Patent application title: Multivariable Antigens Complexed with Targeting Humanized Monoclonal Antibody
Inventors:
Gerard Zurawski (Midlothian, TX, US)
Anne-Laure Flamar (Dallas, TX, US)
Anne-Laure Flamar (Dallas, TX, US)
Eynav Klechevsky (Dallas, TX, US)
Assignees:
Baylor Research Institute
IPC8 Class: AA61K39385FI
USPC Class:
42419611
Class name: Antigen, epitope, or other immunospecific immunoeffector (e.g., immunospecific vaccine, immunospecific stimulator of cell-mediated immunity, immunospecific tolerogen, immunospecific immunosuppressor, etc.) conjugate or complex conjugate or complex includes virus or componenet thereof
Publication date: 2010-12-30
Patent application number: 20100330115
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.-10. (canceled)
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, CD19, 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.
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); oran 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. (canceled)
36. A bivalent and multivalent (rAb.Doc:Coh.cytokine), (rAb.Coh:Doc.cytokine) or (cytokine.sup.1.Coh:cytokine2 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, IL5, 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, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, Il15, IL16, IL17, IL 18, 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, 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, IL5, 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 E1 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 gpI, 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 HIV. 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, CD19, 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-y 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, CD19, 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, CD14, 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-γ 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.
##STR00001##
[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-00001 TABLE 1 rAB-pIRES2 (hIgG4H-Dockerin) or C52. (SEQ ID NO.: 2) ATGGACCTCCTGTGCAAGAACATGAAGCACCTGTGGTTCTTCCTCC TGCTGGTGGCGGCTCCCAGATGGGTCCTGTCCCGGCTGCAGCTGCA GGAGTCGGGCCCAGGCCTGCTGAAGCCTTCGGTGACCCTGTCCCTC ACCTGCACTGTCTCGGGTGACTCCGTCGCCAGTAGTTCTTATTACT GGGGCTGGGTCCGTCAGCCCCCAGGGAAGGGACTCGAGTGGATAGG GACTATCAATTTTAGTGGCAATATGTATTATAGTCCGTCCCTCAGG AGTCGAGTGACCATGTCGGCAGACATGTCCGAGAACTCCTTCTATC TGAAATTGGACTCTGTGACCGCAGCAGACACGGCCGTCTATTATTG TGCGGCAGGACACCTCGTTATGGGATTTGGGGCCCACTGGGGACAG GGAAAACTGGTCTCCGTCTCTCCAGCTTCCACCAAGGGCCCATCCG TCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGC CGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAAC GTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGT CCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGA AGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACT CTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACG TGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGG CGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTC AACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGG ACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGG CCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAG CCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGA TGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTA CCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT TCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGA GGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC CACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGCA ATTCTCCTCAAAATGAAGTACTGTACGGAGATGTGAATGATGACGG AAAAGTAAACTCCACTGACTTGACTTTGTTAAAAAGATATGTTCTT AAAGCCGTCTCAACTCTCCCTTCTTCCAAAGCTGAAAAGAACGCAG ATGTAAATCGTGACGGAAGAGTTAATTCCAGTGATGTCACAATACT TTCAAGATATTTGATAAGGGTAATCGAGAAATTACCAATATAA (SEQ ID NO.: 3) RLQLQESGPGLLKPSVTLSLTCTVSGDSVASSSYYWGWVRQPPGKG LEWIGTINFSGNMYYSPSLRSRVTMSADMSENSFYLKLDSVTAADT AVYYCAAGHLVMGFGAHWGQGKLVSVSPASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCP APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASNSPQNEVLYGD VNDDGKVNSTDLTLLKRYVLKAVSTLPSSKAEKNADVNRDGRV DVTILSRYLIRVIEKLPI
[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-00002 TABLE 2 rAB-pIRES2 (mAnti-DCIR2C9H-LV-hIgG4H-C-Dockerin) or C82. (SEQ ID NO.: 4) ATGAAATGCAGCTGGGTCATCTTCTTCCTGATGGCAGTGGTTACAGG GGTCAATTCAGAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTTGTGA GGCCAGGGGCCTTAGTCAAGTTGTCCTGCAAAGCTTCTGGCTTCAAC ATTAATGACTACTATATCCACTGGGTGAAGCAGCGGCCTGAACAGGG CCTGGAGCGGATTGGATGGATTGATCCTGACAATGGTAATACTATAT ATGACCCGAAGTTCCAGGGCAAGGCCAGTATAACAGCAGACACATCC CCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACAC TGCCGTCTATTACTGTGCTAGAACCCGATCTCCTATGGTTACGACGG GGTTTGTTTACTGGGGCCAAGGGACTGTGGTCACTGTCTCTGCAGCC AAAACGAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAG CACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACT TCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTC CCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGA CCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGAC AAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGC ACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAAC CCAAGGACACTCTCATGATGTCCCGGACCCCTGAGGTCACGTGCGTG GTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTA CGTGGATGGCGTGGAGGTGCATAATGCCAAGACRAAGCCGCGGGAGG AGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTG CACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAA CAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAG GGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAG GAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT CTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC TTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGA GGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC ACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGCAAT TCTCCTCAAAATGAAGTACTGTACGGAGATGTGAATGATGACGGAAA AGTAAACTCCACTGACTTGACTTTGTTAAAAAGATATGTTCTTAAAG CCGTCTCAACTCTCCCTTCTTCCAAAGCTGAAAAGAACGCAGATGTA AATCGTGACGGAAGAGTTAATTCCAGTGATGTCACAATACTTTCAAG ATATTTGATAAGGGTAATCGAGAAATTACCAATATAA (SEQ ID NO.: 5) EVQLQQSGAELVRPGALVKLSCKASGFNINDYYIHWVKQRPEQGLER IGWIDPDNGNTIYDPKFQGKASITADTSPNTAYLQLSSLTSEDTAVY YCARTRSPMVTTGFVYWGQGTVVTVSAAKTKGPSVFPLAPCSRSTSE STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS VVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF EGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLGKASNSPQNEVLYGDVNDDGKVNS TDLTLLKRYVLKAVSTLPSSKAEKNADVNRDGRV VTILSRYLIR VIEKLPI.
[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-00003 TABLE 3 rAB-(mAnti-ASGPR_49C11_7H-SLAML-V- hIgG4H-C-Dockerin) or C153. (SEQ ID NO.: 6) ATGGACCCCAAAGGCTCCCTTTCCTGGAGAATACTTCTGTTTCTCTCC CTGGCTTTTGAGTTGTCGTACGGAGATGTGCAGCTTCAGGAGTCAGGA CCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTC ACTGGCTACTCCATCACCAGTGGTTATAGCTGGCACTGGATCCGGCAG TTTCCAGGAAACAAACTGGAATGGATGGGCTACATACTCTTCAGTGGT AGCACTAACTACAACCCATCTCTGAAAAGTCGAATCTCTATCACTCGA GACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACT GAGGACACAGCCACATATTTCTGTGCAAGATCTAACTATGGTTCCTTT GCTTCCTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACA AAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCC GAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA CCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGC GTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGC AACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAG TCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAA GGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTC ATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGC CAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAG GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACG TACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAC GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCC ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAG GTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTC AGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTG GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACC GTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTG TCTCTGGGTAAAGCTAGCAATTCTCCTCAAAATGAAGTACTGTACGGA GATGTGAATGATGACGGAAAAGTAAACTCCACTGACTTGACTTTGTTA AAAAGATATGTTCTTAAAGCCGTCTCAACTCTCCCTTCTTCCAAAGCT GAAAAGAACGCAGATGTAAATCGTGACGGAAGAGTTAATTCCAGTGAT GTCACAATACTTTCAAGATATTTGATAAGGGTAATCGAGAAATTACCA ATATAA (SEQ ID NO.: 7) DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEW MGYILFSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYFC ARSNYGSFASWGQGTLVTVSAAKTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGKASNSPQNEVLYGDVNDDGKVNSTDLTLLKRYVLKAV STLPSSKAEKNADVNRDGRV VTILSRYLIRVIEKLPI
[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-00004 TABLE 4 rAB-pIRES2 (mAnti-DC-SIGNL16E7H- LV-hIgG4H-C-Dockerin) or C92 (SEQ ID NO.: 8) ATGGAAAGGCACTGGATCTTTCTCTTCCTGTTTTCAGTAACTGCAGG TGTCCACTCCCAGGTCCAGCTTCAGCAGTCTGGGGCTGAGCTGGCAA AACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACC TTTACTACCTACTGGATGCACTGGGTAAAACAGAGGCCTGGACAGGG TCTGGAATGGATTGGATACATTAATCCTATCACTGGTTATACTGAGT ACAATCAGAAGTTCAAGGACAAGGCCACCTTGACTGCAGACAAATCC TCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTC TGCAGTCTATTACTGTGCAAGAGAGGGTTTAAGTGCTATGGACTATT GGGGTCAGGGAACCTCAGTCACCGTCACCTCAGCCAAAACAACGGGC CCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAG CACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACC TTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGT GGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCA ACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAG TCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGA AGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTC TCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTG AGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGT GGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACA GCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG CTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCC GTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAG AGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAG AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGA CATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC AGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTT CTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGA AGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGCAATTCTCCTCAAAAT GAAGTACTGTACGGAGATGTGAATGATGACGGAAAAGTAAACTCCAC TGACTTGACTTTGTTAAAAAGATATGTTCTTAAAGCCGTCTCAACTC TCCCTTCTTCCAAAGCTGAAAAGAACGCAGATGTAAATCGTGACGGA AGAGTTAATTCCAGTGATGTCACAATACTTTCAAGATATTTGATAAG GGTAATCGAGAAATTACCAATATAA (SEQ ID NO.: 9) QVQLQQSGAELAKPGASVKMSCKASGYTFTTYWMHWVKQRPGQGLEW IGYINPITGYTEYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVY YCAREGLSAMDYWGQGTSVTVTSAKTTGPSVFPLAPCSRSTSESTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGKASNSPQNEVLYGDVNDDGKVNSTDLT LLKRYVLKAVSTLPSSKAEKNADVNRDGRV DVTILSRYLIRVIE KLPI
[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-00005 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) DIVMTQSHKFMSTSVGDRVSVTCKASQDVTSAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLT- ISSGQAEDLAL YYCHQYYSAPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS- QESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
[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-00006 TABLE 6 rAB-pCMV (mIgG2bH-Dockerin) or C19. (SEQ ID NO.: 12) ATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACTGCAACTGGAG TACATTCACAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTGAGGCC TGGGACTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGTTACATCTTTACC AGCTACTGGATGCACTGGGTAAAGCAGAGGCCTGGACAAGGCCTTGAGT GGATCGGACTGATTGATCCTTCTGATAGTTATAGTAAGTACAATCAAAA GTTCAAGGGCAAGGCCACATTGACTGTAGACACATCCTCCAGCACAGCC TACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACT GTGCAAGAGGGGAGCTCAGTGACTTCTGGGGCCAAGGCACCACTCTCAC AGTCTCCTCAGCCAAAACAACACCCCCATCAGTCTATCCACTGGCCCCT GGGTGTGGAGATACAACTGGTTCCTCTGTGACTCTGGGATGCCTGGTCA AGGGCTACTTCCCTGAGTCAGTGACTGTGACTTGGAACTCTGGATCCCT GTCCAGCAGTGTGCACACCTTCCCAGCTCTCCTGCAGTCTGGACTCTAC ACTATGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCAAGTCAGA CCGTCACCTGCAGCGTTGCTCACCCAGCCAGCAGCACCACGGTGGACAA AAAACTTGAGCCCAGCGGGCCCATTTCAACAATCAACCCCTGTCCTCCA TGCAAGGAGTGTCACAAATGCCCAGCTCCTAACCTCGAGGGTGGACCAT CCGTCTTCATCTTCCCTCCAAATATCAAGGATGTACTCATGATCTCCCT GACACCCAAGGTCACGTGTGTGGTGGTGGATGTGAGCGAGGATGACCCA GACGTCCGGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTC AGACACAAACCCATAGAGAGGATTACAACAGTACTATCCGGGTGGTCAG TGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAA TGCAAGGTCAACAACAAAGACCTCCCATCACCCATCGAGAGAACCATCT CAAAAATTAAAGGGCTAGTCAGAGCTCCACAAGTATACATCTTGCCGCC ACCAGCAGAGCAGTTGTCCAGGAAAGATGTCAGTCTCACTTGCCTGGTC GTGGGCTTCAACCCTGGAGACATCAGTGTGGAGTGGACCAGCAATGGGC ATACAGAGGAGAACTACAAGGACACCGCACCAGTCCTGGACTCTGACGG TTCTTACTTCATATACAGCAAGCTCGATATAAAAACAAGCAAGTGGGAG AAAACAGATTCCTTCTCATGCAACGTGAGACACGAGGGTCTGAAAAATT ACTACCTGAAGAAGACCATCTCCCGGTCTCCGGGTAAAGCTAGCAATTC TCCTCAAAATGAAGTACTGTACGGAGATGTGAATGATGACGGAAAAGTA AACTCCACTGACTTGACTTTGTTAAAAAGATATGTTCTTAAAGCCGTCT CAACTCTGCCTTCTTCCAAAGCTGAAAAGAACGCAGATGTAAATCGTGA CGGAAGAGTTAATTCCAGTGATGTCACAATACTTTCAAGATATTTGATA AGGGTAATCGAGAAATTACCAATATAA (SEQ ID NO.: 13) QVQLQQPGAELVRPGTSVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIG LIDPSDSYSKYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCAR GELSDFWGQGTTLTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGY FPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVT CSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVF IFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVRISWFVNNVEVHTAQTQ THREDYNSTIRVVSALPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKI KGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTE ENYKDTAPVLDSDGSYFIYSKLDIKTSKWEKTDSFSCNVRHEGLKNYYL KKTISRSPGKASNSPQNEVLYGDVNDDGKVNSTDLTLLKRYVLKAVSTL PSSKAEKNADVNRDGRV DVTILSRYLIRVIEKLPI.
