Patent application title: MULTIVARIABLE ANTIGENS COMPLEXED WITH TARGETING HUMANIZED MONOCLONAL ANTIBODY
Inventors:
Gerard Zurawski (Midlothian, TX, US)
Anne-Laure Flamar (New York, NY, US)
Eynav Klechevsky (Dallas, TX, US)
IPC8 Class: AC07K1628FI
USPC Class:
4241781
Class name: Drug, bio-affecting and body treating compositions conjugate or complex of monoclonal or polyclonal antibody, immunoglobulin, or fragment thereof with nonimmunoglobulin material
Publication date: 2016-02-04
Patent application number: 20160031988
Abstract:
The present invention includes compositions and methods for designing,
making and using modular recombinant antibodies or fragments thereof with
one half of a cohesin-dockerin pair that permits the rapid assembly of
multivariant antigen conjugates.Claims:
1. A modular rAb carrier comprising an antigen-specific binding domain
linked to one or more antigen carrier domains comprising one half of a
cohesin-dockerin binding pair.
2. The rAb of claim 1, wherein the antigen-specific binding domain comprises at least a portion of an antibody.
3. The rAb of claim 1, wherein the antigen-specific binding domain comprises at least a portion of an antibody in a fusion protein with the one half of the cohesin-dockerin binding pair.
4. The rAb of claim 1, further comprising a complementary half of the cohesin-dockerin binding pair bound to an antigen that forms a complex with the modular rAb carrier.
5. The rAb of claim 1, further comprising a complementary half of the cohesin-dockerin binding pair that is a fusion protein with an antigen.
6. The rAb of claim 1, wherein the antigen specific domain comprises a full length antibody, an antibody variable region domain, an Fab fragment, a Fab' fragment, an F(ab)2 fragment, and Fv fragment, and Fabc fragment and/or a Fab fragment with portions of the Fc domain.
7. The rAb of claim 1, wherein the cohesin-dockerin are selected from Clostridium thermocellum, Clostridium josui, Clostridium cellulolyticum and Bacteroides cellulosolvens and combinations thereof.
8. The rAb of claim 1, wherein the antigen-specific binding domain binds a cell surface marker selected from MHC class I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD 19, CD20, CD29, CD31, CD40,CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2, IFN-.gamma. receptor and IL-2 receptor, ICAM-1, Fcγ receptor or other receptor relatively specifically expressed by antigen presenting cells.
9. The rAb of claim 1, wherein the rAb is further defined as: an rAb.Doc; an rAb.Coh; an rAb.(Coh)x; an rAb.(Doc)x; an rAb.(Coh.Doc)x; or an rAb.(Coh)x(Doc)x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
10. The rAb of claim 1, wherein the rAb is further defined as being part of a complex: an rAb.Doc:Coh.antigen; an rAb.Coh:Doc.antigen; an rAb.(Coh)x:(Doc.antigen)x; an rAb.(Doc)x:(Coh.antigen)x; an rAb.(Coh.Doc)x:(Doc.antigen1)(Coh.antigen2); or an rAb.(Coh)x(Doc)x:(Doc.antigen1)x(Coh.antigen2).s- ub.x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
11. A vaccine comprising a modular rAb carrier comprising an antigen specific domain linked to one or more domains comprising one half of the cohesin-dockerin binding pair bound to a complementary half of the cohesin-dockerin binding pair bound to an antigen.
12. The vaccine of claim 11, wherein the antigen specific domain is specific for an immune cell surface protein selected from MHC class I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD 19, CD20, CD29, CD31, CD40,CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2, IFN-.gamma. receptor and IL-2 receptor, ICAM-1, Fcy receptor or other receptor relatively specifically expressed by antigen presenting cells.
13. The vaccine of claim 11, wherein the antigen comprises a bacterial, viral, fungal, protozoan or cancer protein.
14. The vaccine of claim 11, wherein the modular rAb carrier is further defined: an rAb.Doc:Coh.antigen; an rAb.Coh:Doc.antigen; an rAb.(Coh)x:(Doc.antigen)x; an rAb.(Doc)x:(Coh.antigen)x; an rAb.(Coh.Doc)x:(Doc.antigen1)(Coh.antigen2); or an rAb.(Coh)x(Doc)x:(Doc.antigen1)x(Coh.antigen2).s- ub.x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
15. An isolated nucleic acid comprising a coding segment for an target specific domain and one or more domains and one half of a cohesin-dockerin binding pair.
16. The nucleic acid of claim 15, wherein the target is an antigen and the specific domain encodes at least a portion of an antibody.
17. The nucleic acid of claim 15, wherein the one or more domains encodes one or more cohesin domains, one or more dockerin domains or a combination of one or more cohesin and dockerin domains.
18. The nucleic acid of claim 15, wherein the target specific domain comprises an rAb is further defined as: an rAb.Doc; an rAb.Coh; an rAb.(Coh)x; an rAb.(Doc)x; an rAb.(Coh.Doc)x; or an rAb.(Coh)x(Doc)x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
19. A vector comprising a nucleic acid encoding an antigen specific domain and one or more domains that comprise one half of a cohesin-dockerin binding pair, a one half of a cohesin-dockerin binding pair with a protein molecule to be carried and combinations thereof.
20. The vector of claim 19, wherein the one half of a cohesin-dockerin binding pair, a one half of a cohesin-dockerin binding pair with a protein molecule to be carried and combinations thereof are under the control of the same promoter, different promoters, transcribed in-line, transcribed in opposite directions.
21. A host cell comprising a vector comprising a nucleic acid encoding an antigen specific domain and one or more domains and one half of a cohesin-dockerin binding pair.
22. A method of making a modular rAb carrier comprising: combining an antigen specific domain linked to one or more domains comprising one half of a cohesin-dockerin binding pair.
23. The method of claim 22, wherein the rAb is further defined as: an rAb.Doc; an rAb.Coh; an rAb.(Coh)x; an rAb.(Doc)x; an rAb.(Coh.Doc)x; or an rAb.(Coh)x(Doc)x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
24. The method of claim 22, wherein the rAb is complexed with a complementary half of a cohesion:dokerin pair bound to an antigen and is selected from: an rAb.Doc:Coh.antigen; an rAb.Coh:Doc.antigen; an rAb.(Coh)x:(Doc.antigen)x; an rAb.(Doc)x:(Coh.antigen)x; an rAb.(Coh.Doc)x:(Doc.antigen1)(Coh.antigen2); or an rAb.(Coh)x(Doc)x:(Doc.antigen1)x(Coh.antigen2).s- ub.x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
25. An immunotoxin comprising an rAb.Doc:Coh.toxin self-assembled conjugate, wherein the rAb is specific for a cell target.
26. The immunotoxin of claim 25, wherein the toxin is selected from wherein the toxin is selected from the group consisting of a radioactive isotope, metal, enzyme, botulin, tetanus, ricin, cholera, diphtheria, aflatoxins, perfringens toxin, mycotoxins, shigatoxin, staphylococcal enterotoxin B, T2, seguitoxin, saxitoxin, abrin, cyanoginosin, alphatoxin, tetrodotoxin, aconotoxin, snake venom and spider venom.
27. The immunotoxin of claim 25, wherein the cell target comprises a cancer cell selected from hematological cancers, leukemias, lymphomas, neurological tumors, astrocytomas or glioblastomas, melanoma, breast cancer, lung cancer, head and neck cancer, gastrointestinal tumors such as gastric or colon cancer, liver cancer, pancreatic cancer, genitourinary tumors such cervix, uterus, ovarian cancer, vaginal cancer, testicular cancer, prostate cancer or penile cancer, bone tumors, vascular tumors, or cancers of the lip, nasopharynx, pharynx and oral cavity, esophagus, rectum, gall bladder, biliary tree, larynx, lung and bronchus, bladder, kidney, brain and other parts of the nervous system, thyroid, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma and leukemia.
28. The immunotoxin of claim 25, wherein the cell target comprises a pathogen selected from a bacteria, a protozoan, a helminth, a virally-infected cell or a fungus.
29. A method for protein purification, comprising: separating a cohesin or dockerin fusion protein by interacting the fusion protein with a rAb that is conjugated to the complementary cohesin or dockerin bound to a substrate.
30. The method of claim 29, further comprising the step of administering the protein in a therapeutic application comprising transplantation, autoimmune disease, infectious disease or cancer.
31. The use of the cohesin as a fusion partner for toxins for conferring beneficial biochemical properties favoring ready purification of active cohesin.toxin fusion protein.
32. The use of anti-DC rAb.Doc to target DC for therapeutic applications where ablating DC.
33. An anti-DC-SIGN/L antibody provided in an amount that is sufficient to enhance the survival of dendritic cells, wherein the antibody matures and activates the dendritic cells for immunization.
34. The antibody of claim 33, wherein the antibody is targeted in vivo to dendritic cells as an adjuvant in vaccines.
35. A bivalent and multivalent (rAb.sup.1.Doc:Coh.rAb2) self-assembled conjugates as therapeutic, diagnostic, and industrial agents.
36. A bivalent and multivalent (rAb.Doc:Coh.cytokine), (rAb.Coh:Doc.cytokine) or (cytokine.sup.1.Coh:cytokine.sup.2.Doc) self-assembled conjugates as therapeutic, cell proliferation or maturing agents.
37. A method for making modular rAb comprising: screening one or more multivalent rAb and/or rAb.cytokine and/or cytokine.cytokine combinations that are capable of specifically binding to a target cell and delivering the cytokine such that it exerts its effect on the target cell.
38. The method of claim 37, wherein the cytokine comprises interleukins, transforming growth factors (TGFs), fibroblast growth factors (FGFs), platelet derived growth factors (PDGFs), epidermal growth factors (EGFs), connective tissue activated peptides (CTAPs), osteogenic factors, and biologically active analogs, fragments, and derivatives of such growth factors, B/T-cell differentiation factors, B/T-cell growth factors, mitogenic cytokines, chemotactic cytokines, colony stimulating factors, angiogenesis factors, IFN-.alpha., IFN-.beta., IFN-.gamma., IL1, IL2, IL3, IL4, ILS, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, etc., leptin, myostatin, macrophage stimulating protein, platelet-derived growth factor, TNF-.alpha., TNF-.beta., NGF, CD40L, CD137L/4-1BBL, human lymphotoxin-.beta., G-CSF, M-CSF, GM-CSF, PDGF, IL-1.alpha., IL1-.beta., IP-10, PF4, GRO, 9E3, erythropoietin, endostatin, angiostatin, VEGF, transforming growth factor (TGF) supergene family include the beta transforming growth factors (for example TGF-.beta.1, TGF-.beta.2, TGF-.beta.3); bone morphogenetic proteins (for example, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9); heparin-binding growth factors (fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF)); Inhibins (for example, Inhibin A, Inhibin B); growth differentiating factors (for example, GDF-1); and Activins (for example, Activin A, Activin B, Activin AB).
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application Ser. No. 60/888,029, filed Feb. 2, 2007, the contents of which is incorporated by reference herein in its entirety.
TECHNICAL FIELD OF THE INVENTION
[0003] The present invention relates in general to the field of novel vaccines, and more particularly, to the design, manufacture and use of multivariable antigens complexed with targeting humanized monoclonal antibodies.
BACKGROUND OF THE INVENTION
[0004] Without limiting the scope of the invention, its background is described in connection with vaccine development.
[0005] Protein engineering technology relating to monoclonal antibodies is highy advanced regarding humanization (i.e., rendering e.g., a rodent mAb sequence into a human mAb sequence while preserving specific antigen combining sites of the the original mAb) and production (typically secreted from mammalian cell lines). In research and development are new applications of rAbs related to vaccination and are presently based on engineered rAb-antigen fusion proteins (typically with the antigen coding region placed in-frame with the C-terminal codon of the rAb heavy or H chain). A roadblock to this technology is the successful expression and production of fully functional rAb-antigen. In many, perhaps most, cases the desired antigen confounds secretion of the engineered rAb-antigen. Also, the likelihood of poor or null expression is increased if the desired entity includes multiple antigen coding regions.
SUMMARY OF THE INVENTION
[0006] The invention provides methods for the assembly of rAb antigen complexes in a controlled manner by simple mixing components and accomodates the ability to express and produce the rAb and antigen(s) in different expression--production systems that are best suited to the individual rAb and particular antigen. In addition, the invention demonstrates the novel application of the high affinity and high specificity cohesin-dockerin interaction to secreted mammalian expression systems, thus permitting the development of unique protein engineering formats and production of new protein tools for research and clinical application.
[0007] More particularly, the present invention uses the cohesin-dockerin protein domains and their surrounding linker. For example, the invention permits the controlled assembly of recombinant monoclonal antibodies (rAbs) complexed to antigens, toxins, or cellular activating agents. The invention has wide potential application in vaccination and cancer therapy. Also claimed are derivatives of this technology that permit the production of novel proteins with specific affinities for other proteins.
[0008] The invention is based on particular components of the well studied bacterial cellulose degrading protein complex called the cellulosome. Specifically, two protein domains (cohesin and dockerin) and natural protein linker sequences are utilized via the invention in novel contexts and applications.
[0009] The present invention is based on the discovery that particular cohesin and dockerin domains can be sucessfully and efficiently secreted from mammalian cells as fusion proteins while maintaining the specific and high affinity cohesin-dockerin protein-protein interaction. While the extensive cohesin-dockerin literature teaches the expectation that such fusion proteins should have this functionality, it does not describe production of such fusion proteins in mammalian secretion systems. The state of scientific knowledge does not allow the prediction of the discovery since the rules (other than features such as signal peptide) for successful secretion are not fully established. Furthermore, the cohesin linker regions are known to be glycosylated in their native bacteria, and the cohesin and dockerin domains contain predicted glycosylation sites. While this may actually favor secretion from mammalian cells, it is unclear if `unnatural` glyosylation will perturb the cohesis-dockerin interaction.
[0010] While cohesin-dockerin interaction for various commercial applications has been published, the present invention is based on a previously unrealized potential for this interaction built around assembling specific protein complexes unrelated to the controlled assembly enzyme applications.
[0011] The invention includes the use of all cohesin-dockerin sequences from diverse cellulose degrading microbes, but describes the application of specific cohesin and dockerin and linker sequences from the microbe Clostridium thermocellum. For example, the sequence described herein encodes the H chain of a human IgG4 linked at the C-terminal codon to a Clostridium thermocellum dockerin sequence (called rAb.doc). Other embodiments of rAb.doc proteins are described similarly with examples that are engineered by simply transferring the dockerin coding region as a DNA fragment to vectors encoding the different H chain entities.
[0012] More particularly, the present invention includes a modular rAb carrier that includes an antigen-specific binding domain linked to one or more antigen carrier domains and one half of a cohesin-dockerin binding pair. The antigen-specific binding domain may be at least a portion of an antibody and the antibody is a fusion protein with and the binding pair in a fusion protein with one half of a cohesin-dockerin binding pair. The rAb may also include a complementary half of the cohesin-dockerin binding pair bound to an antigen that forms a complex with the modular rAb carrier. The complementary half of the cohesin-dockerin binding pair may itself be a fusion protein with the antigen carried as part of the complex (modular rAb carrier (cohesin/dockerin) antigen complex). Examples of antigen specific domain include a full length antibody, an antibody variable region domain, an Fab fragment, a Fab' fragment, an F(ab)2 fragment, and Fv fragment, and Fabc fragment and/or a Fab fragment with portions of the Fc domain. Non limiting examples of sources for the cohesin-dockerin binding pair include Clostridium thermocellum, Clostridium josui, Clostridium cellulolyticum and Bacteroides cellulosolvens and combinations thereof.
[0013] Non-limiting examples for targeting by the antigen-specific binding domain include: cell surface marker selected from MHC class I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD 19, CD20, CD29, CD31, CD40,CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2, IFN-γ receptor and IL-2 receptor, ICAM-1, Fcγ receptor or other receptor relatively specifically expressed by antigen presenting cells.
[0014] The rAb of the present invention may also includes combinations of the domains that are defined as: an rAb.Doc; an rAb.Coh; an rAb.(Coh)x; an rAb.(Doc)x; an rAb.(Coh.Doc)x; or an rAb.(Coh)x(Doc)x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. Examples of the modular rAb carrier in a complex include:
[0015] an rAb.Doc:Coh.antigen;
[0016] an rAb.Coh:Doc.antigen;
[0017] an rAb.(Coh)x:(Doc.antigen)x;
[0018] an rAb.(Doc)x:(Coh.antigen)x;
[0019] an rAb.(Coh.Doc)x:(Doc.antigen1)(Coh.antigen2); or
[0020] an rAb.(Coh)x(Doc)x:(Doc.antigen1)x(Coh.antigen2).s- ub.x;
[0021] wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
[0022] The present invention also include a vaccine of a modular rAb carrier that includes an antigen specific domain linked to one or more domains comprising one half of the cohesin-dockerin binding pair bound to a complementary half of the cohesin-dockerin binding pair bound to an antigen. Non-limiting examples for targeting the rAb include immune cell surface protein selected from MHC class I, MHC class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD 19, CD20, CD29, CD31, CD40,CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2, IFN-γ receptor and IL-2 receptor, ICAM-1, Fcγ receptor or other receptor relatively specifically expressed by antigen presenting cells. Targets for vaccination with the rAb antigen carrier include, e.g., a bacterial, viral, fungal, protozoan or cancer protein and fragments thereof. The vaccine of claim 11, wherein the modular rAb carrier is further defined: an rAb.Doc:Coh.antigen; an rAb.Coh:Doc.antigen; an rAb.(Coh)x:(Doc.antigen)x; an rAb.(Doc)x:(Coh.antigen)x; an rAb.(Coh.Doc)x: (Doc.antigen1)(Coh.antigen2); or an rAb.(Coh)x(Doc)x:(Doc.antigen1)x(Coh.antigen2).s- ub.x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
[0023] The present invention also includes an isolated nucleic acid comprising a coding segment for a target-specific domain and one or more domains and one half of a cohesin-dockerin binding pair. For example, the target may be an antigen and the target specific domain may encode at least a portion of an antibody. The one or more domains can encode one or more cohesin domains, one or more dockerin domains or a combination of one or more cohesin and dockerin domains. The rAb is further defined as: an rAb.Doc; an rAb.Coh; an rAb.(Coh)x; an rAb.(Doc)x; an rAb.(Coh.Doc)x; or an rAb.(Coh)x(Doc)x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
[0024] The present invention also includes a vector that includes a nucleic acid encoding an antigen specific domain and one or more domains that comprise one half of a cohesin-dockerin binding pair, a one half of a cohesin-dockerin binding pair with a protein molecule to be carried and combinations thereof. The one half of a cohesin-dockerin binding pair, a one half of a cohesin-dockerin binding pair with a protein molecule to be carried and combinations thereof are under the control of the same promoter, different promoters, transcribed in-line, transcribed in opposite directions.
[0025] The present invention also includes a host cell comprising a vector comprising a nucleic acid encoding an antigen specific domain and one or more domains and one half of a cohesin-dockerin binding pair.
[0026] A method of making a modular rAb carrier by combining an antigen specific domain linked to one or more domains of one half of a cohesin-dockerin binding pair. The rAb is further defined as: an rAb.Doc; an rAb.Coh; an rAb.(Coh)x; an rAb.(Doc)x; an rAb.(Coh.Doc)x; or an rAb.(Coh)x(Doc)x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Examples of the rAb is complexed with a complementary half of a cohesion:dokerin pair bound to an antigen and is selected from: an rAb.Doc:Coh.antigen; an rAb.Coh:Doc.antigen; an rAb.(Coh)x:(Doc.antigen)x; an rAb.(Doc)x:(Coh.antigen)x; an rAb.(Coh.Doc)x:(Doc.antigen1)(Coh.antigen2); or an rAb.(Coh)x(Doc)x:(Doc.antigen1)x(Coh.antigen2).s- ub.x; wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
[0027] The present invention may also be an immunotoxin that includes an rAb.Doc:Coh.toxin self-assembled conjugate, wherein the rAb is specific for a cell target. Examples of toxins include a radioactive isotope, metal, enzyme, botulin, tetanus, ricin, cholera, diphtheria, aflatoxins, perfringens toxin, mycotoxins, shigatoxin, staphylococcal enterotoxin B, T2, seguitoxin, saxitoxin, abrin, cyanoginosin, alphatoxin, tetrodotoxin, aconotoxin, snake venom and spider venom. Cell targets for the immunotoxin include diseased or infected cells. Examples of diseased cells for targeting include cancer cell for, e.g., hematological cancers such as leukemias and lymphomas, neurological tumors such as astrocytomas or glioblastomas, melanoma, breast cancer, lung cancer, head and neck cancer, gastrointestinal tumors such as gastric or colon cancer, liver cancer, pancreatic cancer, genitourinary tumors such cervix, uterus, ovarian cancer, vaginal cancer, testicular cancer, prostate cancer or penile cancer, bone tumors, vascular tumors, or cancers of the lip, nasopharynx, pharynx and oral cavity, esophagus, rectum, gall bladder, biliary tree, larynx, lung and bronchus, bladder, kidney, brain and other parts of the nervous system, thyroid, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma and leukemia. The immunotoxin may target pathogens directly, e.g., bacteria, a protozoan, a helminth, a virally-infected cell or a fungus.
[0028] The present invention also includes a method for protein purification by separating a cohesin or dockerin fusion protein by interacting the fusion protein with a rAb that is conjugated to the complementary cohesin or dockerin bound to a substrate. The present invention may also use the cohesin as a fusion partner for toxins for conferring beneficial biochemical properties favoring ready purification of active cohesin.toxin fusion protein. The present invention may also use the anti-DC rAb.Doc to target DC for therapeutic applications where ablating DC. Therapeutic applications include, e.g., transplantation, autoimmune disease, infectious disease or cancer. The invention also includes an anti-DC-SIGN/L antibody provided in an amount that is sufficient to enhance the survival of dendritic cells, wherein the antibody matures and activates the dendritic cells for immunization. The antibody may target cells in vivo, e.g., dendritic cells as an adjuvant in vaccines.
[0029] Also invented is a bivalent and multivalent (rAb1.Doc:Coh.rAb2) self-assembled conjugates as therapeutic, diagnostic, and industrial agents. Alternatively, the invention is a bivalent and multivalent (rAb.Doc:Coh.cytokine), (rAb.Coh:Doc.cytokine) or (cytokine1.Coh:cytokine2.Doc) self-assembled conjugates as therapeutic, cell proliferation or maturing agents. The modular rAbs carrier may be made by method that includes screening one or more multivalent rAb and/or rAb.cytokine and/or cytokine.cytokine combinations that are capable of specifically binding to a target cell and delivering the cytokine such that it exerts its effect on the target cell. Cytokines for use with the present invention include: interleukins, transforming growth factors (TGFs), fibroblast growth factors (FGFs), platelet derived growth factors (PDGFs), epidermal growth factors (EGFs), connective tissue activated peptides (CTAPs), osteogenic factors, and biologically active analogs, fragments, and derivatives of such growth factors, B/T-cell differentiation factors, B/T-cell growth factors, mitogenic cytokines, chemotactic cytokines, colony stimulating factors, angiogenesis factors, IFN-α, IFN-β, IFN-γ, IL1, IL2, IL3, IL4, ILS, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, etc., leptin, myostatin, macrophage stimulating protein, platelet-derived growth factor, TNF-α, TNF-β, NGF, CD40L, CD137L/4-1BBL, human lymphotoxin-β, G-CSF, M-CSF, GM-CSF, PDGF, IL-1a, IL1-β, IP-10, PF4, GRO, 9E3, erythropoietin, endostatin, angiostatin, VEGF, transforming growth factor (TGF) supergene family include the beta transforming growth factors (for example TGF-β1, TGF-β2, TGF-β3); bone morphogenetic proteins (for example, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9); heparin-binding growth factors (fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF)); Inhibins (for example, Inhibin A, Inhibin B); growth differentiating factors (for example, GDF-1); and Activins (for example, Activin A, Activin B, Activin AB).
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:
[0031] FIG. 1 compares the prior art (top portion) with an example of the multiple antigens targeted in a complex simultaneously with the same engineered humanized mAb (MATCHMAB)(bottom portion).
[0032] FIG. 2 shows the use of the present invention to form Bi-specific mAbs.
[0033] FIG. 3 shows Protein G affinity purified secreted rAb proteins analyzed by reducing SDS.PAGE and Coomassie Brilliant Blue staining. Lanes are from left to right.
[0034] FIGS. 4A and 4B show the measurement by anti-human IgFc ELISA of levels of secretion of various rAb.fusion proteins.
[0035] FIG. 5 shows the measurement by anti-human IgFc ELISA (HRP activity) and LOX-1.alkaline phoshatase binding (AP activity) of secreted anti-LOX1--15C4 rAb.(blue symbols) and anti-LOX1--15C4.doc rAb (red symbols) proteins.
[0036] FIG. 6 shows that when co-transfected with a mIgG kappa expression plasmid, rAB-pCMV(mIgG2bH-Dockerin) plasmid directs the efficient secretion of rAB-mIgG2b.Dockerin fusion protein.
[0037] FIGS. 7A and 7B show that the secreted coh.alkaline phosphatase (coh.AP) but not AP binds efficiently and specifically to rAb.Doc immobilized on plastic.
[0038] FIGS. 8A and 8B shows various dilutions of a supernatant containing secreted G.AP bound to immobilized mIgG2a and mIgG2b, but not rAb.doc, while coh.AP bound rAb.doc specifically.
[0039] FIG. 9 shows the differential stability of complexes between a fixed amount of proG.AP or coh.AP or coh2.AP (0.1 ug) and immobilized mIgG2b or rAb.doc (0.25 ug) assembled by incubation for 1 hr in a micro-titre plate.
[0040] FIG. 10 shows the differential stability in human serum of complexes between a fixed amount of proG.AP or coh.AP (0.1 ug) and immobilized mIgG2b or rAb.doc (0.25 ug) were assembled by incubation for 1 hr in a micro-titre plate.
[0041] FIG. 11 is a gel that shows the reduced vs. non-reduced SDS.PAGE analysis of rAb.doc:Coh2.AP complexes produced by sequential application of rAb.doc supernatant and coh.AP supernatant to the same protein G affinity column.
[0042] FIG. 12 is a non-reduced SDS.PAGE analysis of rAb.doc:Coh.Flu HA5-1 complexes produced by sequential application of rAb.doc supernatant and coh.Flu HA5-1 supernatant to the same protein G affinity column.
[0043] FIG. 13 shows that anti-DC_rAb.doc:coh.Flu M1 complex formed by mixing the individual purified components was effective in vitro in expanding Flu M1-specific T cells.
[0044] FIG. 14 shows that Anti-DC_rAb.doc:coh.Flu M1 but not mIgG2b.doc:coh.Flu M1 complexes formed by mixing the individual purified components was effective in vitro in expanding Flu M1-specific T cells.
[0045] FIG. 15 shows CD34+ human DC were sorted into CD1a+ and CD14+ subtypes and cultured with and without 3 nM Anti-DC_rAb.Flu M1 PEP or Anti-DC rAb.
[0046] FIG. 16 shows E. coli harboring expression plasmids directing the synthesis of coh.pep proteins were grown and induced for specific protein production. Cells were harvested and broken by sonication.
[0047] FIG. 17 shows that the DCIR.Doc rAb alone had no effect upon the survival of DCs, but DC-SIGN/L.Doc rAb ehnaces their survival.
[0048] FIG. 18 shows that Coh.PE38 alone slightly increase the number of 7-AAD scored apoptotic cells (from 22.1-29.8%), but when linked to DCIR or DC-SIGN/L.Doc rAbs, Coh.PE38 greatly enhanced the number of 7-AAD scored apoptotic cells.
[0049] FIG. 19 shows the expression of anti-DC-SIGN/L and Anti-DC-ASPGR rAb.Coh and rAb.Doc were efficiently secreted.
[0050] FIG. 20 shows the effect of IL-21 and Coh.IL-21 on the proliferation of human B cells.
DETAILED DESCRIPTION OF THE INVENTION
[0051] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.
[0052] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.
[0053] At present, protein engineering technology enables the ready and controlled addition of an antigen (or different antigens to one of the chains) of a recombinant mAb (H or L, usually the C-terminus of H is often used). If different antigens or different antigen sets need to be linked to the mAb, then the mAb needs to be re-engineered, expressed, and purified as a different entity.
[0054] The present invention provides for the complexing of multiple antigens or proteins (engineered, expressed, and purified independently from the primary mAb) in a controlled, multivariable fashion, to one single primary recombinant mAb. Presently, there are methods for engineering site-specific biotinylation sites that provide for the addition of different proteins (each engineered separately linked to streptavidin) to the one primary mAb. However, the present invention provides for addition to the primary mAb of multiple combinations, in fixed equimolar ratios and locations, of separately engineered proteins.
[0055] As used herein, the term "modular rAb carrier" is used to describe a recombinant antibody system that has been engineered to provide the controlled modular addition of diverse antigens, activating proteins, or other antibodies to a single recombinant monoclonal antibody (mAb). The rAb may be a monoclonal antibody made using standard hybridoma techniques, recombinant antibody display, humanized monoclonal antibodies and the like. The modular rAb carrier can be used to, e.g., target (via one primary recombinant antibody against an internalizing receptor, e.g., a human dendritic cell receptor) multiple antigens and/or antigens and an activating cytokine to dendritic cells (DC). The modular rAb carrier may also be used to join two different recombinant mAbs end-to-end in a controlled and defined manner.
[0056] The antigen binding portion of the "modular rAb carrier" may be one or more variable domains, one or more variable and the first constant domain, an Fab fragment, a Fab' fragment, an F(ab)2 fragment, and Fv fragment, and Fabc fragment and/or a Fab fragment with portions of the Fc domain to which the cognate modular binding portions are added to the amino acid sequence and/or bound. The antibody for use in the modular rAb carrier can be of any isotype or class, subclass or from any source (animal and/or recombinant).
[0057] In one non-limiting example, the modular rAb carrier is engineered to have one or more modular cohesin-dockerin protein domains for making specific and defined protein complexes in the context of engineered recombinant mAbs. The mAb is a portion of a fusion protein that includes one or more modular cohesin-dockerin protein domains carboxy from the antigen binding domains of the mAb. The cohesin-dockerin protein domains may even be attached post-translationally, e.g., by using chemical cross-linkers and/or disulfide bonding.
