Tyr Thr Gly Ala Cys
85 90 95Asn Pro Cys Thr Glu Gly Val
Asp Tyr Thr Ile Ala Ser Asn Asn Leu 100 105
110Pro Ser Cys Leu Leu Cys Thr Val Cys Lys Ser Gly Gln Thr
Asn Lys 115 120 125Ser Ser Cys Thr
Thr Thr Arg Asp Thr Val Cys Gln Cys Glu Lys Gly 130
135 140Ser Phe Gln Asp Lys Asn Ser Pro Glu Met Cys Arg
Thr Cys Arg Thr145 150 155
160Gly Cys Pro Arg Gly Met Val Lys Val Ser Asn Cys Thr Pro Arg Ser
165 170 175Asp Ile Lys Cys Lys
Asn Glu Ser Ala Ala Ser Ser Thr Gly Lys Thr 180
185 190Pro Ala Ala Glu Glu Thr Val Thr Thr Ile Leu Gly
Met Leu Ala Ser 195 200 205Pro Tyr
His Tyr Leu Ile Ile Ile Val Val Leu Val Ile Ile Leu Ala 210
215 220Val Val Val Val Gly Phe Ser Cys Arg Lys Lys
Phe Ile Ser Tyr Leu225 230 235
240Lys Gly Ile Cys Ser Gly Gly Gly Gly Gly Pro Glu Arg Val His Arg
245 250 255Val Leu Phe Arg
Arg Arg Ser Cys Pro Ser Arg Val Pro Gly Ala Glu 260
265 270Asp Asn Ala Arg Asn Glu Thr Leu Ser Asn Arg
Tyr Leu Gln Pro Thr 275 280 285Gln
Val Ser Glu Gln Glu Ile Gln Gly Gln Glu Leu Ala Glu Leu Thr 290
295 300Gly Val Thr Val Glu Ser Pro Glu Glu Pro
Gln Arg Leu Leu Glu Gln305 310 315
320Ala Glu Ala Glu Gly Cys Gln Arg Arg Arg Leu Leu Val Pro Val
Asn 325 330 335Asp Ala Asp
Ser Ala Asp Ile Ser Thr Leu Leu Asp Ala Ser Ala Thr 340
345 350Leu Glu Glu Gly His Ala Lys Glu Thr Ile
Gln Asp Gln Leu Val Gly 355 360
365Ser Glu Lys Leu Phe Tyr Glu Glu Asp Glu Ala Gly Ser Ala Thr Ser 370
375 380Cys Leu3856041PRThumanTrail-R4
extracellular domain (AA 171-211) 60Asn Cys Thr Pro Arg Ser Asp Ile Lys
Cys Lys Asn Glu Ser Ala Ala1 5 10
15Ser Ser Thr Gly Lys Thr Pro Ala Ala Glu Glu Thr Val Thr Thr
Ile 20 25 30Leu Gly Met Leu
Ala Ser Pro Tyr His 35 406159PRTArtificial
SequenceTrail-R4-Fc fusion protein of Trail-R4 extracellular domain
(AA 171-211) and huIgG1 (AA 99-120) with an overlapping amino acid
(TRAIL-R4 AA 209 and huIgG1 AA 100) 61Asn Cys Thr Pro Arg Ser Asp
Ile Lys Cys Lys Asn Glu Ser Ala Ala1 5 10
15Ser Ser Thr Gly Lys Thr Pro Ala Ala Glu Glu Thr Val
Thr Thr Ile 20 25 30Leu Gly
Met Leu Ala Ser Pro Lys Ser Cys Asp Lys Thr His Thr Cys 35
40 45Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly 50 556256PRTArtificial SequenceTrail-R4-Fc fusion
protein of Trail-R4 extracellular domain (AA 171-211) and huIgG1 (AA
99-120) with an overlapping amino acid (TRAIL-R4 AA 208 and
huIgG1 AA 102) 62Asn Cys Thr Pro Arg Ser Asp Ile Lys Cys Lys Asn Glu Ser
Ala Ala1 5 10 15Ser Ser
Thr Gly Lys Thr Pro Ala Ala Glu Glu Thr Val Thr Thr Ile 20
25 30Leu Gly Met Leu Ala Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 35 40
45Pro Ala Pro Glu Leu Leu Gly Gly 50
556345PRTArtificial SequenceTrail-R4-Fc fusion protein of Trail-R4
extracellular domain (AA 171-211) and huIgG1 (AA 99-120) with an
overlapping amino acid (TRAIL-R4 AA 201 and huIgG1 AA 106) 63Asn
Cys Thr Pro Arg Ser Asp Ile Lys Cys Lys Asn Glu Ser Ala Ala1
5 10 15Ser Ser Thr Gly Lys Thr Pro
Ala Ala Glu Glu Thr Val Thr Thr His 20 25
30Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
35 40 456454PRTArtificial
SequenceTrail-R4-Fc fusion protein of Trail-R4 extracellular domain
(AA 171-211) and huIgG1 (AA 99-120) with an overlapping amino acid
(TRAIL-R4 AA 211 and huIgG1 AA 107) 64Asn Cys Thr Pro Arg Ser Asp
Ile Lys Cys Lys Asn Glu Ser Ala Ala1 5 10
15Ser Ser Thr Gly Lys Thr Pro Ala Ala Glu Glu Thr Val
Thr Thr Ile 20 25 30Leu Gly
Met Leu Ala Ser Pro Tyr His Thr Cys Pro Pro Cys Pro Ala 35
40 45Pro Glu Leu Leu Gly Gly
5065455PRThumanTNF-R1 >sp/P19438/TR1A_HUMAN necrosis factor
receptor superfamily member 1A precursor (p60) (TNF-R1) (p55)
(CD120a) [contains Tumor necrosis factor binding protein 1 (TBPI)]
65Met Gly Leu Ser Thr Val Pro Asp Leu Leu Leu Pro Leu Val Leu Leu1
5 10 15Glu Leu Leu Val Gly Ile
Tyr Pro Ser Gly Val Ile Gly Leu Val Pro 20 25
30His Leu Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro
Gln Gly Lys 35 40 45Tyr Ile His
Pro Gln Asn Asn Ser Ile Cys Cys Thr Lys Cys His Lys 50
55 60Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro Gly
Gln Asp Thr Asp65 70 75
80Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu
85 90 95Arg His Cys Leu Ser Cys
Ser Lys Cys Arg Lys Glu Met Gly Gln Val 100
105 110Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val
Cys Gly Cys Arg 115 120 125Lys Asn
Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe 130
135 140Asn Cys Ser Leu Cys Leu Asn Gly Thr Val His
Leu Ser Cys Gln Glu145 150 155
160Lys Gln Asn Thr Val Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu
165 170 175Asn Glu Cys Val
Ser Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr 180
185 190Lys Leu Cys Leu Pro Gln Ile Glu Asn Val Lys
Gly Thr Glu Asp Ser 195 200 205Gly
Thr Thr Val Leu Leu Pro Leu Val Ile Phe Phe Gly Leu Cys Leu 210
215 220Leu Ser Leu Leu Phe Ile Gly Leu Met Tyr
Arg Tyr Gln Arg Trp Lys225 230 235
240Ser Lys Leu Tyr Ser Ile Val Cys Gly Lys Ser Thr Pro Glu Lys
Glu 245 250 255Gly Glu Leu
Glu Gly Thr Thr Thr Lys Pro Leu Ala Pro Asn Pro Ser 260
265 270Phe Ser Pro Thr Pro Gly Phe Thr Pro Thr
Leu Gly Phe Ser Pro Val 275 280
285Pro Ser Ser Thr Phe Thr Ser Ser Ser Thr Tyr Thr Pro Gly Asp Cys 290
295 300Pro Asn Phe Ala Ala Pro Arg Arg
Glu Val Ala Pro Pro Tyr Gln Gly305 310
315 320Ala Asp Pro Ile Leu Ala Thr Ala Leu Ala Ser Asp
Pro Ile Pro Asn 325 330
335Pro Leu Gln Lys Trp Glu Asp Ser Ala His Lys Pro Gln Ser Leu Asp
340 345 350Thr Asp Asp Pro Ala Thr
Leu Tyr Ala Val Val Glu Asn Val Pro Pro 355 360
365Leu Arg Trp Lys Glu Phe Val Arg Arg Leu Gly Leu Ser Asp
His Glu 370 375 380Ile Asp Arg Leu Glu
Leu Gln Asn Gly Arg Cys Leu Arg Glu Ala Gln385 390
395 400Tyr Ser Met Leu Ala Thr Trp Arg Arg Arg
Thr Pro Arg Arg Glu Ala 405 410
415Thr Leu Glu Leu Leu Gly Arg Val Leu Arg Asp Met Asp Leu Leu Gly
420 425 430Cys Leu Glu Asp Ile
Glu Glu Ala Leu Cys Gly Pro Ala Ala Leu Pro 435
440 445Pro Ala Pro Ser Leu Leu Arg 450
4556641PRThumanTNF-R1 extracellular domain (AA 171-211) 66Gly Phe Phe Leu