[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 HA 1 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-00007 TABLE 7 Mam-pCDM8 (Cohesin-SLAML-AP-6xHis) or C16. (SEQ ID NO.: 14) ATGGATCCCAAAGGATCCCTTTCCTGGAGAATACTTCTGTTT CTCTCCCTGGCTTTTGAGTTGAGCTACGGACTCGACGATCTG GATGCAGTAAGGATTAAAGTGGACACAGTAAATGCAAAACCG GGAGACACAGTAAGAATACCTGTAAGATTCAGCGGTATACCA TCCAAGGGAATAGCAAACTGTGACTTTGTATACAGCTATGAC CCGAATGTACTTGAGATAATAGAGATAGAACCGGGAGACATA ATAGTTGACCCGAATCCTGACAAGAGCTTTGATACTGCAGTA TATCCTGACAGAAAGATAATAGTATTCCTGTTTGCAGAAGAC AGCGGAACAGGAGCGTATGCAATAACTAAAGACGGAGTATTT GCTACGATAGTAGCGAAAGTAAAAGAAGGAGCACCTAACGGA CTCAGTGTAATCAAATTTGTAGAAGTAGGCGGATTTGCGAAC AATGACCTTGTAGAACAGAAGACACAGTTCTTTGACGGTGGA GTAAATGTTGGAGATACAACAGAACCTGCAACACCTACAACA CCTGTAACAACACCGACAACAACAGATGATCTGGATGCACTC GAGATCATCCCAGTTGAGGAGGAGAACCCGGACTTCTGGAAC CGCGAGGCAGCCGAGGCCCTGGGTGCCGCCAAGAAGCTGCAG CCTGCACAGACAGCCGCCAAGAACCTCATCATCTTCCTGGGC GATGGGATGGGGGTGTCTACGGTGACAGCTGCCAGGATCCTA AAAGGGCAGAAGAAGGACAAACTGGGGCCTGAGTTACCCCTG GCCATGGACCGCTTCCCATATGTGGCTCTGTCCAAGACATAC AATGTAGACAAACATGTGCCAGACAGTGGAGCCACAGCCACG GCCTACCTGTGCGGGGTCAAGGGCAACTTCCAGACCATTGGC TTGAGTGCAGCCGCCCGCTTTAACCAGTGCAACACGACACGC GGCAACGAGGTCATCTCCGTGATGAATCGGGCCAAGAAAGCA GGGAAGTCAGTGGGAGTGGTAACCACCACACGAGTGCAGCAC GCCTCGCCAGCCGGCACCTACGCCCACACGGTGAACCGCAAC TGGTACTCGGACGCCGACGTGCCTGCCTCGGCCCGCCAGGAG GGGTGCCAGGACATCGCTACGCAGCTCATCTCCAACATGGAC ATTGACGTGATCCTAGGTGGAGGCCGAAAGTACATGTTTCGC ATGGGAACCCCAGACCCTGAGTACCCAGATGACTACAGCCAA GGTGGGACCAGGCTGGACGGGAAGAATCTGGTGCAGGAATGG CTGGCGAAGCGCCAGGGTGCCCGGTACGTGTGGAACCGCACT GAGCTCATGCAGGCTTCCCTGGACCCGTCTGTGACCCATCTC ATGGGTCTCTTTGAGCCTGGAGACATGAAATACGAGATCCAC CGAGACTCCACACTGGACCCCTCCCTGATGGAGATGACAGAG GCTGCCCTGCGCCTGCTGAGCAGGAACCCCCGCGGCTTCTTC CTCTTCGTGGAGGGTGGTCGCATCGACCATGGTCATCATGAA AGCAGGGCTTACCGGGCACTGACTGAGACGATCATGTTCGAC GACGCCATTGAGAGGGCGGGCCAGCTCACCAGCGAGGAGGAC ACGCTGAGCCTCGTCACTGCCGACCACTCCCACGTCTTCTCC TTCGGAGGCTACCCCCTGCGAGGGAGCTCCATCTTCGGGCTG GCCCCTGGCAAGGCCCGGGACAGGAAGGCCTACACGGTCCTC CTATACGGAAACGGTCCAGGCTATGTGCTCAAGGACGGCGCC CGGCCGGATGTTACCGAGAGCGAGAGCGGGAGCCCCGAGTAT CGGCAGCAGTCAGCAGTGCCCCTGGACGAAGAGACCCACGCA GGCGAGGACGTGGCGGTGTTCGCGCGCGGCCCGCAGGCGCAC CTGGTTCACGGCGTGCAGGAGCAGACCTTCATAGCGCACGTC ATGGCCTTCGCCGCCTGCCTGGAGCCCTACACCGCCTGCGAC CTGGCGCCCCCCGCCGGCACCACCCACCATCACCATCACCAT TGA (SEQ ID NO.: 15) LDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIANCDFV YSYDPNVLEIIEIEPGELIVDPN KSFDTAVYPDRKMIVF LFAEDSGTGAYAITEDGVFATIVAKVKSGAPNGLSVIKFVEV GGFANNDLVEQKTQFFDGGVNVGD EPA P P V P DDLDALEIIPVEEENPDFWNREAAEALGAAKK LQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPEL PLAMDRFPYVALSKTYNVDKHVPDSGATATAYLCGVKGNFQT IGLSAAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRV QRASPAGTYAHTVNRNWYSDADVPASARQEGCQDIATQLISN MDIDVILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQ EWLAKRQGARYVWNRTELMQASLDPSVTHLMGLFEPGDMKYE IHRDSTLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGH HESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHSHV FSFGGYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPGYVLKD GARPDVTESESGSPEYRQQSAVPLDEETHAGEDVAVFARGPQ AHLVHGVQEQTFIAHVMAFAACLEPYTACDLAPPAGT T
[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-00008 TABLE 8 Mam-pCDM8 (Cohesin-Cohesin-SLAML-AP-6xHis) or C17. (SEQ ID NO.: 16) ATGGATCCCAAAGGATCCCTTTCCTGGAGAATACTTCTGTTT CTCTCCCTGGCTTTTGAGTTGAGCTACGGACTCGACGATCTG GATGCAGTAAGGATTAAAGTGGACACAGTAAATGCAAAACCG GGAGACACAGTAAGAATACCTGTAAGATTCAGCGGTATACCA TCCAAGGGAATAGCAAACTGTGACTTTGTATACAGCTATGAC CCGAATGTACTTGAGATAATAGAGATAAAACCGGGAGAATTG ATAGTTGACCCGAATCCTGACAAGAGCTTTGATACTGCAGTA TATCCTGACAGAAAGATAATAGTATTCCTGTTTGCAGAAGAC AGCGGAACAGGAGCGTATGCAATAACTAAAGACGGAGTATTT GCTACGATAGTAGCGAAAGTAAAATCCGGAGCACCTAACGGA CTCAGTGTAATCAAATTTGTAGAAGTAGGCGGATTTGCGAAT AATGACCTTGTAGAACAGAAGACACAGTTCTTTGACGGTGGA GTAAATGTTGGAGATACAACAGAACCTGCAACACCTACAACA CCTGTAACAACACCGACAACAACAGATGATCTGGATGCAGTA AGGATTAAAGTGGACACAGTAAATGCAAAACCGGGAGACACA GTAAATATACCTGTAAGATTCAGTGGTATACCATCCAAGGGA ATAGCAAACTGTGACTTTGTATACAGCTATGACCCGAATGTA CTTGAGATAATAGAGATAAAACCGGGAGAATTGATAGTTGAC CCGAATCCTACCAAGAGCTTTGATACTGCAGTATATCCTGAC AGAAAGATGATAGTATTCCTGTTTGCGGAAGACAGCGGAACA GGAGCGTATGCAATAACTAAAGACGGAGTATTTGCTACGATA GTAGCGAAAGTAAAAGAAGGAGCACCTAACGGACTCAGTGTA ATCAAATTTGTAGAAGTAGGCGGATTTGCGAACAATGACCTT GTAGAACAGAAGACACAGTTCTTTGACGGTGGAGTAAATGTT GGAGATACAACAGAACCTGCAACACCTACAACACCTGTAACA ACACCGACAACAACAGATGATCTGGATGCACTCGAGATCATC CCAGTTGAGGAGGAGAACCCGGACTTCTGGAACCGCGAGGCA GCCGAGGCCCTGGGTGCCGCCAAGAAGCTGCAGCCTGCACAG ACAGCCGCCAAGAACCTCATCATCTTCCTGGGCGATGGGATG GGGGTGTCTACGGTGACAGCTGCCAGGATCCTAAAAGGGCAG AAGAAGGACAAACTGGGGCCTGAGTTACCCCTGGCCATGGAC CGCTTCCCATATGTGGCTCTGTCCAAGACATACAATGTAGAC AAACATGTGCCAGACAGTGGAGCCACAGCCACGGCCTACCTG TGCGGGGTCAAGGGCAACTTCCAGACCATTGGCTTGAGTGCA GCCGCCCGCTTTAACCAGTGCAACACGACACGCGGCAACGAG GTCATCTCCGTGATGAATCGGGCCAAGAAAGCAGGGAAGTCA GTGGGAGTGGTAACCACCACACGAGTGCAGCACGCCTCGCCA GCCGGCACCTACGCCCACACGGTGAACCGCAACTGGTACTCG GACGCCGACGTGCCTGCCTCGGCCCGCCAGGAGGGGTGCCAG GACATCGCTACGCAGCTCATCTCCAACATGGACATTGACGTG ATCCTAGGTGGAGGCCGAAAGTACATGTTTCGCATGGGAACC CCAGACCCTGAGTACCCAGATGACTACAGCCAAGGTGGGACC AGGCTGGACGGGAAGAATCTGGTGCAGGAATGGCTGGCGAAG CGCCAGGGTGCCCGGTACGTGTGGAACCGCACTGAGCTCATG CAGGCTTCCCTGGACCCGTCTGTGACCCATCTCATGGGTCTC TTTGAGCCTGGAGACATGAAATACGAGATCCACCGAGACTCC ACACTGGACCCCTCCCTGATGGAGATGACAGAGGCTGCCCTG CGCCTGCTGAGCAGGAACCCCCGCGGCTTCTTCCTCTTCGTG GAGGGTGGTCGCATCGACCATGGTCATCATGAAAGCAGGGCT TACCGGGCACTGACTGAGACGATCATGTTCGACGACGCCATT GAGAGGGCGGGCCAGCTCACCAGCGAGGAGGACACGCTGAGC CTCGTCACTGCCGACCACTCCCACGTCTTCTCCTTCGGAGGC TACCCCCTGCGAGGGAGCTCCATCTTCGGGCTGGCCCCTGGC AAGGCCCGGGACAGGAAGGCCTACACGGTCCTCCTATACGGA AACGGTCCAGGCTATGTGCTCAAGGACGGCGCCCGGCCGGAT GTTACCGAGAGCGAGAGCGGGAGCCCCGAGTATCGGCAGCAG TCAGCAGTGCCCCTGGACGAAGAGACCCACGCAGGCGAGGAC