[0058] The modular rAb carrier will be used to carry a separate molecule, e.g., a peptide, protein, lipid, carbohydrate, nucleic acid (oligonucleotide, aptamer, vector with or without base or backbone modifications) or combinations thereof by binding that separate molecule to the complementary half of the cohesion:dockerin pair. For example, either the dockerin or cohesin made be made into a fusion protein or chemically bound to an antigen, a peptide, a protein, a toxin, a cytokine, an enzyme, a structural protein, an extracellular matrix protein, another antibody, a cell or fragments thereof. The modular rAb carrier may have one or more cohesin, dockerin or both cohesin and dockerin domains that allow the formation of a complex with one or more complementary cohesin/dockerin-molecules for delivery via the antigen recognition domain of the modular rAb carrier.
[0059] The term "antigen" as used herein refers to a molecule that can initiate a humoral and/or cellular immune response in a recipient of the antigen. Antigen may be used in two different contexts with the present invention: as a target for the antibody or other antigen recognition domain of the rAb or as the molecule that is carried to and/or into a cell or target by the rAb as part of a dockerin/cohesin-molecule complement to the modular rAb carrier. The antigen is usually an agent that causes a disease for which a vaccination would be advantageous treatment. When the antigen is presented on MHC, the peptide is often about 8 to about 25 amino acids. Antigens include any type of biologic molecule, including, for example, simple intermediary metabolites, sugars, lipids and hormones as well as macromolecules such as complex carbohydrates, phospholipids, nucleic acids and proteins. Common categories of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoal and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, and other miscellaneous antigens.
[0060] The modular rAb carrier is able to carry any number of active agents, e.g., antibiotics, anti-infective agents, antiviral agents, anti-tumoral agents, antipyretics, analgesics, anti-inflammatory agents, therapeutic agents for osteoporosis, enzymes, cytokines, anticoagulants, polysaccharides, collagen, cells, and combinations of two or more of the foregoing active agents. Examples of antibiotics for delivery using the present invention include, without limitation, tetracycline, aminoglycosides, penicillins, cephalosporins, sulfonamide drugs, chloramphenicol sodium succinate, erythromycin, vancomycin, lincomycin, clindamycin, nystatin, amphotericin B, amantidine, idoxuridine, p-amino salicyclic acid, isoniazid, rifampin, antinomycin D, mithramycin, daunomycin, adriamycin, bleomycin, vinblastine, vincristine, procarbazine, imidazole carboxamide, and the like.
[0061] Examples of anti-tumor agents for delivery using the present invention include, without limitation, doxorubicin, Daunorubicin, taxol, methotrexate, and the like. Examples of antipyretics and analgesics include aspirin, Motrin®, Ibuprofen®, naprosyn, acetaminophen, and the like.
[0062] Examples of anti-inflammatory agents for delivery using the present invention include, without limitation, include NSAIDS, aspirin, steroids, dexamethasone, hydrocortisone, prednisolone, Diclofenac Na, and the like.
[0063] Examples of therapeutic agents for treating osteoporosis and other factors acting on bone and skeleton include for delivery using the present invention include, without limitation, calcium, alendronate, bone GLa peptide, parathyroid hormone and its active fragments, histone H4-related bone formation and proliferation peptide and mutations, derivatives and analogs thereof.
[0064] Examples of enzymes and enzyme cofactors for delivery using the present invention include, without limitation, pancrease, L-asparaginase, hyaluronidase, chymotrypsin, trypsin, tPA, streptokinase, urokinase, pancreatin, collagenase, trypsinogen, chymotrypsinogen, plasminogen, streptokinase, adenyl cyclase, superoxide dismutase (SOD), and the like.
[0065] Examples of cytokines for delivery using the present invention include, without limitation, interleukins, transforming growth factors (TGFs), fibroblast growth factors (FGFs), platelet derived growth factors (PDGFs), epidermal growth factors (EGFs), connective tissue activated peptides (CTAPs), osteogenic factors, and biologically active analogs, fragments, and derivatives of such growth factors. Cytokines may be B/T-cell differentiation factors, B/T-cell growth factors, mitogenic cytokines, chemotactic cytokines, colony stimulating factors, angiogenesis factors, IFN-α, IFN-β, IFN-γ, IL1, IL2, IL3, IL4, ILS, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, etc., leptin, myostatin, macrophage stimulating protein, platelet-derived growth factor, TNF-α, TNF-β, NGF, CD40L, CD137L/4-1BBL, human lymphotoxin-β, G-CSF, M-CSF, GM-CSF, PDGF, IL-1α, IL1-β, IP-10, PF4, GRO, 9E3, erythropoietin, endostatin, angiostatin, VEGF or any fragments or combinations thereof. Other cytokines include members of the transforming growth factor (TGF) supergene family include the beta transforming growth factors (for example TGF-β1, TGF-β2, TGF-β3); bone morphogenetic proteins (for example, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9); heparin-binding growth factors (for example, fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF)); Inhibins (for example, Inhibin A, Inhibin B); growth differentiating factors (for example, GDF-1); and Activins (for example, Activin A, Activin B, Activin AB).
[0066] Examples of growth factors for delivery using the present invention include, without limitation, growth factors that can be isolated from native or natural sources, such as from mammalian cells, or can be prepared synthetically, such as by recombinant DNA techniques or by various chemical processes. In addition, analogs, fragments, or derivatives of these factors can be used, provided that they exhibit at least some of the biological activity of the native molecule. For example, analogs can be prepared by expression of genes altered by site-specific mutagenesis or other genetic engineering techniques.
[0067] Examples of anticoagulants for delivery using the present invention include, without limitation, include warfarin, heparin, Hirudin, and the like. Examples of factors acting on the immune system include for delivery using the present invention include, without limitation, factors which control inflammation and malignant neoplasms and factors which attack infective microorganisms, such as chemotactic peptides and bradykinins.
[0068] Examples of viral antigens and/or viral antigenic targets include, but are not limited to, e.g., retroviral antigens such as retroviral antigens from the human immunodeficiency virus (HIV) antigens such as gene products of the gag, pol, and env genes, the Nef protein, reverse transcriptase, and other HIV components; hepatitis viral antigens such as the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, and other hepatitis, e.g., hepatitis A, B, and C, viral components such as hepatitis C viral RNA; influenza viral antigens such as hemagglutinin and neuraminidase and other influenza viral components; measles viral antigens such as the measles virus fusion protein and other measles virus components; rubella viral antigens such as proteins El and E2 and other rubella virus components; rotaviral antigens such as VP7sc and other rotaviral components; cytomegaloviral antigens such as envelope glycoprotein B and other cytomegaloviral antigen components; respiratory syncytial viral antigens such as the RSV fusion protein, the M2 protein and other respiratory syncytial viral antigen components; herpes simplex viral antigens such as immediate early proteins, glycoprotein D, and other herpes simplex viral antigen components; varicella zoster viral antigens such as gpl, gpII, and other varicella zoster viral antigen components; Japanese encephalitis viral antigens such as proteins E, M-E, M-E-NS1, NS1, NS1-NS2A, 80% E, and other Japanese encephalitis viral antigen components; rabies viral antigens such as rabies glycoprotein, rabies nucleoprotein and other rabies viral antigen components. See Fundamental Virology, Second Edition, eds. Fields, B. N. and Knipe, D. M. (Raven Press, New York, 1991) for additional examples of viral antigens.
[0069] Antigens and/or antigenic targets that may be delivered using the rAb-DC/DC-antigen vaccines of the present invention include genes encoding antigens such as viral antigens, bacterial antigens, fungal antigens or parasitic antigens. Viruses include picornavirus, coronavirus, togavirus, flavirvirus, rhabdovirus, paramyxovirus, orthomyxovirus, bunyavirus, arenavirus, reovirus, retrovirus, papilomavirus, parvovirus, herpesvirus, poxvirus, hepadnavirus, and spongiform virus. Other viral targets include influenza, herpes simplex virus 1 and 2, measles, dengue, smallpox, polio or HW. Pathogens include trypanosomes, tapeworms, roundworms, helminthes, malaria. Tumor markers, such as fetal antigen or prostate specific antigen, may be targeted in this manner. Other examples include: HIV env proteins and hepatitis B surface antigen. Administration of a vector according to the present invention for vaccination purposes would require that the vector-associated antigens be sufficiently non-immunogenic to enable long term expression of the transgene, for which a strong immune response would be desired. In some cases, vaccination of an individual may only be required infrequently, such as yearly or biennially, and provide long term immunologic protection against the infectious agent. Specific examples of organisms, allergens and nucleic and amino sequences for use in vectors and ultimately as antigens with the present invention may be found in U.S. Pat. No. 6,541,011, relevant portions incorporated herein by reference, in particular, the tables that match organisms and specific sequences that may be used with the present invention.
[0070] Bacterial antigens for use with the rAb vaccine disclosed herein include, but are not limited to, e.g., bacterial antigens such as pertussis toxin, filamentous hemagglutinin, pertactin, FIM2, FIM3, adenylate cyclase and other pertussis bacterial antigen components; diptheria bacterial antigens such as diptheria toxin or toxoid and other diptheria bacterial antigen components; tetanus bacterial antigens such as tetanus toxin or toxoid and other tetanus bacterial antigen components; streptococcal bacterial antigens such as M proteins and other streptococcal bacterial antigen components; gram-negative bacilli bacterial antigens such as lipopolysaccharides and other gram-negative bacterial antigen components, Mycobacterium tuberculosis bacterial antigens such as mycolic acid, heat shock protein 65 (HSP65), the 30 kDa major secreted protein, antigen 85A and other mycobacterial antigen components; Helicobacter pylori bacterial antigen components; pneumococcal bacterial antigens such as pneumolysin, pneumococcal capsular polysaccharides and other pneumococcal bacterial antigen components; haemophilus influenza bacterial antigens such as capsular polysaccharides and other haemophilus influenza bacterial antigen components; anthrax bacterial antigens such as anthrax protective antigen and other anthrax bacterial antigen components; rickettsiae bacterial antigens such as rompA and other rickettsiae bacterial antigen component. Also included with the bacterial antigens described herein are any other bacterial, mycobacterial, mycoplasmal, rickettsial, or chlamydial antigens. Partial or whole pathogens may also be: haemophilus influenza; Plasmodium falciparum; neisseria meningitidis; streptococcus pneumoniae; neisseria gonorrhoeae; salmonella serotype typhi; shigella; vibrio cholerae; Dengue Fever; Encephalitides; Japanese Encephalitis; lyme disease; Yersinia pestis; west nile virus; yellow fever; tularemia; hepatitis (viral; bacterial); RSV (respiratory syncytial virus); HPIV 1 and HPIV 3; adenovirus; small pox; allergies and cancers.
[0071] Fungal antigens for use with compositions and methods of the invention include, but are not limited to, e.g., candida fungal antigen components; histoplasma fungal antigens such as heat shock protein 60 (HSP60) and other histoplasma fungal antigen components; cryptococcal fungal antigens such as capsular polysaccharides and other cryptococcal fungal antigen components; coccidiodes fungal antigens such as spherule antigens and other coccidiodes fungal antigen components; and tinea fungal antigens such as trichophytin and other coccidiodes fungal antigen components.
[0072] Examples of protozoal and other parasitic antigens include, but are not limited to, e.g., plasmodium falciparum antigens such as merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte/gamete surface antigens, blood-stage antigen pf 155/RESA and other plasmodial antigen components; toxoplasma antigens such as SAG-1, p30 and other toxoplasmal antigen components; schistosomae antigens such as glutathione-S-transferase, paramyosin, and other schistosomal antigen components; leishmania major and other leishmaniae antigens such as gp63, lipophosphoglycan and its associated protein and other leishmanial antigen components; and trypanosoma cruzi antigens such as the 75-77 kDa antigen, the 56 kDa antigen and other trypanosomal antigen components.
[0073] Target antigens on immune cell surfaces that can be targeted using the antigen recognition site of the antibody portion of the rAb of the present invention will generally be selected based on a number of factors, including: likelihood of internalization, level of immune cell specificity, type of immune cell targeted, level of immune cell maturity and/or activation and the like. Examples of cell surface markers for dendritic cells include, but are not limited to, MHC class I, MHC Class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD 19, CD20, CD29, CD31, CD40,CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2, IFN-γ receptor and IL-2 receptor, ICAM-1, Fcγ receptor or other receptor relatively specifically expressed by antigen presenting cells. Examples of cell surface markers for antigen presenting cells include, but are not limited to, MHC class I, MHC Class II, CD1, CD2, CD3, CD4, CD8, CD11b, CD14, CD15, CD16, CD 19, CD20, CD29, CD31, CD40,CD43, CD44, CD45, CD54, CD56, CD57, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR, DC-ASPGR, CLEC-6, CD40, BDCA-2, MARCO, DEC-205, mannose receptor, Langerin, DECTIN-1, B7-1, B7-2, IFN-γ receptor and IL-2 receptor, ICAM-1, Fcγ receptor or other receptor relatively specifically expressed by antigen presenting cells. Examples of cell surface markers for T cells include, but are not limited to, CD3, CD4, CD8, CD 14, CD20, CD11b, CD16, CD45 and HLA-DR.
[0074] Target antigens on cell surfaces for delivery includes those characteristic of tumor antigens typically will be derived from the cell surface, cytoplasm, nucleus, organelles and the like of cells of tumor tissue. Examples of tumor targets for the antibody portion of the present invention include, without limitation, hematological cancers such as leukemias and lymphomas, neurological tumors such as astrocytomas or glioblastomas, melanoma, breast cancer, lung cancer, head and neck cancer, gastrointestinal tumors such as gastric or colon cancer, liver cancer, pancreatic cancer, genitourinary tumors such cervix, uterus, ovarian cancer, vaginal cancer, testicular cancer, prostate cancer or penile cancer, bone tumors, vascular tumors, or cancers of the lip, nasopharynx, pharynx and oral cavity, esophagus, rectum, gall bladder, biliary tree, larynx, lung and bronchus, bladder, kidney, brain and other parts of the nervous system, thyroid, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma and leukemia.
[0075] Examples of antigens that may be delivered alone or in combination to immune cells for antigen presentation using the present invention include tumor proteins, e.g., mutated oncogenes; viral proteins associated with tumors; and tumor mucins and glycolipids. The antigens may be viral proteins associated with tumors would be those from the classes of viruses noted above. Certain antigens may be characteristic of tumors (one subset being proteins not usually expressed by a tumor precursor cell), or may be a protein which is normally expressed in a tumor precursor cell, but having a mutation characteristic of a tumor. Other antigens include mutant variant(s) of the normal protein having an altered activity or subcellular distribution, e.g., mutations of genes giving rise to tumor antigens.
[0076] Specific non-limiting examples of tumor antigens include: CEA, prostate specific antigen (PSA), HER-2/neu, BAGE, GAGE, MAGE 1-4, 6 and 12, MUC (Mucin) (e.g., MUC-1, MUC-2, etc.), GM2 and GD2 gangliosides, ras, myc, tyrosinase, MART (melanoma antigen), Pmel 17(gp100), GnT-V intron V sequence (N-acetylglucoaminyltransferase V intron V sequence), Prostate Ca psm, PRAME (melanoma antigen), β-catenin, MUM-1-B (melanoma ubiquitous mutated gene product), GAGE (melanoma antigen) 1, BAGE (melanoma antigen) 2-10, c-ERB2 (Her2/neu), EBNA (Epstein-Barr Virus nuclear antigen) 1-6, gp75, human papilloma virus (HPV) E6 and E7, p53, lung resistance protein (LRP), Bcl-2, and Ki-67. In addition, the immunogenic molecule can be an autoantigen involved in the initiation and/or propagation of an autoimmune disease, the pathology of which is largely due to the activity of antibodies specific for a molecule expressed by the relevant target organ, tissue, or cells, e.g., SLE or MG. In such diseases, it can be desirable to direct an ongoing antibody-mediated (i.e., a Th2-type) immune response to the relevant autoantigen towards a cellular (i.e., a Th1-type) immune response. Alternatively, it can be desirable to prevent onset of or decrease the level of a Th2 response to the autoantigen in a subject not having, but who is suspected of being susceptible to, the relevant autoimmune disease by prophylactically inducing a Th1 response to the appropriate autoantigen. Autoantigens of interest include, without limitation: (a) with respect to SLE, the Smith protein, RNP ribonucleoprotein, and the SS-A and SS-B proteins; and (b) with respect to MG, the acetylcholine receptor.Examples of other miscellaneous antigens involved in one or more types of autoimmune response include, e.g., endogenous hormones such as luteinizing hormone, follicular stimulating hormone, testosterone, growth hormone, prolactin, and other hormones.
[0077] Antigens involved in autoimmune diseases, allergy, and graft rejection can be used in the compositions and methods of the invention. For example, an antigen involved in any one or more of the following autoimmune diseases or disorders can be used in the present invention: diabetes, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's Syndrome, including keratoconjunctivitis sicca secondary to Sjogren's Syndrome, alopecia areata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Crohn's disease, Graves ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and interstitial lung fibrosis. Examples of antigens involved in autoimmune disease include glutamic acid decarboxylase 65 (GAD 65), native DNA, myelin basic protein, myelin proteolipid protein, acetylcholine receptor components, thyroglobulin, and the thyroid stimulating hormone (TSH) receptor. Examples of antigens involved in allergy include pollen antigens such as Japanese cedar pollen antigens, ragweed pollen antigens, rye grass pollen antigens, animal derived antigens such as dust mite antigens and feline antigens, histocompatiblity antigens, and penicillin and other therapeutic drugs. Examples of antigens involved in graft rejection include antigenic components of the graft to be transplanted into the graft recipient such as heart, lung, liver, pancreas, kidney, and neural graft components. The antigen may be an altered peptide ligand useful in treating an autoimmune disease.
[0078] As used herein, the term "epitope(s)" refer to a peptide or protein antigen that includes a primary, secondary or tertiary structure similar to an epitope located within any of a number of pathogen polypeptides encoded by the pathogen DNA or RNA. The level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against such polypeptides will also bind to, react with, or otherwise recognize, the peptide or protein antigen. Various immunoassay methods may be employed in conjunction with such antibodies, such as, for example, Western blotting, ELISA, RIA, and the like, all of which are known to those of skill in the art. The identification of pathogen epitopes, and/or their functional equivalents, suitable for use in vaccines is part of the present invention. Once isolated and identified, one may readily obtain functional equivalents. For example, one may employ the methods of Hopp, as taught in U.S. Pat. No. 4,554,101, incorporated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity. The methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences (see, for example, Jameson and Wolf, 1988; Wolf et al., 1988; U.S. Pat. No. 4,554,101). The amino acid sequence of these "epitopic core sequences" may then be readily incorporated into peptides, either through the application of peptide synthesis or recombinant technology.
[0079] As used herein, the term "promoter" describes a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors. The phrases "operatively positioned," "operatively linked," "under control," and "under transcriptional control" mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence (i.e., ORF) to control transcriptional initiation and/or expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. A listing of promoters and/or enhancers that may be used with the present invention is described in, e.g., U.S. Pat. No. 6,410,241, relevant descriptions and tables incorporated herein by reference.
[0080] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations, in vivo, ex vivo or in vitro. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, "host cell" refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of expressing a heterologous gene encoded by a vector as delivered using the rAb protein vector of the present invention. A host cell can, and has been, used as a recipient for vectors. A host cell may be "transfected" or "transformed," which refers to a process by which the exogenous nucleic acid expressing an antigen, as disclosed herein, is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny.
[0081] The preparation of vaccine compositions that includes the nucleic acids that encode antigens of the invention as the active ingredient, may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to infection can also be prepared. The preparation may be emulsified, encapsulated in liposomes. The active immunogenic ingredients are often mixed with carriers which are pharmaceutically acceptable and compatible with the active ingredient.
[0082] The term "pharmaceutically acceptable carrier" refers to a carrier that does not cause an allergic reaction or other untoward effect in subjects to whom it is administered. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants that may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, MTP-PE and RIBI, which contains three components extracted from bacteria, monophosporyl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. Other examples of adjuvants include DDA (dimethyldioctadecylammonium bromide), Freund's complete and incomplete adjuvants and QuilA. In addition, immune modulating substances such as lymphokines (e.g., IFN-γ, IL-2 and IL-12) or synthetic IFN-γ inducers such as poly I:C can be used in combination with adjuvants described herein.
[0083] Pharmaceutical products that may include a naked polynucleotide with a single or multiple copies of the specific nucleotide sequences that bind to specific DNA-binding sites of the apolipoproteins present on plasma lipoproteins as described in the current invention. The polynucleotide may encode a biologically active peptide, antisense RNA, or ribozyme and will be provided in a physiologically acceptable administrable form. Another pharmaceutical product that may spring from the current invention may include a highly purified plasma lipoprotein fraction, isolated according to the methodology, described herein from either the patients blood or other source, and a polynucleotide containing single or multiple copies of the specific nucleotide sequences that bind to specific DNA-binding sites of the apolipoproteins present on plasma lipoproteins, prebound to the purified lipoprotein fraction in a physiologically acceptable, administrable form.
[0084] Yet another pharmaceutical product may include a highly purified plasma lipoprotein fraction which contains recombinant apolipoprotein fragments containing single or multiple copies of specific DNA-binding motifs, prebound to a polynucleotide containing single or multiple copies of the specific nucleotide sequences, in a physiologically acceptable administrable form. Yet another pharmaceutical product may include a highly purified plasma lipoprotein fraction which contains recombinant apolipoprotein fragments containing single or multiple copies of specific DNA-binding motifs, prebound to a polynucleotide containing single or multiple copies of the specific nucleotide sequences, in a physiologically acceptable administrable form.
[0085] The dosage to be administered depends to a great extent on the body weight and physical condition of the subject being treated as well as the route of administration and frequency of treatment. A pharmaceutical composition that includes the naked polynucleotide prebound to a highly purified lipoprotein fraction may be administered in amounts ranging from 1 μg to 1 mg polynucleotide and 1 μg to 100 mg protein.
[0086] Administration of the therapeutic virus particle to a patient will follow general protocols for the administration of chemotherapeutics, taking into account the toxicity, if any, of the vector. It is anticipated that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described gene therapy.
[0087] Where clinical application of a gene therapy is contemplated, it will be necessary to prepare the complex as a pharmaceutical composition appropriate for the intended application. Generally this will entail preparing a pharmaceutical composition that is essentially free of pyrogens, as well as any other impurities that could be harmful to humans or animals. One also will generally desire to employ appropriate salts and buffers to render the complex stable and allow for complex uptake by target cells.
[0088] Aqueous compositions of the present invention may include an effective amount of the compound, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions can also be referred to as inocula. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. The compositions of the present invention may include classic pharmaceutical preparations. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
[0089] Disease States. Depending on the particular disease to be treated, administration of therapeutic compositions according to the present invention will be via any common route so long as the target tissue is available via that route in order to maximize the delivery of antigen to a site for maximum (or in some cases minimum) immune response. Administration will generally be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Other areas for delivery include: oral, nasal, buccal, rectal, vaginal or topical. Topical administration would be particularly advantageous for treatment of skin cancers. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.
[0090] Vaccine or treatment compositions of the invention may be administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories, and in some cases, oral formulations or formulations suitable for distribution as aerosols. In the case of the oral formulations, the manipulation of T-cell subsets employing adjuvants, antigen packaging, or the addition of individual cytokines to various formulation that result in improved oral vaccines with optimized immune responses. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25-70%.
[0091] The antigen encoding nucleic acids of the invention may be formulated into the vaccine or treatment compositions as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or with organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
[0092] Vaccine or treatment compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective. The quantity to be administered depends on the subject to be treated, including, e.g., capacity of the subject's immune system to synthesize antibodies, and the degree of protection or treatment desired. Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a range from about 0.1 mg to 1000 mg, such as in the range from about 1 mg to 300 mg, and preferably in the range from about 10 mg to 50 mg. Suitable regiments for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and may be peculiar to each subject. It will be apparent to those of skill in the art that the therapeutically effective amount of nucleic acid molecule or fusion polypeptides of this invention will depend, inter alia, upon the administration schedule, the unit dose of antigen administered, whether the nucleic acid molecule or fusion polypeptide is administered in combination with other therapeutic agents, the immune status and health of the recipient, and the therapeutic activity of the particular nucleic acid molecule or fusion polypeptide.
[0093] The compositions can be given in a single dose schedule or in a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may include, e.g., 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months. Periodic boosters at intervals of 1-5 years, usually 3 years, are desirable to maintain the desired levels of protective immunity. The course of the immunization can be followed by in vitro proliferation assays of peripheral blood lymphocytes (PBLs) co-cultured with ESAT6 or ST-CF, and by measuring the levels of IFN-y released from the primed lymphocytes. The assays may be performed using conventional labels, such as radionuclides, enzymes, fluorescent labels and the like. These techniques are known to one skilled in the art and can be found in U.S. Pat. Nos. 3,791,932, 4,174,384 and 3,949,064, relevant portions incorporated by reference.
[0094] The modular rAb carrier and/or conjugated rAb carrier-(cohesion/dockerin and/or dockerin-cohesin)-antigen complex (rAb-DC/DC-antigen vaccine) may be provided in one or more "unit doses" depending on whether the nucleic acid vectors are used, the final purified proteins, or the final vaccine form is used. Unit dose is defined as containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts. The subject to be treated may also be evaluated, in particular, the state of the subject's immune system and the protection desired. A unit dose need not be administered as a single injection but may include continuous infusion over a set period of time. Unit dose of the present invention may conveniently may be described in terms of DNA/kg (or protein/Kg) body weight, with ranges between about 0.05, 0.10, 0.15, 0.20, 0.25, 0.5, 1, 10, 50, 100, 1,000 or more mg/DNA or protein/kg body weight are administered. Likewise the amount of rAb-DC/DC-antigen vaccine delivered can vary from about 0.2 to about 8.0 mg/kg body weight. Thus, in particular embodiments, 0.4 mg, 0.5 mg, 0.8 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 3.0 mg, 4.0 mg, 5.0 mg, 5.5 mg, 6.0 mg, 6.5 mg, 7.0 mg and 7.5 mg of the vaccine may be delivered to an individual in vivo. The dosage of rAb-DC/DC-antigen vaccine to be administered depends to a great extent on the weight and physical condition of the subject being treated as well as the route of administration and the frequency of treatment. A pharmaceutical composition that includes a naked polynucleotide prebound to a liposomal or viral delivery vector may be administered in amounts ranging from 1 μg to 1 mg polynucleotide to 1 μg to 100 mg protein. Thus, particular compositions may include between about 1 μg, 5 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 100 μg, 150 μg, 200 μg, 250 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg or 1,000 μg polynucleotide or protein that is bound independently to 1 μg, 5 μg, 10 μg, 20 μg, 3.0 μg, 40 μg 50 μg, 60 μg, 70 μg, 80 μg, 100 μg, 150 μg, 200 μg, 250 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 1.5 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg or 100 mg vector.
[0095] The present invention was tested in an in vitro cellular system that measures immune stimulation of human Flu-specific T cells by dendritic cells to which Flu antigen has been targeted. The results shown herein demonstrate the specific expansion of such antigen specific cells at doses of the antigen which are by themselves ineffective in this system.
[0096] The present invention may also be used to make a modular rAb carrier that is, e.g., a recombinant humanized mAb (directed to a specific human dendritic cell receptor) complexed with protective antigens from Ricin, Anthrax toxin, and Staphylococcus B enterotoxin. The potential market for this entity is vaccination of all military personel and stored vaccine held in reserve to administer to large population centers in response to any biothreat related to these agents. The invention has broad application to the design of vaccines in general, both for human and animal use. Industries of interest is pharmaceutical and biotechnology
[0097] One commercial application of the invention is a recombinant humanized mAb (directed to the specific human dendritic cell receptor DCIR) fused through the Ab heavy chain to antigens known or suspected to encode protective antigens. These include as examples for vaccination against various agents--hemagglutinins from Influenza H5N1; HIV gag from attenuated toxins from Ricin, Anthrax toxin, and Staphylococcus B enterotoxin; `strings` of antigenic peptides from melanona anigens, etc. The potential market for this entity is preventative or therapeutic vaccination of at risk or infected people. The invention has broad application for vaccination against many diseases and cancers, both for human and animal use. Industries of interest are pharmaceutical and biotechnology. In addition, this invention has implications beyond anti-DCIR application since it describes a method to identify particularly favorable sequences to enhance secretion of recombinant antibodies.
[0098] The application of anti-DCIR combining regions for making engineered recombinant monoclonal antibodies fused to antigens as potent therapeutic or preventative vaccination agents. Use of different V-region sequences against the same combining specificity to find those most compatible with efficient expression of a H chain C-terminal linked antigen or other protein sequence.
EXAMPLE 1
Multiple Antigens Targeted in a Complex Simultaneously with the Same Engineered Humanized mAb (MATCHMAB)
[0099] One type of therapeutic (in this case, vaccination) entity envisioned is a humanized DC-targeting mAb-antigen fusion protein, where the antibody variable region specificity is directed against an internalizing human dendritic cell receptor. The present state-of-the art is to engineer the fusion of the desired antigen to the C-terminus of the mAb H chain. This paradigm obviously allows different antigens (A1, A2, A3) to be engineered to the same proven targeting mAb backbone (Y in the figure below), thus extending the utility of the one mAb to immunizing against different pathogenic agents. This concept can be further extended by engineering, e.g., the A1, A2, A3 coding regions end-to-end fused to the IgGFc C-terminal coding region.
[0100] The present invention disclosed a new paradigm for linking the antigen to the targeting mAb that extends the concept for the first time to multiple antigens targeted in a complex simultaneously with the same engineered humanized mAb (MATCHMAB).
[0101] FIG. 1 compares the prior art (top portion) with an example of the multiple antigens targeted in a complex simultaneously with the same engineered humanized mAb (MATCHMAB)(bottom portion). Y represents the humanized anti-DC targeting mAb; A1, A2, A3 are independent protective antigens, or any other desired protein domains; C1, C2, C2 are specific high affinity capture domains for, respectively, docking domains D1, D2. D3; and DnAn are the corresponding docking-antigen fusion proteins. Note that the various domains are not drawn to scale. The mAb itself is ˜150 kDa, C is ˜17 Da, D is ˜8 kDa and A varies, but is usually >20 kDa).