Arg Glu Asn Glu Cys Val Ser Cys Ser Asn Cys Lys1 5
10 15Lys Ser Leu Glu Cys Thr Lys Leu Cys Leu
Pro Gln Ile Glu Asn Val 20 25
30Lys Gly Thr Glu Asp Ser Gly Thr Thr 35
406757PRTArtificial SequenceTNF-R1-Fc fusion protein of TNF-R1
extracellular domain (AA 171-211) and huIgG1 (AA 99-120) with an
overlapping amino acid (TNF-R1 AA 206 and huIgG1 AA 99) 67Gly Phe Phe Leu
Arg Glu Asn Glu Cys Val Ser Cys Ser Asn Cys Lys1 5
10 15Lys Ser Leu Glu Cys Thr Lys Leu Cys Leu
Pro Gln Ile Glu Asn Val 20 25
30Lys Gly Thr Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
35 40 45Cys Pro Ala Pro Glu Leu Leu Gly
Gly 50 556852PRTArtificial SequenceTNF-R1-Fc fusion
protein of TNF-R1 extracellular domain (AA 171-211) and huIgG1 (AA
99-120) with an overlapping amino acid (TNF-R1 AA 203 and huIgG1 AA
101) 68Gly Phe Phe Leu Arg Glu Asn Glu Cys Val Ser Cys Ser Asn Cys Lys1
5 10 15Lys Ser Leu Glu Cys
Thr Lys Leu Cys Leu Pro Gln Ile Glu Asn Val 20
25 30Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu 35 40 45Leu Leu
Gly Gly 506948PRTArtificial SequenceTNF-R1-Fc fusion protein of TNF-R1
extracellular domain (AA 171-211) and huIgG1 (AA 99-120) with an
overlapping amino acid (TNF-R1 AA 203 and huIgG1 AA 105) 69Gly Phe Phe
Leu Arg Glu Asn Glu Cys Val Ser Cys Ser Asn Cys Lys1 5
10 15Lys Ser Leu Glu Cys Thr Lys Leu Cys
Leu Pro Gln Ile Glu Asn Val 20 25
30Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
35 40 457056PRTArtificial
SequenceTNF-R1-Fc fusion protein of TNF-R1 extracellular domain (AA
171-211) and huIgG1 (AA 99-120) with an overlapping amino acid
(TNF-R1 AA 208 and huIgG1 AA 102) 70Gly Phe Phe Leu Arg Glu Asn Glu Cys
Val Ser Cys Ser Asn Cys Lys1 5 10
15Lys Ser Leu Glu Cys Thr Lys Leu Cys Leu Pro Gln Ile Glu Asn
Val 20 25 30Lys Gly Thr Glu
Asp Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 35
40 45Pro Ala Pro Glu Leu Leu Gly Gly 50
557153PRTArtificial SequenceTNF-R1-Fc fusion protein of TNF-R1
extracellular domain (AA 171-211) and huIgG1 (AA 99-120) with an
overlapping amino acid (TNF-R1 AA 207 and huIgG1 AA 104) 71Gly Phe Phe
Leu Arg Glu Asn Glu Cys Val Ser Cys Ser Asn Cys Lys1 5
10 15Lys Ser Leu Glu Cys Thr Lys Leu Cys
Leu Pro Gln Ile Glu Asn Val 20 25
30Lys Gly Thr Glu Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
35 40 45Glu Leu Leu Gly Gly
507255PRTArtificial SequenceTNF-R1-Fc fusion protein of TNF-R1
extracellular domain (AA 171-211) and huIgG1 (AA 99-120) with an
overlapping amino acid (TNF-R1 AA 211 and huIgG1 AA 106) 72Gly Phe Phe
Leu Arg Glu Asn Glu Cys Val Ser Cys Ser Asn Cys Lys1 5
10 15Lys Ser Leu Glu Cys Thr Lys Leu Cys
Leu Pro Gln Ile Glu Asn Val 20 25
30Lys Gly Thr Glu Asp Ser Gly Thr Thr His Thr Cys Pro Pro Cys Pro
35 40 45Ala Pro Glu Leu Leu Gly Gly
50 557354PRTArtificial SequenceTNF-R1-Fc fusion
protein of TNF-R1 extracellular domain (AA 171-211) and huIgG1 (AA
99-120) with an overlapping amino acid (TNF-R1 AA 210 and huIgG1 AA