GTGGCGGTGTTCGCGCGCGGCCCGCAGGCGCACCTGGTTCAC GGCGTGCAGGAGCAGACCTTCATAGCGCACGTCATGGCCTTC GCCGCCTGCCTGGAGCCCTACACCGCCTGCGACCTGGCGCCC CCCGCCGGCACCACCCACCATCACCATCACCATTGA (SEQ ID NO.: 17) LDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIANCDFVY SYDPNVLEIIEIKPGELIVDPNPDKSFDTAVYPDRKIIVFLF AEDSGTGAYAITKDGVFATIVAKVKSGAPNGLSVIKFVEVGG FANNDLVEQKTQFFDGGVNVGD EPA P PV P DDLDAVRIKVDTVNAKPGDTVNIPVRFSGIPSK GIANCDFVYSYDPNVLEIIEIKPGELIVDP KSFDTAVYP DRKMIVFLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLS VIKFVEVGGFANNDLVEQKTQFFDGGVNVGD EPA P PV P DDLDALEIIPVEEENPDFWNREAAEALGAAKKLQPA QTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPELPLAM DRFPYVALSKTYNVDKHVPDSGATATAYLCGVKGNFQTIGLS AAARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHAS PAGTYAHTVNRNWYSDADVPASARQEGCQDIATQLISNMDID VILGGGRKYMFRMGTPDPEYPDDYSQGGTRLDGKNLVQEWLA KRQGARYVWNRTELMQASLDPSVTHLMGLFEPGDMKYEIHRD STLDPSLMEMTEAALRLLSRNPRGFFLFVEGGRIDHGHHESR AYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHSHVFSFG GYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPGYVLKDGARP DVTESESGSPEYRQQSAVPLDEETHAGEDVAVFARGPQAHLV HGVQEQTFIAHVMAFAACLEPYTACDLAPPAGTT
[0129]TABLE 9 shows the nucleic acid and amino acid sequences for Mam-pCDM8(SLAML-Cohesin-hPSA) or C149. 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-00009 TABLE 9 Mam-pCDM8 (SLAML-Cohesin-hPSA) or C149. (SEQ ID NO.: 18) ATGGATCCCAAAGGATCCCTTTCCTGGAGAATACTTCTGTTTCT CTCCCTGGCTTTTGAGTTGAGCTACGGACTCGACGATCTGGATG CAGTAAGGATTAAAGTGGACACAGTAAATGCAAAACCGGGAGAC ACAGTAAGAATACCTGTAAGATTCAGCGGTATACCATCCAGGGA ATAGCAAACTGTGACTTTGTATACAGCTATGACCCGAATGTACT TGAGATAATAGAGATAGAACCGGGAGACATAATAGTTGACCCGA ATCCTGACAAGAGCTTTGATACTGCAGTATATCCTGACAGAAAG ATAATAGTATTCCTGTTTGCAGAAGACAGCGGAACAGGAGCGTA TGCAATAACTAAAGACGGAGTATTTGCTACGATAGTAGCGAAAG TAAAAGAAGGAGCACCTAACGGACTCAGTGTAATCAAATTTGTA GAAGTAGGCGGATTTGCGAACAATGACCTTGTAGAACAGAAGAC ACAGTTCTTTGACGGTGGAGTAAATGTTGGAGATACAACAGAAC CTGCAACACCTACAACACCTGTAACAACACCGACAACAACAGAT GATCTGGATGCACTCGAGGCGCCCCTCATCCTGTCTCGGATTGT GGGAGGCTGGGAGTGCGAGAAGCATTCCCAACCCTGGCAGGTGC TTGTGGCCTCTCGTGGCAGGGCAGTCTGCGGCGGTGTTCTGGTG CACCCCCAGTGGGTCCTCACAGCTGCCCACTGCATCAGGAACAA AAGCGTGATCTTGCTGGGTCGGCACAGCCTGTTTCATCCTGAAG ACACAGGCCAGGTATTTCAGGTCAGCCACAGCTTCCCACACCCG CTCTACGATATGAGCCTCCTGAAGAATCGATTCCTCAGGCCAGG TGATGACTCCAGCCACGACCTCATGCTGCTCCGCCTGTCAGAGC CTGCCGAGCTCACGGATGCTGTGAAGGTCATGGACCTGCCCACC CAGGAGCCAGCACTGGGGACCACCTGCTACGCCTCAGGCTGGGG CAGCATTGAACCAGAGGAGTTCTTGACCCCAAAGAAACTTCAGT GTGTGGACCTCCATGTTATTTCCAATGACGTGTGCGCGCAAGTT CACCCTCAGAAGGTGACCAAGTTCATGCTGTGTGCTGGACGCTG GACAGGGGGCAAAAGCACCTGCTCGGGTGATTCTGGGGGCCCAC TTGTCTGTAATGGTGTGCTTCAAGGTATCACGTCATGGGGCAGT GAACCATGTGCCCTGCCCGAAAGGCCTTCCCTGTACACCAAGGT GGTGCATTACCGGAAGTGGATCAAGGACACCATCGTGGCCAACC CCTGA (SEQ ID NO.: 19) LDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIANCDFVYS YDPNVLEIIEIEPGELIVDPNP KSFDTAVYPDRKMIVFLFA EDSGTGAYAITEDGVFATIVAKVKSGAPNGLSVIKFVEVGGFAN NDLVEQKTQFFDGGVNVGD EPA P PV P DDLDALEAPLILSRIVGGWECEKHSQPWQVLVASRGRA VCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQV SHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAV KVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVIS NDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQ GITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP
[0130]TABLE 10 shows the nucleic acid and amino acid sequences for Mam-pCDM8(SLAML-Cohesin-FluHA5-1-6× 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-00010 TABLE 10 Mam-pCDM8 (SLAML-Cohesin-FluHA5-1-6x-His) or C24. (SEQ ID NO.: 20) ATGGATCCCAAAGGATCCCTTTCCTGGAGAATACTTCTG TTTCTCTCCCTGGCTTTTGAGTTGAGCTACGGACTCGAC GATCTGGATGCAGTAAGGATTAAAGTGGACACAGTAAAT GCAAAACCGGGAGACACAGTAAGAATACCTGTAAGATTC AGCGGTATACCATCCAAGGGAATAGCAAACTGTGACTTT GTATACAGCTATGACCCGAATGTACTTGAGATAATAGAG ATAGAACCGGGAGACATAATAGTTGACCCGAATCCTGAC AAGAGCTTTGATACTGCAGTATATCCTGACAGAAAGATA ATAGTATTCCTGTTTGCAGAAGACAGCGGAACAGGAGCG TATGCAATAACTAAAGACGGAGTATTTGCTACGATAGTA GCGAAAGTAAAAGAAGGAGCACCTAACGGACTCAGTGTA ATCAAATTTGTAGAAGTAGGCGGATTTGCGAACAATGAC CTTGTAGAACAGAAGACACAGTTCTTTGACGGTGGAGTA AATGTTGGAGATACAACAGAACCTGCAACACCTACAACA CCTGTAACAACACCGACAACAACAGATGATCTGGATGCA CTCGAGGATCAGATTTGCATTGGTTACCATGCAAACAAC TCGACAGAGCAGGTTGACACAATAATGGAAAAGAACGTT ACTGTTACACATGCCCAAGACATACTGGAAAAGAAACAC AACGGGAAGCTCTGCGATCTAGATGGAGTGAAGCCTCTA ATTTTGAGAGATTGTAGCGTAGCTGGATGGCTCCTCGGA AACCCAATGTGTGACGAATTCATCAATGTGCCGGAATGG TCTTACATAGTGGAGAAGGCCAATCCAGTCAATGACCTC TGTTACCCAGGGGATTTCAATGACTATGAAAAATTGAAA CACCTATTGAGCAGAATAAACCATTTTGAGAAAATTCAG ATCATCCCCAAAAGTTCTTGGTCCAGTCATGAAGCCTCA TTAGGGGTGAGCTCAGCATGTCCATACCAGGGAAAGTCC TCCTTTTTCAGAAATGTGGTATGGCTTATCAAAAAGAAC AGTACATACCCAACAATAAAGAGGAGCTACAATAATACC AACCAAGAAGATCTTTTGGTACTGTGGGGGATTCACCAT CCTAATGATGCGGCAGAGCAGACAAAGCTCTATCAAAAC CCAACCACCTATATTTCCGTTGGGACATCAACACTAAAC CAGAGATTGGTACCAAGAATAGCTACTAGATCCAAAGTA AACGGGCAAAGTGGAAGGATGGAGTTCTTCTGGACAATT TTAAAGCCGAATGATGCAATCAACTTCGAGAGTAATGGA AATTTCATTGCTCCAGAATATGCATACAAAATTGTCAAG AAAGGGGACTCAACAATTATGAAAAGTGAATTGGAATAT GGTAACTGCAACACCAAGTGTCAAACTCCAATGGGGGCG ATAAACTCTAGCATGCCATTCCACAATATACACCCTCTC ACCATTGGGGAATGCCCCAAATATGTGAAATCAAACAGA TTAGTCCTTGCGCACCATCACCATCACCATTGA (SEQ ID NO.: 21) LDDLDAVRIKVDTVNAKPGDTVRIPVRFSGIPSKGIANC DFVYSYDPNVLEIIEIEPGELIVDPNP KSFDTAVYP DRKMIVFLFAEDSGTGAYAITEDGVFATIVAKVKSGAPN GLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGD E PA P PV P DDLDALEDQICIGYHANN STEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPL ILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDL CYPGDFNDYEKLKHLLSRINHFEKIQIIPKSSWSSHEAS LGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNT NQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLN QRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNG NFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGA INSSMPFHNIHPLTIGECPKYVKSNRLVLA .