[0102] The MATCHMAB is based on using cellulosome-assembly cohesin-dockerin sequences to form modular non-covalent targeting mAb-antigen complexes. The relatively small and specific cohesin-dockerin protein-protein interaction domains can allow simple customized formulation of targeting mAb-antigen complexes. Thus, a single manufactured humanized mAb (in the above notation: Y.C1.C2.C3.Cn) can be use as the basis of delivering multiple antigens in various, yet strictly defined, combinations.
[0103] Example of sequence encoding C1.C2.C3.Cn is taken from the public sequence >gi|50656899|gb|AAT79550.1| of cellulosomal anchoring scaffoldin B precursor (Bacteroides cellulosolvens). Below with blue showing the leader secretion sequence and yellow and grey highlighting various cohesin domains. Red regions are linkers spacing some of the cohesin domains.
TABLE-US-00001 (SEQ ID NO.: 1) ##STR00001## ##STR00002## ##STR00003## ##STR00004## ##STR00005## ##STR00006## ##STR00007## ##STR00008## ##STR00009## ##STR00010## ##STR00011## ##STR00012## ##STR00013## ##STR00014## ##STR00015## ##STR00016## ##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022## ##STR00023## ##STR00024## PTVTPNVASPTPTKVVAEPTSNQPAGPGITGTIPTATTTATATPTKASVATATPTATPIVVVEPTIVRP GYNKDADLAVFISSDKSRYEESSIITYSIEYKNIGKVNATNVKIAAQIPKFTKVYDAAKGAVKGSEIVWM IGNLAVGESYTKEYKVKVDSLTKSEEYTDNTVTISSDQTVDIPENITTGNDDKSTIRVMLYSNRFTPGSH SSYILGYKDKTFKPKQNVTRAEVAAMFARIMGLTVKDGAKSSYKDVSNKHWALKYIEAVTKSGIFKGYKD STFHPNAPITRAELSTVIFNYLHLNNIAPSKVHFTDINKHWAKNYIEEIYRFKLIQGYSDGSFKPNNNIT RAEVVTMINRMLYRGPLKVKVGSFPDVSPKYWAYGDIEEASRNHKYTRDEKDGSEILIE
[0104] The cohesin domains (C) interact with small domains (e.g., 56 residues) called dockerins (D). These are Ca++ containing structures with two-fold symmetry and they can bind to a cognate cohesin with various affinities (e.g., 6E6 M, 2E7M). Affinities between dockerin and multiple cohesins (as found on scaffoldins) can be much higher (e.g., >E9 M). The interaction is non-covalent and is well defined (by structure analysis) for at least one C-D pair. Dockerins are designed to be domains linked to different domain (enzyme in nature), and cohesions are designed to function in linear arrays (either directly end-to-end, or joined by flexible PT-rich linkers of various sizes (e.g., 12, 17, 25, 28, 36). It is known that a particular dockerin can have specificity for a particular cohesin (e.g., a C-D pair from one bacterial species may not be interchangeable with a C-D pair from a different species). This feature makes it is possible to ensure the specific and precise interaction of various D-antigen fusion proteins with an engineered mAb containing cohesin domains of various specificities.
[0105] In practice, this invention includes adapting C-D pairs known from the literature, newly gleamed from nature, or developed with new specificities using phage display technology. The latter technology can also be used to enhance (`mature`) the affinity of a C-D interaction, should this be desired. Also, engineering cysteine residues at opposing faces of the C-D interaction (based on modeling from the published C-D structures) could be used to make a covalent bond between C-D to strengthen the interaction. Furthermore, the dimeric nature of the mAb (and therefore the linked C-domains) can be used to advantage for affinity enhancement purposes. In this embodiment, e.g., the D-antigen fusion protein is engineered either with a second identical dockerin domain (D-antigen-D, or D-D-antigen), or with a homodimerization domain. This configuration, provided the linkers between domains were not constraining, will result in the preferred simultaneous binding of both D domains to the same mAb, with greatly enhanced stability compared to the single interaction.
[0106] Based on the crystal structure of the cohesin-dockerin complex (e.g., see PNAS 2003,13809-13814, Cellulosome assembly revealed by the crystal structure of the cohesin-dockerin complex. Ana L. Carvalho *, Fernando M. V. Dias, Jose A. M. Prates, Tibor Nagy, Harry J. Gilbert, Gideon J. Davies, Luis M. A. Ferreira, Maria J. Romao and Carlos M. G. A. Fonte), it is apparent that one embodiment is an antigen-dockerin fusion proteins (i.e., antigen fused to the N-terminus of a dockerin). However, both from the structure and from the nature of cohesin domain organization within scaffoldins, it is apparent that cohesions can be fused end-to-end, even without spacer sequences. Furthermore, it is apparent that well-described techniques are available to engineer miniaturized versions of the cohesin and dockerin domains (see for example, Proc. Natl. Acad. Sci. USA Vol. 94, pp. 10080-10085, September 1997. Structural mimicry of a native protein by a minimized binding domain. Melissa A. Starovasnik, Andrew C. Braisted, And James A. Wells).
[0107] It is recognized herein that the linker sequences have a propensity for O-linked glycosylation resulting from ST richness. Also, both the C and D domains can have potential N-linked sites. These features can be advantageous in enhancing the solubility of the mammalian cell-expressed engineered mAb through decoration with carbohydrates. Of course, the consequences of glycosylation of the C domains needs to be check by function (binding to the cognate D), and if needed rectified by site directed mutagenesis. An attractive feature of this invention is that D-A can be expressed in whatever system is known to be best. For example, the tumor antigen MART1 is a membrane protein and is best prepared in high yield via E. coli inclusion bodies. Schema using antigens directly fused to the mAb are restricted to antigens that are compatible with mammalian-cell expression.
[0108] Another embodiment of the invention is the use of the D-C interaction to make bi-specific mAbs joined tail-to-tail. FIG. 2 shows the use of the present invention to form Bi-specific mAbs. mAb1 (black) is expressed with C-terminal C1 and mAb2 (magenta) is expressed with C-terminal D1. Mixing equimolar mAb1 and mAb2 will result in a bi-specific 1:1 complex. Note that, since each mAb molecule contains two molar equivalents of C or D (the mAb is itself a dimeric structure), the bi-specific mAb will be greatly stabilized by two concurrent C-D interactions. Especially at lower (mAb), this will be the most stable configuration.
EXAMPLE 2
Combination of Antibody and Cohesion/Dockerin Domains and Antigens
[0109] Example 2 shows that particular cohesin and dockerin domains can be sucessfully and efficiently secreted from mammalian cells as fusion proteins while maintaining the specific and high affinity cohesin-dockerin protein-protein interaction. While the extensive cohesin-dockerin literature teaches the expectation that such fusion proteins should have this functionality, it does not describe production of such fusion proteins in mammalian secretion systems. The state of scientific knowledge does not allow the prediction of the discovery since the rules (other than features such as signal peptide) for successful secretion are not fully established. Furthermore, the cohesin linker regions are known to be glycosylated in their native bacteria, and the cohesin and dockerin domains contain predicted glycosylation sites. While this may actually favor secretion from mammalian cells, it is unclear if `unnatural` glyosylation will perturb the cohesis-dockerin interaction.
[0110] While cohesin-dockerin interaction for various commercial applications has been published, the present invention is based on a previously unrealized utility for this interaction built around assembling specific protein complexes unrelated to the envisioned controlled assembly enzyme applications.
[0111] The invention includes the use of all cohesin-dockerin sequences from diverse cellulose degrading microbes, but describes the application of specific cohesin and dockerin and linker sequences from the microbe Clostridium thermocellum. For example, the sequence described in Table 1 encodes the H chain of a human IgG4 linked at the C-terminal codon to a Clostridium thermocellum dockerin sequence (called rAb.doc). Other embodiments of rAb.doc proteins are described similarly in Table 2 and these are engineered by simply transferring the dockerin coding region as a DNA fragment to vectors encoding the different H chain entities.
[0112] TABLE 1 shows the nucleic acid and amino acid sequences for rAB-pIRES2(hIgG4H-Dockerin) or C52. DNA (entire coding region) and amino acid sequence (the predicted secreted product) of human IgG4H.doc fusion protein is shown below. The dockerin domain (taken from Clostridium thermocellum celD is highlighted in yellow and the H chain and dockerin joining sequence is underlined. The highly predicted N-linked glycosylation site within the dockerin domain is highlighted in red.
TABLE-US-00002 TABLE 1 rAB-pIRES2(hIgG4H-Dockerin) or C52. (SEQ ID NO.: 2) ATGGACCTCCTGTGCAAGAACATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCT- GTCCCGGCTGC AGCTGCAGGAGTCGGGCCCAGGCCTGCTGAAGCCTTCGGTGACCCTGTCCCTCACCTGCACTGTCTCGGGTGAC- TCCGTCGCCAG TAGTTCTTATTACTGGGGCTGGGTCCGTCAGCCCCCAGGGAAGGGACTCGAGTGGATAGGGACTATCAATTTTA- GTGGCAATATG TATTATAGTCCGTCCCTCAGGAGTCGAGTGACCATGTCGGCAGACATGTCCGAGAACTCCTTCTATCTGAAATT- GGACTCTGTGA CCGCAGCAGACACGGCCGTCTATTATTGTGCGGCAGGACACCTCGTTATGGGATTTGGGGCCCACTGGGGACAG- GGAAAACTGGT CTCCGTCTCTCCAGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGA- GCACAGCCGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG- CGTGCACACCT TCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGC- ACGAAGACCTA CACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCAT- GCCCACCCTGC CCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTC- CCGGACCCCTG AGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTG- GAGGTGCATAA TGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC- AGGACTGGCTG AACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGC- CAAAGGGCAGC CCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGC- CTGGTCAAAGG CTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTC- CCGTGCTGGAC TCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTC- ATGCTCCGTGA TGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGCAATTCTCCT- CAAAATGAAGT ACTGTACGGAGATGTGAATGATGACGGAAAAGTAAACTCCACTGACTTGACTTTGTTAAAAAGATATGTTCTTA- AAGCCGTCTCA ACTCTCCCTTCTTCCAAAGCTGAAAAGAACGCAGATGTAAATCGTGACGGAAGAGTTAATTCCAGTGATGTCAC- AATACTTTCAA GATATTTGATAAGGGTAATCGAGAAATTACCAATATAA (SEQ ID NO.: 3) RLQLQESGPGLLKPSVTLSLTCTVSGDSVASSSYYWGWVRQPPGKGLEWIGTINFSGNMYYSPSLRSRVTMSAD- MSENSFYLKLD SVTAADTAVYYCAAGHLVMGFGAHWGQGKLVSVSPASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV- SWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFP- PKPKDTLMISR TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP- SSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK- SRWQEGNVFSC ##STR00025##
[0113] TABLE 2 shows the nucleic acid and amino acid sequences for rAB-pIRES2(mAnti-DCIR2C9H-LV-hIgG4H-C-Dockerin) or C82. DNA (entire coding region) and amino acid sequence (the predicted secreted product) is shown below. The dockerin domain is highlighted in yellow and the H chain and dockerin joining sequence is underlined. The IgG variable region is highlighted in blue. The highly predicted N-linked glycosylation site within the dockerin domain is highlighted in red.
TABLE-US-00003 TABLE 2 rAB-pIRES2(mAnti-DCIR2C9H-LV-hIgG4H-C-Dockerin) or C82. (SEQ ID NO.: 4) ATGAAATGCAGCTGGGTCATCTTCTTCCTGATGGCAGTGGTTACAGGGGTCAATTCAGAGGTTCAGCTGCAGCA- GTCTGGGGCTG AGCTTGTGAGGCCAGGGGCCTTAGTCAAGTTGTCCTGCAAAGCTTCTGGCTTCAACATTAATGACTACTATATC- CACTGGGTGAA GCAGCGGCCTGAACAGGGCCTGGAGCGGATTGGATGGATTGATCCTGACAATGGTAATACTATATATGACCCGA- AGTTCCAGGGC AAGGCCAGTATAACAGCAGACACATCCCCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACAC- TGCCGTCTATT ACTGTGCTAGAACCCGATCTCCTATGGTTACGACGGGGTTTGTTTACTGGGGCCAAGGGACTGTGGTCACTGTC- TCTGCAGCCAA AACGAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCT- GCCTGGTCAAG GACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGC- TGTCCTACAGT CCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGC- AACGTAGATCA CAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCAC- CTGAGTTCGAA GGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCAC- GTGCGTGGTGG TGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAG- ACRAAGCCGCG GGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCA- AGGAGTACAAG TGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGA- GCCACAGGTGT ACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAC- CCCAGCGACAT CGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG- GCTCCTTCTTC CTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGA- GGCTCTGCACA ACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGCAATTCTCCTCAAAATGAAGTACTGTAC- GGAGATGTGAA TGATGACGGAAAAGTAAACTCCACTGACTTGACTTTGTTAAAAAGATATGTTCTTAAAGCCGTCTCAACTCTCC- CTTCTTCCAAA GCTGAAAAGAACGCAGATGTAAATCGTGACGGAAGAGTTAATTCCAGTGATGTCACAATACTTTCAAGATATTT- GATAAGGGTAA TCGAGAAATTACCAATATAA (SEQ ID NO.: 5) ##STR00026## ##STR00027## VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLF- PPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL- PSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD- KSRWQEGNVFS ##STR00028##
[0114] TABLE 3 shows the nucleic acid and amino acid sequences for rAB-(mAnti-ASGPR--49C11--7H-SLAML-V-hIgG4H-C-Dockerin) or C153. DNA (entire coding region) and amino acid sequence (the predicted secreted product) is shown below. The dockerin domain is highlighted in yellow and the H chain and dockerin joining sequence is underlined. The IgG variable region is highlighted in blue. The highly predicted N-linked glycosylation site within the dockerin domain is highlighted in red.
TABLE-US-00004 TABLE 3 rAB-(mAnti-ASGPR_49C11_7H-SLAML-V-hIgG4H-C-Dockerin) or C153. (SEQ ID NO.: 6) ATGGACCCCAAAGGCTCCCTTTCCTGGAGAATACTTCTGTTTCTCTCCCTGGCTTTTGAGTTGTCGTACGGAGA- TGTGCAGCTTC AGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCACTGGCTACTCCATC- ACCAGTGGTTA TAGCTGGCACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATACTCTTCAGTGGTAGCA- CTAACTACAAC CCATCTCTGAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGT- GACTACTGAGG ACACAGCCACATATTTCTGTGCAAGATCTAACTATGGTTCCTTTGCTTCCTGGGGCCAAGGGACTCTGGTCACT- GTCTCTGCAGC CAAAACAAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGG- GCTGCCTGGTC AAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCC- GGCTGTCCTAC AGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACC- TGCAACGTAGA TCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAG- CACCTGAGTTC GAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGT- CACGTGCGTGG TGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCC- AAGACAAAGCC GCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACG- GCAAGGAGTAC AAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG- AGAGCCACAGG TGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC- TACCCCAGCGA CATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCG- ACGGCTCCTTC TTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCA- TGAGGCTCTGC ACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGCAATTCTCCTCAAAATGAAGTACTG- TACGGAGATGT GAATGATGACGGAAAAGTAAACTCCACTGACTTGACTTTGTTAAAAAGATATGTTCTTAAAGCCGTCTCAACTC- TCCCTTCTTCC AAAGCTGAAAAGAACGCAGATGTAAATCGTGACGGAAGAGTTAATTCCAGTGATGTCACAATACTTTCAAGATA- TTTGATAAGGG TAATCGAGAAATTACCAATATAA (SEQ ID NO.: 7) ##STR00029## ##STR00030## PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKP- KDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI- EKTISKAKGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW- QEGNVFSCSVM ##STR00031##
[0115] TABLE 4 shows the nucleic acid and amino acid sequences for rAB-pIRES2(mAnti-DC-SIGNL16E7H-LV-hIgG4H-C-Dockerin) or C92. DNA (entire coding region) and amino acid sequence (the predicted secreted product) is shown below. The dockerin domain is highlighted in yellow and the H chain and dockerin joining sequence is underlined. The IgG variable region is highlighted in blue. The highly predicted N-linked glycosylation site within the dockerin domain is highlighted in red.
TABLE-US-00005 TABLE 4 rAB-pIRES2(mAnti-DC-SIGNL16E7H-LV-hIgG4H-C-Dockerin) or C92 (SEQ ID NO.: 8) ATGGAAAGGCACTGGATCTTTCTCTTCCTGTTTTCAGTAACTGCAGGTGTCCACTCCCAGGTCCAGCTTCAGCA- GTCTGGGGCTG AGCTGGCAAAACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTACCTACTGGATG- CACTGGGTAAA ACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTATCACTGGTTATACTGAGTACAATCAGA- AGTTCAAGGAC AAGGCCACCTTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTC- TGCAGTCTATT ACTGTGCAAGAGAGGGTTTAAGTGCTATGGACTATTGGGGTCAGGGAACCTCAGTCACCGTCACCTCAGCCAAA- ACAACGGGCCC ATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGG- ACTACTTCCCC GAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTC- CTCAGGACTCT ACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCAC- AAGCCCAGCAA CACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAG- GGGGACCATCA GTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGT- GGACGTGAGCC AGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG- GAGGAGCAGTT CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGT- GCAAGGTCTCC AACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTA- CACCCTGCCCC CATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATC- GCCGTGGAGTG GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC- TCTACAGCAGG CTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAA- CCACTACACAC AGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGCAATTCTCCTCAAAATGAAGTACTGTACGGAGATGTGAAT- GATGACGGAAA AGTAAACTCCACTGACTTGACTTTGTTAAAAAGATATGTTCTTAAAGCCGTCTCAACTCTCCCTTCTTCCAAAG- CTGAAAAGAAC GCAGATGTAAATCGTGACGGAAGAGTTAATTCCAGTGATGTCACAATACTTTCAAGATATTTGATAAGGGTAAT- CGAGAAATTAC CAATATAA (SEQ ID NO.: 9) ##STR00032## ##STR00033## PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKP- KDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI- EKTISKAKGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW- QEGNVFSCSVM ##STR00034##
[0116] Mammalian expression plasmids encoding such rAb.doc IgG H chain proteins are created using standard molecular biology techniques and can be based on commercially available expression plasmid vectors such as pIRES2-DsRed2 (BD Biosciences). To produce secreted rAb.doc, mammalian cells are co-transfected with this expression plasmid and an expression plasmid encoding a complimentary IgG L chain (exemplified in Table 3). Standard protocols (such as the FreeStyle® 293 Expression System, Invitrogen) are used as for mammalian cells, transfection reagents, and culture media. Transfected cells are cultured for 3-7 days and the culture supernatant is harvested by centrifugation, clarified by filtration, and the rAB.doc protein purified by Protein G affinity chromatography using protocols from the column manufacturer (GE Pharmacia).
[0117] FIG. 3 shows analysis of typical secreted rAb.doc products by reducing SDS.PAGE with staining by Coomassie Brilliant Blue. This analysis shows that the rAb.doc is efficiently produced as a secreted H+L chain dimer. Heterogeneity in the H chain likely reflects N-linked glycosylation at a highly predicted (Potential 0.6426, NetNGlyc 1.0 Server--Technical University of Denmark) site within the dockerin sequence.
[0118] TABLE 5 shows the nucleic acid and amino acid sequences for rAB-pIRES2(mAnti-DC-SIGNL16E7K-LV-hIgGK-C) or C73. DNA (entire coding region) and amino acid sequence (the predicted secreted product) of IgG Kappa protein fusing the V region from the mAnti-DC-SIGNL16E7 hybridoma (highlighted in blue)to a human C region (highlighted in yellow).
TABLE-US-00006 TABLE 5 rAB-pIRES2(mAnti-DC-SIGNL16E7K-LV-hIgGK-C) or C73. (SEQ ID NO.: 10) ATGCATCGCACCAGCATGGGCATCAAGATGGAGTCACAGATTCAGGCATTTGTATTCGTGTTTCTCTGGTTGTC- TGGTGTTGGCG GAGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAG- GCCAGTCAGGA TGTGACTTCTGCTGTAGCCTGGTATCAACAAAAACCAGGGCAATCTCCTAAACTACTGATTTACTGGGCATCCA- CCCGGCACACT GGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTATACTCTCACCATCAGCAGTGGGCAGGCTGA- AGACCTGGCAC TTTATTACTGTCACCAATATTATAGCGCTCCTCGGACGTTCGGTGGAGGCACCAAGCTCGAGATCAAACGAACT- GTGGCTGCACC ATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATA- ACTTCTATCCC AGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCA- GGACAGCAAGG ACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGC- GAAGTCACCCA TCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO.: 11) ##STR00035## ##STR00036## ##STR00037##
[0119] FIG. 3 shows Protein G affinity purified secreted rAb proteins analyzed by reducing SDS.PAGE and Coomassie Brilliant Blue staining. Lanes are from left to right.
[0120] The invention enbodies the unanticipated presence and use of this glycosylation site that likely confers onto mammalian cell-secreted dockerin fusion proteins desirable solubility and pharmacokinetic properties well known to be associated with glycosylation. FIG. 2 shows that rAb.antigen fusion proteins employing identical IgG H and L sequences can differ dramatically in efficiency of secretion. In both sited examples, rAb.doc entities are well expressed compared to rAb fused to Influenza HAS sequences which typically express very poorly. The invention also embodies the unanticipated capacity of the dockerin domain to not significantly hinder the secretion of the associated rAb entity. Furthermore, the invention embodies the property of the dockerin domain to not hinder the functionality of the rAb specific antigen combining regions. This property is exemplified in FIG. 5 which shows concordance between IgFc reactivity and LOX-1 reactivity between anti-LOX1--15C4 rAb proteins and anti-LOX1--15C4.doc.
[0121] FIGS. 4A and 4B show the measurement by anti-human IgFc ELISA of levels of secretion of various rAb.fusion proteins. 2.5 ug each of the H and L chain expression plasmids were transfected into 293F cells and two-fold dilutions of supernatant samples were tested after three days of culture. Y axis values are arbitrary HRP activity.
[0122] FIG. 5 shows the measurement by anti-human IgFc ELISA (HRP activity) and LOX-1.alkaline phoshatase binding (AP activity) of secreted anti-LOX1--15C4 rAb.(blue symbols)and anti-LOX1--15C4.doc rAb (red symbols) proteins. Different ratios totalling 5 ug of the H and L chain expression plasmids were transfected into 293F cells and supernatant samples were tested after three days of culture.
[0123] The invention embodies the property of the dockerin domain to be efficiently and functionally expressed in the context of fusion proteins other than hIgG4 and its close derivatives. For example, Table 6 shows the sequence of a rAb.doc entity based on a mouse IgG2b H chain fusion protein.
[0124] TABLE 6 shows the nucleic acid and amino acid sequences for rAB-pCMV(mIgG2bH-Dockerin) or C19. DNA (entire coding region) and amino acid sequence (the predicted secreted product) is shown. The dockerin domain is highlighted in yellow and the H chain and dockerin joining sequence is underlined. The highly predicted N-linked glycosylation site within the dockerin domain is highlighted in red.
TABLE-US-00007 TABLE 6 rAB-pCMV(mIgG2bH-Dockerin) or C19. (SEQ ID NO.: 12) ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAGTACATTCACAGGTCCAACTGCAGCA- GCCTGGGGCTG AGCTGGTGAGGCCTGGGACTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGTTACATCTTTACCAGCTACTGGATG- CACTGGGTAAA GCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGACTGATTGATCCTTCTGATAGTTATAGTAAGTACAATCAAA- AGTTCAAGGGC AAGGCCACATTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTC- TGCGGTCTATT ACTGTGCAAGAGGGGAGCTCAGTGACTTCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACAACA- CCCCCATCAGT CTATCCACTGGCCCCTGGGTGTGGAGATACAACTGGTTCCTCTGTGACTCTGGGATGCCTGGTCAAGGGCTACT- TCCCTGAGTCA GTGACTGTGACTTGGAACTCTGGATCCCTGTCCAGCAGTGTGCACACCTTCCCAGCTCTCCTGCAGTCTGGACT- CTACACTATGA GCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCAAGTCAGACCGTCACCTGCAGCGTTGCTCACCCAGCCAGC- AGCACCACGGT GGACAAAAAACTTGAGCCCAGCGGGCCCATTTCAACAATCAACCCCTGTCCTCCATGCAAGGAGTGTCACAAAT- GCCCAGCTCCT AACCTCGAGGGTGGACCATCCGTCTTCATCTTCCCTCCAAATATCAAGGATGTACTCATGATCTCCCTGACACC- CAAGGTCACGT GTGTGGTGGTGGATGTGAGCGAGGATGACCCAGACGTCCGGATCAGCTGGTTTGTGAACAACGTGGAAGTACAC- ACAGCTCAGAC ACAAACCCATAGAGAGGATTACAACAGTACTATCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGA- TGAGTGGCAAG GAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCATCACCCATCGAGAGAACCATCTCAAAAATTAAAGGGCT- AGTCAGAGCTC CACAAGTATACATCTTGCCGCCACCAGCAGAGCAGTTGTCCAGGAAAGATGTCAGTCTCACTTGCCTGGTCGTG- GGCTTCAACCC TGGAGACATCAGTGTGGAGTGGACCAGCAATGGGCATACAGAGGAGAACTACAAGGACACCGCACCAGTCCTGG- ACTCTGACGGT TCTTACTTCATATACAGCAAGCTCGATATAAAAACAAGCAAGTGGGAGAAAACAGATTCCTTCTCATGCAACGT- GAGACACGAGG GTCTGAAAAATTACTACCTGAAGAAGACCATCTCCCGGTCTCCGGGTAAAGCTAGCAATTCTCCTCAAAATGAA- GTACTGTACGG AGATGTGAATGATGACGGAAAAGTAAACTCCACTGACTTGACTTTGTTAAAAAGATATGTTCTTAAAGCCGTCT- CAACTCTGCCT TCTTCCAAAGCTGAAAAGAACGCAGATGTAAATCGTGACGGAAGAGTTAATTCCAGTGATGTCACAATACTTTC- AAGATATTTGA TAAGGGTAATCGAGAAATTACCAATATAA (SEQ ID NO.: 13) QVQLQQPGAELVRPGTSVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIGLIDPSDSYSKYNQKFKGKATLTVDT- SSSTAYMQLSS LTSEDSAVYYCARGELSDFWGQGTTLTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSG- SLSSSVHTFPA LLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSV- FIFPPNIKDVL MISLTPKVTCVVVDVSEDDPDVRISWFVNNVEVHTAQTQTHREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNN- KDLPSPIERTI SKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKL- DIKTSKWEKTD ##STR00038##
[0125] FIG. 6 shows that when co-transfected with a mIgG kappa expression plasmid, rAB-pCMV(mIgG2bH-Dockerin) plasmid directs the efficient secretion of rAB-mIgG2b.Dockerin fusion protein. In FIG. 6, a Protein G affinity purified rAb proteins secreted from transfected 293 F cells analyzed by reducing SDS.PAGE and Coomassie Brilliant Blue staining. Lanes 11 and 12 show mIgG2b.doc products.
[0126] The use of the rAb.doc invention detailed above is the assembly of rAb-antigen or toxin or activator or enzyme complexes via the specificity and tenacity of the dockerin-cohesin interaction. Table 5 shows one embodiment of the invention in the form of a cohesin.alkaline phosphatase fusion protein (coh.AP). Also described are additional embodiments such as an alkaline phosphatase fusion protein containing two cohesion domains (coh.coh.AP) and other proteins are examples of the generality of the invention such as the single cohesin domain fused to other sequences such as the mature sequence of human prostate specific antigen (coh.hPSA) and to the HA1 domain of influenza A HAS (coh.Flu HA5-1).
[0127] TABLE 7 shows the nucleic acid and amino acid sequences for Mam-pCDM8(Cohesin-SLAML-AP-6× His) or C16. DNA (entire coding region) and amino acid sequence (the predicted secreted product) is shown below. The cohesin domain is highlighted in yellow and the cohesin and alkaline phosphatase joining sequence is underlined. The highly predicted (G-score>0.5, NetOGlyc 3.1 Server--Technical University of Denmark) O-linked glycosylation sites within the cohesin domain and the linker distal to the cohesin domain are highlighted in red. Residues highlighted grey are a C-terminal His tag to facilitate purification via metal affinity chromatography.