106) 73Gly Phe Phe Leu Arg Glu Asn Glu Cys Val Ser Cys Ser Asn Cys Lys1
5 10 15Lys Ser Leu Glu Cys
Thr Lys Leu Cys Leu Pro Gln Ile Glu Asn Val 20
25 30Lys Gly Thr Glu Asp Ser Gly Thr His Thr Cys Pro
Pro Cys Pro Ala 35 40 45Pro Glu
Leu Leu Gly Gly 5074461PRThumanTNF-R2 >sp/P20333/TR1B_HUMAN
necrosis factor receptor superfamily member 1B precursor (p80)
(TNF-R2) (p75) (CD120b) [contains Tumor necrosis factor binding
protein 2 (TBPII)] 74Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly
Leu Glu Leu1 5 10 15Trp
Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr 20
25 30Ala Pro Glu Pro Gly Ser Thr Cys
Arg Leu Arg Glu Tyr Tyr Asp Gln 35 40
45Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys
50 55 60Val Phe Cys Thr Lys Thr Ser Asp
Thr Val Cys Asp Ser Cys Glu Asp65 70 75
80Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys
Leu Ser Cys 85 90 95Gly
Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg
100 105 110Glu Gln Asn Arg Ile Cys Thr
Cys Arg Pro Gly Trp Tyr Cys Ala Leu 115 120
125Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys
Arg 130 135 140Pro Gly Phe Gly Val Ala
Arg Pro Gly Thr Glu Thr Ser Asp Val Val145 150
155 160Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn
Thr Thr Ser Ser Thr 165 170
175Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly
180 185 190Asn Ala Ser Met Asp Ala
Val Cys Thr Ser Thr Ser Pro Thr Arg Ser 195 200
205Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr
Arg Ser 210 215 220Gln His Thr Gln Pro
Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser225 230
235 240Phe Leu Leu Pro Met Gly Pro Ser Pro Pro
Ala Glu Gly Ser Thr Gly 245 250
255Asp Phe Ala Leu Pro Val Gly Leu Ile Val Gly Val Thr Ala Leu Gly
260 265 270Leu Leu Ile Ile Gly
Val Val Asn Cys Val Ile Met Thr Gln Val Lys 275
280 285Lys Lys Pro Leu Cys Leu Gln Arg Glu Ala Lys Val
Pro His Leu Pro 290 295 300Ala Asp Lys
Ala Arg Gly Thr Gln Gly Pro Glu Gln Gln His Leu Leu305
310 315 320Ile Thr Ala Pro Ser Ser Ser
Ser Ser Ser Leu Glu Ser Ser Ala Ser 325
330 335Ala Leu Asp Arg Arg Ala Pro Thr Arg Asn Gln Pro
Gln Ala Pro Gly 340 345 350Val
Glu Ala Ser Gly Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser 355
360 365Asp Ser Ser Pro Gly Gly His Gly Thr
Gln Val Asn Val Thr Cys Ile 370 375
380Val Asn Val Cys Ser Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln385
390 395 400Ala Ser Ser Thr
Met Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro 405
410 415Lys Asp Glu Gln Val Pro Phe Ser Lys Glu
Glu Cys Ala Phe Arg Ser 420 425
430Gln Leu Glu Thr Pro Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro
435 440 445Leu Pro Leu Gly Val Pro Asp
Ala Gly Met Lys Pro Ser 450 455
4607537PRThumanTNF-R2 extracellular domain (AA 221-257) 75Ser Thr Arg Ser
Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala1 5
10 15Pro Ser Thr Ser Phe Leu Leu Pro Met Gly
Pro Ser Pro Pro Ala Glu 20 25
30Gly Ser Thr Gly Asp 357653PRTArtificial SequenceTNF-R2-Fc