[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-00011 TABLE 11 E coli-pET28 (Cohesin-FluM1-6x-His) or C32. (SEQ ID NO.: 22) ATGGATCTGGATGCAGTAAGGATTAAAGTGGACACAGTAAATGCAA AACCGGGAGACACAGTAAATATACCTGTAAGATTCAGTGGTATACC ATCCAAGGGAATAGCAAACTGTGACTTTGTATACAGCTATGACCCG AATGTACTTGAGATAATAGAGATAAAACCGGGAGAATTGATAGTTG ACCCGAATCCTACCAAGAGCTTTGATACTGCAGTATATCCTGACAG AAAGATGATAGTATTCCTGTTTGCGGAAGACAGCGGAACAGGAGCG TATGCAATAACTAAAGACGGAGTATTTGCTACGATAGTAGCGAAAG TAAAAGAAGGAGCACCTAACGGGCTCAGTGTAATCAAATTTGTAGA AGTAGGCGGATTTGCGAACAATGACCTTGTAGAACAGAAGACACAG TTCTTTGACGGTGGAGTAAATGTTGGAGATACAACAGAACCTGCAA CACCTACAACACCTGTAACAACACCGACAACAACAGATGATCTGGA TGCAGCTAGCCTTCTAACCGAGGTCGAAACGTACGTTCTCTCTATC ATCCCGTCAGGCCCCCTCAAAGCCGAGATCGCACAGAGACTTGAAG ATGTCTTTGCAGGGAAGAACACCGATCTTGAGGTTCTCATGGAATG GCTAAAGACAAGACCAATCCTGTCACCTCTGACTAAGGGGATTTTA GGATTTGTGTTCACGCTCACCGTGCCCAGTGAGCGGGGACTGCAGC GTAGACGCTTTGTCCAAAATGCTCTTAATGGGAACGGAGATCCAAA TAACATGGACAAAGCAGTTAAACTGTATAGGAAGCTTAAGAGGGAG ATAACATTCCATGGGGCCAAAGAAATAGCACTCAGTTATTCTGCTG GTGCACTTGCCAGTTGTATGGGCCTCATATACAACAGGATGGGGGC TGTGACCACTGAAGTGGCATTTGGCCTGGTATGCGCAACCTGTGAA CAGATTGCTGACTCCCAGCATCGGTCTCATAGGCAAATGGTGACAA CAACCAATCCACTAATCAGACATGAGAACAGAATGGTTCTAGCCAG CACTACAGCTAAGGCTATGGAGCAAATGGCTGGATCGAGTGAGCAA GCAGCAGAGGCCATGGATATTGCTAGTCAGGCCAGGCAAATGGTGC AGGCGATGAGAACCATTGGGACTCATCCTAGCTCCAGTGCTGGTCT AAAAGATGATCTTCTTGAAAATTTGCAGGCTTACCAGAAACGGATG GGGGTGCAGATGCAGCGATTCAAGCTCGAGCACCACCACCACCACC ACTGA (SEQ ID NO.: 23) MDLDAVRIKVDTVNAKPGDTVNIPVRFSGIPSKGIANCDFVYSYDP NVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIVELFAEDSGTGA YAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQ FFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASLLTEVETYVLSI IPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGIL GFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKRE ITFEIGAKEIALSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATC EQIADSQHRSHRQMVTTTNPLIRHENRINVLASTTAKAMEQMAGSS EQAAEAMDIASQARQMVQAMRTIGTHPSSSAGLKDDLLENLQAYQK RMGVQMQRFKLE
[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 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.
[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 MI-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.
##STR00002##
[0152]The HLA-A0201 Restricted Peptide Sequences are:
TABLE-US-00012 GP100 WT: 154-162: KTWGQYWQV (SEQ ID NO.: 26) GP100 M: 209-217 (2M): IMDQVPFSV; (SEQ ID NO.: 27) 209-217 WT: ITDQVPFSV (SEQ ID NO.: 28) GP100 M: 280-288 (9V): YLEPGPVTV (SEQ ID NO.: 29) 280-288 WT: YLEPGPVTA (SEQ ID NO.: 30)
[0153]C180 is E. coli-pET28(Cohesin-hgp100-PeptideA-6× His):
##STR00003##
[0154]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.
[0155]MART-1 is:
##STR00004##
[0156]The HLA-A0201 Restricted Peptides Sequences are:
##STR00005##
[0157]C181 is E. coli-pET28(Cohesin-hMART-1-PeptideB-6× His)
##STR00006##
[0158]C186 is E. coli-pET28(Cohesin-Flex-hMART-1-PeptideA-6× His)
##STR00007##
[0159]C172 is rAB-pIRES2(mAnti-ASGPR--49C11--7H-LV-hIgG4H-hMART-1-PeptideA)
[0160]C174 is rAB-pIRES2(hIgG4H-hMART-1-PeptideA)
##STR00008##
[0161]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.
[0162]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.
[0163]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.
[0164]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
[0165]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 (SS IP). 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.
[0166]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.
[0167]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).
[0168]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.
[0169]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.
[0170]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. NCl, NIH) was used as a template for PCR with primers
TABLE-US-00013 PE38-N3 (cacggtcaccgtctccaaagcttccggagctagcGAGGGCGGCAGCCTG GCCGCGCT (SEQ ID NO.: 39)) and PE38-C3 (GGCCGGCTCCTGCGAAGGGAGCCGGCCGGTCGCGGCCGCTTACTTCAGG TCCTCGCGCGGCGGTTTGCCG (SEQ ID NO.: 40)).
[0171]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).
##STR00009##
[0172]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.
[0173]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.
[0174]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. 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.
[0175]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):
##STR00010##
[0176]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.
[0177]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).
[0178]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.
[0179]Mam-pCDM8(SLAML-Cohesin-hIL-21)
##STR00011##
[0180]Thus rAb.Doc:Coh.IL-21 can deliver concomitant proliferation and activation signals to a B cell (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.
[0181]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.
[0182]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.
[0183]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.
[0184]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.
[0185]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.
[0186]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.
[0187]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
4312299PRTBacteroides cellulosolvens 1Met Gln Ser Pro Arg Leu Lys Arg Lys
Ile Leu Ser Val Ile Leu Ala1 5 10
15Val Cys Tyr Ile Ile Ser Ser Phe Ser Ile Gln Phe Ala Ala Thr
Pro 20 25 30Gln Val Asn Ile
Ile Ile Gly Ser Ala Gln Gly Ile Pro Gly Ser Thr 35
40 45Val Lys Val Pro Ile Asn Leu Gln Asn Val Pro Glu
Ile Gly Ile Asn 50 55 60Asn Cys Asp
Phe Thr Ile Lys Phe Asp Ser Asp Ile Leu Asp Phe Asn65 70
75 80Ser Val Glu Ala Gly Asp Ile Val
Pro Leu Pro Val Ala Ser Phe Ser 85 90
95Ser Asn Asn Ser Lys Asp Ile Ile Lys Phe Leu Phe Ser Asp
Ala Thr 100 105 110Gln Gly Asn
Met Pro Ile Asn Glu Asn Gly Leu Phe Ala Val Ile Ser 115
120 125Phe Lys Ile Lys Asp Asn Ala Gln Lys Gly Ile
Ser Asn Ile Lys Val 130 135 140Ser Ser
Tyr Gly Ser Phe Ser Gly Met Ser Gly Lys Glu Met Gln Ser145
150 155 160Leu Ser Pro Thr Phe Phe Ser
Gly Ser Ile Asp Val Ser Asp Val Ser 165
170 175Thr Ser Lys Leu Asp Val Lys Val Gly Asn Val Glu
Gly Ile Ala Gly 180 185 190Thr
Glu Val Asn Val Pro Ile Thr Phe Glu Asn Val Pro Asp Asn Gly 195
200 205Ile Asn Asn Cys Asn Phe Thr Leu Ser
Tyr Asp Ser Asn Ala Leu Glu 210 215
220Phe Leu Thr Thr Glu Ala Gly Asn Ile Ile Pro Leu Ala Ile Ala Asp225
230 235 240Tyr Ser Ser Tyr
Arg Ser Met Glu Gly Lys Ile Lys Phe Leu Phe Ser 245
250 255Asp Ser Ser Gln Gly Thr Arg Ser Ile Lys
Asn Asp Gly Val Phe Ala 260 265
270Asn Ile Lys Phe Lys Ile Lys Gly Asn Ala Ile Arg Asp Thr Tyr Arg
275 280 285Ile Asp Leu Ser Glu Leu Gly
Ser Phe Ser Ser Lys Gln Asn Asn Asn 290 295
300Leu Lys Ser Ile Ala Thr Gln Phe Leu Ser Gly Ser Val Asn Val
Lys305 310 315 320Asp Ile
Glu Ser Ser Val Ser Pro Thr Thr Ser Val His Pro Thr Pro
325 330 335Thr Ser Val Pro Pro Thr Pro
Thr Lys Ser Ser Pro Gly Asn Lys Met 340 345
350Lys Ile Gln Ile Gly Asp Val Lys Ala Asn Gln Gly Asp Thr
Val Ile 355 360 365Val Pro Ile Thr
Phe Asn Glu Val Pro Val Met Gly Val Asn Asn Cys 370
375 380Asn Phe Thr Leu Ala Tyr Asp Lys Asn Ile Met Glu
Phe Ile Ser Ala385 390 395
400Asp Ala Gly Asp Ile Val Thr Leu Pro Met Ala Asn Tyr Ser Tyr Asn
405 410 415Met Pro Ser Asp Gly
Leu Val Lys Phe Leu Tyr Asn Asp Gln Ala Gln 420
425 430Gly Ala Met Ser Ile Lys Glu Asp Gly Thr Phe Ala
Asn Val Lys Phe 435 440 445Lys Ile
Lys Gln Ser Ala Ala Phe Gly Lys Tyr Ser Val Gly Ile Lys 450
455 460Ala Ile Gly Ser Ile Ser Ala Leu Ser Asn Ser
Lys Leu Ile Pro Ile465 470 475
480Glu Ser Ile Phe Lys Asp Gly Ser Ile Thr Val Thr Asn Lys Pro Ile
485 490 495Val Asn Ile Glu
Ile Gly Lys Val Lys Val Lys Ala Gly Asp Lys Ile 500
505 510Lys Val Pro Val Glu Ile Lys Asp Ile Pro Ser
Ile Gly Ile Asn Asn 515 520 525Cys
Asn Phe Thr Leu Lys Tyr Asn Ser Asn Val Leu Lys Tyr Val Ser 530
535 540Asn Glu Ala Gly Thr Ile Val Pro Ala Pro
Leu Ala Asn Leu Ser Ile545 550 555
560Asn Lys Pro Asp Glu Gly Ile Ile Lys Leu Leu Phe Ser Asp Ala
Ser 565 570 575Gln Gly Gly
Met Pro Ile Lys Asp Asn Gly Ile Phe Val Asn Leu Glu 580
585 590Phe Gln Ala Val Asn Asp Ala Asn Ile Gly
Val Tyr Gly Leu Glu Leu 595 600
605Asp Thr Ile Gly Ala Phe Ser Gly Ile Ser Ser Ala Lys Met Thr Ser 610
615 620Ile Glu Pro Gln Phe Asn Asn Gly
Ser Ile Glu Ile Phe Asn Ser Ala625 630
635 640Gln Thr Pro Val Pro Ser Asn Thr Glu Val Gln Thr
Pro Thr Asn Thr 645 650
655Ile Ser Val Thr Pro Thr Asn Asn Ser Thr Pro Thr Asn Asn Ser Thr
660 665 670Pro Lys Pro Asn Pro Leu