TABLE-US-00008 TABLE 7 Mam-pCDM8(Cohesin-SLAML-AP-6xHis) or C16. (SEQ ID NO.: 14) ATGGATCCCAAAGGATCCCTTTCCTGGAGAATACTTCTGTTTCTCTCCCTGGCTTTTGAGTTGAGCTACGGACT- CGACGATCTGG ATGCAGTAAGGATTAAAGTGGACACAGTAAATGCAAAACCGGGAGACACAGTAAGAATACCTGTAAGATTCAGC- GGTATACCATC CAAGGGAATAGCAAACTGTGACTTTGTATACAGCTATGACCCGAATGTACTTGAGATAATAGAGATAGAACCGG- GAGACATAATA GTTGACCCGAATCCTGACAAGAGCTTTGATACTGCAGTATATCCTGACAGAAAGATAATAGTATTCCTGTTTGC- AGAAGACAGCG GAACAGGAGCGTATGCAATAACTAAAGACGGAGTATTTGCTACGATAGTAGCGAAAGTAAAAGAAGGAGCACCT- AACGGACTCAG TGTAATCAAATTTGTAGAAGTAGGCGGATTTGCGAACAATGACCTTGTAGAACAGAAGACACAGTTCTTTGACG- GTGGAGTAAAT GTTGGAGATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACAGATGATCTGGATGC- ACTCGAGATCA TCCCAGTTGAGGAGGAGAACCCGGACTTCTGGAACCGCGAGGCAGCCGAGGCCCTGGGTGCCGCCAAGAAGCTG- CAGCCTGCACA GACAGCCGCCAAGAACCTCATCATCTTCCTGGGCGATGGGATGGGGGTGTCTACGGTGACAGCTGCCAGGATCC- TAAAAGGGCAG AAGAAGGACAAACTGGGGCCTGAGTTACCCCTGGCCATGGACCGCTTCCCATATGTGGCTCTGTCCAAGACATA- CAATGTAGACA AACATGTGCCAGACAGTGGAGCCACAGCCACGGCCTACCTGTGCGGGGTCAAGGGCAACTTCCAGACCATTGGC- TTGAGTGCAGC CGCCCGCTTTAACCAGTGCAACACGACACGCGGCAACGAGGTCATCTCCGTGATGAATCGGGCCAAGAAAGCAG- GGAAGTCAGTG GGAGTGGTAACCACCACACGAGTGCAGCACGCCTCGCCAGCCGGCACCTACGCCCACACGGTGAACCGCAACTG- GTACTCGGACG CCGACGTGCCTGCCTCGGCCCGCCAGGAGGGGTGCCAGGACATCGCTACGCAGCTCATCTCCAACATGGACATT- GACGTGATCCT AGGTGGAGGCCGAAAGTACATGTTTCGCATGGGAACCCCAGACCCTGAGTACCCAGATGACTACAGCCAAGGTG- GGACCAGGCTG GACGGGAAGAATCTGGTGCAGGAATGGCTGGCGAAGCGCCAGGGTGCCCGGTACGTGTGGAACCGCACTGAGCT- CATGCAGGCTT CCCTGGACCCGTCTGTGACCCATCTCATGGGTCTCTTTGAGCCTGGAGACATGAAATACGAGATCCACCGAGAC- TCCACACTGGA CCCCTCCCTGATGGAGATGACAGAGGCTGCCCTGCGCCTGCTGAGCAGGAACCCCCGCGGCTTCTTCCTCTTCG- TGGAGGGTGGT CGCATCGACCATGGTCATCATGAAAGCAGGGCTTACCGGGCACTGACTGAGACGATCATGTTCGACGACGCCAT- TGAGAGGGCGG GCCAGCTCACCAGCGAGGAGGACACGCTGAGCCTCGTCACTGCCGACCACTCCCACGTCTTCTCCTTCGGAGGC- TACCCCCTGCG AGGGAGCTCCATCTTCGGGCTGGCCCCTGGCAAGGCCCGGGACAGGAAGGCCTACACGGTCCTCCTATACGGAA- ACGGTCCAGGC TATGTGCTCAAGGACGGCGCCCGGCCGGATGTTACCGAGAGCGAGAGCGGGAGCCCCGAGTATCGGCAGCAGTC- AGCAGTGCCCC TGGACGAAGAGACCCACGCAGGCGAGGACGTGGCGGTGTTCGCGCGCGGCCCGCAGGCGCACCTGGTTCACGGC- GTGCAGGAGCA GACCTTCATAGCGCACGTCATGGCCTTCGCCGCCTGCCTGGAGCCCTACACCGCCTGCGACCTGGCGCCCCCCG- CCGGCACCACC CACCATCACCATCACCATTGA (SEQ ID NO.: 15) ##STR00039## ##STR00040## ALEIIPVEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPELPLAM- DRFPYVALSKT YNVDKHVPDSGATATAYLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASP- AGTYAHTVNRN WYSDADVPASARQEGCQDIATQLISNMDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKR- QGARYVWNRTE LMQASLDPSVTHLMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESRAYR- ALTETIMFDDA IERAGQLTSEEDTLSLVTADHSHVFSFGGYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPGYVLKDGARPDVTE- SESGSPEYRQQ ##STR00041##
[0128] TABLE 8 shows the nucleic acid and amino acid sequences for Mam-pCDM8(Cohesin-Cohesin-SLAML-AP-6× His) or C17. DNA (entire coding region) and amino acid sequence (the predicted secreted product) is shown below. The cohesin domain is highlighted in yellow and the cohesin and alkaline phosphatase joining sequence is underlined. The highly predicted O-linked glycosylation sites within the linker distal to the cohesin domains are highlighted in red as is a single highy predicted N-linked glycosylation site (NPT). Residues highlighted grey are a C-terminal His tag to facilitate purificaytion via metal affinity chromatography.
TABLE-US-00009 TABLE 8 Mam-pCDM8(Cohesin-Cohesin-SLAML-AP-6xHis) or C17. ATGGATCCCAAAGGATCCCTTTCCTGGAGAATACTTCTGTTTCTCTCCCTGGCTTTTGAGTTGAGCTACGGACT- CGACGATCTGG ATGCAGTAAGGATTAAAGTGGACACAGTAAATGCAAAACCGGGAGACACAGTAAGAATACCTGTAAGATTCAGC- GGTATACCATC CAAGGGAATAGCAAACTGTGACTTTGTATACAGCTATGACCCGAATGTACTTGAGATAATAGAGATAAAACCGG- GAGAATTGATA GTTGACCCGAATCCTGACAAGAGCTTTGATACTGCAGTATATCCTGACAGAAAGATAATAGTATTCCTGTTTGC- AGAAGACAGCG GAACAGGAGCGTATGCAATAACTAAAGACGGAGTATTTGCTACGATAGTAGCGAAAGTAAAATCCGGAGCACCT- AACGGACTCAG TGTAATCAAATTTGTAGAAGTAGGCGGATTTGCGAATAATGACCTTGTAGAACAGAAGACACAGTTCTTTGACG- GTGGAGTAAAT GTTGGAGATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACAGATGATCTGGATGC- AGTAAGGATTA AAGTGGACACAGTAAATGCAAAACCGGGAGACACAGTAAATATACCTGTAAGATTCAGTGGTATACCATCCAAG- GGAATAGCAAA CTGTGACTTTGTATACAGCTATGACCCGAATGTACTTGAGATAATAGAGATAAAACCGGGAGAATTGATAGTTG- ACCCGAATCCT ACCAAGAGCTTTGATACTGCAGTATATCCTGACAGAAAGATGATAGTATTCCTGTTTGCGGAAGACAGCGGAAC- AGGAGCGTATG CAATAACTAAAGACGGAGTATTTGCTACGATAGTAGCGAAAGTAAAAGAAGGAGCACCTAACGGACTCAGTGTA- ATCAAATTTGT AGAAGTAGGCGGATTTGCGAACAATGACCTTGTAGAACAGAAGACACAGTTCTTTGACGGTGGAGTAAATGTTG- GAGATACAACA GAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACAGATGATCTGGATGCACTCGAGATCATCCC- AGTTGAGGAGG AGAACCCGGACTTCTGGAACCGCGAGGCAGCCGAGGCCCTGGGTGCCGCCAAGAAGCTGCAGCCTGCACAGACA- GCCGCCAAGAA CCTCATCATCTTCCTGGGCGATGGGATGGGGGTGTCTACGGTGACAGCTGCCAGGATCCTAAAAGGGCAGAAGA- AGGACAAACTG GGGCCTGAGTTACCCCTGGCCATGGACCGCTTCCCATATGTGGCTCTGTCCAAGACATACAATGTAGACAAACA- TGTGCCAGACA GTGGAGCCACAGCCACGGCCTACCTGTGCGGGGTCAAGGGCAACTTCCAGACCATTGGCTTGAGTGCAGCCGCC- CGCTTTAACCA GTGCAACACGACACGCGGCAACGAGGTCATCTCCGTGATGAATCGGGCCAAGAAAGCAGGGAAGTCAGTGGGAG- TGGTAACCACC ACACGAGTGCAGCACGCCTCGCCAGCCGGCACCTACGCCCACACGGTGAACCGCAACTGGTACTCGGACGCCGA- CGTGCCTGCCT CGGCCCGCCAGGAGGGGTGCCAGGACATCGCTACGCAGCTCATCTCCAACATGGACATTGACGTGATCCTAGGT- GGAGGCCGAAA GTACATGTTTCGCATGGGAACCCCAGACCCTGAGTACCCAGATGACTACAGCCAAGGTGGGACCAGGCTGGACG- GGAAGAATCTG GTGCAGGAATGGCTGGCGAAGCGCCAGGGTGCCCGGTACGTGTGGAACCGCACTGAGCTCATGCAGGCTTCCCT- GGACCCGTCTG TGACCCATCTCATGGGTCTCTTTGAGCCTGGAGACATGAAATACGAGATCCACCGAGACTCCACACTGGACCCC- TCCCTGATGGA GATGACAGAGGCTGCCCTGCGCCTGCTGAGCAGGAACCCCCGCGGCTTCTTCCTCTTCGTGGAGGGTGGTCGCA- TCGACCATGGT CATCATGAAAGCAGGGCTTACCGGGCACTGACTGAGACGATCATGTTCGACGACGCCATTGAGAGGGCGGGCCA- GCTCACCAGCG AGGAGGACACGCTGAGCCTCGTCACTGCCGACCACTCCCACGTCTTCTCCTTCGGAGGCTACCCCCTGCGAGGG- AGCTCCATCTT CGGGCTGGCCCCTGGCAAGGCCCGGGACAGGAAGGCCTACACGGTCCTCCTATACGGAAACGGTCCAGGCTATG- TGCTCAAGGAC GGCGCCCGGCCGGATGTTACCGAGAGCGAGAGCGGGAGCCCCGAGTATCGGCAGCAGTCAGCAGTGCCCCTGGA- CGAAGAGACCC ACGCAGGCGAGGACGTGGCGGTGTTCGCGCGCGGCCCGCAGGCGCACCTGGTTCACGGCGTGCAGGAGCAGACC- TTCATAGCGCA CGTCATGGCCTTCGCCGCCTGCCTGGAGCCCTACACCGCCTGCGACCTGGCGCCCCCCGCCGGCACCACCCACC- ATCACCATCAC CATTGA (SEQ ID NO.:16) ##STR00042## ##STR00043## ##STR00044## ##STR00045## VEEENPDFWNREAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPELPLAMDRFPYV- ALSKTYNVDKH VPDSGATATAYLCGVKGNFQTIGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAH- TVRNWYSDAD VPASARQEGCQDIATQLISNMDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKRQGARYV- WNRTELMQASL DPSVTHLMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALRLLSRNPRGFFGFVEGGRIDHGHHESRAYRALTETI- MFDDAIERAGQ LTSEEDTLSLVTADHSHVFSFGGYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSP- EYRQQSAVPLD ##STR00046##
[0129] TABLE 9 shows the nucleic acid and amino acid sequences for Mam-pCDM8(SLAML-Cohesin-hPSA) or C 149. DNA (entire coding region) and amino acid sequence (the predicted secreted product) is shown below. The cohesin domain is highlighted in yellow and the cohesin and hPSA joining sequence is underlined. The highly predicted O-linked glycosylation sites within the linker distal to the cohesin domains and a single highly predicted N-linked glycosylation site within the cohesin domain are highlighted in red.
TABLE-US-00010 TABLE 9 Mam-pCDM8(SLAML-Cohesin-hPSA)or C149. ATGGATCCCAAAGGATCCCTTTCCTGGAGAATACTTCTGTTTCTCTCCCTGGCTTTTGAGTTGAGCTACGGACT- CGACGATCTGG ATGCAGTAAGGATTAAAGTGGACACAGTAAATGCAAAACCGGGAGACACAGTAAGAATACCTGTAAGATTCAGC- GGTATACCATC CAAGGGAATAGCAAACTGTGACTTTGTATACAGCTATGACCCGAATGTACTTGAGATAATAGAGATAGAACCGG- GAGACATAATA GTTGACCCGAATCCTGACAAGAGCTTTGATACTGCAGTATATCCTGACAGAAAGATAATAGTATTCCTGTTTGC- AGAAGACAGCG GAACAGGAGCGTATGCAATAACTAAAGACGGAGTATTTGCTACGATAGTAGCGAAAGTAAAAGAAGGAGCACCT- AACGGACTCAG TGTAATCAAATTTGTAGAAGTAGGCGGATTTGCGAACAATGACCTTGTAGAACAGAAGACACAGTTCTTTGACG- GTGGAGTAAAT GTTGGAGATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACAGATGATCTGGATGC- ACTCGAGGCGC CCCTCATCCTGTCTCGGATTGTGGGAGGCTGGGAGTGCGAGAAGCATTCCCAACCCTGGCAGGTGCTTGTGGCC- TCTCGTGGCAG GGCAGTCTGCGGCGGTGTTCTGGTGCACCCCCAGTGGGTCCTCACAGCTGCCCACTGCATCAGGAACAAAAGCG- TGATCTTGCTG GGTCGGCACAGCCTGTTTCATCCTGAAGACACAGGCCAGGTATTTCAGGTCAGCCACAGCTTCCCACACCCGCT- CTACGATATGA GCCTCCTGAAGAATCGATTCCTCAGGCCAGGTGATGACTCCAGCCACGACCTCATGCTGCTCCGCCTGTCAGAG- CCTGCCGAGCT CACGGATGCTGTGAAGGTCATGGACCTGCCCACCCAGGAGCCAGCACTGGGGACCACCTGCTACGCCTCAGGCT- GGGGCAGCATT GAACCAGAGGAGTTCTTGACCCCAAAGAAACTTCAGTGTGTGGACCTCCATGTTATTTCCAATGACGTGTGCGC- GCAAGTTCACC CTCAGAAGGTGACCAAGTTCATGCTGTGTGCTGGACGCTGGACAGGGGGCAAAAGCACCTGCTCGGGTGATTCT- GGGGGCCCACT TGTCTGTAATGGTGTGCTTCAAGGTATCACGTCATGGGGCAGTGAACCATGTGCCCTGCCCGAAAGGCCTTCCC- TGTACACCAAG GTGGTGCATTACCGGAAGTGGATCAAGGACACCATCGTGGCCAACCCCTGA (SEQ ID NO.: 18) ##STR00047## ##STR00048## ALEAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGV- FQVSHSFPHP LYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQ- CVDLHVISNDVC AQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTI- VANP (SEQ ID NO.: 19)
[0130] TABLE 10 shows the nucleic acid and amino acid sequences for Mam-pCDM8(SLAML-Cohesin-F1uHA5-1-6× His)or C24. DNA (entire coding region) and amino acid sequence (the predicted secreted product) is shown below. The cohesin domain is highlighted in yellow and the cohesin and Flu HA5-1 joining sequence is underlined. The highly predicted O-linked glycosylation sites within the linker distal to the cohesin domains and a single highly predicted N-linked glycosylation site within the cohesin domain are highlighted in red. Residues highlighted grey are a C-terminal His tag to facilitate purification via metal affinity chromatography.
TABLE-US-00011 TABLE 10 Mam-pCDM8(SLAML-Cohesin-FluHA5-1-6xHis)or C24. ATGGATCCCAAAGGATCCCTTTCCTGGAGAATACTTCTGTTTCTCTCCCTGGCTTTTGAGTTGAGCTACGGACT- CGACGATCTGG ATGCAGTAAGGATTAAAGTGGACACAGTAAATGCAAAACCGGGAGACACAGTAAGAATACCTGTAAGATTCAGC- GGTATACCATC CAAGGGAATAGCAAACTGTGACTTTGTATACAGCTATGACCCGAATGTACTTGAGATAATAGAGATAGAACCGG- GAGACATAATA GTTGACCCGAATCCTGACAAGAGCTTTGATACTGCAGTATATCCTGACAGAAAGATAATAGTATTCCTGTTTGC- AGAAGACAGCG GAACAGGAGCGTATGCAATAACTAAAGACGGAGTATTTGCTACGATAGTAGCGAAAGTAAAAGAAGGAGCACCT- AACGGACTCAG TGTAATCAAATTTGTAGAAGTAGGCGGATTTGCGAACAATGACCTTGTAGAACAGAAGACACAGTTCTTTGACG- GTGGAGTAAAT GTTGGAGATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACAGATGATCTGGATGC- ACTCGAGGATC AGATTTGCATTGGTTACCATGCAAACAACTCGACAGAGCAGGTTGACACAATAATGGAAAAGAACGTTACTGTT- ACACATGCCCA AGACATACTGGAAAAGAAACACAACGGGAAGCTCTGCGATCTAGATGGAGTGAAGCCTCTAATTTTGAGAGATT- GTAGCGTAGCT GGATGGCTCCTCGGAAACCCAATGTGTGACGAATTCATCAATGTGCCGGAATGGTCTTACATAGTGGAGAAGGC- CAATCCAGTCA ATGACCTCTGTTACCCAGGGGATTTCAATGACTATGAAAAATTGAAACACCTATTGAGCAGAATAAACCATTTT- GAGAAAATTCA GATCATCCCCAAAAGTTCTTGGTCCAGTCATGAAGCCTCATTAGGGGTGAGCTCAGCATGTCCATACCAGGGAA- AGTCCTCCTTT TTCAGAAATGTGGTATGGCTTATCAAAAAGAACAGTACATACCCAACAATAAAGAGGAGCTACAATAATACCAA- CCAAGAAGATC TTTTGGTACTGTGGGGGATTCACCATCCTAATGATGCGGCAGAGCAGACAAAGCTCTATCAAAACCCAACCACC- TATATTTCCGT TGGGACATCAACACTAAACCAGAGATTGGTACCAAGAATAGCTACTAGATCCAAAGTAAACGGGCAAAGTGGAA- GGATGGAGTTC TTCTGGACAATTTTAAAGCCGAATGATGCAATCAACTTCGAGAGTAATGGAAATTTCATTGCTCCAGAATATGC- ATACAAAATTG TCAAGAAAGGGGACTCAACAATTATGAAAAGTGAATTGGAATATGGTAACTGCAACACCAAGTGTCAAACTCCA- ATGGGGGCGAT AAACTCTAGCATGCCATTCCACAATATACACCCTCTCACCATTGGGGAATGCCCCAAATATGTGAAATCAAACA- GATTAGTCCTT GCGCACCATCACCATCACCATTGA (SEQ ID NO.: 20) ##STR00049## ##STR00050## ALEDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFI- NVPEWSYIVEK ANPVNDLCYPGDFNDYEKLKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNST- YPTIKRSYNNT NQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINF- ESNGNGIAPEY ##STR00051## No.: 21)
[0131] Similar to the above mentioned rAb.doc constructs, the invention embodies the efficient secretion from mammalian cells of functional cohesin fusion proteins (called herein coh.fusions). It was not obvious that cohesin domains could be so successfully secreted while retaining dockerin-binding function. FIG. 5 demonstrates that supernatant containing secreted coh.alkaline phosphatase (coh.AP) binds specifically to a rAb.doc protein immobilized on a plastic surface.
[0132] FIGS. 7A and 7B show that the Expression plasmids encoding secreted alkaline phosphatase (AP) or coh.AP directed secretion of functional proteins from transfected 293F cells. After 3 days of culture supernatants were harvested and tested for their ability to bind 0.25 ug of either rAb.doc (top panel) or rAb (lower panel) bound to a 96 well micro-titre plate. After 1 hr of incubation the plates were washed and developed with a chromogenic AP substrate.
[0133] The invention embodies the application of assembly of specific protein complexes based on the cohesin.dockerin interaction. Specific antibody.antigen complexes can also be assembled using the established interaction of protein A or protein G IgFc binding domains. The invention embodies unique properties of the cohesin.dockerin interaction that result in greatly superior complex formation compared to the e.g., protein G interaction with IgG. In FIGS. 6 and 7 the interaction of a cohesin.AP (called Coh.AP) protein is shown to be specific for a rAb.Doc protein.
[0134] FIGS. 8A and 8B shows various dilutions of a supernatant containing secreted G.AP were incubated for 1 hr in micro-titre wells containing 0.25 ug of immobilized mIgG2a, mIgG2b, or a mIgG2b-based rAb.doc. After washing the bound AP activity was developed using chromogenic AP substrate. The proG.AP did not bind to the rAb.doc since it was an isotype variant of mIgG2b that did not interact with the particular protein G domain used in the proG.AP construct.
[0135] FIG. 8B shows an identical study, but employing dilutions of a supernatant containing secreted Coh.AP. Coh.AP binds only to rAb.doc, again demonstrating the specificity of the coh.doc interaction.
[0136] FIG. 9 demonstrates the vastly superior stability of preassembled complexes based on coh.doc interaction compared to proG.IgGFc interaction. FIG. 9 shows the formation of complexes between a fixed amount of proG.AP or coh.AP or coh2.AP (0.1 ug) and immobilized mIgG2b or rAb.doc (0.25 ug) were assembled by incubation for 1 hr in a micro-titre plate. At various times a 20-fold excess of soluble mIgG2b or rAb.doc were added and incubation continued for various times. Plates were then washed and bound AP activity accessed by addition of chromogenic AP substrate.
[0137] This example shows the use of such coh.doc complexes in settings containing serum (e.g., tissue culture media and in vivo administration). FIG. 10 demonstrates the vast superiority of coh.doc complexes compared to proG.IgGFc complexes in such a setting. Under the conditions used, ˜15 ug/ml Ig was sufficient to completely displace bound proG.AP, while the coh.AP remained stably bound to rAb.doc even in the presence of pure serum (15 mg/ml Ig)
[0138] FIG. 10 shows the formation of complexes between a fixed amount of proG.AP or coh.AP (0.1 ug) and immobilized mIgG2b or rAb.doc (0.25 ug) were assembled by incubation for 1 hr in a micro-titre plate. Various dilutions of human serum were added and incubation continued for 4 hrs. Plates were then washed and bound AP activity accessed by addition of chromogenic AP substrate.
[0139] The invention also embodies a particular utility of the coh.doc interaction that permits a production process that ensures complete complex formation and that can be concomitant with a purification process for the coh.fusion protein entity. This invention is exemplified in FIGS. 11 and 12, which illustrate this process via sequential capture of rAb.doc from culture supernatant by protein G affinity chromatography, followed by capture of coh.antigen from culture supernatant by the proteinG:rAb.doc column. Elution with low pH then releases pure rAb.doc:coh.antigen. If there is an excess of coh.antigen over rAb.doc, them full and complete complex should result. A related embodiment of this invention would be application to the protein G captured rAb.doc of excess pure or partially purified coh.fusion protein.
[0140] FIG. 11 shows a gel of reduced vs. non-reduced SDS.PAGE analysis of rAb.doc:Coh2.AP complexes produced by sequential application of rAb.doc supernatant and coh.AP supernatant to the same protein G affinity column. Lanes 2 and 4 show that Coh2.AP co-purifies with rAb.doc.
[0141] FIG. 12 is a non-reduced SDS.PAGE analysis of rAb.doc:Coh.Flu HA5-1 complexes produced by sequential application of rAb.doc supernatant and coh.Flu HA5-1 supernatant to the same protein G affinity column. Lanes 1 to 4 left to right show that Coh.Flu HA5-1 co-purifies with rAb.doc.
[0142] A well described feature of cohesin domains is their compatibility with the standard E. coli bacterial expression system. The invention embodies the novel use of expression of dockerin fusion proteins in mammalian secretion systems, and it also encompasses the formation of coh.doc complexes where the different components (i.e., coh and doc) are expressed in different systems. This is a great advantage since it affords the possibility of using the most favorable expression system for each component. For example, coh.Flu M1 expression constructs failed to efficiently direct the synthesis of secreted product from transfected mammalian cells. However, coh.Flu M1 was very efficiently expressed as a soluble protein in E. coli. Table 6 shows the sequence of the coh.Flu M1 used in this example.
[0143] TABLE 11 shows the nucleic and amino acid sequence for E coli-pET28(Cohesin-FluM1-6× His) or C32 is shown below. In the amino acid sequence the cohesin domain is highlighted in yellow and the point of fusion between cohesion and influenza A M1 protein is underlined. Residues highliighted grey are a C-terminal His tag to facilitate purificaytion via metal affinity chromatography.
TABLE-US-00012 TABLE 11 E. coli-pET28(Cohesin-FluM1-6xHis) or C32. ATGGATCTGGATGCAGTAAGGATTAAAGTGGACACAGTAAATGCAAAACCGGGAGACACAGTAAATATACCTGT- AAGATTCAGTG GTATACCATCCAAGGGAATAGCAAACTGTGACTTTGTATACAGCTATGACCCGAATGTACTTGAGATAATAGAG- ATAAAACCGGG AGAATTGATAGTTGACCCGAATCCTACCAAGAGCTTTGATACTGCAGTATATCCTGACAGAAAGATGATAGTAT- TCCTGTTTGCG GAAGACAGCGGAACAGGAGCGTATGCAATAACTAAAGACGGAGTATTTGCTACGATAGTAGCGAAAGTAAAAGA- AGGAGCACCTA ACGGGCTCAGTGTAATCAAATTTGTAGAAGTAGGCGGATTTGCGAACAATGACCTTGTAGAACAGAAGACACAG- TTCTTTGACGG TGGAGTAAATGTTGGAGATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACAGATG- ATCTGGATGCA GCTAGCCTTCTAACCGAGGTCGAAACGTACGTTCTCTCTATCATCCCGTCAGGCCCCCTCAAAGCCGAGATCGC- ACAGAGACTTG AAGATGTCTTTGCAGGGAAGAACACCGATCTTGAGGTTCTCATGGAATGGCTAAAGACAAGACCAATCCTGTCA- CCTCTGACTAA GGGGATTTTAGGATTTGTGTTCACGCTCACCGTGCCCAGTGAGCGGGGACTGCAGCGTAGACGCTTTGTCCAAA- ATGCTCTTAAT GGGAACGGAGATCCAAATAACATGGACAAAGCAGTTAAACTGTATAGGAAGCTTAAGAGGGAGATAACATTCCA- TGGGGCCAAAG AAATAGCACTCAGTTATTCTGCTGGTGCACTTGCCAGTTGTATGGGCCTCATATACAACAGGATGGGGGCTGTG- ACCACTGAAGT GGCATTTGGCCTGGTATGCGCAACCTGTGAACAGATTGCTGACTCCCAGCATCGGTCTCATAGGCAAATGGTGA- CAACAACCAAT CCACTAATCAGACATGAGAACAGAATGGTTCTAGCCAGCACTACAGCTAAGGCTATGGAGCAAATGGCTGGATC- GAGTGAGCAAG CAGCAGAGGCCATGGATATTGCTAGTCAGGCCAGGCAAATGGTGCAGGCGATGAGAACCATTGGGACTCATCCT- AGCTCCAGTGC TGGTCTAAAAGATGATCTTCTTGAAAATTTGCAGGCTTACCAGAAACGGATGGGGGTGCAGATGCAGCGATTCA- AGCTCGAGCAC CACCACCACCACCACTGA (SEQ ID NO.: 22) ##STR00052## ##STR00053## ASLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERG- LQRRRFVQNALN GNGDPNNMDKAVKLYRKLKREITFHGAKEIALSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQ- HRSHRQMVTTTN ##STR00054## ##STR00055##
[0144] The invention embodies the use of the dockerin.cohesin interaction to assemble ordered and specific complexes for various therapeutic or vaccination purposes. An example is the use of rAb.doc with binding specificity to an internalizing human Dendritic Cell (DC) receptor complexed with coh.Flu M1 protein. FIG. 11 demonstrates this utility by an in vitro study. DC cultured with anti-DC_rAb.doc:coh.Flu M1, then co-cultured with autologous T cells, directed the expansion of T cells with specific memory of Flu M1. Equivalent doses of coh.Flu M1 alone had no such effect. The study shows at least a 50-fold enhancement of Flu M1-specific T cell expansion via the anti-DC_rAb.doc:coh.Flu M1 compared to coh.Flu M1 alone.
[0145] FIG. 13 shows that functional anti-DC_rAb.doc:coh.Flu M1 complex was formed by mixing the individual purified components. Various amounts of the complex, or coh.Flu M1 alone, were incubated in culture medium with 5E4 human DC (from a HLA201 donor) and 10E5 autologous T cells. After 24 hr, the DC were activated with CD4OL and incubation was continued for an additional 9 days. Cells were harvested and stained with a PE-labeled Flu M1 peptide GILGFVFTL (SEQ ID NO.:24) HLA-A2 tetramer and analyzed for the frequency of antigen-specific CD8+ cells.
[0146] FIG. 14 shows a similar example incorporating the additional control of coh.Flu M1 complexed to an isotype-matched mAb.doc with no binding to the human DC. FIG. 12 shows that Anti-DC_rAb directly linked via an H chain fusion to a peptide fragment spanning the Flu M1 GILGFVFTL epitope is also effective in eliciting DC targeted antigen delivery resulting in expansion of Flu M1-specific T cells. However the Anti-DC_rAb.Flu M1 PEP entity was secreted very poorly from mammalian cells, likely precluding production of such a vaccine. This problem illustrates the embodiment of the invention that allows production issues to be solved by employing expression systems appropriate for the (in this case) vaccine antigen.
[0147] FIG. 14 shows that Anti-DC_rAb.doc:coh.Flu M1 or mIgG2b.doc:coh.Flu M1 complexes were formed by mixing the individual purified components. Various amounts of the complexes, or coh.Flu M1 alone, were incubated in culture medium with 5E4 human DC (from a HLA201 donor) and 10E5 autologous T cells. After 24 hr, the DC were activated with CD40L and incubation was continued for an additional 9 days. Cells were harvested and stained with a PE-labeled Flu M1 peptide GILGFVFTL (SEQ ID NO.:24) HLA-A2 tetramer and analyzed for the frequency of antigen-specific CD8+ cells. Concentrations for mIgG2.doc complexes were the same as those for Anti-DC_rAb complexes.
[0148] FIG. 15 shows CD34+ human DC were sorted into CD1a+ and CD14+ subtypes and cultured with and without 3 nM Anti-DC_rAb.Flu M1 PEP or Anti-DC_rAb. Autologous T cells were added after 1 day and culture continued for a further 8 days. Analysis was as described above. The CD1a+ cells were very efficient in expanding Flu M1-specific CD8+ cells only with Anti-DC_rAb.Flu M1 PEP treatment.
[0149] While one type of embodiment of the invention is a vaccine composed of an Anti-DC-rAb.doc:coh.antigen complex, it is envisioned that in some cases a preferred DC-targeting vaccine will be Anti-DC-rAb.antigen where antigen is likely a string of protective antigens. Identification of such antigens in efficacious combinations compatible with efficient expression in production systems is extremely problematic. One embodiment of the invention affords a method to streamline testing of antigen epitope combinations for the development of such vaccines. Specifically, the invention teaches a method to screen likely antigen epitopes alone and in combinations for efficacy as a prelude to addressing production of the desired Anti-DC-rAb.antigen. For example, TABLE 13 shows the sequences of exemplative cohesin.peptide constructs which can be readily expressed via E. coli systems. Using techniques similar to those described in FIG. 11, diverse collections of coh.pep proteins can be readily tested for efficacy as complexes with a single anti-DC_rAb.doc entity. The most efficacious coh.pep compounds can then be engineered directly as anti-DC_rAb.peptide fusion proteins. FIG. 16 shows examples of purified coh.PEP proteins expressed in E. coli.