fusion protein of TNF-R2 extracellular domain (AA 221-257) and
huIgG1 (AA 99-120) with an overlapping amino acid (TNF-R2 AA 252 and
huIgG1 AA 99) 76Ser Thr Arg Ser Gln His Thr Gln Pro Thr Pro Glu Pro Ser
Thr Ala1 5 10 15Pro Ser
Thr Ser Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu 20
25 30Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro 35 40
45Glu Leu Leu Gly Gly 507750PRTArtificial SequenceTNF-R2-Fc fusion
protein of TNF-R2 extracellular domain (AA 221-257) and huIgG1 (AA
99-120) with an overlapping amino acid (TNF-R2 AA 250 and huIgG1 AA
100) 77Ser Thr Arg Ser Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala1
5 10 15Pro Ser Thr Ser Phe
Leu Leu Pro Met Gly Pro Ser Pro Pro Lys Ser 20
25 30Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu 35 40 45Gly Gly
507849PRTArtificial SequenceTNF-R2-Fc fusion protein of TNF-R2
extracellular domain (AA 221-257) and huIgG1 (AA 99-120) with an
overlapping amino acid (TNF-R2 AA 249 and huIgG1 AA 100) 78Ser Thr Arg
Ser Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala1 5
10 15Pro Ser Thr Ser Phe Leu Leu Pro Met
Gly Pro Ser Pro Lys Ser Cys 20 25
30Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
35 40 45Gly7952PRTArtificial
SequenceTNF-R2-Fc fusion protein of TNF-R2 extracellular domain (AA
221-257) and huIgG1 (AA 99-120) with an overlapping amino acid
(TNF-R2 AA 254 and huIgG1 AA 102) 79Ser Thr Arg Ser Gln His Thr Gln Pro
Thr Pro Glu Pro Ser Thr Ala1 5 10
15Pro Ser Thr Ser Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala
Glu 20 25 30Gly Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 35
40 45Leu Leu Gly Gly 508046PRTArtificial
SequenceTNF-R2-Fc fusion protein of TNF-R2 extracellular domain (AA
221-257) and huIgG1 (AA 99-120) with an overlapping amino acid
(TNF-R2 AA 248 and huIgG1 AA 102) 80Ser Thr Arg Ser Gln His Thr Gln Pro
Thr Pro Glu Pro Ser Thr Ala1 5 10
15Pro Ser Thr Ser Phe Leu Leu Pro Met Gly Pro Ser Cys Asp Lys
Thr 20 25 30His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 35 40
458153PRTArtificial SequenceTNF-R2-Fc fusion protein of
TNF-R2 extracellular domain (AA 221-257) and huIgG1 (AA 99-120) with
an overlapping amino acid (TNF-R2 AA 257 and huIgG1 AA 104) 81Ser
Thr Arg Ser Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala1
5 10 15Pro Ser Thr Ser Phe Leu Leu
Pro Met Gly Pro Ser Pro Pro Ala Glu 20 25
30Gly Ser Thr Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro 35 40 45Glu Leu Leu Gly
Gly 508249PRTArtificial SequenceTNF-R2-Fc fusion protein of TNF-R2
extracellular domain (AA 221-257) and huIgG1 (AA 99-120) with an
overlapping amino acid (TNF-R2 AA 255 and huIgG1 AA 106) 82Ser Thr Arg
Ser Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala1 5
10 15Pro Ser Thr Ser Phe Leu Leu Pro Met
Gly Pro Ser Pro Pro Ala Glu 20 25
30Gly Ser Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
35 40 45Gly
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