Tyr Asn Leu Asn Val Asn Ile Gly Glu Ile 675 680
685Ser Gly Glu Ala Gly Gly Val Ile Glu Val Pro Ile Glu Phe
Lys Asn 690 695 700Val Pro Asp Phe Gly
Ile Asn Asn Cys Asp Phe Ser Val Lys Tyr Asp705 710
715 720Lys Ser Ile Phe Glu Tyr Val Thr Tyr Glu
Ala Gly Ser Ile Val Lys 725 730
735Asp Ser Ile Val Asn Leu Ala Cys Met Glu Asn Ser Gly Ile Ile Asn
740 745 750Leu Leu Phe Asn Asp
Ala Thr Gln Ser Ser Ser Pro Ile Lys Asn Asn 755
760 765Gly Val Phe Ala Lys Leu Lys Phe Lys Ile Asn Ser
Asn Ala Ala Ser 770 775 780Gly Thr Tyr
Gln Ile Asn Ala Glu Gly Tyr Gly Lys Phe Ser Gly Asn785
790 795 800Leu Asn Gly Lys Leu Thr Ser
Ile Asn Pro Ile Phe Glu Asn Gly Ile 805
810 815Ile Asn Ile Gly Asn Val Thr Val Lys Pro Thr Ser
Thr Pro Ala Asp 820 825 830Ser
Ser Thr Ile Thr Pro Thr Ala Thr Pro Thr Ala Thr Pro Thr Ile 835
840 845Lys Gly Thr Pro Thr Val Thr Pro Ile
Tyr Trp Met Asn Val Leu Ile 850 855
860Gly Asn Met Asn Ala Ala Ile Gly Glu Glu Val Val Val Pro Ile Glu865
870 875 880Phe Lys Asn Val
Pro Pro Phe Gly Ile Asn Asn Cys Asp Phe Lys Leu 885
890 895Val Tyr Asp Ser Asn Ala Leu Glu Leu Lys
Lys Val Glu Ala Gly Asp 900 905
910Ile Val Pro Glu Pro Leu Ala Asn Leu Ser Ser Asn Lys Ser Glu Gly
915 920 925Lys Ile Gln Phe Leu Phe Asn
Asp Ala Ser Gln Gly Ser Met Gln Ile 930 935
940Glu Asn Gly Gly Val Phe Ala Lys Ile Thr Phe Lys Val Lys Ser
Thr945 950 955 960Ala Ala
Ser Gly Ile Tyr Asn Ile Arg Lys Asp Ser Val Gly Ser Phe
965 970 975Ser Gly Leu Ile Asp Asn Lys
Met Thr Ser Ile Gly Pro Lys Phe Thr 980 985
990Asp Gly Ser Ile Val Val Gly Thr Val Thr Pro Thr Ala Thr
Ala Thr 995 1000 1005Pro Ser Ala
Ile Val Thr Thr Ile Thr Pro Thr Ala Thr Thr Lys 1010
1015 1020Pro Ile Ala Thr Pro Thr Ile Lys Gly Thr Pro
Thr Ala Thr Pro 1025 1030 1035Met Tyr
Trp Met Asn Val Val Ile Gly Lys Met Asn Ala Glu Val 1040
1045 1050Gly Gly Glu Val Val Val Pro Ile Glu Phe
Asn Asn Val Pro Ser 1055 1060 1065Phe
Gly Ile Asn Asn Cys Asp Phe Lys Leu Val Tyr Asp Ala Thr 1070
1075 1080Ala Leu Glu Leu Lys Asn Val Glu Ala
Gly Asp Ile Ile Lys Thr 1085 1090
1095Pro Leu Ala Asn Phe Ser Asn Asn Lys Ser Glu Glu Gly Lys Ile
1100 1105 1110Ser Phe Leu Phe Asn Asp
Ala Ser Gln Gly Ser Met Gln Ile Glu 1115 1120
1125Asn Gly Gly Val Phe Ala Lys Ile Thr Phe Lys Val Lys Ser
Thr 1130 1135 1140Thr Ala Thr Gly Val
Tyr Asp Leu Arg Lys Asp Leu Val Gly Ser 1145 1150
1155Phe Ser Gly Leu Lys Asp Asn Lys Met Thr Ser Ile Gly
Ala Glu 1160 1165 1170Phe Thr Asn Gly
Ser Ile Thr Val Ala Ala Thr Ala Pro Thr Val 1175
1180 1185Thr Pro Thr Val Asn Ala Thr Pro Ser Ala Ala
Thr Pro Thr Val 1190 1195 1200Thr Pro
Thr Ala Thr Ala Thr Pro Ser Val Thr Ile Pro Thr Val 1205
1210 1215Thr Pro Thr Ala Thr Ala Thr Pro Ser Val
Thr Ile Pro Thr Val 1220 1225 1230Thr
Pro Thr Ala Thr Ala Thr Pro Ser Ala Ala Thr Pro Thr Val 1235
1240 1245Thr Pro Thr Ala Thr Ala Thr Pro Ser
Val Thr Ile Pro Thr Val 1250 1255
1260Thr Pro Thr Val Thr Ala Thr Pro Ser Asp Thr Ile Pro Thr Val
1265 1270 1275Thr Pro Thr Ala Thr Ala
Thr Pro Ser Ala Ile Val Thr Thr Ile 1280 1285
1290Thr Pro Thr Ala Thr Ala Lys Pro Ile Ala Thr Pro Thr Ile
Lys 1295 1300 1305Gly Thr Pro Thr Ala
Thr Pro Met Tyr Trp Met Asn Val Val Ile 1310 1315
1320Gly Lys Met Asn Ala Glu Val Gly Gly Glu Val Val Val
Pro Ile 1325 1330 1335Glu Phe Lys Asn
Val Pro Ser Phe Gly Ile Asn Asn Cys Asp Phe 1340
1345 1350Lys Leu Val Tyr Asp Ala Thr Ala Leu Glu Leu
Lys Asn Val Glu 1355 1360 1365Ala Gly
Asp Ile Ile Lys Thr Pro Leu Ala Asn Phe Ser Asn Asn 1370
1375 1380Lys Ser Glu Glu Gly Lys Ile Ser Phe Leu
Phe Asn Asp Ala Ser 1385 1390 1395Gln
Gly Ser Met Gln Ile Glu Asn Gly Gly Val Ser Ala Lys Ile 1400
1405 1410Thr Phe Lys Val Lys Ser Thr Thr Ala
Ile Gly Val Tyr Asp Ile 1415 1420
1425Arg Lys Asp Leu Ile Gly Ser Phe Ser Gly Leu Lys Asp Ser Lys
1430 1435 1440Met Thr Ser Ile Gly Ala
Glu Phe Thr Asn Gly Ser Ile Thr Val 1445 1450
1455Ala Thr Thr Ala Pro Thr Val Thr Pro Thr Ala Thr Ala Thr
Pro 1460 1465 1470Ser Val Thr Ile Pro
Thr Val Thr Pro Thr Ala Thr Ala Thr Pro 1475 1480
1485Gly Thr Ala Thr Pro Gly Thr Ala Thr Pro Thr Ala Thr
Ala Thr 1490 1495 1500Pro Gly Ala Ala
Thr Pro Thr Glu Thr Ala Thr Pro Ser Val Met 1505
1510 1515Ile Pro Thr Val Thr Pro Thr Ala Thr Ala Thr
Pro Thr Ala Thr 1520 1525 1530Ala Thr
Pro Thr Val Lys Gly Thr Pro Thr Ile Lys Pro Val Tyr 1535
1540 1545Lys Met Asn Val Val Ile Gly Arg Val Asn
Val Val Ala Gly Glu 1550 1555 1560Glu
Val Val Val Pro Val Glu Phe Lys Asn Ile Pro Ala Ile Gly 1565
1570 1575Val Asn Asn Cys Asn Phe Val Leu Glu
Tyr Asp Ala Asn Val Leu 1580 1585
1590Glu Val Lys Lys Val Asp Ala Gly Glu Ile Val Pro Asp Ala Leu
1595 1600 1605Ile Asn Phe Gly Ser Asn
Asn Ser Asp Glu Gly Lys Val Tyr Phe 1610 1615
1620Leu Phe Asn Asp Ala Leu Gln Gly Arg Met Gln Ile Ala Asn
Asp 1625 1630 1635Gly Ile Phe Ala Asn
Ile Thr Phe Lys Val Lys Ser Ser Ala Ala 1640 1645
1650Ala Gly Ile Tyr Asn Ile Arg Lys Asp Ser Val Gly Ala
Phe Ser 1655 1660 1665Gly Leu Val Asp
Lys Leu Val Pro Ile Ser Ala Glu Phe Thr Asp 1670
1675 1680Gly Ser Ile Ser Val Glu Ser Ala Lys Ser Thr
Pro Thr Ala Thr 1685 1690 1695Ala Thr
Gly Thr Asn Val Thr Pro Thr Val Ala Ala Thr Val Thr 1700
1705 1710Pro Thr Ala Thr Pro Ala Ser Thr Thr Pro
Thr Ala Thr Pro Thr 1715 1720 1725Ala
Thr Ser Thr Val Lys Gly Thr Pro Thr Ala Thr Pro Leu Tyr 1730
1735 1740Ser Met Asn Val Ile Ile Gly Lys Val
Asn Ala Glu Ala Ser Gly 1745 1750
1755Glu Val Val Val Pro Val Glu Phe Lys Asp Val Pro Ser Ile Gly
1760 1765 1770Ile Asn Asn Cys Asn Phe
Ile Leu Glu Tyr Asp Ala Ser Ala Leu 1775 1780
1785Glu Leu Asp Ser Ala Glu Ala Gly Glu Ile Val Pro Val Pro
Leu 1790 1795 1800Gly Asn Phe Ser Ser
Asn Asn Lys Asp Glu Gly Lys Ile Tyr Phe 1805 1810
1815Leu Phe Ser Asp Gly Thr Gln Gly Arg Met Gln Ile Val
Asn Asp 1820 1825 1830Gly Ile Phe Ala
Lys Ile Lys Phe Lys Val Lys Ser Thr Ala Ser 1835
1840 1845Asp Gly Thr Tyr Tyr Ile Arg Lys Asp Ser Val
Gly Ala Phe Ser 1850 1855 1860Gly Leu
Ile Glu Lys Lys Ile Ile Lys Ile Gly Ala Glu Phe Thr 1865
1870 1875Asp Gly Ser Ile Thr Val Arg Ser Leu Thr
Pro Thr Pro Thr Val 1880 1885 1890Thr
Pro Asn Val Ala Ser Pro Thr Pro Thr Lys Val Val Ala Glu 1895
1900 1905Pro Thr Ser Asn Gln Pro Ala Gly Pro
Gly Pro Ile Thr Gly Thr 1910 1915
1920Ile Pro Thr Ala Thr Thr Thr Ala Thr Ala Thr Pro Thr Lys Ala
1925 1930 1935Ser Val Ala Thr Ala Thr
Pro Thr Ala Thr Pro Ile Val Val Val 1940 1945
1950Glu Pro Thr Ile Val Arg Pro Gly Tyr Asn Lys Asp Ala Asp
Leu 1955 1960 1965Ala Val Phe Ile Ser
Ser Asp Lys Ser Arg Tyr Glu Glu Ser Ser 1970 1975
1980Ile Ile Thr Tyr Ser Ile Glu Tyr Lys Asn Ile Gly Lys
Val Asn 1985 1990 1995Ala Thr Asn Val
Lys Ile Ala Ala Gln Ile Pro Lys Phe Thr Lys 2000
2005 2010Val Tyr Asp Ala Ala Lys Gly Ala Val Lys Gly
Ser Glu Ile Val 2015 2020 2025Trp Met
Ile Gly Asn Leu Ala Val Gly Glu Ser Tyr Thr Lys Glu 2030
2035 2040Tyr Lys Val Lys Val Asp Ser Leu Thr Lys
Ser Glu Glu Tyr Thr 2045 2050 2055Asp
Asn Thr Val Thr Ile Ser Ser Asp Gln Thr Val Asp Ile Pro 2060
2065 2070Glu Asn Ile Thr Thr Gly Asn Asp Asp
Lys Ser Thr Ile Arg Val 2075 2080
2085Met Leu Tyr Ser Asn Arg Phe Thr Pro Gly Ser His Ser Ser Tyr
2090 2095 2100Ile Leu Gly Tyr Lys Asp
Lys Thr Phe Lys Pro Lys Gln Asn Val 2105 2110
2115Thr Arg Ala Glu Val Ala Ala Met Phe Ala Arg Ile Met Gly
Leu 2120 2125 2130Thr Val Lys Asp Gly
Ala Lys Ser Ser Tyr Lys Asp Val Ser Asn 2135 2140
2145Lys His Trp Ala Leu Lys Tyr Ile Glu Ala Val Thr Lys
Ser Gly 2150 2155 2160Ile Phe Lys Gly
Tyr Lys Asp Ser Thr Phe His Pro Asn Ala Pro 2165
2170 2175Ile Thr Arg Ala Glu Leu Ser Thr Val Ile Phe
Asn Tyr Leu His 2180 2185 2190Leu Asn
Asn Ile Ala Pro Ser Lys Val His Phe Thr Asp Ile Asn 2195
2200 2205Lys His Trp Ala Lys Asn Tyr Ile Glu Glu
Ile Tyr Arg Phe Lys 2210 2215 2220Leu
Ile Gln Gly Tyr Ser Asp Gly Ser Phe Lys Pro Asn Asn Asn 2225
2230 2235Ile Thr Arg Ala Glu Val Val Thr Met
Ile Asn Arg Met Leu Tyr 2240 2245
2250Arg Gly Pro Leu Lys Val Lys Val Gly Ser Phe Pro Asp Val Ser
2255 2260 2265Pro Lys Tyr Trp Ala Tyr
Gly Asp Ile Glu Glu Ala Ser Arg Asn 2270 2275
2280His Lys Tyr Thr Arg Asp Glu Lys Asp Gly Ser Glu Ile Leu
Ile 2285 2290 2295Glu
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 Val1 5
10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser
Val Ala Ser Ser 20 25 30Ser
Tyr Tyr Trp Gly Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu 35
40 45Trp Ile Gly Thr Ile Asn Phe Ser Gly
Asn Met Tyr Tyr Ser Pro Ser 50 55
60Leu Arg Ser Arg Val Thr Met Ser Ala Asp Met Ser Glu Asn Ser Phe65
70 75 80Tyr Leu Lys Leu Asp
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85
90 95Cys Ala Ala Gly His Leu Val Met Gly Phe Gly
Ala His Trp Gly Gln 100 105