[0150] TABLE 12 shows the amino acid sequence of the melanoma-associated antigen gp100. Well known HLA-A201-restricted dominant peptides are shaded and detailed below the sequence. Peptide sequences labeled M are variants with enhanced affinity for HLA-A201. C180 is an E. coli expression construct that encodes the sequence shown below in which the cohesin domain is shaded blue and the gp100 peptide is shaded grey. Underlined residues bounding the peptide are native to gp100. C-terminal His tags are to facilitate purification via metal affinity chromatography.
[0151] Shown below is the gp100 sequence and the associated peptides referred to above.
TABLE-US-00013 (SEQ ID NO.: 25) MDLVLKRCLLHLAVIGALLAVGATKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSLKVSNDG- PTLIGANASFSI ##STR00056## ##STR00057## ##STR00058## PSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSIVVLSGTTAAQV- TTTEWVETTARE LPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFSVTLDIVQGIESAEILQAVPS- GEGDAFELTVSC QGGLPKEACMEISSPGCQPPAQRLCQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIMPGQE- AGLGQVPLTVGI LLVLMAVVLASLTYRRRLMKQDFSVPQLPHSSSHWLRLPRIFCSCPIGENSPLLSGQQV
[0152] The HLA-A0201 restricted peptide sequences are:
[0153] GP100 WT: 154-162: KTWGQYWQV (SEQ ID NO.:26)
[0154] GP100 M: 209-217 (2M): IMDQVPFSV (SEQ ID NO.:27); 209-217 WT: ITDQVPFSV (SEQ ID NO.:28)
[0155] GP100 M: 280-288 (9V): YLEPGPVTV (SEQ ID NO.:29) 280-288 WT: YLEPGPVTA (SEQ ID NO.:30)
[0156] C180 is E. coli-pET28(Cohesin-hgp100-PeptideA-6× His):
TABLE-US-00014 (SEQ ID NO.: 31) ##STR00059## ##STR00060## ##STR00061##
[0157] TABLE 13 shows the amino acid sequence of the melanoma antigen MART-1. Well known HLA-A201-restricted dominant peptides are shaded and detailed below the sequence. M peptides show peptide sequence variants with enhanced affinity for HLA-A201. C181 is an E. coli expression construct that encodes the sequence shown below in which the cohesin domain is shaded yellow and the MART-1 peptide is shaded grey. Underlined residues bounding the peptide are native to MART-1. C172 and C174 are two constructs directing the expression of anti-DC_rAb.MART-1 peptide and a matching control rAb.MART-1 peptide H chain. Only the sequences appended to the C-terminal residue are shown. C-terminal His tags are to facilitate purification via metal affinity chromatography.
[0158] MART-1 is:
TABLE-US-00015 (SEQ ID NO.: 32) ##STR00062## SLQEKNCEPVVPNAPPAYEKLSAEQSPPYSP
[0159] The HLA-A0201 restricted peptides sequences are:
TABLE-US-00016 (SEQ ID NO.: 33) ##STR00063## (SEQ ID NO.: 34) ##STR00064## (SEQ ID NO.: 35) ##STR00065##
[0160] C181 is E. coli-pET28(Cohesin-hMART-1-PeptideB-6× His)
TABLE-US-00017 (SEQ ID NO.: 36) ##STR00066## ##STR00067## ##STR00068##
[0161] C186 is E. coli-pET28(Cohesin-Flex-hMART-1-PeptideA-6× His)
TABLE-US-00018 (SEQ ID NO.: 37) ##STR00069## ##STR00070## ##STR00071##
[0162] C172 is rAB-pIRES2(mAnti-ASGPR--49C11--7H-LV-hIgG4H-hMART-1-PeptideA)
[0163] C174 is rAB-pIRES2(hIgG4H-hMART-1-PeptideA)
TABLE-US-00019 (SEQ ID NO.: 38) ##STR00072##
[0164] FIG. 16 shows E. coli harboring expression plasmids directing the synthesis of coh.pep proteins were grown and induced for specific protein production. Cells were harvested and broken by sonication. The supernatant fractions were applied purified by metal affinity chromatography. Analysis was by reducing SDS.PAGE gel stained by Coomassie Brilliant Blue. The figure shows typical product coh.pep proteins labeled from left to right.
[0165] This Example shows the successful use of cohesin and dockerin fusion proteins secreted from mammalian cells. If both fusion partners are rAbs with different specificities (i.e., rAb1.doc and rAb2.coh), then simple mixing results in rAb1.doc:rAb2.coh which is a bi-specific antibody. Bispecific antibodies have many potential therapeutic and technical applications. The invention provides a simple and predictable means to assemble such entities through the doc:coh interaction. Alternately, if rAb1.doc:rAb1.coh were assembled such entities represent controlled cross-linked mAbs with potentially unique biological properties.
[0166] Cohesin.dockerin modules exist in diverse cellulose degrading species. While they have sequence similarities, they can have specificities that do not cross between species. This affords an opportunity to build novel scaffolds composed of cohesins with different specificities and use this scaffold to assemble high order complexes in a spatially and numerically controlled manner. Others have described the core technology for using this notion for biotechnology applications (see Fierobe, H.-P., Mechaly, A., Tardif, C., Belaich, A., Lamed, R., Shoham, Y., Belaich, J.-P., and Bayer, E. A. (2001) Design and production of active cellulosome chimeras: Selective incorporation of dockerin-containing enzymes into defined functional complexes. J. Biol. Chem. 276, 21257-21261.). The invention embodies the specific use of this technology for applications related to manufacture of rAb.(doc:coh.fusion)n complexes where n represents >1 pairings of doc:coh interactions with unique specificities. Thus, the invention envisions the assembly (by simple mixing of components) of spatially ordered complexes between rAb.doc1.doc2.doc3.etc. and coh1.fusionA, coh2.fusionB, coh3.fusion3, etc. The coh.fusion proteins could represent different antigens, or combinations of antigens and activating agents like cytokines.
[0167] By extension multiple coh:doc specificities could also be used to make bivalent rAbs with higher order antigen specificities. Cellulose degrading bacteria and similar organisms also use cellulose binding domains (CBD) to organize the degradation machinery. The structure of a CBD from Clostridium thermocellum shows that the N and C-termini are in close proximity and are not an integral part of the CBD functional structure. In fact CBD typically occurs linked to other domains such as coh.CBD.coh in cipA. The invention encompasses the use of entities such as coh.CBD.coh to assemble spatially and numerically ordered complexes mimicking antibodies and multi subunit receptors. For example, a IgG kappa chain v region fused to doc1 and a IgG H chain V region linked to doc2 can assemble with coh1.CBD.coh2 to yield VL.doc1:coh1.CBD.coh2:VH.doc2 to yield an entity with affinity and binding specificity analogous to the original mAb. Such entities should be e.g., very useful screening tools for refining mAb specificities through mutagenesis procedures, particularly since the VL and VH component could be mutated independently and combined by mixing in various combinations. As described above, this technology can be readily extended to multiple controlled coh:V.doc combinations potentially yielding binding entities with extremely high specificities and affinities. An extension of this would be using e.g., coh1.coh2.CBD.coh3 as a template for assembly of cytoR1.doc+cytoR2.doc+cytoR3.doc (where cytoR represents the ectodomain of one subunit of a complex cytokine receptor). Such entities will have utility for blocking cytokine interactions for therapy and in biotechnology for measuring cytokines in complex supernatants.
EXAMPLE 3
Using Cohesin-Dockerin Technology for Immunotoxin Therapy
[0168] Currently 1.2 million Americans develop cancer each year and about 500,000 die from the disease, because most cancers cannot be cured once they have metastasized. To develop a new treatment for metastatic cancer, genetic engineering has been used to modify a powerful bacterial toxin, Pseudomonas exotoxin A (PE), so that instead of killing normal cells it selectively kills cancer cells. PE is a three domain protein composed of 613 amino acids. Anti-cancer agents are produced by deleting its binding domain (aa 1-252) and replacing it with the Fv fragment of an antibody or with a growth factor that binds to antigens present on cancer cells. These agents are termed recombinant immunotoxins (RITs). RITs have been made that target Ley present on colon, breast, lung and other epithelial cancers (B3(Fv)-PE38), that target the EGF receptor overexpressed on glioblastomas (TGF-alpha-PE38), that target mutant EGF receptors present on glioblastomas (MR-1(Fv)-PE38KDEL), and that target the IL-2 receptor present on many T and B cell leukemias and lymphomas LMB-2 or anti-Tac(Fv)-PE38 and that target CD22 on B cell malignancies and that target BL22 or RFB4(dsFv)-PE38 ovarian cancers and mesotheliomas (SS1P). These agents are produced in E. coli because large amounts can be readily purified from this source and because the toxin itself would kill mammalian cells expressing it. When administered to mice with the appropriate human cancer xenograft, all these RITs produce complete tumor regressions. Most of these agents are now in clinical trials in humans and several have produced complete and partial remissions in humans with cancer.
[0169] An ideal immunotoxin should be very active so that only small amounts need to be given to cause tumor regressions, stable so it remains functional during the 5-10 hours required to reach the interior of a tumor, and non immunogenic so it can be given repeatedly. Initially, recombinant immunotoxins contained amino acids 253-613 of PE (domains II and III). It has been determined that amino acids 364-395 can be deleted without loss of activity. Increased stability can be addressed by linking the toxin to a whole antibody, which are well known to have long half-lives and the technology in the invention provides this solution.
[0170] While the rAb.Doc:Coh.toxin technology can be applied to known cancer antigens, it can also be tested to kill intra-tumoral DC that are suspected to foster escape of the tumor from immune surveillance. In this latter case, anti-DC toxin therapy could be doubly advantageous since build up of immunity against the administered toxin itself should be suppressed (that is because DC themselves are key to the initiation of this immune response via uptake and processing of the antigen. In this therapy, the DCs that uptake the antigen die and cannot mount the anti-toxin response).
[0171] Frankel (Clinical Cancer Research, 8, 942-944, 2002) describes issues hindering the wider application of immunotoxins. These include production problems which often require refolding of E. coli inclusion body expressed material where misfolding contaminents are problematic. Also, affinity of the immunotoxin for its target is often difficult to obtain in sufficient strength. The technology basis of this invention addresses both these issues--firstly, we found that cohesin. PE38 fusion protein is expressed in E. coli as a soluble protein that can be purified in a fully functional state (with both cohesin and toxin activities in tact) by simple biochemical means without complex refolding. Secondly, high affinity monoclonal antibodies against target antigens can be routinely obtained by one practiced in the art. What is difficult is engineering the antibody variable regions in a form that is fused with toxin and fully functional for target binding. The usual means (e.g., sFv forms) of engineering invarably lead to significant loss of affinity against the target compared to the initial monoclonal antibody. The rAb.Doc:Coh.toxin technology circumvents this issue affording a means to preserve both the high affinity binding sites of the initial mAb (note that humanization of mouse mAb V regions while maintaining high and specific binding activity is routine to one practiced in the art), as well as the beneficial properties of long half-life and non-antigenicity of a full recombinant hIgG context.
[0172] Furthermore since the cohesin.toxin is produced independently, one formulation of the toxin can be conjugated to any number of separately produced targeting rAb.Doc proteins by simple mixing of the component prior to injection of the patient. This greatly simplifies manufacturing as well as research development time. The technology described in the invention can be readily applied to any toxin and any rAb specificity.
[0173] Details of the rAb.Doc:Coh.toxin technology. pRB 391 (from Dr. Pastan) Pastan, Chief of the Laboratory of Molecular Biology, Division of Basic Sciences. NCI, NIH) was used as a template for PCR with primers
TABLE-US-00020 PE38-N3 (cacggtcaccgtctccaaagcttccggagctagcGAGGGCGGCAGCCTG GCCGCGCT (SEQ ID NO.: 39)) and PE38-C3 (GGCCGGCTCCTGCGAAGGGAGCCGGCCGGTCGCGGCCGCTTACTTCAGG TCCTCGCGCGGCGGTTTGCCG (SEQ ID NO.: 40)).
[0174] Cloning was into the previously established construct C21 or E. coli-pET28(Cohesin-6× His) to generate a fusion protein encoding Cohesin-PE38 corresponding to the amino acid sequence shown below (grey residues are cohesin; yellow residues are PE38, separated by a linker sequence native to the cohesin domain).
TABLE-US-00021 (SEQ ID NO.: 41) ##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077## ##STR00078## ##STR00079##
[0175] Expression and purification of recombinant Coh.PE38 protein--E. coli cells from each 1 L fermentation were resuspended in 25 ml ice-cold 50 mM Tris, 1 mM EDTA pH 8.0 with 0.1 ml of protease inhibitor Cocktail II (Calbiochem). The cells were sonicated on ice 2×5 min at setting 18 (Fisher Sonic Dismembrator 60) with a 5 min rest period and then spun at 17,000 r.p.m. (Sorvall SA-600) for 20 min at 4° C. The supernatant was passed through 1 ml ANX Sepharose column equilibrated in 50 mM Tris, 1 mM EDTA pH 8.0 and and eluted with a 0-1 M NaCl gradient in Buffer B. Fractions containing Cohesin.PE38 sere identified by SDS.PAGE and pooled fractions were further purified by purification via anti-cohesin mAb affinity chromatography with elution by 0.1 M glycine pH 2.7.
[0176] Selective killing of human DC by rAb.Doc targeted Coh.PE38--Human DC were prepared from blood monocytes by culture for 6 days in with GM-CSF and IL-4. The DCs were then cultured with either Coh.PE38 alone, anti-DC-SIGN/L 16E7 rAb.Doc alone, anti-DCIR 24A5.Doc alone, or the rAb.Docs together with Coh.PE38 (1.25 ug/ml of agents were added). After 48 hr the cells were stained with a reagent (7-AAD) that detects apoptotic cells and analyzed by FACS scoring forward versus side scatter and 7-AAD fluorescence.
[0177] FIG. 17 shows that the DCIR.Doc rAb alone had no effect upon the survival of DCs. However, DC-SIGN/L alone has a survival enhancing effect upon the DC (evidenced both by the scatter analysis and the 7-AAD staining. FIG. 18 shows that Coh.PE38 alone slightly increase the number of 7-AAD scored apoptotic cells (from 22.1-29.8%). However, targeting the Coh.PE38 toxin via DCIR.Doc increased the 7-AAD positive population to 55.3%. The scatter analysis even more dramatically revealed an almost complete loss of the population characteristic of viable DC. Targeting the Coh.PE38 toxin via DC-SIGN/L.Doc increased the 7-AAD positive population to 53.7%.with a similar loss of the viable DC scatter population.
[0178] However, this latter result should be viewed in the context of the survival effect of the DC-SIGN/L.Doc rAb, meaning that the killing can be viewed as from 3.1-53.1% 7-AAD positive.
[0179] Using Cohesin-Dockerin Technology to make Multivalent Antibodies. A Cohesin domain was engineered in-frame with the C-terminus of a rAb H chain using PCR based on C17 (Mam-pCDM8(Cohesin-Cohesin-SLAML-AP-6× His))as template. The resulting secreted H chain sequence is shown below (the cohesin domain is highlighted in grey and the C-terminal H chain residue is in bold):
TABLE-US-00022 (SEQ ID NO.: 42) QIQLVQSGPELKKPGETVKISCKASGYSFTNYGMNWVKQAPGKGLKWMGWINTYTGESTYADDFKGRFAFSLET- SASTAYLQISH LKNEDMATYFCARGDFRYYYFDYWGQGTTLTGSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVIVS- WNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPP- KPKDTLMISRI PEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS- SIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYGRLTVDKS- RWQEGNVFSCS ##STR00080## ##STR00081##
[0180] This expression construct was co-transfected with the appropriate rAb L chain into 293F cells and expression of secreted rAb was appraised by anti-hIgGFc ELISA at day 3. FIG. 19 shows the expression of anti-DC-SIGN/L and Anti-DC-ASPGR rAb.Coh were efficiently secreted.
[0181] Thus both Cohesin and Dockerin domains are readily expressed as rAb fusion proteins. This property is essential for the use of (rAb1.Coh:rAb2.Doc) complexes as bivalent antibodies (i.e., having two different combining specificities in one protein). Bivalent antibodies have many desirable features suited to industrial, analytic, and therapeutic applications. They are, however, difficult to develop and molecular tools used to engineer them typically adulterate desirable features of high affinity and specificity inherent to the parent monoclonal antibodies. The (rAb1.Coh:rAb2.Doc) technology circumvents this obstacle and is, moreover, extensible to higher (than 2) valency of combining power by incorporating multiple Cohesin or Dockerin strings with pair wise specificities as described elsewhere in this application. Furthermore, this technology can be extended to using, e.g., a cytokine to provide the additional valency (i.e., rAb1.Doc:Coh.cytokine).
[0182] For example, a fusion protein between a Cohesin domain and IL-21 was engineered as an expression construct and the Coh.IL-21 protein was efficiently secreted from transiently transfected 293F cells and easily purified by sequential Q Sepharose and anti-Cohesin affinity chromatography. The sequence of the secreted product is shown below with the cohesin domain shown in grey and the IL-21 domain in yellow. This product was fully functional as determined by it's efficacy in sustaining proliferation of human B cells.
[0183] Mam-pCDM8(SLAML-Cohesin-hIL-21)
TABLE-US-00023 (SEQ ID NO.: 43) ##STR00082## ##STR00083## ##STR00084## ##STR00085##
[0184] Thus rAb.Doc:Coh.IL-21 can deliver concomitant proliferation and activation signals to a B cel (i.e., if the rAb itself has activation properties). This notion can be extended to any rAb with biological properties directed to a particular cell type and any cytokine with activity directed to the same cell type. FIG. 20 shows the effect of IL-21 and Coh:IL-21 on the proliferation of human B cells.
[0185] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.
[0186] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
[0187] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
[0188] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
[0189] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
[0190] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
[0191] All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Sequence CWU
1
1
4312299PRTBacteroides cellulosolvens 1Met Gln Ser Pro Arg Leu Lys Arg Lys
Ile Leu Ser Val Ile Leu Ala 1 5 10
15 Val Cys Tyr Ile Ile Ser Ser Phe Ser Ile Gln Phe Ala Ala
Thr Pro 20 25 30
Gln Val Asn Ile Ile Ile Gly Ser Ala Gln Gly Ile Pro Gly Ser Thr
35 40 45 Val Lys Val Pro
Ile Asn Leu Gln Asn Val Pro Glu Ile Gly Ile Asn 50
55 60 Asn Cys Asp Phe Thr Ile Lys Phe
Asp Ser Asp Ile Leu Asp Phe Asn 65 70
75 80 Ser Val Glu Ala Gly Asp Ile Val Pro Leu Pro Val
Ala Ser Phe Ser 85 90
95 Ser Asn Asn Ser Lys Asp Ile Ile Lys Phe Leu Phe Ser Asp Ala Thr
100 105 110 Gln Gly Asn
Met Pro Ile Asn Glu Asn Gly Leu Phe Ala Val Ile Ser 115
120 125 Phe Lys Ile Lys Asp Asn Ala Gln
Lys Gly Ile Ser Asn Ile Lys Val 130 135
140 Ser Ser Tyr Gly Ser Phe Ser Gly Met Ser Gly Lys Glu
Met Gln Ser 145 150 155
160 Leu Ser Pro Thr Phe Phe Ser Gly Ser Ile Asp Val Ser Asp Val Ser
165 170 175 Thr Ser Lys Leu
Asp Val Lys Val Gly Asn Val Glu Gly Ile Ala Gly 180
185 190 Thr Glu Val Asn Val Pro Ile Thr Phe
Glu Asn Val Pro Asp Asn Gly 195 200
205 Ile Asn Asn Cys Asn Phe Thr Leu Ser Tyr Asp Ser Asn Ala
Leu Glu 210 215 220
Phe Leu Thr Thr Glu Ala Gly Asn Ile Ile Pro Leu Ala Ile Ala Asp 225
230 235 240 Tyr Ser Ser Tyr Arg
Ser Met Glu Gly Lys Ile Lys Phe Leu Phe Ser 245
250 255 Asp Ser Ser Gln Gly Thr Arg Ser Ile Lys
Asn Asp Gly Val Phe Ala 260 265
270 Asn Ile Lys Phe Lys Ile Lys Gly Asn Ala Ile Arg Asp Thr Tyr
Arg 275 280 285 Ile
Asp Leu Ser Glu Leu Gly Ser Phe Ser Ser Lys Gln Asn Asn Asn 290
295 300 Leu Lys Ser Ile Ala Thr
Gln Phe Leu Ser Gly Ser Val Asn Val Lys 305 310
315 320 Asp Ile Glu Ser Ser Val Ser Pro Thr Thr Ser
Val His Pro Thr Pro 325 330
335 Thr Ser Val Pro Pro Thr Pro Thr Lys Ser Ser Pro Gly Asn Lys Met
340 345 350 Lys Ile
Gln Ile Gly Asp Val Lys Ala Asn Gln Gly Asp Thr Val Ile 355
360 365 Val Pro Ile Thr Phe Asn Glu
Val Pro Val Met Gly Val Asn Asn Cys 370 375
380 Asn Phe Thr Leu Ala Tyr Asp Lys Asn Ile Met Glu
Phe Ile Ser Ala 385 390 395
400 Asp Ala Gly Asp Ile Val Thr Leu Pro Met Ala Asn Tyr Ser Tyr Asn
405 410 415 Met Pro Ser
Asp Gly Leu Val Lys Phe Leu Tyr Asn Asp Gln Ala Gln 420
425 430 Gly Ala Met Ser Ile Lys Glu Asp
Gly Thr Phe Ala Asn Val Lys Phe 435 440
445 Lys Ile Lys Gln Ser Ala Ala Phe Gly Lys Tyr Ser Val
Gly Ile Lys 450 455 460
Ala Ile Gly Ser Ile Ser Ala Leu Ser Asn Ser Lys Leu Ile Pro Ile 465
470 475 480 Glu Ser Ile Phe
Lys Asp Gly Ser Ile Thr Val Thr Asn Lys Pro Ile 485
490 495 Val Asn Ile Glu Ile Gly Lys Val Lys
Val Lys Ala Gly Asp Lys Ile 500 505
510 Lys Val Pro Val Glu Ile Lys Asp Ile Pro Ser Ile Gly Ile
Asn Asn 515 520 525
Cys Asn Phe Thr Leu Lys Tyr Asn Ser Asn Val Leu Lys Tyr Val Ser 530
535 540 Asn Glu Ala Gly Thr
Ile Val Pro Ala Pro Leu Ala Asn Leu Ser Ile 545 550
555 560 Asn Lys Pro Asp Glu Gly Ile Ile Lys Leu
Leu Phe Ser Asp Ala Ser 565 570
575 Gln Gly Gly Met Pro Ile Lys Asp Asn Gly Ile Phe Val Asn Leu
Glu 580 585 590 Phe
Gln Ala Val Asn Asp Ala Asn Ile Gly Val Tyr Gly Leu Glu Leu 595
600 605 Asp Thr Ile Gly Ala Phe
Ser Gly Ile Ser Ser Ala Lys Met Thr Ser 610 615
620 Ile Glu Pro Gln Phe Asn Asn Gly Ser Ile Glu
Ile Phe Asn Ser Ala 625 630 635
640 Gln Thr Pro Val Pro Ser Asn Thr Glu Val Gln Thr Pro Thr Asn Thr
645 650 655 Ile Ser
Val Thr Pro Thr Asn Asn Ser Thr Pro Thr Asn Asn Ser Thr 660
665 670 Pro Lys Pro Asn Pro Leu Tyr
Asn Leu Asn Val Asn Ile Gly Glu Ile 675 680
685 Ser Gly Glu Ala Gly Gly Val Ile Glu Val Pro Ile
Glu Phe Lys Asn 690 695 700
Val Pro Asp Phe Gly Ile Asn Asn Cys Asp Phe Ser Val Lys Tyr Asp 705
710 715 720 Lys Ser Ile
Phe Glu Tyr Val Thr Tyr Glu Ala Gly Ser Ile Val Lys 725
730 735 Asp Ser Ile Val Asn Leu Ala Cys
Met Glu Asn Ser Gly Ile Ile Asn 740 745
750 Leu Leu Phe Asn Asp Ala Thr Gln Ser Ser Ser Pro Ile
Lys Asn Asn 755 760 765
Gly Val Phe Ala Lys Leu Lys Phe Lys Ile Asn Ser Asn Ala Ala Ser 770
775 780 Gly Thr Tyr Gln
Ile Asn Ala Glu Gly Tyr Gly Lys Phe Ser Gly Asn 785 790
795 800 Leu Asn Gly Lys Leu Thr Ser Ile Asn
Pro Ile Phe Glu Asn Gly Ile 805 810
815 Ile Asn Ile Gly Asn Val Thr Val Lys Pro Thr Ser Thr Pro
Ala Asp 820 825 830
Ser Ser Thr Ile Thr Pro Thr Ala Thr Pro Thr Ala Thr Pro Thr Ile
835 840 845 Lys Gly Thr Pro
Thr Val Thr Pro Ile Tyr Trp Met Asn Val Leu Ile 850
855 860 Gly Asn Met Asn Ala Ala Ile Gly
Glu Glu Val Val Val Pro Ile Glu 865 870
875 880 Phe Lys Asn Val Pro Pro Phe Gly Ile Asn Asn Cys
Asp Phe Lys Leu 885 890
895 Val Tyr Asp Ser Asn Ala Leu Glu Leu Lys Lys Val Glu Ala Gly Asp
900 905 910 Ile Val Pro
Glu Pro Leu Ala Asn Leu Ser Ser Asn Lys Ser Glu Gly 915
920 925 Lys Ile Gln Phe Leu Phe Asn Asp
Ala Ser Gln Gly Ser Met Gln Ile 930 935
940 Glu Asn Gly Gly Val Phe Ala Lys Ile Thr Phe Lys Val
Lys Ser Thr 945 950 955
960 Ala Ala Ser Gly Ile Tyr Asn Ile Arg Lys Asp Ser Val Gly Ser Phe
965 970 975 Ser Gly Leu Ile