110Gly Lys Leu Val Ser Val Ser Pro Ala Ser Thr Lys Gly Pro Ser Val
115 120 125Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135
140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser145 150 155 160Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro 180 185
190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp
His Lys 195 200 205Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210
215 220Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly
Gly Pro Ser Val225 230 235
240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255Pro Glu Val Thr Cys
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260
265 270Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys 275 280 285Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290
295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340
345 350Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370
375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser385 390 395
400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
Arg 405 410 415Trp Gln Glu
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420
425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Leu Gly Lys Ala 435 440
445Ser Asn Ser Pro Gln Asn Glu Val Leu Tyr Gly Asp Val Asn Asp Asp 450
455 460Gly Lys Val Asn Ser Thr Asp Leu
Thr Leu Leu Lys Arg Tyr Val Leu465 470
475 480Lys Ala Val Ser Thr Leu Pro Ser Ser Lys Ala Glu
Lys Asn Ala Asp 485 490
495Val Asn Arg Asp Gly Arg Val Asn Ser Ser Asp Val Thr Ile Leu Ser
500 505 510Arg Tyr Leu Ile Arg Val
Ile Glu Lys Leu Pro Ile 515 52041635DNAArtificial
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
Ala1 5 10 15Leu Val Lys
Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Asn Asp Tyr 20
25 30Tyr Ile His Trp Val Lys Gln Arg Pro Glu
Gln Gly Leu Glu Arg Ile 35 40
45Gly Trp Ile Asp Pro Asp Asn Gly Asn Thr Ile Tyr Asp Pro Lys Phe 50
55 60Gln Gly Lys Ala Ser Ile Thr Ala Asp
Thr Ser Pro Asn Thr Ala Tyr65 70 75
80Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95Ala Arg
Thr Arg Ser Pro Met Val Thr Thr Gly Phe Val Tyr Trp Gly 100
105 110Gln Gly Thr Val Val Thr Val Ser Ala
Ala Lys Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val145 150
155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly
Thr Lys Thr Tyr Thr Cys Asn Val Asp His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys
Tyr Gly 210 215 220Pro Pro Cys Pro Pro
Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser225 230
235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg 245 250
255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
260 265 270Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275
280 285Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305
310 315 320Lys Cys Lys Val Ser Asn Lys
Gly Leu Pro Ser Ser Ile Glu Lys Thr 325
330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu 340 345 350Pro
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355
360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375
380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385
390 395 400Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405
410 415Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala 420 425
430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440 445Ala Ser Asn Ser Pro Gln Asn
Glu Val Leu Tyr Gly Asp Val Asn Asp 450 455
460Asp Gly Lys Val Asn Ser Thr Asp Leu Thr Leu Leu Lys Arg Tyr
Val465 470 475 480Leu Lys
Ala Val Ser Thr Leu Pro Ser Ser Lys Ala Glu Lys Asn Ala
485 490 495Asp Val Asn Arg Asp Gly Arg
Val Asn Ser Ser Asp Val Thr Ile Leu 500 505
510Ser Arg Tyr Leu Ile Arg Val Ile Glu Lys Leu Pro Ile
515 520 52561638DNAArtificial
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
Gln1 5 10 15Ser Leu Ser
Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Gly 20
25 30Tyr Ser Trp His Trp Ile Arg Gln Phe Pro
Gly Asn Lys Leu Glu Trp 35 40
45Met Gly Tyr Ile Leu Phe Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu 50
55 60Lys Ser Arg Ile Ser Ile Thr Arg Asp
Thr Ser Lys Asn Gln Phe Phe65 70 75
80Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr
Phe Cys 85 90 95Ala Arg
Ser Asn Tyr Gly Ser Phe Ala Ser Trp Gly Gln Gly Thr Leu 100
105 110Val Thr Val Ser Ala Ala Lys Thr Lys
Gly Pro Ser Val Phe Pro Leu 115 120
125Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
130 135 140Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser145 150
155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser 165 170
175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
180 185 190Leu Gly Thr Lys Thr Tyr
Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200
205Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro
Cys Pro 210 215 220Pro Cys Pro Ala Pro
Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe225 230
235 240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val 245 250
255Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
260 265 270Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275
280 285Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr 290 295 300Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val305
310 315 320Ser Asn Lys Gly Leu Pro Ser
Ser Ile Glu Lys Thr Ile Ser Lys Ala 325
330 335Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Gln 340 345 350Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355
360 365Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro 370 375
380Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser385
390 395 400Phe Phe Leu Tyr
Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405
410 415Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His 420 425
430Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Ala Ser Asn Ser
435 440 445Pro Gln Asn Glu Val Leu Tyr
Gly Asp Val Asn Asp Asp Gly Lys Val 450 455
460Asn Ser Thr Asp Leu Thr Leu Leu Lys Arg Tyr Val Leu Lys Ala
Val465 470 475 480Ser Thr
Leu Pro Ser Ser Lys Ala Glu Lys Asn Ala Asp Val Asn Arg
485 490 495Asp Gly Arg Val Asn Ser Ser
Asp Val Thr Ile Leu Ser Arg Tyr Leu 500 505
510Ile Arg Val Ile Glu Lys Leu Pro Ile 515
52081623DNAArtificial 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 Ala1 5
10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Thr Tyr 20 25 30Trp
Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45Gly Tyr Ile Asn Pro Ile Thr Gly Tyr
Thr Glu Tyr Asn Gln Lys Phe 50 55
60Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65
70 75 80Met Gln Leu Ser Ser
Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95Ala Arg Glu Gly Leu Ser Ala Met Asp Tyr Trp
Gly Gln Gly Thr Ser 100 105
110Val Thr Val Thr Ser Ala Lys Thr Thr Gly Pro Ser Val Phe Pro Leu
115 120 125Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135
140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser145 150 155 160Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser 180 185
190Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro
Ser Asn 195 200 205Thr Lys Val Asp
Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210
215 220Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser
Val Phe Leu Phe225 230 235
240Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260
265 270Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro 275 280 285Arg Glu
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290
295 300Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val305 310 315
320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340
345 350Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly 355 360 365Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370
375 380Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser385 390 395
400Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu 405 410 415Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420
425 430Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu
Gly Lys Ala Ser Asn Ser 435 440
445Pro Gln Asn Glu Val Leu Tyr Gly Asp Val Asn Asp Asp Gly Lys Val 450
455 460Asn Ser Thr Asp Leu Thr Leu Leu
Lys Arg Tyr Val Leu Lys Ala Val465 470
475 480Ser Thr Leu Pro Ser Ser Lys Ala Glu Lys Asn Ala
Asp Val Asn Arg 485 490
495Asp Gly Arg Val Asn Ser Ser Asp Val Thr Ile Leu Ser Arg Tyr Leu
500 505 510Ile Arg Val Ile Glu Lys
Leu Pro Ile 515 52010732DNAArtificial
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
Gly1 5 10 15Asp Arg Val
Ser Val Thr Cys Lys Ala Ser Gln Asp Val Thr Ser Ala 20
25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ser Pro Lys Leu Leu Ile 35 40
45Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50
55 60Ser Gly Ser Gly Thr Asp Tyr Thr Leu
Thr Ile Ser Ser Gly Gln Ala65 70 75