Asp Asn Lys Met Thr Ser Ile Gly Pro Lys Phe Thr 980
985 990 Asp Gly Ser Ile Val Val Gly Thr
Val Thr Pro Thr Ala Thr Ala Thr 995 1000
1005 Pro Ser Ala Ile Val Thr Thr Ile Thr Pro Thr
Ala Thr Thr Lys 1010 1015 1020
Pro Ile Ala Thr Pro Thr Ile Lys Gly Thr Pro Thr Ala Thr Pro
1025 1030 1035 Met Tyr Trp
Met Asn Val Val Ile Gly Lys Met Asn Ala Glu Val 1040
1045 1050 Gly Gly Glu Val Val Val Pro Ile
Glu Phe Asn Asn Val Pro Ser 1055 1060
1065 Phe Gly Ile Asn Asn Cys Asp Phe Lys Leu Val Tyr Asp
Ala Thr 1070 1075 1080
Ala Leu Glu Leu Lys Asn Val Glu Ala Gly Asp Ile Ile Lys Thr 1085
1090 1095 Pro Leu Ala Asn Phe
Ser Asn Asn Lys Ser Glu Glu Gly Lys Ile 1100 1105
1110 Ser Phe Leu Phe Asn Asp Ala Ser Gln Gly
Ser Met Gln Ile Glu 1115 1120 1125
Asn Gly Gly Val Phe Ala Lys Ile Thr Phe Lys Val Lys Ser Thr
1130 1135 1140 Thr Ala
Thr Gly Val Tyr Asp Leu Arg Lys Asp Leu Val Gly Ser 1145
1150 1155 Phe Ser Gly Leu Lys Asp Asn
Lys Met Thr Ser Ile Gly Ala Glu 1160 1165
1170 Phe Thr Asn Gly Ser Ile Thr Val Ala Ala Thr Ala
Pro Thr Val 1175 1180 1185
Thr Pro Thr Val Asn Ala Thr Pro Ser Ala Ala Thr Pro Thr Val 1190
1195 1200 Thr Pro Thr Ala Thr
Ala Thr Pro Ser Val Thr Ile Pro Thr Val 1205 1210
1215 Thr Pro Thr Ala Thr Ala Thr Pro Ser Val
Thr Ile Pro Thr Val 1220 1225 1230
Thr Pro Thr Ala Thr Ala Thr Pro Ser Ala Ala Thr Pro Thr Val
1235 1240 1245 Thr Pro
Thr Ala Thr Ala Thr Pro Ser Val Thr Ile Pro Thr Val 1250
1255 1260 Thr Pro Thr Val Thr Ala Thr
Pro Ser Asp Thr Ile Pro Thr Val 1265 1270
1275 Thr Pro Thr Ala Thr Ala Thr Pro Ser Ala Ile Val
Thr Thr Ile 1280 1285 1290
Thr Pro Thr Ala Thr Ala Lys Pro Ile Ala Thr Pro Thr Ile Lys 1295
1300 1305 Gly Thr Pro Thr Ala
Thr Pro Met Tyr Trp Met Asn Val Val Ile 1310 1315
1320 Gly Lys Met Asn Ala Glu Val Gly Gly Glu
Val Val Val Pro Ile 1325 1330 1335
Glu Phe Lys Asn Val Pro Ser Phe Gly Ile Asn Asn Cys Asp Phe
1340 1345 1350 Lys Leu
Val Tyr Asp Ala Thr Ala Leu Glu Leu Lys Asn Val Glu 1355
1360 1365 Ala Gly Asp Ile Ile Lys Thr
Pro Leu Ala Asn Phe Ser Asn Asn 1370 1375
1380 Lys Ser Glu Glu Gly Lys Ile Ser Phe Leu Phe Asn
Asp Ala Ser 1385 1390 1395
Gln Gly Ser Met Gln Ile Glu Asn Gly Gly Val Ser Ala Lys Ile 1400
1405 1410 Thr Phe Lys Val Lys
Ser Thr Thr Ala Ile Gly Val Tyr Asp Ile 1415 1420
1425 Arg Lys Asp Leu Ile Gly Ser Phe Ser Gly
Leu Lys Asp Ser Lys 1430 1435 1440
Met Thr Ser Ile Gly Ala Glu Phe Thr Asn Gly Ser Ile Thr Val
1445 1450 1455 Ala Thr
Thr Ala Pro Thr Val Thr Pro Thr Ala Thr Ala Thr Pro 1460
1465 1470 Ser Val Thr Ile Pro Thr Val
Thr Pro Thr Ala Thr Ala Thr Pro 1475 1480
1485 Gly Thr Ala Thr Pro Gly Thr Ala Thr Pro Thr Ala
Thr Ala Thr 1490 1495 1500
Pro Gly Ala Ala Thr Pro Thr Glu Thr Ala Thr Pro Ser Val Met 1505
1510 1515 Ile Pro Thr Val Thr
Pro Thr Ala Thr Ala Thr Pro Thr Ala Thr 1520 1525
1530 Ala Thr Pro Thr Val Lys Gly Thr Pro Thr
Ile Lys Pro Val Tyr 1535 1540 1545
Lys Met Asn Val Val Ile Gly Arg Val Asn Val Val Ala Gly Glu
1550 1555 1560 Glu Val
Val Val Pro Val Glu Phe Lys Asn Ile Pro Ala Ile Gly 1565
1570 1575 Val Asn Asn Cys Asn Phe Val
Leu Glu Tyr Asp Ala Asn Val Leu 1580 1585
1590 Glu Val Lys Lys Val Asp Ala Gly Glu Ile Val Pro
Asp Ala Leu 1595 1600 1605
Ile Asn Phe Gly Ser Asn Asn Ser Asp Glu Gly Lys Val Tyr Phe 1610
1615 1620 Leu Phe Asn Asp Ala
Leu Gln Gly Arg Met Gln Ile Ala Asn Asp 1625 1630
1635 Gly Ile Phe Ala Asn Ile Thr Phe Lys Val
Lys Ser Ser Ala Ala 1640 1645 1650
Ala Gly Ile Tyr Asn Ile Arg Lys Asp Ser Val Gly Ala Phe Ser
1655 1660 1665 Gly Leu
Val Asp Lys Leu Val Pro Ile Ser Ala Glu Phe Thr Asp 1670
1675 1680 Gly Ser Ile Ser Val Glu Ser
Ala Lys Ser Thr Pro Thr Ala Thr 1685 1690
1695 Ala Thr Gly Thr Asn Val Thr Pro Thr Val Ala Ala
Thr Val Thr 1700 1705 1710
Pro Thr Ala Thr Pro Ala Ser Thr Thr Pro Thr Ala Thr Pro Thr 1715
1720 1725 Ala Thr Ser Thr Val
Lys Gly Thr Pro Thr Ala Thr Pro Leu Tyr 1730 1735
1740 Ser Met Asn Val Ile Ile Gly Lys Val Asn
Ala Glu Ala Ser Gly 1745 1750 1755
Glu Val Val Val Pro Val Glu Phe Lys Asp Val Pro Ser Ile Gly
1760 1765 1770 Ile Asn
Asn Cys Asn Phe Ile Leu Glu Tyr Asp Ala Ser Ala Leu 1775
1780 1785 Glu Leu Asp Ser Ala Glu Ala
Gly Glu Ile Val Pro Val Pro Leu 1790 1795
1800 Gly Asn Phe Ser Ser Asn Asn Lys Asp Glu Gly Lys
Ile Tyr Phe 1805 1810 1815
Leu Phe Ser Asp Gly Thr Gln Gly Arg Met Gln Ile Val Asn Asp 1820
1825 1830 Gly Ile Phe Ala Lys
Ile Lys Phe Lys Val Lys Ser Thr Ala Ser 1835 1840
1845 Asp Gly Thr Tyr Tyr Ile Arg Lys Asp Ser
Val Gly Ala Phe Ser 1850 1855 1860
Gly Leu Ile Glu Lys Lys Ile Ile Lys Ile Gly Ala Glu Phe Thr
1865 1870 1875 Asp Gly
Ser Ile Thr Val Arg Ser Leu Thr Pro Thr Pro Thr Val 1880
1885 1890 Thr Pro Asn Val Ala Ser Pro
Thr Pro Thr Lys Val Val Ala Glu 1895 1900
1905 Pro Thr Ser Asn Gln Pro Ala Gly Pro Gly Pro Ile
Thr Gly Thr 1910 1915 1920
Ile Pro Thr Ala Thr Thr Thr Ala Thr Ala Thr Pro Thr Lys Ala 1925
1930 1935 Ser Val Ala Thr Ala
Thr Pro Thr Ala Thr Pro Ile Val Val Val 1940 1945
1950 Glu Pro Thr Ile Val Arg Pro Gly Tyr Asn
Lys Asp Ala Asp Leu 1955 1960 1965
Ala Val Phe Ile Ser Ser Asp Lys Ser Arg Tyr Glu Glu Ser Ser
1970 1975 1980 Ile Ile
Thr Tyr Ser Ile Glu Tyr Lys Asn Ile Gly Lys Val Asn 1985
1990 1995 Ala Thr Asn Val Lys Ile Ala
Ala Gln Ile Pro Lys Phe Thr Lys 2000 2005
2010 Val Tyr Asp Ala Ala Lys Gly Ala Val Lys Gly Ser
Glu Ile Val 2015 2020 2025
Trp Met Ile Gly Asn Leu Ala Val Gly Glu Ser Tyr Thr Lys Glu 2030
2035 2040 Tyr Lys Val Lys Val
Asp Ser Leu Thr Lys Ser Glu Glu Tyr Thr 2045 2050
2055 Asp Asn Thr Val Thr Ile Ser Ser Asp Gln
Thr Val Asp Ile Pro 2060 2065 2070
Glu Asn Ile Thr Thr Gly Asn Asp Asp Lys Ser Thr Ile Arg Val
2075 2080 2085 Met Leu
Tyr Ser Asn Arg Phe Thr Pro Gly Ser His Ser Ser Tyr 2090
2095 2100 Ile Leu Gly Tyr Lys Asp Lys
Thr Phe Lys Pro Lys Gln Asn Val 2105 2110
2115 Thr Arg Ala Glu Val Ala Ala Met Phe Ala Arg Ile
Met Gly Leu 2120 2125 2130
Thr Val Lys Asp Gly Ala Lys Ser Ser Tyr Lys Asp Val Ser Asn 2135
2140 2145 Lys His Trp Ala Leu
Lys Tyr Ile Glu Ala Val Thr Lys Ser Gly 2150 2155
2160 Ile Phe Lys Gly Tyr Lys Asp Ser Thr Phe
His Pro Asn Ala Pro 2165 2170 2175
Ile Thr Arg Ala Glu Leu Ser Thr Val Ile Phe Asn Tyr Leu His
2180 2185 2190 Leu Asn
Asn Ile Ala Pro Ser Lys Val His Phe Thr Asp Ile Asn 2195
2200 2205 Lys His Trp Ala Lys Asn Tyr
Ile Glu Glu Ile Tyr Arg Phe Lys 2210 2215
2220 Leu Ile Gln Gly Tyr Ser Asp Gly Ser Phe Lys Pro
Asn Asn Asn 2225 2230 2235
Ile Thr Arg Ala Glu Val Val Thr Met Ile Asn Arg Met Leu Tyr 2240
2245 2250 Arg Gly Pro Leu Lys
Val Lys Val Gly Ser Phe Pro Asp Val Ser 2255 2260
2265 Pro Lys Tyr Trp Ala Tyr Gly Asp Ile Glu
Glu Ala Ser Arg Asn 2270 2275 2280
His Lys Tyr Thr Arg Asp Glu Lys Asp Gly Ser Glu Ile Leu Ile
2285 2290 2295 Glu
21653DNAArtificial SequenceSynthetic oligonucleotide 2atggacctcc
tgtgcaagaa catgaagcac ctgtggttct tcctcctgct ggtggcggct 60cccagatggg
tcctgtcccg gctgcagctg caggagtcgg gcccaggcct gctgaagcct 120tcggtgaccc
tgtccctcac ctgcactgtc tcgggtgact ccgtcgccag tagttcttat 180tactggggct
gggtccgtca gcccccaggg aagggactcg agtggatagg gactatcaat 240tttagtggca
atatgtatta tagtccgtcc ctcaggagtc gagtgaccat gtcggcagac 300atgtccgaga
actccttcta tctgaaattg gactctgtga ccgcagcaga cacggccgtc 360tattattgtg
cggcaggaca cctcgttatg ggatttgggg cccactgggg acagggaaaa 420ctggtctccg
tctctccagc ttccaccaag ggcccatccg tcttccccct ggcgccctgc 480tccaggagca
cctccgagag cacagccgcc ctgggctgcc tggtcaagga ctacttcccc 540gaaccggtga
cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca caccttcccg 600gctgtcctac
agtcctcagg actctactcc ctcagcagcg tggtgaccgt gccctccagc 660agcttgggca
cgaagaccta cacctgcaac gtagatcaca agcccagcaa caccaaggtg 720gacaagagag
ttgagtccaa atatggtccc ccatgcccac cctgcccagc acctgagttc 780gaagggggac
catcagtctt cctgttcccc ccaaaaccca aggacactct catgatctcc 840cggacccctg
aggtcacgtg cgtggtggtg gacgtgagcc aggaagaccc cgaggtccag 900ttcaactggt
acgtggatgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 960cagttcaaca
gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 1020aacggcaagg
agtacaagtg caaggtctcc aacaaaggcc tcccgtcctc catcgagaaa 1080accatctcca
aagccaaagg gcagccccga gagccacagg tgtacaccct gcccccatcc 1140caggaggaga
tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctacccc 1200agcgacatcg
ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1260cctcccgtgc
tggactccga cggctccttc ttcctctaca gcaggctaac cgtggacaag 1320agcaggtggc
aggaggggaa tgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1380cactacacac
agaagagcct ctccctgtct ctgggtaaag ctagcaattc tcctcaaaat 1440gaagtactgt
acggagatgt gaatgatgac ggaaaagtaa actccactga cttgactttg 1500ttaaaaagat
atgttcttaa agccgtctca actctccctt cttccaaagc tgaaaagaac 1560gcagatgtaa
atcgtgacgg aagagttaat tccagtgatg tcacaatact ttcaagatat 1620ttgataaggg
taatcgagaa attaccaata taa
16533524PRTArtificial SequenceSynthetic peptide. 3Arg Leu Gln Leu Gln Glu
Ser Gly Pro Gly Leu Leu Lys Pro Ser Val 1 5
10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp
Ser Val Ala Ser Ser 20 25
30 Ser Tyr Tyr Trp Gly Trp Val Arg Gln Pro Pro Gly Lys Gly Leu
Glu 35 40 45 Trp
Ile Gly Thr Ile Asn Phe Ser Gly Asn Met Tyr Tyr Ser Pro Ser 50
55 60 Leu Arg Ser Arg Val Thr
Met Ser Ala Asp Met Ser Glu Asn Ser Phe 65 70
75 80 Tyr Leu Lys Leu Asp Ser Val Thr Ala Ala Asp
Thr Ala Val Tyr Tyr 85 90
95 Cys Ala Ala Gly His Leu Val Met Gly Phe Gly Ala His Trp Gly Gln
100 105 110 Gly Lys
Leu Val Ser Val Ser Pro Ala Ser Thr Lys Gly Pro Ser Val 115
120 125 Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135
140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser 145 150 155
160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175 Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180
185 190 Ser Ser Ser Leu Gly Thr Lys Thr
Tyr Thr Cys Asn Val Asp His Lys 195 200
205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys
Tyr Gly Pro 210 215 220
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val 225
230 235 240 Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245
250 255 Pro Glu Val Thr Cys Val Val Val Asp
Val Ser Gln Glu Asp Pro Glu 260 265
270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290
295 300 Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310
315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
Ser Ile Glu Lys Thr Ile 325 330
335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro 340 345 350 Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355
360 365 Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser 385 390 395
400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
405 410 415 Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420
425 430 His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Leu Gly Lys Ala 435 440
445 Ser Asn Ser Pro Gln Asn Glu Val Leu Tyr Gly Asp
Val Asn Asp Asp 450 455 460
Gly Lys Val Asn Ser Thr Asp Leu Thr Leu Leu Lys Arg Tyr Val Leu 465
470 475 480 Lys Ala Val
Ser Thr Leu Pro Ser Ser Lys Ala Glu Lys Asn Ala Asp 485
490 495 Val Asn Arg Asp Gly Arg Val Asn
Ser Ser Asp Val Thr Ile Leu Ser 500 505
510 Arg Tyr Leu Ile Arg Val Ile Glu Lys Leu Pro Ile
515 520 4 1635DNAArtificial
SequenceSynthetic oligonucleotide. 4atgaaatgca gctgggtcat cttcttcctg
atggcagtgg ttacaggggt caattcagag 60gttcagctgc agcagtctgg ggctgagctt
gtgaggccag gggccttagt caagttgtcc 120tgcaaagctt ctggcttcaa cattaatgac
tactatatcc actgggtgaa gcagcggcct 180gaacagggcc tggagcggat tggatggatt
gatcctgaca atggtaatac tatatatgac 240ccgaagttcc agggcaaggc cagtataaca
gcagacacat cccccaacac agcctacctg 300cagctcagca gcctgacatc tgaggacact
gccgtctatt actgtgctag aacccgatct 360cctatggtta cgacggggtt tgtttactgg
ggccaaggga ctgtggtcac tgtctctgca 420gccaaaacga agggcccatc cgtcttcccc
ctggcgccct gctccaggag cacctccgag 480agcacagccg ccctgggctg cctggtcaag
gactacttcc ccgaaccggt gacggtgtcg 540tggaactcag gcgccctgac cagcggcgtg
cacaccttcc cggctgtcct acagtcctca 600ggactctact ccctcagcag cgtggtgacc
gtgccctcca gcagcttggg cacgaagacc 660tacacctgca acgtagatca caagcccagc
aacaccaagg tggacaagag agttgagtcc 720aaatatggtc ccccatgccc accctgccca
gcacctgagt tcgaaggggg accatcagtc 780ttcctgttcc ccccaaaacc caaggacact
ctcatgatct cccggacccc tgaggtcacg 840tgcgtggtgg tggacgtgag ccaggaagac
cccgaggtcc agttcaactg gtacgtggat 900ggcgtggagg tgcataatgc caagacraag
ccgcgggagg agcagttcaa cagcacgtac 960cgtgtggtca gcgtcctcac cgtcctgcac
caggactggc tgaacggcaa ggagtacaag 1020tgcaaggtct ccaacaaagg cctcccgtcc
tccatcgaga aaaccatctc caaagccaaa 1080gggcagcccc gagagccaca ggtgtacacc
ctgcccccat cccaggagga gatgaccaag 1140aaccaggtca gcctgacctg cctggtcaaa
ggcttctacc ccagcgacat cgccgtggag 1200tgggagagca atgggcagcc ggagaacaac
tacaagacca cgcctcccgt gctggactcc 1260gacggctcct tcttcctcta cagcaggcta
accgtggaca agagcaggtg gcaggagggg 1320aatgtcttct catgctccgt gatgcatgag
gctctgcaca accactacac acagaagagc 1380ctctccctgt ctctgggtaa agctagcaat
tctcctcaaa atgaagtact gtacggagat 1440gtgaatgatg acggaaaagt aaactccact
gacttgactt tgttaaaaag atatgttctt 1500aaagccgtct caactctccc ttcttccaaa
gctgaaaaga acgcagatgt aaatcgtgac 1560ggaagagtta attccagtga tgtcacaata
ctttcaagat atttgataag ggtaatcgag 1620aaattaccaa tataa
16355525PRTArtificial SequenceSynthetic
peptide. 5Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15 Leu Val
Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Asn Asp Tyr 20
25 30 Tyr Ile His Trp Val Lys Gln
Arg Pro Glu Gln Gly Leu Glu Arg Ile 35 40
45 Gly Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr
Asp Pro Lys Phe 50 55 60
Gln Gly Lys Ala Ser Ile Thr Ala Asp Thr Ser Pro Asn Thr Ala Tyr 65
70 75 80 Leu Gln Leu
Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Thr Arg Ser Pro Met Val
Thr Thr Gly Phe Val Tyr Trp Gly 100 105
110 Gln Gly Thr Val Val Thr Val Ser Ala Ala Lys Thr Lys
Gly Pro Ser 115 120 125
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 130
135 140 Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150
155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170
175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190
Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
195 200 205 Lys Pro Ser Asn
Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly 210
215 220 Pro Pro Cys Pro Pro Cys Pro Ala
Pro Glu Phe Glu Gly Gly Pro Ser 225 230
235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg 245 250
255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
260 265 270 Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275
280 285 Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Tyr Arg Val Val 290 295
300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr 305 310 315
320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
325 330 335 Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340
345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys 355 360
365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser 370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385
390 395 400 Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405
410 415 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala 420 425
430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys 435 440 445 Ala
Ser Asn Ser Pro Gln Asn Glu Val Leu Tyr Gly Asp Val Asn Asp 450
455 460 Asp Gly Lys Val Asn Ser
Thr Asp Leu Thr Leu Leu Lys Arg Tyr Val 465 470
475 480 Leu Lys Ala Val Ser Thr Leu Pro Ser Ser Lys
Ala Glu Lys Asn Ala 485 490
495 Asp Val Asn Arg Asp Gly Arg Val Asn Ser Ser Asp Val Thr Ile Leu
500 505 510 Ser Arg
Tyr Leu Ile Arg Val Ile Glu Lys Leu Pro Ile 515
520 525 6 1638DNAArtificial SequenceSynthetic
oligonucleotide. 6atggacccca aaggctccct ttcctggaga atacttctgt ttctctccct
ggcttttgag 60ttgtcgtacg gagatgtgca gcttcaggag tcaggacctg acctggtgaa
accttctcag 120tcactttcac tcacctgcac tgtcactggc tactccatca ccagtggtta
tagctggcac 180tggatccggc agtttccagg aaacaaactg gaatggatgg gctacatact
cttcagtggt 240agcactaact acaacccatc tctgaaaagt cgaatctcta tcactcgaga
cacatccaag 300aaccagttct tcctgcagtt gaattctgtg actactgagg acacagccac
atatttctgt 360gcaagatcta actatggttc ctttgcttcc tggggccaag ggactctggt
cactgtctct 420gcagccaaaa caaagggccc atccgtcttc cccctggcgc cctgctccag
gagcacctcc 480gagagcacag ccgccctggg ctgcctggtc aaggactact tccccgaacc
ggtgacggtg 540tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt
cctacagtcc 600tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt
gggcacgaag 660acctacacct gcaacgtaga tcacaagccc agcaacacca aggtggacaa
gagagttgag 720tccaaatatg gtcccccatg cccaccctgc ccagcacctg agttcgaagg
gggaccatca 780gtcttcctgt tccccccaaa acccaaggac actctcatga tctcccggac
ccctgaggtc 840acgtgcgtgg tggtggacgt gagccaggaa gaccccgagg tccagttcaa
ctggtacgtg 900gatggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagtt
caacagcacg 960taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaacgg
caaggagtac 1020aagtgcaagg tctccaacaa aggcctcccg tcctccatcg agaaaaccat
ctccaaagcc 1080aaagggcagc cccgagagcc acaggtgtac accctgcccc catcccagga
ggagatgacc 1140aagaaccagg tcagcctgac ctgcctggtc aaaggcttct accccagcga
catcgccgtg 1200gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc
cgtgctggac 1260tccgacggct ccttcttcct ctacagcagg ctaaccgtgg acaagagcag
gtggcaggag 1320gggaatgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta
cacacagaag 1380agcctctccc tgtctctggg taaagctagc aattctcctc aaaatgaagt
actgtacgga 1440gatgtgaatg atgacggaaa agtaaactcc actgacttga ctttgttaaa
aagatatgtt 1500cttaaagccg tctcaactct cccttcttcc aaagctgaaa agaacgcaga
tgtaaatcgt 1560gacggaagag ttaattccag tgatgtcaca atactttcaa gatatttgat
aagggtaatc 1620gagaaattac caatataa
16387521PRTArtificial SequenceSynthetic peptide. 7Asp Val Gln
Leu Gln Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln 1 5
10 15 Ser Leu Ser Leu Thr Cys Thr Val
Thr Gly Tyr Ser Ile Thr Ser Gly 20 25
30 Tyr Ser Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys
Leu Glu Trp 35 40 45
Met Gly Tyr Ile Leu Phe Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu 50
55 60 Lys Ser Arg Ile
Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70
75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu
Asp Thr Ala Thr Tyr Phe Cys 85 90
95 Ala Arg Ser Asn Tyr Gly Ser Phe Ala Ser Trp Gly Gln Gly
Thr Leu 100 105 110
Val Thr Val Ser Ala Ala Lys Thr Lys Gly Pro Ser Val Phe Pro Leu
115 120 125 Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130
135 140 Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser 145 150
155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170
175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190 Leu Gly Thr
Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195
200 205 Thr Lys Val Asp Lys Arg Val Glu
Ser Lys Tyr Gly Pro Pro Cys Pro 210 215
220 Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val
Phe Leu Phe 225 230 235
240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255 Thr Cys Val Val
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260
265 270 Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro 275 280
285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr 290 295 300
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305
310 315 320 Ser Asn Lys Gly Leu
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325
330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Gln 340 345
350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly 355 360 365 Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370
375 380 Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390
395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
Ser Arg Trp Gln Glu 405 410
415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430 Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ser Asn Ser 435
440 445 Pro Gln Asn Glu Val Leu Tyr
Gly Asp Val Asn Asp Asp Gly Lys Val 450 455
460 Asn Ser Thr Asp Leu Thr Leu Leu Lys Arg Tyr Val
Leu Lys Ala Val 465 470 475
480 Ser Thr Leu Pro Ser Ser Lys Ala Glu Lys Asn Ala Asp Val Asn Arg
485 490 495 Asp Gly Arg
Val Asn Ser Ser Asp Val Thr Ile Leu Ser Arg Tyr Leu 500
505 510 Ile Arg Val Ile Glu Lys Leu Pro
Ile 515 520 8 1623DNAArtificial
SequenceSynthetic oligonucleotide. 8atggaaaggc actggatctt tctcttcctg
ttttcagtaa ctgcaggtgt ccactcccag 60gtccagcttc agcagtctgg ggctgagctg
gcaaaacctg gggcctcagt gaagatgtcc 120tgcaaggctt ctggctacac ctttactacc
tactggatgc actgggtaaa acagaggcct 180ggacagggtc tggaatggat tggatacatt
aatcctatca ctggttatac tgagtacaat 240cagaagttca aggacaaggc caccttgact
gcagacaaat cctccagcac agcctacatg 300caactgagca gcctgacatc tgaggactct
gcagtctatt actgtgcaag agagggttta 360agtgctatgg actattgggg tcagggaacc
tcagtcaccg tcacctcagc caaaacaacg 420ggcccatccg tcttccccct ggcgccctgc
tccaggagca cctccgagag cacagccgcc 480ctgggctgcc tggtcaagga ctacttcccc
gaaccggtga cggtgtcgtg gaactcaggc 540gccctgacca gcggcgtgca caccttcccg
gctgtcctac agtcctcagg actctactcc 600ctcagcagcg tggtgaccgt gccctccagc
agcttgggca cgaagaccta cacctgcaac 660gtagatcaca agcccagcaa caccaaggtg
gacaagagag ttgagtccaa atatggtccc 720ccatgcccac cctgcccagc acctgagttc
gaagggggac catcagtctt cctgttcccc 780ccaaaaccca aggacactct catgatctcc
cggacccctg aggtcacgtg cgtggtggtg 840gacgtgagcc aggaagaccc cgaggtccag
ttcaactggt acgtggatgg cgtggaggtg 900cataatgcca agacaaagcc gcgggaggag
cagttcaaca gcacgtaccg tgtggtcagc 960gtcctcaccg tcctgcacca ggactggctg
aacggcaagg agtacaagtg caaggtctcc 1020aacaaaggcc tcccgtcctc catcgagaaa
accatctcca aagccaaagg gcagccccga 1080gagccacagg tgtacaccct gcccccatcc
caggaggaga tgaccaagaa ccaggtcagc 1140ctgacctgcc tggtcaaagg cttctacccc
agcgacatcg ccgtggagtg ggagagcaat 1200gggcagccgg agaacaacta caagaccacg
cctcccgtgc tggactccga cggctccttc 1260ttcctctaca gcaggctaac cgtggacaag
agcaggtggc aggaggggaa tgtcttctca 1320tgctccgtga tgcatgaggc tctgcacaac
cactacacac agaagagcct ctccctgtct 1380ctgggtaaag ctagcaattc tcctcaaaat
gaagtactgt acggagatgt gaatgatgac 1440ggaaaagtaa actccactga cttgactttg
ttaaaaagat atgttcttaa agccgtctca 1500actctccctt cttccaaagc tgaaaagaac
gcagatgtaa atcgtgacgg aagagttaat 1560tccagtgatg tcacaatact ttcaagatat
ttgataaggg taatcgagaa attaccaata 1620taa
16239521PRTArtificial SequenceSynthetic
peptide. 9Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala
1 5 10 15 Ser Val
Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr 20
25 30 Trp Met His Trp Val Lys Gln
Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45 Gly Tyr Ile Asn Pro Ile Thr Gly Tyr Thr Glu Tyr
Asn Gln Lys Phe 50 55 60
Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65
70 75 80 Met Gln Leu
Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Glu Gly Leu Ser Ala Met
Asp Tyr Trp Gly Gln Gly Thr Ser 100 105
110 Val Thr Val Thr Ser Ala Lys Thr Thr Gly Pro Ser Val
Phe Pro Leu 115 120 125
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130
135 140 Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150
155 160 Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser 165 170
175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser 180 185 190
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
195 200 205 Thr Lys Val Asp
Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210
215 220 Pro Cys Pro Ala Pro Glu Phe Glu
Gly Gly Pro Ser Val Phe Leu Phe 225 230
235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val 245 250
255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
260 265 270 Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275
280 285 Arg Glu Glu Gln Phe Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr 290 295
300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val 305 310 315
320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335 Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340
345 350 Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly 355 360
365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro 370 375 380
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385
390 395 400 Phe Phe Leu Tyr Ser
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405
410 415 Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His 420 425