80Glu Asp Leu Ala Leu Tyr Tyr Cys His Gln Tyr Tyr Ser Ala
Pro Arg 85 90 95Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100
105 110Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150
155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170
175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 210121644DNAArtificial
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
Thr1 5 10 15Ser Val Lys
Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr 20
25 30Trp Met His Trp Val Lys Gln Arg Pro Gly
Gln Gly Leu Glu Trp Ile 35 40
45Gly Leu Ile Asp Pro Ser Asp Ser Tyr Ser Lys Tyr Asn Gln Lys Phe 50
55 60Lys Gly Lys Ala Thr Leu Thr Val Asp
Thr Ser Ser Ser Thr Ala Tyr65 70 75
80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
Tyr Cys 85 90 95Ala Arg
Gly Glu Leu Ser Asp Phe Trp Gly Gln Gly Thr Thr Leu Thr 100
105 110Val Ser Ser Ala Lys Thr Thr Pro Pro
Ser Val Tyr Pro Leu Ala Pro 115 120
125Gly Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val
130 135 140Lys Gly Tyr Phe Pro Glu Ser
Val Thr Val Thr Trp Asn Ser Gly Ser145 150
155 160Leu Ser Ser Ser Val His Thr Phe Pro Ala Leu Leu
Gln Ser Gly Leu 165 170
175Tyr Thr Met Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser
180 185 190Gln Thr Val Thr Cys Ser
Val Ala His Pro Ala Ser Ser Thr Thr Val 195 200
205Asp Lys Lys Leu Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn
Pro Cys 210 215 220Pro Pro Cys Lys Glu
Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly225 230
235 240Gly Pro Ser Val Phe Ile Phe Pro Pro Asn
Ile Lys Asp Val Leu Met 245 250
255Ile Ser Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Val Ser Glu
260 265 270Asp Asp Pro Asp Val
Arg Ile Ser Trp Phe Val Asn Asn Val Glu Val 275
280 285His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr
Asn Ser Thr Ile 290 295 300Arg Val Val
Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly305
310 315 320Lys Glu Phe Lys Cys Lys Val
Asn Asn Lys Asp Leu Pro Ser Pro Ile 325
330 335Glu Arg Thr Ile Ser Lys Ile Lys Gly Leu Val Arg
Ala Pro Gln Val 340 345 350Tyr
Ile Leu Pro Pro Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser 355
360 365Leu Thr Cys Leu Val Val Gly Phe Asn
Pro Gly Asp Ile Ser Val Glu 370 375
380Trp Thr Ser Asn Gly His Thr Glu Glu Asn Tyr Lys Asp Thr Ala Pro385
390 395 400Val Leu Asp Ser
Asp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asp Ile 405
410 415Lys Thr Ser Lys Trp Glu Lys Thr Asp Ser
Phe Ser Cys Asn Val Arg 420 425
430His Glu Gly Leu Lys Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser
435 440 445Pro Gly Lys Ala Ser Asn Ser
Pro Gln Asn Glu Val Leu Tyr Gly Asp 450 455
460Val Asn Asp Asp Gly Lys Val Asn Ser Thr Asp Leu Thr Leu Leu
Lys465 470 475 480Arg Tyr
Val Leu Lys Ala Val Ser Thr Leu Pro Ser Ser Lys Ala Glu
485 490 495Lys Asn Ala Asp Val Asn Arg
Asp Gly Arg Val Asn Ser Ser Asp Val 500 505
510Thr Ile Leu Ser Arg Tyr Leu Ile Arg Val Ile Glu Lys Leu
Pro Ile 515 520
525142061DNAArtificial 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 Ala1 5
10 15Lys Pro Gly Asp Thr Val Arg Ile Pro Val Arg
Phe Ser Gly Ile Pro 20 25
30Ser Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn
35 40 45Val Leu Glu Ile Ile Glu Ile Glu
Pro Gly Glu Leu Ile Val Asp Pro 50 55
60Asn Pro Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro Asp Arg Lys Met65
70 75 80Ile Val Phe Leu Phe
Ala Glu Asp Ser Gly Thr Gly Ala Tyr Ala Ile 85
90 95Thr Glu Asp Gly Val Phe Ala Thr Ile Val Ala
Lys Val Lys Ser Gly 100 105
110Ala Pro Asn Gly Leu Ser Val Ile Lys Phe Val Glu Val Gly Gly Phe
115 120 125Ala Asn Asn Asp Leu Val Glu
Gln Lys Thr Gln Phe Phe Asp Gly Gly 130 135
140Val Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro
Val145 150 155 160Thr Thr
Pro Thr Thr Thr Asp Asp Leu Asp Ala Leu Glu Ile Ile Pro
165 170 175Val Glu Glu Glu Asn Pro Asp
Phe Trp Asn Arg Glu Ala Ala Glu Ala 180 185
190Leu Gly Ala Ala Lys Lys Leu Gln Pro Ala Gln Thr Ala Ala
Lys Asn 195 200 205Leu Ile Ile Phe
Leu Gly Asp Gly Met Gly Val Ser Thr Val Thr Ala 210
215 220Ala Arg Ile Leu Lys Gly Gln Lys Lys Asp Lys Leu
Gly Pro Glu Leu225 230 235
240Pro Leu Ala Met Asp Arg Phe Pro Tyr Val Ala Leu Ser Lys Thr Tyr
245 250 255Asn Val Asp Lys His
Val Pro Asp Ser Gly Ala Thr Ala Thr Ala Tyr 260
265 270Leu Cys Gly Val Lys Gly Asn Phe Gln Thr Ile Gly
Leu Ser Ala Ala 275 280 285Ala Arg
Phe Asn Gln Cys Asn Thr Thr Arg Gly Asn Glu Val Ile Ser 290
295 300Val Met Asn Arg Ala Lys Lys Ala Gly Lys Ser
Val Gly Val Val Thr305 310 315
320Thr Thr Arg Val Gln His Ala Ser Pro Ala Gly Thr Tyr Ala His Thr
325 330 335Val Asn Arg Asn
Trp Tyr Ser Asp Ala Asp Val Pro Ala Ser Ala Arg 340
345 350Gln Glu Gly Cys Gln Asp Ile Ala Thr Gln Leu
Ile Ser Asn Met Asp 355 360 365Ile
Asp Val Ile Leu Gly Gly Gly Arg Lys Tyr Met Phe Arg Met Gly 370
375 380Thr Pro Asp Pro Glu Tyr Pro Asp Asp Tyr
Ser Gln Gly Gly Thr Arg385 390 395
400Leu Asp Gly Lys Asn Leu Val Gln Glu Trp Leu Ala Lys Arg Gln
Gly 405 410 415Ala Arg Tyr
Val Trp Asn Arg Thr Glu Leu Met Gln Ala Ser Leu Asp 420
425 430Pro Ser Val Thr His Leu Met Gly Leu Phe
Glu Pro Gly Asp Met Lys 435 440
445Tyr Glu Ile His Arg Asp Ser Thr Leu Asp Pro Ser Leu Met Glu Met 450
455 460Thr Glu Ala Ala Leu Arg Leu Leu
Ser Arg Asn Pro Arg Gly Phe Phe465 470
475 480Leu Phe Val Glu Gly Gly Arg Ile Asp His Gly His
His Glu Ser Arg 485 490
495Ala Tyr Arg Ala Leu Thr Glu Thr Ile Met Phe Asp Asp Ala Ile Glu
500 505 510Arg Ala Gly Gln Leu Thr
Ser Glu Glu Asp Thr Leu Ser Leu Val Thr 515 520
525Ala Asp His Ser His Val Phe Ser Phe Gly Gly Tyr Pro Leu
Arg Gly 530 535 540Ser Ser Ile Phe Gly
Leu Ala Pro Gly Lys Ala Arg Asp Arg Lys Ala545 550
555 560Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro
Gly Tyr Val Leu Lys Asp 565 570
575Gly Ala Arg Pro Asp Val Thr Glu Ser Glu Ser Gly Ser Pro Glu Tyr
580 585 590Arg Gln Gln Ser Ala
Val Pro Leu Asp Glu Glu Thr His Ala Gly Glu 595
600 605Asp Val Ala Val Phe Ala Arg Gly Pro Gln Ala His
Leu Val His Gly 610 615 620Val Gln Glu
Gln Thr Phe Ile Ala His Val Met Ala Phe Ala Ala Cys625
630 635 640Leu Glu Pro Tyr Thr Ala Cys
Asp Leu Ala Pro Pro Ala Gly Thr Thr 645
650 655His His His His His His
660162556DNAArtificial 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 Lys1 5
10 15Pro Gly Asp Thr Val Arg Ile Pro Val Arg Phe
Ser Gly Ile Pro Ser 20 25
30Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn Val
35 40 45Leu Glu Ile Ile Glu Ile Lys Pro
Gly Glu Leu Ile Val Asp Pro Asn 50 55
60Pro Asp Lys Ser Phe Asp Thr Ala Val Tyr Pro Asp Arg Lys Ile Ile65
70 75 80Val Phe Leu Phe Ala
Glu Asp Ser Gly Thr Gly Ala Tyr Ala Ile Thr 85
90 95Lys Asp Gly Val Phe Ala Thr Ile Val Ala Lys
Val Lys Ser Gly Ala 100 105
110Pro Asn Gly Leu Ser Val Ile Lys Phe Val Glu Val Gly Gly Phe Ala
115 120 125Asn Asn Asp Leu Val Glu Gln
Lys Thr Gln Phe Phe Asp Gly Gly Val 130 135
140Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro Val
Thr145 150 155 160Thr Pro
Thr Thr Thr Asp Asp Leu Asp Ala Val Arg Ile Lys Val Asp
165 170 175Thr Val Asn Ala Lys Pro Gly
Asp Thr Val Asn Ile Pro Val Arg Phe 180 185
190Ser Gly Ile Pro Ser Lys Gly Ile Ala Asn Cys Asp Phe Val
Tyr Ser 195 200 205Tyr Asp Pro Asn
Val Leu Glu Ile Ile Glu Ile Lys Pro Gly Glu Leu 210
215 220Ile Val Asp Pro Asn Pro Thr Lys Ser Phe Asp Thr
Ala Val Tyr Pro225 230 235
240Asp Arg Lys Met Ile Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly
245 250 255Ala Tyr Ala Ile Thr
Lys Asp Gly Val Phe Ala Thr Ile Val Ala Lys 260
265 270Val Lys Glu Gly Ala Pro Asn Gly Leu Ser Val Ile
Lys Phe Val Glu 275 280 285Val Gly
Gly Phe Ala Asn Asn Asp Leu Val Glu Gln Lys Thr Gln Phe 290
295 300Phe Asp Gly Gly Val Asn Val Gly Asp Thr Thr
Glu Pro Ala Thr Pro305 310 315
320Thr Thr Pro Val Thr Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala Leu
325 330 335Glu Ile Ile Pro
Val Glu Glu Glu Asn Pro Asp Phe Trp Asn Arg Glu 340
345 350Ala Ala Glu Ala Leu Gly Ala Ala Lys Lys Leu
Gln Pro Ala Gln Thr 355 360 365Ala
Ala Lys Asn Leu Ile Ile Phe Leu Gly Asp Gly Met Gly Val Ser 370
375 380Thr Val Thr Ala Ala Arg Ile Leu Lys Gly
Gln Lys Lys Asp Lys Leu385 390 395
400Gly Pro Glu Leu Pro Leu Ala Met Asp Arg Phe Pro Tyr Val Ala
Leu 405 410 415Ser Lys Thr
Tyr Asn Val Asp Lys His Val Pro Asp Ser Gly Ala Thr 420
425 430Ala Thr Ala Tyr Leu Cys Gly Val Lys Gly
Asn Phe Gln Thr Ile Gly 435 440
445Leu Ser Ala Ala Ala Arg Phe Asn Gln Cys Asn Thr Thr Arg Gly Asn 450
455 460Glu Val Ile Ser Val Met Asn Arg
Ala Lys Lys Ala Gly Lys Ser Val465 470
475 480Gly Val Val Thr Thr Thr Arg Val Gln His Ala Ser
Pro Ala Gly Thr 485 490
495Tyr Ala His Thr Val Asn Arg Asn Trp Tyr Ser Asp Ala Asp Val Pro