430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ser Asn
Ser 435 440 445 Pro
Gln Asn Glu Val Leu Tyr Gly Asp Val Asn Asp Asp Gly Lys Val 450
455 460 Asn Ser Thr Asp Leu Thr
Leu Leu Lys Arg Tyr Val Leu Lys Ala Val 465 470
475 480 Ser Thr Leu Pro Ser Ser Lys Ala Glu Lys Asn
Ala Asp Val Asn Arg 485 490
495 Asp Gly Arg Val Asn Ser Ser Asp Val Thr Ile Leu Ser Arg Tyr Leu
500 505 510 Ile Arg
Val Ile Glu Lys Leu Pro Ile 515 520
10732DNAArtificial SequenceSynthetic oligonucleotide. 10atgcatcgca
ccagcatggg catcaagatg gagtcacaga ttcaggcatt tgtattcgtg 60tttctctggt
tgtctggtgt tggcggagac attgtgatga cccagtctca caaattcatg 120tccacatcag
taggagacag ggtcagcgtc acctgcaagg ccagtcagga tgtgacttct 180gctgtagcct
ggtatcaaca aaaaccaggg caatctccta aactactgat ttactgggca 240tccacccggc
acactggagt ccctgatcgc ttcacaggca gtggatctgg gacagattat 300actctcacca
tcagcagtgg gcaggctgaa gacctggcac tttattactg tcaccaatat 360tatagcgctc
ctcggacgtt cggtggaggc accaagctcg agatcaaacg aactgtggct 420gcaccatctg
tcttcatctt cccgccatct gatgagcagt tgaaatctgg aactgcctct 480gttgtgtgcc
tgctgaataa cttctatccc agagaggcca aagtacagtg gaaggtggat 540aacgccctcc
aatcgggtaa ctcccaggag agtgtcacag agcaggacag caaggacagc 600acctacagcc
tcagcagcac cctgacgctg agcaaagcag actacgagaa acacaaagtc 660tatgcctgcg
aagtcaccca tcagggcctg agctcgcccg tcacaaagag cttcaacagg 720ggagagtgtt
ag
73211214PRTArtificial SequenceSynthetic peptide. 11Asp Ile Val Met Thr
Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5
10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser
Gln Asp Val Thr Ser Ala 20 25
30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu
Ile 35 40 45 Tyr
Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50
55 60 Ser Gly Ser Gly Thr Asp
Tyr Thr Leu Thr Ile Ser Ser Gly Gln Ala 65 70
75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys His Gln Tyr
Tyr Ser Ala Pro Arg 85 90
95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110 Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115
120 125 Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln 145 150 155
160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175 Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180
185 190 Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200
205 Phe Asn Arg Gly Glu Cys 210
121644DNAArtificial SequenceSynthetic oligonucleotide. 12atgggatggt
catgtatcat cctttttcta gtagcaactg caactggagt acattcacag 60gtccaactgc
agcagcctgg ggctgagctg gtgaggcctg ggacttcagt gaagttgtcc 120tgcaaggctt
ctggttacat ctttaccagc tactggatgc actgggtaaa gcagaggcct 180ggacaaggcc
ttgagtggat cggactgatt gatccttctg atagttatag taagtacaat 240caaaagttca
agggcaaggc cacattgact gtagacacat cctccagcac agcctacatg 300cagctcagca
gcctgacatc tgaggactct gcggtctatt actgtgcaag aggggagctc 360agtgacttct
ggggccaagg caccactctc acagtctcct cagccaaaac aacaccccca 420tcagtctatc
cactggcccc tgggtgtgga gatacaactg gttcctctgt gactctggga 480tgcctggtca
agggctactt ccctgagtca gtgactgtga cttggaactc tggatccctg 540tccagcagtg
tgcacacctt cccagctctc ctgcagtctg gactctacac tatgagcagc 600tcagtgactg
tcccctccag cacctggcca agtcagaccg tcacctgcag cgttgctcac 660ccagccagca
gcaccacggt ggacaaaaaa cttgagccca gcgggcccat ttcaacaatc 720aacccctgtc
ctccatgcaa ggagtgtcac aaatgcccag ctcctaacct cgagggtgga 780ccatccgtct
tcatcttccc tccaaatatc aaggatgtac tcatgatctc cctgacaccc 840aaggtcacgt
gtgtggtggt ggatgtgagc gaggatgacc cagacgtccg gatcagctgg 900tttgtgaaca
acgtggaagt acacacagct cagacacaaa cccatagaga ggattacaac 960agtactatcc
gggtggtcag tgccctcccc atccagcacc aggactggat gagtggcaag 1020gagttcaaat
gcaaggtcaa caacaaagac ctcccatcac ccatcgagag aaccatctca 1080aaaattaaag
ggctagtcag agctccacaa gtatacatct tgccgccacc agcagagcag 1140ttgtccagga
aagatgtcag tctcacttgc ctggtcgtgg gcttcaaccc tggagacatc 1200agtgtggagt
ggaccagcaa tgggcataca gaggagaact acaaggacac cgcaccagtc 1260ctggactctg
acggttctta cttcatatac agcaagctcg atataaaaac aagcaagtgg 1320gagaaaacag
attccttctc atgcaacgtg agacacgagg gtctgaaaaa ttactacctg 1380aagaagacca
tctcccggtc tccgggtaaa gctagcaatt ctcctcaaaa tgaagtactg 1440tacggagatg
tgaatgatga cggaaaagta aactccactg acttgacttt gttaaaaaga 1500tatgttctta
aagccgtctc aactctgcct tcttccaaag ctgaaaagaa cgcagatgta 1560aatcgtgacg
gaagagttaa ttccagtgat gtcacaatac tttcaagata tttgataagg 1620gtaatcgaga
aattaccaat ataa
164413528PRTArtificial SequenceSynthetic peptide. 13Gln Val Gln Leu Gln
Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5
10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly
Tyr Ile Phe Thr Ser Tyr 20 25
30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45 Gly
Leu Ile Asp Pro Ser Asp Ser Tyr Ser Lys Tyr Asn Gln Lys Phe 50
55 60 Lys Gly Lys Ala Thr Leu
Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70
75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Glu Leu Ser Asp Phe Trp Gly Gln Gly Thr Thr Leu Thr
100 105 110 Val Ser
Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro 115
120 125 Gly Cys Gly Asp Thr Thr Gly
Ser Ser Val Thr Leu Gly Cys Leu Val 130 135
140 Lys Gly Tyr Phe Pro Glu Ser Val Thr Val Thr Trp
Asn Ser Gly Ser 145 150 155
160 Leu Ser Ser Ser Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu
165 170 175 Tyr Thr Met
Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser 180
185 190 Gln Thr Val Thr Cys Ser Val Ala
His Pro Ala Ser Ser Thr Thr Val 195 200
205 Asp Lys Lys Leu Glu Pro Ser Gly Pro Ile Ser Thr Ile
Asn Pro Cys 210 215 220
Pro Pro Cys Lys Glu Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly 225
230 235 240 Gly Pro Ser Val
Phe Ile Phe Pro Pro Asn Ile Lys Asp Val Leu Met 245
250 255 Ile Ser Leu Thr Pro Lys Val Thr Cys
Val Val Val Asp Val Ser Glu 260 265
270 Asp Asp Pro Asp Val Arg Ile Ser Trp Phe Val Asn Asn Val
Glu Val 275 280 285
His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Ile 290
295 300 Arg Val Val Ser Ala
Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly 305 310
315 320 Lys Glu Phe Lys Cys Lys Val Asn Asn Lys
Asp Leu Pro Ser Pro Ile 325 330
335 Glu Arg Thr Ile Ser Lys Ile Lys Gly Leu Val Arg Ala Pro Gln
Val 340 345 350 Tyr
Ile Leu Pro Pro Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser 355
360 365 Leu Thr Cys Leu Val Val
Gly Phe Asn Pro Gly Asp Ile Ser Val Glu 370 375
380 Trp Thr Ser Asn Gly His Thr Glu Glu Asn Tyr
Lys Asp Thr Ala Pro 385 390 395
400 Val Leu Asp Ser Asp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asp Ile
405 410 415 Lys Thr
Ser Lys Trp Glu Lys Thr Asp Ser Phe Ser Cys Asn Val Arg 420
425 430 His Glu Gly Leu Lys Asn Tyr
Tyr Leu Lys Lys Thr Ile Ser Arg Ser 435 440
445 Pro Gly Lys Ala Ser Asn Ser Pro Gln Asn Glu Val
Leu Tyr Gly Asp 450 455 460
Val Asn Asp Asp Gly Lys Val Asn Ser Thr Asp Leu Thr Leu Leu Lys 465
470 475 480 Arg Tyr Val
Leu Lys Ala Val Ser Thr Leu Pro Ser Ser Lys Ala Glu 485
490 495 Lys Asn Ala Asp Val Asn Arg Asp
Gly Arg Val Asn Ser Ser Asp Val 500 505
510 Thr Ile Leu Ser Arg Tyr Leu Ile Arg Val Ile Glu Lys
Leu Pro Ile 515 520 525
14 2061DNAArtificial SequenceSynthetic oligonucleotide. 14atggatccca
aaggatccct ttcctggaga atacttctgt ttctctccct ggcttttgag 60ttgagctacg
gactcgacga tctggatgca gtaaggatta aagtggacac agtaaatgca 120aaaccgggag
acacagtaag aatacctgta agattcagcg gtataccatc caagggaata 180gcaaactgtg
actttgtata cagctatgac ccgaatgtac ttgagataat agagatagaa 240ccgggagaca
taatagttga cccgaatcct gacaagagct ttgatactgc agtatatcct 300gacagaaaga
taatagtatt cctgtttgca gaagacagcg gaacaggagc gtatgcaata 360actaaagacg
gagtatttgc tacgatagta gcgaaagtaa aagaaggagc acctaacgga 420ctcagtgtaa
tcaaatttgt agaagtaggc ggatttgcga acaatgacct tgtagaacag 480aagacacagt
tctttgacgg tggagtaaat gttggagata caacagaacc tgcaacacct 540acaacacctg
taacaacacc gacaacaaca gatgatctgg atgcactcga gatcatccca 600gttgaggagg
agaacccgga cttctggaac cgcgaggcag ccgaggccct gggtgccgcc 660aagaagctgc
agcctgcaca gacagccgcc aagaacctca tcatcttcct gggcgatggg 720atgggggtgt
ctacggtgac agctgccagg atcctaaaag ggcagaagaa ggacaaactg 780gggcctgagt
tacccctggc catggaccgc ttcccatatg tggctctgtc caagacatac 840aatgtagaca
aacatgtgcc agacagtgga gccacagcca cggcctacct gtgcggggtc 900aagggcaact
tccagaccat tggcttgagt gcagccgccc gctttaacca gtgcaacacg 960acacgcggca
acgaggtcat ctccgtgatg aatcgggcca agaaagcagg gaagtcagtg 1020ggagtggtaa
ccaccacacg agtgcagcac gcctcgccag ccggcaccta cgcccacacg 1080gtgaaccgca
actggtactc ggacgccgac gtgcctgcct cggcccgcca ggaggggtgc 1140caggacatcg
ctacgcagct catctccaac atggacattg acgtgatcct aggtggaggc 1200cgaaagtaca
tgtttcgcat gggaacccca gaccctgagt acccagatga ctacagccaa 1260ggtgggacca
ggctggacgg gaagaatctg gtgcaggaat ggctggcgaa gcgccagggt 1320gcccggtacg
tgtggaaccg cactgagctc atgcaggctt ccctggaccc gtctgtgacc 1380catctcatgg
gtctctttga gcctggagac atgaaatacg agatccaccg agactccaca 1440ctggacccct
ccctgatgga gatgacagag gctgccctgc gcctgctgag caggaacccc 1500cgcggcttct
tcctcttcgt ggagggtggt cgcatcgacc atggtcatca tgaaagcagg 1560gcttaccggg
cactgactga gacgatcatg ttcgacgacg ccattgagag ggcgggccag 1620ctcaccagcg
aggaggacac gctgagcctc gtcactgccg accactccca cgtcttctcc 1680ttcggaggct
accccctgcg agggagctcc atcttcgggc tggcccctgg caaggcccgg 1740gacaggaagg
cctacacggt cctcctatac ggaaacggtc caggctatgt gctcaaggac 1800ggcgcccggc
cggatgttac cgagagcgag agcgggagcc ccgagtatcg gcagcagtca 1860gcagtgcccc
tggacgaaga gacccacgca ggcgaggacg tggcggtgtt cgcgcgcggc 1920ccgcaggcgc
acctggttca cggcgtgcag gagcagacct tcatagcgca cgtcatggcc 1980ttcgccgcct
gcctggagcc ctacaccgcc tgcgacctgg cgccccccgc cggcaccacc 2040caccatcacc
atcaccattg a
206115662PRTArtificial SequenceSynthetic peptide. 15Leu Asp Asp Leu Asp
Ala Val Arg Ile Lys Val Asp Thr Val Asn Ala 1 5
10 15 Lys Pro Gly Asp Thr Val Arg Ile Pro Val
Arg Phe Ser Gly Ile Pro 20 25
30 Ser Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro
Asn 35 40 45 Val
Leu Glu Ile Ile Glu Ile Glu Pro Gly Glu Leu Ile Val Asp Pro 50
55 60 Asn Pro Thr Lys Ser Phe
Asp Thr Ala Val Tyr Pro Asp Arg Lys Met 65 70
75 80 Ile Val Phe Leu Phe Ala Glu Asp Ser Gly Thr
Gly Ala Tyr Ala Ile 85 90
95 Thr Glu Asp Gly Val Phe Ala Thr Ile Val Ala Lys Val Lys Ser Gly
100 105 110 Ala Pro
Asn Gly Leu Ser Val Ile Lys Phe Val Glu Val Gly Gly Phe 115
120 125 Ala Asn Asn Asp Leu Val Glu
Gln Lys Thr Gln Phe Phe Asp Gly Gly 130 135
140 Val Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro
Thr Thr Pro Val 145 150 155
160 Thr Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala Leu Glu Ile Ile Pro
165 170 175 Val Glu Glu
Glu Asn Pro Asp Phe Trp Asn Arg Glu Ala Ala Glu Ala 180
185 190 Leu Gly Ala Ala Lys Lys Leu Gln
Pro Ala Gln Thr Ala Ala Lys Asn 195 200
205 Leu Ile Ile Phe Leu Gly Asp Gly Met Gly Val Ser Thr
Val Thr Ala 210 215 220
Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp Lys Leu Gly Pro Glu Leu 225
230 235 240 Pro Leu Ala Met
Asp Arg Phe Pro Tyr Val Ala Leu Ser Lys Thr Tyr 245
250 255 Asn Val Asp Lys His Val Pro Asp Ser
Gly Ala Thr Ala Thr Ala Tyr 260 265
270 Leu Cys Gly Val Lys Gly Asn Phe Gln Thr Ile Gly Leu Ser
Ala Ala 275 280 285
Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg Gly Asn Glu Val Ile Ser 290
295 300 Val Met Asn Arg Ala
Lys Lys Ala Gly Lys Ser Val Gly Val Val Thr 305 310
315 320 Thr Thr Arg Val Gln His Ala Ser Pro Ala
Gly Thr Tyr Ala His Thr 325 330
335 Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp Val Pro Ala Ser Ala
Arg 340 345 350 Gln
Glu Gly Cys Gln Asp Ile Ala Thr Gln Leu Ile Ser Asn Met Asp 355
360 365 Ile Asp Val Ile Leu Gly
Gly Gly Arg Lys Tyr Met Phe Arg Met Gly 370 375
380 Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr Ser
Gln Gly Gly Thr Arg 385 390 395
400 Leu Asp Gly Lys Asn Leu Val Gln Glu Trp Leu Ala Lys Arg Gln Gly
405 410 415 Ala Arg
Tyr Val Trp Asn Arg Thr Glu Leu Met Gln Ala Ser Leu Asp 420
425 430 Pro Ser Val Thr His Leu Met
Gly Leu Phe Glu Pro Gly Asp Met Lys 435 440
445 Tyr Glu Ile His Arg Asp Ser Thr Leu Asp Pro Ser
Leu Met Glu Met 450 455 460
Thr Glu Ala Ala Leu Arg Leu Leu Ser Arg Asn Pro Arg Gly Phe Phe 465
470 475 480 Leu Phe Val
Glu Gly Gly Arg Ile Asp His Gly His His Glu Ser Arg 485
490 495 Ala Tyr Arg Ala Leu Thr Glu Thr
Ile Met Phe Asp Asp Ala Ile Glu 500 505
510 Arg Ala Gly Gln Leu Thr Ser Glu Glu Asp Thr Leu Ser
Leu Val Thr 515 520 525
Ala Asp His Ser His Val Phe Ser Phe Gly Gly Tyr Pro Leu Arg Gly 530
535 540 Ser Ser Ile Phe
Gly Leu Ala Pro Gly Lys Ala Arg Asp Arg Lys Ala 545 550
555 560 Tyr Thr Val Leu Leu Tyr Gly Asn Gly
Pro Gly Tyr Val Leu Lys Asp 565 570
575 Gly Ala Arg Pro Asp Val Thr Glu Ser Glu Ser Gly Ser Pro
Glu Tyr 580 585 590
Arg Gln Gln Ser Ala Val Pro Leu Asp Glu Glu Thr His Ala Gly Glu
595 600 605 Asp Val Ala Val
Phe Ala Arg Gly Pro Gln Ala His Leu Val His Gly 610
615 620 Val Gln Glu Gln Thr Phe Ile Ala
His Val Met Ala Phe Ala Ala Cys 625 630
635 640 Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala Pro Pro
Ala Gly Thr Thr 645 650
655 His His His His His His 660 16
2556DNAArtificial SequenceSynthetic oligonucleotide. 16atggatccca
aaggatccct ttcctggaga atacttctgt ttctctccct ggcttttgag 60ttgagctacg
gactcgacga tctggatgca gtaaggatta aagtggacac agtaaatgca 120aaaccgggag
acacagtaag aatacctgta agattcagcg gtataccatc caagggaata 180gcaaactgtg
actttgtata cagctatgac ccgaatgtac ttgagataat agagataaaa 240ccgggagaat
tgatagttga cccgaatcct gacaagagct ttgatactgc agtatatcct 300gacagaaaga
taatagtatt cctgtttgca gaagacagcg gaacaggagc gtatgcaata 360actaaagacg
gagtatttgc tacgatagta gcgaaagtaa aatccggagc acctaacgga 420ctcagtgtaa
tcaaatttgt agaagtaggc ggatttgcga ataatgacct tgtagaacag 480aagacacagt
tctttgacgg tggagtaaat gttggagata caacagaacc tgcaacacct 540acaacacctg
taacaacacc gacaacaaca gatgatctgg atgcagtaag gattaaagtg 600gacacagtaa
atgcaaaacc gggagacaca gtaaatatac ctgtaagatt cagtggtata 660ccatccaagg
gaatagcaaa ctgtgacttt gtatacagct atgacccgaa tgtacttgag 720ataatagaga
taaaaccggg agaattgata gttgacccga atcctaccaa gagctttgat 780actgcagtat
atcctgacag aaagatgata gtattcctgt ttgcggaaga cagcggaaca 840ggagcgtatg
caataactaa agacggagta tttgctacga tagtagcgaa agtaaaagaa 900ggagcaccta
acggactcag tgtaatcaaa tttgtagaag taggcggatt tgcgaacaat 960gaccttgtag
aacagaagac acagttcttt gacggtggag taaatgttgg agatacaaca 1020gaacctgcaa
cacctacaac acctgtaaca acaccgacaa caacagatga tctggatgca 1080ctcgagatca
tcccagttga ggaggagaac ccggacttct ggaaccgcga ggcagccgag 1140gccctgggtg
ccgccaagaa gctgcagcct gcacagacag ccgccaagaa cctcatcatc 1200ttcctgggcg
atgggatggg ggtgtctacg gtgacagctg ccaggatcct aaaagggcag 1260aagaaggaca
aactggggcc tgagttaccc ctggccatgg accgcttccc atatgtggct 1320ctgtccaaga
catacaatgt agacaaacat gtgccagaca gtggagccac agccacggcc 1380tacctgtgcg
gggtcaaggg caacttccag accattggct tgagtgcagc cgcccgcttt 1440aaccagtgca
acacgacacg cggcaacgag gtcatctccg tgatgaatcg ggccaagaaa 1500gcagggaagt
cagtgggagt ggtaaccacc acacgagtgc agcacgcctc gccagccggc 1560acctacgccc
acacggtgaa ccgcaactgg tactcggacg ccgacgtgcc tgcctcggcc 1620cgccaggagg
ggtgccagga catcgctacg cagctcatct ccaacatgga cattgacgtg 1680atcctaggtg
gaggccgaaa gtacatgttt cgcatgggaa ccccagaccc tgagtaccca 1740gatgactaca
gccaaggtgg gaccaggctg gacgggaaga atctggtgca ggaatggctg 1800gcgaagcgcc
agggtgcccg gtacgtgtgg aaccgcactg agctcatgca ggcttccctg 1860gacccgtctg
tgacccatct catgggtctc tttgagcctg gagacatgaa atacgagatc 1920caccgagact
ccacactgga cccctccctg atggagatga cagaggctgc cctgcgcctg 1980ctgagcagga
acccccgcgg cttcttcctc ttcgtggagg gtggtcgcat cgaccatggt 2040catcatgaaa
gcagggctta ccgggcactg actgagacga tcatgttcga cgacgccatt 2100gagagggcgg
gccagctcac cagcgaggag gacacgctga gcctcgtcac tgccgaccac 2160tcccacgtct
tctccttcgg aggctacccc ctgcgaggga gctccatctt cgggctggcc 2220cctggcaagg
cccgggacag gaaggcctac acggtcctcc tatacggaaa cggtccaggc 2280tatgtgctca
aggacggcgc ccggccggat gttaccgaga gcgagagcgg gagccccgag 2340tatcggcagc
agtcagcagt gcccctggac gaagagaccc acgcaggcga ggacgtggcg 2400gtgttcgcgc
gcggcccgca ggcgcacctg gttcacggcg tgcaggagca gaccttcata 2460gcgcacgtca
tggccttcgc cgcctgcctg gagccctaca ccgcctgcga cctggcgccc 2520cccgccggca
ccacccacca tcaccatcac cattga
255617826PRTArtificial SequenceSynthetic peptide. 17Leu Asp Leu Asp Ala
Val Arg Ile Lys Val Asp Thr Val Asn Ala Lys 1 5
10 15 Pro Gly Asp Thr Val Arg Ile Pro Val Arg
Phe Ser Gly Ile Pro Ser 20 25
30 Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn
Val 35 40 45 Leu
Glu Ile Ile Glu Ile Lys Pro Gly Glu Leu Ile Val Asp Pro Asn 50
55 60 Pro Asp Lys Ser Phe Asp
Thr Ala Val Tyr Pro Asp Arg Lys Ile Ile 65 70
75 80 Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly
Ala Tyr Ala Ile Thr 85 90
95 Lys Asp Gly Val Phe Ala Thr Ile Val Ala Lys Val Lys Ser Gly Ala
100 105 110 Pro Asn
Gly Leu Ser Val Ile Lys Phe Val Glu Val Gly Gly Phe Ala 115
120 125 Asn Asn Asp Leu Val Glu Gln
Lys Thr Gln Phe Phe Asp Gly Gly Val 130 135
140 Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro Thr
Thr Pro Val Thr 145 150 155
160 Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala Val Arg Ile Lys Val Asp
165 170 175 Thr Val Asn
Ala Lys Pro Gly Asp Thr Val Asn Ile Pro Val Arg Phe 180
185 190 Ser Gly Ile Pro Ser Lys Gly Ile
Ala Asn Cys Asp Phe Val Tyr Ser 195 200
205 Tyr Asp Pro Asn Val Leu Glu Ile Ile Glu Ile Lys Pro
Gly Glu Leu 210 215 220
Ile Val Asp Pro Asn Pro Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro 225
230 235 240 Asp Arg Lys Met
Ile Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly 245
250 255 Ala Tyr Ala Ile Thr Lys Asp Gly Val
Phe Ala Thr Ile Val Ala Lys 260 265
270 Val Lys Glu Gly Ala Pro Asn Gly Leu Ser Val Ile Lys Phe
Val Glu 275 280 285
Val Gly Gly Phe Ala Asn Asn Asp Leu Val Glu Gln Lys Thr Gln Phe 290
295 300 Phe Asp Gly Gly Val
Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro 305 310
315 320 Thr Thr Pro Val Thr Thr Pro Thr Thr Thr
Asp Asp Leu Asp Ala Leu 325 330
335 Glu Ile Ile Pro Val Glu Glu Glu Asn Pro Asp Phe Trp Asn Arg
Glu 340 345 350 Ala
Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu Gln Pro Ala Gln Thr 355
360 365 Ala Ala Lys Asn Leu Ile
Ile Phe Leu Gly Asp Gly Met Gly Val Ser 370 375
380 Thr Val Thr Ala Ala Arg Ile Leu Lys Gly Gln
Lys Lys Asp Lys Leu 385 390 395
400 Gly Pro Glu Leu Pro Leu Ala Met Asp Arg Phe Pro Tyr Val Ala Leu
405 410 415 Ser Lys
Thr Tyr Asn Val Asp Lys His Val Pro Asp Ser Gly Ala Thr 420
425 430 Ala Thr Ala Tyr Leu Cys Gly
Val Lys Gly Asn Phe Gln Thr Ile Gly 435 440
445 Leu Ser Ala Ala Ala Arg Phe Asn Gln Cys Asn Thr
Thr Arg Gly Asn 450 455 460
Glu Val Ile Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys Ser Val 465
470 475 480 Gly Val Val
Thr Thr Thr Arg Val Gln His Ala Ser Pro Ala Gly Thr 485
490 495 Tyr Ala His Thr Val Asn Arg Asn
Trp Tyr Ser Asp Ala Asp Val Pro 500 505
510 Ala Ser Ala Arg Gln Glu Gly Cys Gln Asp Ile Ala Thr
Gln Leu Ile 515 520 525
Ser Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys Tyr Met 530
535 540 Phe Arg Met Gly
Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr Ser Gln 545 550
555 560 Gly Gly Thr Arg Leu Asp Gly Lys Asn
Leu Val Gln Glu Trp Leu Ala 565 570
575 Lys Arg Gln Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu
Met Gln 580 585 590
Ala Ser Leu Asp Pro Ser Val Thr His Leu Met Gly Leu Phe Glu Pro
595 600 605 Gly Asp Met Lys
Tyr Glu Ile His Arg Asp Ser Thr Leu Asp Pro Ser 610
615 620 Leu Met Glu Met Thr Glu Ala Ala
Leu Arg Leu Leu Ser Arg Asn Pro 625 630
635 640 Arg Gly Phe Phe Leu Phe Val Glu Gly Gly Arg Ile
Asp His Gly His 645 650
655 His Glu Ser Arg Ala Tyr Arg Ala Leu Thr Glu Thr Ile Met Phe Asp
660 665 670 Asp Ala Ile
Glu Arg Ala Gly Gln Leu Thr Ser Glu Glu Asp Thr Leu 675
680 685 Ser Leu Val Thr Ala Asp His Ser
His Val Phe Ser Phe Gly Gly Tyr 690 695
700 Pro Leu Arg Gly Ser Ser Ile Phe Gly Leu Ala Pro Gly
Lys Ala Arg 705 710 715
720 Asp Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro Gly Tyr
725 730 735 Val Leu Lys Asp
Gly Ala Arg Pro Asp Val Thr Glu Ser Glu Ser Gly 740
745 750 Ser Pro Glu Tyr Arg Gln Gln Ser Ala
Val Pro Leu Asp Glu Glu Thr 755 760
765 His Ala Gly Glu Asp Val Ala Val Phe Ala Arg Gly Pro Gln
Ala His 770 775 780
Leu Val His Gly Val Gln Glu Gln Thr Phe Ile Ala His Val Met Ala 785
790 795 800 Phe Ala Ala Cys Leu
Glu Pro Tyr Thr Ala Cys Asp Leu Ala Pro Pro 805
810 815 Ala Gly Thr Thr His His His His His His
820 825 18 1326DNAArtificial
SequenceSynthetic oligonucleotide. 18atggatccca aaggatccct ttcctggaga
atacttctgt ttctctccct ggcttttgag 60ttgagctacg gactcgacga tctggatgca
gtaaggatta aagtggacac agtaaatgca 120aaaccgggag acacagtaag aatacctgta
agattcagcg gtataccatc caagggaata 180gcaaactgtg actttgtata cagctatgac
ccgaatgtac ttgagataat agagatagaa 240ccgggagaca taatagttga cccgaatcct
gacaagagct ttgatactgc agtatatcct 300gacagaaaga taatagtatt cctgtttgca
gaagacagcg gaacaggagc gtatgcaata 360actaaagacg gagtatttgc tacgatagta
gcgaaagtaa aagaaggagc acctaacgga 420ctcagtgtaa tcaaatttgt agaagtaggc
ggatttgcga acaatgacct tgtagaacag 480aagacacagt tctttgacgg tggagtaaat
gttggagata caacagaacc tgcaacacct 540acaacacctg taacaacacc gacaacaaca
gatgatctgg atgcactcga ggcgcccctc 600atcctgtctc ggattgtggg aggctgggag
tgcgagaagc attcccaacc ctggcaggtg 660cttgtggcct ctcgtggcag ggcagtctgc
ggcggtgttc tggtgcaccc ccagtgggtc 720ctcacagctg cccactgcat caggaacaaa
agcgtgatct tgctgggtcg gcacagcctg 780tttcatcctg aagacacagg ccaggtattt
caggtcagcc acagcttccc acacccgctc 840tacgatatga gcctcctgaa gaatcgattc
ctcaggccag gtgatgactc cagccacgac 900ctcatgctgc tccgcctgtc agagcctgcc
gagctcacgg atgctgtgaa ggtcatggac 960ctgcccaccc aggagccagc actggggacc
acctgctacg cctcaggctg gggcagcatt 1020gaaccagagg agttcttgac cccaaagaaa
cttcagtgtg tggacctcca tgttatttcc 1080aatgacgtgt gcgcgcaagt tcaccctcag
aaggtgacca agttcatgct gtgtgctgga 1140cgctggacag ggggcaaaag cacctgctcg
ggtgattctg ggggcccact tgtctgtaat 1200ggtgtgcttc aaggtatcac gtcatggggc
agtgaaccat gtgccctgcc cgaaaggcct 1260tccctgtaca ccaaggtggt gcattaccgg
aagtggatca aggacaccat cgtggccaac 1320ccctga
132619417PRTArtificial SequenceSynthetic
peptide. 19Leu Asp Asp Leu Asp Ala Val Arg Ile Lys Val Asp Thr Val Asn
Ala 1 5 10 15 Lys
Pro Gly Asp Thr Val Arg Ile Pro Val Arg Phe Ser Gly Ile Pro
20 25 30 Ser Lys Gly Ile Ala
Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn 35
40 45 Val Leu Glu Ile Ile Glu Ile Glu Pro
Gly Glu Leu Ile Val Asp Pro 50 55
60 Asn Pro Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro Asp
Arg Lys Met 65 70 75
80 Ile Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly Ala Tyr Ala Ile
85 90 95 Thr Glu Asp Gly
Val Phe Ala Thr Ile Val Ala Lys Val Lys Ser Gly 100
105 110 Ala Pro Asn Gly Leu Ser Val Ile Lys
Phe Val Glu Val Gly Gly Phe 115 120
125 Ala Asn Asn Asp Leu Val Glu Gln Lys Thr Gln Phe Phe Asp
Gly Gly 130 135 140
Val Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro Val 145
150 155 160 Thr Thr Pro Thr Thr
Thr Asp Asp Leu Asp Ala Leu Glu Ala Pro Leu 165
170 175 Ile Leu Ser Arg Ile Val Gly Gly Trp Glu
Cys Glu Lys His Ser Gln 180 185
190 Pro Trp Gln Val Leu Val Ala Ser Arg Gly Arg Ala Val Cys Gly
Gly 195 200 205 Val
Leu Val His Pro Gln Trp Val Leu Thr Ala Ala His Cys Ile Arg 210
215 220 Asn Lys Ser Val Ile Leu
Leu Gly Arg His Ser Leu Phe His Pro Glu 225 230
235 240 Asp Thr Gly Gln Val Phe Gln Val Ser His Ser
Phe Pro His Pro Leu 245 250
255 Tyr Asp Met Ser Leu Leu Lys Asn Arg Phe Leu Arg Pro Gly Asp Asp
260 265 270 Ser Ser
His Asp Leu Met Leu Leu Arg Leu Ser Glu Pro Ala Glu Leu 275
280 285 Thr Asp Ala Val Lys Val Met
Asp Leu Pro Thr Gln Glu Pro Ala Leu 290 295
300 Gly Thr Thr Cys Tyr Ala Ser Gly Trp Gly Ser Ile
Glu Pro Glu Glu 305 310 315
320 Phe Leu Thr Pro Lys Lys Leu Gln Cys Val Asp Leu His Val Ile Ser
325 330 335 Asn Asp Val
Cys Ala Gln Val His Pro Gln Lys Val Thr Lys Phe Met 340
345 350 Leu Cys Ala Gly Arg Trp Thr Gly
Gly Lys Ser Thr Cys Ser Gly Asp 355 360
365 Ser Gly Gly Pro Leu Val Cys Asn Gly Val Leu Gln Gly
Ile Thr Ser 370 375 380
Trp Gly Ser Glu Pro Cys Ala Leu Pro Glu Arg Pro Ser Leu Tyr Thr 385
390 395 400 Lys Val Val His
Tyr Arg Lys Trp Ile Lys Asp Thr Ile Val Ala Asn 405
410 415 Pro 201554DNAArtificial
SequenceSynthetic oligonucleotide. 20atggatccca aaggatccct ttcctggaga
atacttctgt ttctctccct ggcttttgag 60ttgagctacg gactcgacga tctggatgca
gtaaggatta aagtggacac agtaaatgca 120aaaccgggag acacagtaag aatacctgta
agattcagcg gtataccatc caagggaata 180gcaaactgtg actttgtata cagctatgac
ccgaatgtac ttgagataat agagatagaa 240ccgggagaca taatagttga cccgaatcct
gacaagagct ttgatactgc agtatatcct 300gacagaaaga taatagtatt cctgtttgca
gaagacagcg gaacaggagc gtatgcaata 360actaaagacg gagtatttgc tacgatagta
gcgaaagtaa aagaaggagc acctaacgga 420ctcagtgtaa tcaaatttgt agaagtaggc
ggatttgcga acaatgacct tgtagaacag 480aagacacagt tctttgacgg tggagtaaat
gttggagata caacagaacc tgcaacacct 540acaacacctg taacaacacc gacaacaaca
gatgatctgg atgcactcga ggatcagatt 600tgcattggtt accatgcaaa caactcgaca
gagcaggttg acacaataat ggaaaagaac 660gttactgtta cacatgccca agacatactg
gaaaagaaac acaacgggaa gctctgcgat 720ctagatggag tgaagcctct aattttgaga
gattgtagcg tagctggatg gctcctcgga 780aacccaatgt gtgacgaatt catcaatgtg
ccggaatggt cttacatagt ggagaaggcc 840aatccagtca atgacctctg ttacccaggg
gatttcaatg actatgaaaa attgaaacac 900ctattgagca gaataaacca ttttgagaaa
attcagatca tccccaaaag ttcttggtcc 960agtcatgaag cctcattagg ggtgagctca
gcatgtccat accagggaaa gtcctccttt 1020ttcagaaatg tggtatggct tatcaaaaag
aacagtacat acccaacaat aaagaggagc 1080tacaataata ccaaccaaga agatcttttg
gtactgtggg ggattcacca tcctaatgat 1140gcggcagagc agacaaagct ctatcaaaac
ccaaccacct atatttccgt tgggacatca 1200acactaaacc agagattggt accaagaata
gctactagat ccaaagtaaa cgggcaaagt 1260ggaaggatgg agttcttctg gacaatttta
aagccgaatg atgcaatcaa cttcgagagt 1320aatggaaatt tcattgctcc agaatatgca
tacaaaattg tcaagaaagg ggactcaaca 1380attatgaaaa gtgaattgga atatggtaac
tgcaacacca agtgtcaaac tccaatgggg 1440gcgataaact ctagcatgcc attccacaat
atacaccctc tcaccattgg ggaatgcccc 1500aaatatgtga aatcaaacag attagtcctt
gcgcaccatc accatcacca ttga 155421493PRTArtificial
SequenceSynthetic peptide. 21Leu Asp Asp Leu Asp Ala Val Arg Ile Lys Val
Asp Thr Val Asn Ala 1 5 10
15 Lys Pro Gly Asp Thr Val Arg Ile Pro Val Arg Phe Ser Gly Ile Pro
20 25 30 Ser Lys
Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn 35
40 45 Val Leu Glu Ile Ile Glu Ile
Glu Pro Gly Glu Leu Ile Val Asp Pro 50 55
60 Asn Pro Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro
Asp Arg Lys Met 65 70 75
80 Ile Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly Ala Tyr Ala Ile
85 90 95 Thr Glu Asp
Gly Val Phe Ala Thr Ile Val Ala Lys Val Lys Ser Gly 100
105 110 Ala Pro Asn Gly Leu Ser Val Ile
Lys Phe Val Glu Val Gly Gly Phe 115 120
125 Ala Asn Asn Asp Leu Val Glu Gln Lys Thr Gln Phe Phe
Asp Gly Gly 130 135 140
Val Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro Val 145
150 155 160 Thr Thr Pro Thr
Thr Thr Asp Asp Leu Asp Ala Leu Glu Asp Gln Ile 165
170 175 Cys Ile Gly Tyr His Ala Asn Asn Ser
Thr Glu Gln Val Asp Thr Ile 180 185
190 Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile Leu
Glu Lys 195 200 205
Lys His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys Pro Leu Ile 210
215 220 Leu Arg Asp Cys Ser
Val Ala Gly Trp Leu Leu Gly Asn Pro Met Cys 225 230
235 240 Asp Glu Phe Ile Asn Val Pro Glu Trp Ser
Tyr Ile Val Glu Lys Ala 245 250
255 Asn Pro Val Asn Asp Leu Cys Tyr Pro Gly Asp Phe Asn Asp Tyr
Glu 260 265 270 Lys
Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu Lys Ile Gln 275
280 285 Ile Ile Pro Lys Ser Ser
Trp Ser Ser His Glu Ala Ser Leu Gly Val 290 295
300 Ser Ser Ala Cys Pro Tyr Gln Gly Lys Ser Ser
Phe Phe Arg Asn Val 305 310 315
320 Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr Ile Lys Arg Ser
325 330 335 Tyr Asn
Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp Gly Ile His 340
345 350 His Pro Asn Asp Ala Ala Glu
Gln Thr Lys Leu Tyr Gln Asn Pro Thr 355 360
365 Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln
Arg Leu Val Pro 370 375 380
Arg Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly Arg Met Glu 385
390 395 400 Phe Phe Trp
Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn Phe Glu Ser 405
410 415 Asn Gly Asn Phe Ile Ala Pro Glu
Tyr Ala Tyr Lys Ile Val Lys Lys 420 425
430 Gly Asp Ser Thr Ile Met Lys Ser Glu Leu Glu Tyr Gly
Asn Cys Asn 435 440 445
Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser Met Pro Phe 450
455 460 His Asn Ile His
Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys 465 470
475 480 Ser Asn Arg Leu Val Leu Ala His His
His His His His 485 490
221293DNAArtificial SequenceSynthetic oligonucleotide. 22atggatctgg
atgcagtaag gattaaagtg gacacagtaa atgcaaaacc gggagacaca 60gtaaatatac
ctgtaagatt cagtggtata ccatccaagg gaatagcaaa ctgtgacttt 120gtatacagct
atgacccgaa tgtacttgag ataatagaga taaaaccggg agaattgata 180gttgacccga
atcctaccaa gagctttgat actgcagtat atcctgacag aaagatgata 240gtattcctgt
ttgcggaaga cagcggaaca ggagcgtatg caataactaa agacggagta 300tttgctacga
tagtagcgaa agtaaaagaa ggagcaccta acgggctcag tgtaatcaaa 360tttgtagaag
taggcggatt tgcgaacaat gaccttgtag aacagaagac acagttcttt 420gacggtggag
taaatgttgg agatacaaca gaacctgcaa cacctacaac acctgtaaca 480acaccgacaa
caacagatga tctggatgca gctagccttc taaccgaggt cgaaacgtac 540gttctctcta
tcatcccgtc aggccccctc aaagccgaga tcgcacagag acttgaagat 600gtctttgcag
ggaagaacac cgatcttgag gttctcatgg aatggctaaa gacaagacca 660atcctgtcac
ctctgactaa ggggatttta ggatttgtgt tcacgctcac cgtgcccagt 720gagcggggac
tgcagcgtag acgctttgtc caaaatgctc ttaatgggaa cggagatcca 780aataacatgg
acaaagcagt taaactgtat aggaagctta agagggagat aacattccat 840ggggccaaag
aaatagcact cagttattct gctggtgcac ttgccagttg tatgggcctc 900atatacaaca
ggatgggggc tgtgaccact gaagtggcat ttggcctggt atgcgcaacc 960tgtgaacaga
ttgctgactc ccagcatcgg tctcataggc aaatggtgac aacaaccaat 1020ccactaatca
gacatgagaa cagaatggtt ctagccagca ctacagctaa ggctatggag 1080caaatggctg
gatcgagtga gcaagcagca gaggccatgg atattgctag tcaggccagg 1140caaatggtgc
aggcgatgag aaccattggg actcatccta gctccagtgc tggtctaaaa 1200gatgatcttc
ttgaaaattt gcaggcttac cagaaacgga tgggggtgca gatgcagcga 1260ttcaagctcg
agcaccacca ccaccaccac tga
129323430PRTArtificial SequenceSynthetic peptide. 23Met Asp Leu Asp Ala
Val Arg Ile Lys Val Asp Thr Val Asn Ala Lys 1 5
10 15 Pro Gly Asp Thr Val Asn Ile Pro Val Arg
Phe Ser Gly Ile Pro Ser 20 25
30 Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn
Val 35 40 45 Leu
Glu Ile Ile Glu Ile Lys Pro Gly Glu Leu Ile Val Asp Pro Asn 50
55 60 Pro Thr Lys Ser Phe Asp
Thr Ala Val Tyr Pro Asp Arg Lys Met Ile 65 70
75 80 Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly
Ala Tyr Ala Ile Thr 85 90
95 Lys Asp Gly Val Phe Ala Thr Ile Val Ala Lys Val Lys Glu Gly Ala
100 105 110 Pro Asn
Gly Leu Ser Val Ile Lys Phe Val Glu Val Gly Gly Phe Ala 115
120 125 Asn Asn Asp Leu Val Glu Gln
Lys Thr Gln Phe Phe Asp Gly Gly Val 130 135
140 Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro Thr
Thr Pro Val Thr 145 150 155
160 Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala Ala Ser Leu Leu Thr Glu
165 170 175 Val Glu Thr
Tyr Val Leu Ser Ile Ile Pro Ser Gly Pro Leu Lys Ala 180
185 190 Glu Ile Ala Gln Arg Leu Glu Asp
Val Phe Ala Gly Lys Asn Thr Asp 195 200
205 Leu Glu Val Leu Met Glu Trp Leu Lys Thr Arg Pro Ile
Leu Ser Pro 210 215 220
Leu Thr Lys Gly Ile Leu Gly Phe Val Phe Thr Leu Thr Val Pro Ser 225
230 235 240 Glu Arg Gly Leu
Gln Arg Arg Arg Phe Val Gln Asn Ala Leu Asn Gly 245
250 255 Asn Gly Asp Pro Asn Asn Met Asp Lys
Ala Val Lys Leu Tyr Arg Lys 260 265
270 Leu Lys Arg Glu Ile Thr Phe His Gly Ala Lys Glu Ile Ala
Leu Ser 275 280 285
Tyr Ser Ala Gly Ala Leu Ala Ser Cys Met Gly Leu Ile Tyr Asn Arg 290
295 300 Met Gly Ala Val Thr
Thr Glu Val Ala Phe Gly Leu Val Cys Ala Thr 305 310
315 320 Cys Glu Gln Ile Ala Asp Ser Gln His Arg
Ser His Arg Gln Met Val 325 330
335 Thr Thr Thr Asn Pro Leu Ile Arg His Glu Asn Arg Met Val Leu
Ala 340 345 350 Ser
Thr Thr Ala Lys Ala Met Glu Gln Met Ala Gly Ser Ser Glu Gln 355
360 365 Ala Ala Glu Ala Met Asp
Ile Ala Ser Gln Ala Arg Gln Met Val Gln 370 375
380 Ala Met Arg Thr Ile Gly Thr His Pro Ser Ser
Ser Ala Gly Leu Lys 385 390 395
400 Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr Gln Lys Arg Met Gly Val
405 410 415 Gln Met
Gln Arg Phe Lys Leu Glu His His His His His His 420
425 430 249PRTArtificial SequenceSynthetic peptide.
24Gly Ile Leu Gly Phe Val Phe Thr Leu 1 5
25661PRTArtificial SequenceSynthetic peptide. 25Met Asp Leu Val Leu Lys
Arg Cys Leu Leu His Leu Ala Val Ile Gly 1 5
10 15 Ala Leu Leu Ala Val Gly Ala Thr Lys Val Pro
Arg Asn Gln Asp Trp 20 25
30 Leu Gly Val Ser Arg Gln Leu Arg Thr Lys Ala Trp Asn Arg Gln
Leu 35 40 45 Tyr
Pro Glu Trp Thr Glu Ala Gln Arg Leu Asp Cys Trp Arg Gly Gly 50
55 60 Gln Val Ser Leu Lys Val
Ser Asn Asp Gly Pro Thr Leu Ile Gly Ala 65 70
75 80 Asn Ala Ser Phe Ser Ile Ala Leu Asn Phe Pro
Gly Ser Gln Lys Val 85 90
95 Leu Pro Asp Gly Gln Val Ile Trp Val Asn Asn Thr Ile Ile Asn Gly
100 105 110 Ser Gln
Val Trp Gly Gly Gln Pro Val Tyr Pro Gln Glu Thr Asp Asp 115
120 125 Ala Cys Ile Phe Pro Asp Gly
Gly Pro Cys Pro Ser Gly Ser Trp Ser 130 135
140 Gln Lys Arg Ser Phe Val Tyr Val Trp Lys Thr Trp
Gly Gln Tyr Trp 145 150 155
160 Gln Val Leu Gly Gly Pro Val Ser Gly Leu Ser Ile Gly Thr Gly Arg
165 170 175 Ala Met Leu
Gly Thr His Thr Met Glu Val Thr Val Tyr His Arg Arg 180
185 190 Gly Ser Arg Ser Tyr Val Pro Leu
Ala His Ser Ser Ser Ala Phe Thr 195 200
205 Ile Thr Asp Gln Val Pro Phe Ser Val Ser Val Ser Gln
Leu Arg Ala 210 215 220
Leu Asp Gly Gly Asn Lys His Phe Leu Arg Asn Gln Pro Leu Thr Phe 225
230 235 240 Ala Leu Gln Leu
His Asp Pro Ser Gly Tyr Leu Ala Glu Ala Asp Leu 245
250 255 Ser Tyr Thr Trp Asp Phe Gly Asp Ser
Ser Gly Thr Leu Ile Ser Arg 260 265
270 Ala Leu Val Val Thr His Thr Tyr Leu Glu Pro Gly Pro Val
Thr Ala 275 280 285
Gln Val Val Leu Gln Ala Ala Ile Pro Leu Thr Ser Cys Gly Ser Ser 290
295 300 Pro Val Pro Gly Thr
Thr Asp Gly His Arg Pro Thr Ala Glu Ala Pro 305 310
315 320 Asn Thr Thr Ala Gly Gln Val Pro Thr Thr
Glu Val Val Gly Thr Thr 325 330
335 Pro Gly Gln Ala Pro Thr Ala Glu Pro Ser Gly Thr Thr Ser Val
Gln 340 345 350 Val
Pro Thr Thr Glu Val Ile Ser Thr Ala Pro Val Gln Met Pro Thr 355
360 365 Ala Glu Ser Thr Gly Met
Thr Pro Glu Lys Val Pro Val Ser Glu Val 370 375
380 Met Gly Thr Thr Leu Ala Glu Met Ser Thr Pro
Glu Ala Thr Gly Met 385 390 395
400 Thr Pro Ala Glu Val Ser Ile Val Val Leu Ser Gly Thr Thr Ala Ala
405 410 415 Gln Val
Thr Thr Thr Glu Trp Val Glu Thr Thr Ala Arg Glu Leu Pro 420
425 430 Ile Pro Glu Pro Glu Gly Pro
Asp Ala Ser Ser Ile Met Ser Thr Glu 435 440
445 Ser Ile Thr Gly Ser Leu Gly Pro Leu Leu Asp Gly
Thr Ala Thr Leu 450 455 460
Arg Leu Val Lys Arg Gln Val Pro Leu Asp Cys Val Leu Tyr Arg Tyr 465
470 475 480 Gly Ser Phe
Ser Val Thr Leu Asp Ile Val Gln Gly Ile Glu Ser Ala 485
490 495 Glu Ile Leu Gln Ala Val Pro Ser
Gly Glu Gly Asp Ala Phe Glu Leu 500 505
510 Thr Val Ser Cys Gln Gly Gly Leu Pro Lys Glu Ala Cys
Met Glu Ile 515 520 525
Ser Ser Pro Gly Cys Gln Pro Pro Ala Gln Arg Leu Cys Gln Pro Val 530
535 540 Leu Pro Ser Pro
Ala Cys Gln Leu Val Leu His Gln Ile Leu Lys Gly 545 550
555 560 Gly Ser Gly Thr Tyr Cys Leu Asn Val
Ser Leu Ala Asp Thr Asn Ser 565 570
575 Leu Ala Val Val Ser Thr Gln Leu Ile Met Pro Gly Gln Glu
Ala Gly 580 585 590
Leu Gly Gln Val Pro Leu Ile Val Gly Ile Leu Leu Val Leu Met Ala
595 600 605 Val Val Leu Ala
Ser Leu Ile Tyr Arg Arg Arg Leu Met Lys Gln Asp 610
615 620 Phe Ser Val Pro Gln Leu Pro His
Ser Ser Ser His Trp Leu Arg Leu 625 630
635 640 Pro Arg Ile Phe Cys Ser Cys Pro Ile Gly Glu Asn
Ser Pro Leu Leu 645 650
655 Ser Gly Gln Gln Val 660 269PRTArtificial
SequenceSynthetic peptide. 26Lys Thr Trp Gly Gln Tyr Trp Gln Val 1
5 279PRTArtificial SequenceSynthetic peptide.
27Ile Met Asp Gln Val Pro Phe Ser Val 1 5
289PRTArtificialChemically synthesized peptide 28Ile Thr Asp Gln Val Pro
Phe Ser Val 1 5 299PRTArtificial
SequenceSynthetic peptide. 29Tyr Leu Glu Pro Gly Pro Val Thr Val 1
5 309PRTArtificialChemically synthesized peptide
30Tyr Leu Glu Pro Gly Pro Val Thr Ala 1 5
31204PRTArtificial SequenceSynthetic peptide. 31Met Asp Leu Asp Ala Val
Arg Ile Lys Val Asp Thr Val Asn Ala Lys 1 5
10 15 Pro Gly Asp Thr Val Asn Ile Pro Val Arg Phe
Ser Gly Ile Pro Ser 20 25
30 Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn
Val 35 40 45 Leu
Glu Ile Ile Glu Ile Lys Pro Gly Glu Leu Ile Val Asp Pro Asn 50
55 60 Pro Thr Lys Ser Phe Asp
Thr Ala Val Tyr Pro Asp Arg Lys Met Ile 65 70
75 80 Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly
Ala Tyr Ala Ile Thr 85 90
95 Lys Asp Gly Val Phe Ala Thr Ile Val Ala Lys Val Lys Glu Gly Ala
100 105 110 Pro Asn
Gly Leu Ser Val Ile Lys Phe Val Glu Val Gly Gly Phe Ala 115
120 125 Asn Asn Asp Leu Val Glu Gln
Lys Thr Gln Phe Phe Asp Gly Gly Val 130 135
140 Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro Thr
Thr Pro Val Thr 145 150 155
160 Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala Ala Arg Ser Ala Phe Thr
165 170 175 Ile Met Asp
Gln Val Pro Phe Ser Val Ser Val Ser Ala Ser Arg Lys 180
185 190 Gly Ala Ala Ala Leu Glu His His
His His His His 195 200
32118PRTArtificial SequenceSynthetic peptide. 32Met Pro Arg Glu Asp Ala
His Phe Ile Tyr Gly Tyr Pro Lys Lys Gly 1 5
10 15 His Gly His Ser Tyr Thr Thr Ala Glu Glu Ala
Ala Gly Ile Gly Ile 20 25
30 Leu Thr Val Ile Leu Gly Val Leu Leu Leu Ile Gly Cys Trp Tyr
Cys 35 40 45 Arg
Arg Arg Asn Gly Tyr Arg Ala Leu Met Asp Lys Ser Leu His Val 50
55 60 Gly Thr Gln Cys Ala Leu
Thr Arg Arg Cys Pro Gln Glu Gly Phe Asp 65 70
75 80 His Arg Asp Ser Lys Val Ser Leu Gln Glu Lys
Asn Cys Glu Pro Val 85 90
95 Val Pro Asn Ala Pro Pro Ala Tyr Glu Lys Leu Ser Ala Glu Gln Ser
100 105 110 Pro Pro
Pro Tyr Ser Pro 115 339PRTArtificial
SequenceSynthetic peptide. 33Ala Ala Gly Ile Gly Ile Leu Thr Val 1
5 3410PRTArtificial SequenceSynthetic peptide.
34Glu Ala Ala Gly Ile Gly Ile Leu Thr Val 1 5
10 3510PRTArtificial SequenceSynthetic peptide. 35Glu Leu Ala Gly
Ile Gly Ile Leu Thr Val 1 5 10
36204PRTArtificial SequenceSynthetic peptide. 36Met Asp Leu Asp Ala Val
Arg Ile Lys Val Asp Thr Val Asn Ala Lys 1 5
10 15 Pro Gly Asp Thr Val Asn Ile Pro Val Arg Phe
Ser Gly Ile Pro Ser 20 25
30 Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn
Val 35 40 45 Leu
Glu Ile Ile Glu Ile Lys Pro Gly Glu Leu Ile Val Asp Pro Asn 50
55 60 Pro Thr Lys Ser Phe Asp
Thr Ala Val Tyr Pro Asp Arg Lys Met Ile 65 70
75 80 Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly
Ala Tyr Ala Ile Thr 85 90
95 Lys Asp Gly Val Phe Ala Thr Ile Val Ala Lys Val Lys Glu Gly Ala
100 105 110 Pro Asn
Gly Leu Ser Val Ile Lys Phe Val Glu Val Gly Gly Phe Ala 115
120 125 Asn Asn Asp Leu Val Glu Gln
Lys Thr Gln Phe Phe Asp Gly Gly Val 130 135
140 Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro Thr
Thr Pro Val Thr 145 150 155
160 Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala Ala Arg Thr Ala Glu Glu
165 170 175 Leu Ala Gly
Ile Gly Ile Leu Thr Val Ile Leu Gly Ala Ser Arg Lys 180
185 190 Gly Ala Ala Ala Leu Glu His His
His His His His 195 200
37241PRTArtificial SequenceSynthetic peptide. 37Met Asp Leu Asp Ala Val
Arg Ile Lys Val Asp Thr Val Asn Ala Lys 1 5
10 15 Pro Gly Asp Thr Val Asn Ile Pro Val Arg Phe
Ser Gly Ile Pro Ser 20 25
30 Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn
Val 35 40 45 Leu
Glu Ile Ile Glu Ile Lys Pro Gly Glu Leu Ile Val Asp Pro Asn 50
55 60 Pro Thr Lys Ser Phe Asp
Thr Ala Val Tyr Pro Asp Arg Lys Met Ile 65 70
75 80 Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly
Ala Tyr Ala Ile Thr 85 90
95 Lys Asp Gly Val Phe Ala Thr Ile Val Ala Lys Val Lys Glu Gly Ala
100 105 110 Pro Asn
Gly Leu Ser Val Ile Lys Phe Val Glu Val Gly Gly Phe Ala 115
120 125 Asn Asn Asp Leu Val Glu Gln
Lys Thr Gln Phe Phe Asp Gly Gly Val 130 135
140 Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro Thr
Thr Pro Val Thr 145 150 155
160 Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala Ala Ser Asp Thr Thr Glu
165 170 175 Ala Arg His
Pro His Pro Pro Val Thr Thr Pro Thr Thr Asp Arg Lys 180
185 190 Gly Thr Thr Ala Glu Glu Leu Ala
Gly Ile Gly Ile Leu Thr Val Ile 195 200
205 Leu Gly Gly Lys Arg Thr Asn Asn Ser Thr Pro Thr Lys
Gly Glu Phe 210 215 220
Cys Arg Tyr Pro Ser His Trp Arg Pro Leu Glu His His His His His 225
230 235 240 His
3866PRTArtificial SequenceSynthetic peptide. 38Ala Ser Asp Thr Thr Glu
Ala Arg His Pro His Pro Pro Val Thr Thr 1 5
10 15 Pro Thr Thr Thr Asp Arg Lys Gly Thr Thr Ala
Glu Glu Leu Ala Gly 20 25
30 Ile Gly Ile Leu Thr Val Ile Leu Gly Gly Lys Arg Thr Asn Asn
Ser 35 40 45 Thr
Pro Thr Lys Gly Glu Phe Cys Arg Tyr Pro Ser His Trp Arg Pro 50
55 60 Arg Leu 65
3957DNAArtificial SequenceSynthetic oligonucleotide. 39cacggtcacc
gtctccaaag cttccggagc tagcgagggc ggcagcctgg ccgcgct
574070DNAArtificial SequenceSynthetic oligonucleotide. 40ggccggctcc
tgcgaaggga gccggccggt cgcggccgct tacttcaggt cctcgcgcgg 60cggtttgccg
7041518PRTArtificial SequenceSynthetic peptide. 41Met Asp Leu Asp Ala Val
Arg Ile Lys Val Asp Thr Val Asn Ala Lys 1 5
10 15 Pro Gly Asp Thr Val Asn Ile Pro Val Arg Phe
Ser Gly Ile Pro Ser 20 25
30 Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn
Val 35 40 45 Leu
Glu Ile Ile Glu Ile Lys Pro Gly Glu Leu Ile Val Asp Pro Asn 50
55 60 Pro Thr Lys Ser Phe Asp
Thr Ala Val Tyr Pro Asp Arg Lys Met Ile 65 70
75 80 Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly
Ala Tyr Ala Ile Thr 85 90
95 Lys Asp Gly Val Phe Ala Thr Ile Val Ala Lys Val Lys Glu Gly Ala
100 105 110 Pro Asn
Gly Leu Ser Val Ile Lys Phe Val Glu Val Gly Gly Phe Ala 115
120 125 Asn Asn Asp Leu Val Glu Gln
Lys Thr Gln Phe Phe Asp Gly Gly Val 130 135
140 Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro Thr
Thr Pro Val Thr 145 150 155
160 Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala Ala Ser Glu Gly Gly Ser
165 170 175 Leu Ala Ala
Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Thr 180
185 190 Phe Thr Arg His Arg Gln Pro Arg
Gly Trp Glu Gln Leu Glu Gln Cys 195 200
205 Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala
Ala Arg Leu 210 215 220
Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro 225
230 235 240 Gly Ser Gly Gly
Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln 245
250 255 Ala Arg Leu Ala Leu Thr Leu Ala Ala
Ala Glu Ser Glu Arg Phe Val 260 265
270 Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Gly
Pro Ala 275 280 285
Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu 290
295 300 Phe Leu Gly Asp Gly
Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln 305 310
315 320 Asn Trp Thr Val Glu Arg Leu Leu Gln Ala
His Arg Gln Leu Glu Glu 325 330
335 Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala
Ala 340 345 350 Gln
Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp 355
360 365 Ala Ile Trp Arg Gly Phe
Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr 370 375
380 Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg
Gly Arg Ile Arg Asn 385 390 395
400 Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe
405 410 415 Tyr Arg
Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val 420
425 430 Glu Arg Leu Ile Gly His Pro
Leu Pro Leu Arg Leu Asp Ala Ile Thr 435 440
445 Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile
Leu Gly Trp Pro 450 455 460
Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro 465
470 475 480 Arg Asn Val
Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu 485
490 495 Gln Ala Ile Ser Ala Leu Pro Asp
Tyr Ala Ser Gln Pro Gly Lys Pro 500 505
510 Pro Arg Glu Asp Leu Lys 515
42614PRTArtificial SequenceSynthetic peptide. 42Gln Ile Gln Leu Val Gln
Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5
10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr
Ser Phe Thr Asn Tyr 20 25
30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp
Met 35 40 45 Gly
Trp Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr Ala Asp Asp Phe 50
55 60 Lys Gly Arg Phe Ala Phe
Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70
75 80 Leu Gln Ile Ser Asn Leu Lys Asn Glu Asp Met
Ala Thr Tyr Phe Cys 85 90
95 Ala Arg Gly Asp Phe Arg Tyr Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Thr
Leu Thr Gly Ser Ser Ala Lys Thr Lys Gly Pro Ser Val Phe 115
120 125 Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp 145 150 155
160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180
185 190 Ser Ser Leu Gly Thr Lys Thr Tyr
Thr Cys Asn Val Asp His Lys Pro 195 200
205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr
Gly Pro Pro 210 215 220
Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe 225
230 235 240 Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245
250 255 Glu Val Thr Cys Val Val Val Asp Val
Ser Gln Glu Asp Pro Glu Val 260 265
270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr 275 280 285
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290
295 300 Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310
315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
Ile Glu Lys Thr Ile Ser 325 330
335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro 340 345 350 Ser
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355
360 365 Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375
380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp 385 390 395
400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp
405 410 415 Gln Glu
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420
425 430 Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Leu Gly Lys Ala Ser 435 440
445 Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro Val Thr
Thr Pro Thr Thr 450 455 460
Thr Asp Asp Leu Asp Ala Val Arg Ile Lys Val Asp Thr Val Asn Ala 465
470 475 480 Lys Pro Gly
Asp Thr Val Asn Ile Pro Val Arg Phe Ser Gly Ile Pro 485
490 495 Ser Lys Gly Ile Ala Asn Cys Asp
Phe Val Tyr Ser Tyr Asp Pro Asn 500 505
510 Val Leu Glu Ile Ile Glu Ile Lys Pro Gly Glu Leu Ile
Val Asp Pro 515 520 525
Asn Pro Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro Asp Arg Lys Met 530
535 540 Ile Val Phe Leu
Phe Ala Glu Asp Ser Gly Thr Gly Ala Tyr Ala Ile 545 550
555 560 Thr Lys Asp Gly Val Phe Ala Thr Ile
Val Ala Lys Val Lys Glu Gly 565 570
575 Ala Pro Asn Gly Leu Ser Val Ile Lys Phe Val Glu Val Gly
Gly Phe 580 585 590
Ala Asn Asn Asp Leu Val Glu Gln Lys Thr Gln Phe Phe Asp Gly Gly
595 600 605 Val Asn Val Gly
Asp Thr 610 43308PRTArtificial SequenceSynthetic
peptide. 43Leu Asp Asp Leu Asp Ala Val Arg Ile Lys Val Asp Thr Val Asn
Ala 1 5 10 15 Lys
Pro Gly Asp Thr Val Arg Ile Pro Val Arg Phe Ser Gly Ile Pro
20 25 30 Ser Lys Gly Ile Ala
Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn 35
40 45 Val Leu Glu Ile Ile Glu Ile Glu Pro
Gly Asp Ile Ile Val Asp Pro 50 55
60 Asn Pro Asp Lys Ser Phe Asp Thr Ala Val Tyr Pro Asp
Arg Lys Ile 65 70 75
80 Ile Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly Ala Tyr Ala Ile
85 90 95 Thr Lys Asp Gly
Val Phe Ala Thr Ile Val Ala Lys Val Lys Glu Gly 100
105 110 Ala Pro Asn Gly Leu Ser Val Ile Lys
Phe Val Glu Val Gly Gly Phe 115 120
125 Ala Asn Asn Asp Leu Val Glu Gln Lys Thr Gln Phe Phe Asp
Gly Gly 130 135 140
Val Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro Val 145
150 155 160 Thr Thr Pro Thr Thr
Thr Asp Asp Leu Asp Ala Leu Glu Ala Asp Gln 165
170 175 Gly Gln Asp Arg His Met Ile Arg Met Arg
Gln Leu Ile Asp Ile Val 180 185
190 Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu
Pro 195 200 205 Ala
Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys 210
215 220 Phe Gln Lys Ala Gln Leu
Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg 225 230
235 240 Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg
Lys Pro Pro Ser Thr 245 250
255 Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp
260 265 270 Ser Tyr
Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser 275
280 285 Leu Leu Gln Lys Met Ile His
Gln His Leu Ser Ser Arg Thr His Gly 290 295
300 Ser Glu Asp Ser 305
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