500 505 510Ala Ser Ala Arg Gln Glu
Gly Cys Gln Asp Ile Ala Thr Gln Leu Ile 515 520
525Ser Asn Met Asp Ile Asp Val Ile Leu Gly Gly Gly Arg Lys
Tyr Met 530 535 540Phe Arg Met Gly Thr
Pro Asp Pro Glu Tyr Pro Asp Asp Tyr Ser Gln545 550
555 560Gly Gly Thr Arg Leu Asp Gly Lys Asn Leu
Val Gln Glu Trp Leu Ala 565 570
575Lys Arg Gln Gly Ala Arg Tyr Val Trp Asn Arg Thr Glu Leu Met Gln
580 585 590Ala Ser Leu Asp Pro
Ser Val Thr His Leu Met Gly Leu Phe Glu Pro 595
600 605Gly Asp Met Lys Tyr Glu Ile His Arg Asp Ser Thr
Leu Asp Pro Ser 610 615 620Leu Met Glu
Met Thr Glu Ala Ala Leu Arg Leu Leu Ser Arg Asn Pro625
630 635 640Arg Gly Phe Phe Leu Phe Val
Glu Gly Gly Arg Ile Asp His Gly His 645
650 655His Glu Ser Arg Ala Tyr Arg Ala Leu Thr Glu Thr
Ile Met Phe Asp 660 665 670Asp
Ala Ile Glu Arg Ala Gly Gln Leu Thr Ser Glu Glu Asp Thr Leu 675
680 685Ser Leu Val Thr Ala Asp His Ser His
Val Phe Ser Phe Gly Gly Tyr 690 695
700Pro Leu Arg Gly Ser Ser Ile Phe Gly Leu Ala Pro Gly Lys Ala Arg705
710 715 720Asp Arg Lys Ala
Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro Gly Tyr 725
730 735Val Leu Lys Asp Gly Ala Arg Pro Asp Val
Thr Glu Ser Glu Ser Gly 740 745
750Ser Pro Glu Tyr Arg Gln Gln Ser Ala Val Pro Leu Asp Glu Glu Thr
755 760 765His Ala Gly Glu Asp Val Ala
Val Phe Ala Arg Gly Pro Gln Ala His 770 775
780Leu Val His Gly Val Gln Glu Gln Thr Phe Ile Ala His Val Met
Ala785 790 795 800Phe Ala
Ala Cys Leu Glu Pro Tyr Thr Ala Cys Asp Leu Ala Pro Pro
805 810 815Ala Gly Thr Thr His His His
His His His 820 825181326DNAArtificial
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
Ala1 5 10 15Lys Pro Gly
Asp Thr Val Arg Ile Pro Val Arg Phe Ser Gly Ile Pro 20
25 30Ser Lys Gly Ile Ala Asn Cys Asp Phe Val
Tyr Ser Tyr Asp Pro Asn 35 40
45Val Leu Glu Ile Ile Glu Ile Glu Pro Gly Glu Leu Ile Val Asp Pro 50
55 60Asn Pro Thr Lys Ser Phe Asp Thr Ala
Val Tyr Pro Asp Arg Lys Met65 70 75
80Ile Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly Ala Tyr
Ala Ile 85 90 95Thr Glu
Asp Gly Val Phe Ala Thr Ile Val Ala Lys Val Lys Ser Gly 100
105 110Ala Pro Asn Gly Leu Ser Val Ile Lys
Phe Val Glu Val Gly Gly Phe 115 120
125Ala Asn Asn Asp Leu Val Glu Gln Lys Thr Gln Phe Phe Asp Gly Gly
130 135 140Val Asn Val Gly Asp Thr Thr
Glu Pro Ala Thr Pro Thr Thr Pro Val145 150
155 160Thr Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala Leu
Glu Ala Pro Leu 165 170
175Ile Leu Ser Arg Ile Val Gly Gly Trp Glu Cys Glu Lys His Ser Gln
180 185 190Pro Trp Gln Val Leu Val
Ala Ser Arg Gly Arg Ala Val Cys Gly Gly 195 200
205Val Leu Val His Pro Gln Trp Val Leu Thr Ala Ala His Cys
Ile Arg 210 215 220Asn Lys Ser Val Ile
Leu Leu Gly Arg His Ser Leu Phe His Pro Glu225 230
235 240Asp Thr Gly Gln Val Phe Gln Val Ser His
Ser Phe Pro His Pro Leu 245 250
255Tyr Asp Met Ser Leu Leu Lys Asn Arg Phe Leu Arg Pro Gly Asp Asp
260 265 270Ser Ser His Asp Leu
Met Leu Leu Arg Leu Ser Glu Pro Ala Glu Leu 275
280 285Thr Asp Ala Val Lys Val Met Asp Leu Pro Thr Gln
Glu Pro Ala Leu 290 295 300Gly Thr Thr
Cys Tyr Ala Ser Gly Trp Gly Ser Ile Glu Pro Glu Glu305
310 315 320Phe Leu Thr Pro Lys Lys Leu
Gln Cys Val Asp Leu His Val Ile Ser 325
330 335Asn Asp Val Cys Ala Gln Val His Pro Gln Lys Val
Thr Lys Phe Met 340 345 350Leu
Cys Ala Gly Arg Trp Thr Gly Gly Lys Ser Thr Cys Ser Gly Asp 355
360 365Ser Gly Gly Pro Leu Val Cys Asn Gly
Val Leu Gln Gly Ile Thr Ser 370 375
380Trp Gly Ser Glu Pro Cys Ala Leu Pro Glu Arg Pro Ser Leu Tyr Thr385
390 395 400Lys Val Val His
Tyr Arg Lys Trp Ile Lys Asp Thr Ile Val Ala Asn 405
410 415Pro201554DNAArtificial 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 Ala1 5
10 15Lys Pro Gly Asp Thr Val Arg Ile
Pro Val Arg Phe Ser Gly Ile Pro 20 25
30Ser Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro
Asn 35 40 45Val Leu Glu Ile Ile
Glu Ile Glu Pro Gly Glu Leu Ile Val Asp Pro 50 55
60Asn Pro Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro Asp Arg
Lys Met65 70 75 80Ile
Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly Ala Tyr Ala Ile
85 90 95Thr Glu Asp Gly Val Phe Ala
Thr Ile Val Ala Lys Val Lys Ser Gly 100 105
110Ala Pro Asn Gly Leu Ser Val Ile Lys Phe Val Glu Val Gly
Gly Phe 115 120 125Ala Asn Asn Asp
Leu Val Glu Gln Lys Thr Gln Phe Phe Asp Gly Gly 130
135 140Val Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro
Thr Thr Pro Val145 150 155
160Thr Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala Leu Glu Asp Gln Ile
165 170 175Cys Ile Gly Tyr His
Ala Asn Asn Ser Thr Glu Gln Val Asp Thr Ile 180
185 190Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp
Ile Leu Glu Lys 195 200 205Lys His
Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys Pro Leu Ile 210
215 220Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu
Gly Asn Pro Met Cys225 230 235
240Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val Glu Lys Ala
245 250 255Asn Pro Val Asn
Asp Leu Cys Tyr Pro Gly Asp Phe Asn Asp Tyr Glu 260
265 270Lys Leu Lys His Leu Leu Ser Arg Ile Asn His
Phe Glu Lys Ile Gln 275 280 285Ile
Ile Pro Lys Ser Ser Trp Ser Ser His Glu Ala Ser Leu Gly Val 290
295 300Ser Ser Ala Cys Pro Tyr Gln Gly Lys Ser
Ser Phe Phe Arg Asn Val305 310 315
320Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro Thr Ile Lys Arg
Ser 325 330 335Tyr Asn Asn
Thr Asn Gln Glu Asp Leu Leu Val Leu Trp Gly Ile His 340
345 350His Pro Asn Asp Ala Ala Glu Gln Thr Lys
Leu Tyr Gln Asn Pro Thr 355 360
365Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg Leu Val Pro 370
375 380Arg Ile Ala Thr Arg Ser Lys Val
Asn Gly Gln Ser Gly Arg Met Glu385 390
395 400Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile
Asn Phe Glu Ser 405 410
415Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile Val Lys Lys
420 425 430Gly Asp Ser Thr Ile Met
Lys Ser Glu Leu Glu Tyr Gly Asn Cys Asn 435 440
445Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser Met
Pro Phe 450 455 460His Asn Ile His Pro
Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys465 470
475 480Ser Asn Arg Leu Val Leu Ala His His His
His His His 485 490221293DNAArtificial
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 Lys1 5 10
15Pro Gly Asp Thr Val Asn Ile Pro Val Arg Phe Ser Gly Ile Pro Ser
20 25 30Lys Gly Ile Ala Asn Cys Asp
Phe Val Tyr Ser Tyr Asp Pro Asn Val 35 40
45Leu Glu Ile Ile Glu Ile Lys Pro Gly Glu Leu Ile Val Asp Pro
Asn 50 55 60Pro Thr Lys Ser Phe Asp
Thr Ala Val Tyr Pro Asp Arg Lys Met Ile65 70
75 80Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly
Ala Tyr Ala Ile Thr 85 90
95Lys Asp Gly Val Phe Ala Thr Ile Val Ala Lys Val Lys Glu Gly Ala
100 105 110Pro Asn Gly Leu Ser Val
Ile Lys Phe Val Glu Val Gly Gly Phe Ala 115 120
125Asn Asn Asp Leu Val Glu Gln Lys Thr Gln Phe Phe Asp Gly
Gly Val 130 135 140Asn Val Gly Asp Thr
Thr Glu Pro Ala Thr Pro Thr Thr Pro Val Thr145 150
155 160Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala
Ala Ser Leu Leu Thr Glu 165 170
175Val Glu Thr Tyr Val Leu Ser Ile Ile Pro Ser Gly Pro Leu Lys Ala
180 185 190Glu Ile Ala Gln Arg
Leu Glu Asp Val Phe Ala Gly Lys Asn Thr Asp 195
200 205Leu Glu Val Leu Met Glu Trp Leu Lys Thr Arg Pro
Ile Leu Ser Pro 210 215 220Leu Thr Lys
Gly Ile Leu Gly Phe Val Phe Thr Leu Thr Val Pro Ser225
230 235 240Glu Arg Gly Leu Gln Arg Arg
Arg Phe Val Gln Asn Ala Leu Asn Gly 245
250 255Asn Gly Asp Pro Asn Asn Met Asp Lys Ala Val Lys
Leu Tyr Arg Lys 260 265 270Leu
Lys Arg Glu Ile Thr Phe His Gly Ala Lys Glu Ile Ala Leu Ser 275
280 285Tyr Ser Ala Gly Ala Leu Ala Ser Cys
Met Gly Leu Ile Tyr Asn Arg 290 295
300Met Gly Ala Val Thr Thr Glu Val Ala Phe Gly Leu Val Cys Ala Thr305
310 315 320Cys Glu Gln Ile
Ala Asp Ser Gln His Arg Ser His Arg Gln Met Val 325
330 335Thr Thr Thr Asn Pro Leu Ile Arg His Glu
Asn Arg Met Val Leu Ala 340 345
350Ser Thr Thr Ala Lys Ala Met Glu Gln Met Ala Gly Ser Ser Glu Gln
355 360 365Ala Ala Glu Ala Met Asp Ile
Ala Ser Gln Ala Arg Gln Met Val Gln 370 375
380Ala Met Arg Thr Ile Gly Thr His Pro Ser Ser Ser Ala Gly Leu
Lys385 390 395 400Asp Asp
Leu Leu Glu Asn Leu Gln Ala Tyr Gln Lys Arg Met Gly Val
405 410 415Gln Met Gln Arg Phe Lys Leu
Glu His His His His His His 420 425
430249PRTArtificial SequenceSynthetic peptide. 24Gly Ile Leu Gly Phe
Val Phe Thr Leu1 525661PRTArtificial SequenceSynthetic
peptide. 25Met Asp Leu Val Leu Lys Arg Cys Leu Leu His Leu Ala Val Ile
Gly1 5 10 15Ala Leu Leu
Ala Val Gly Ala Thr Lys Val Pro Arg Asn Gln Asp Trp 20
25 30Leu Gly Val Ser Arg Gln Leu Arg Thr Lys
Ala Trp Asn Arg Gln Leu 35 40
45Tyr Pro Glu Trp Thr Glu Ala Gln Arg Leu Asp Cys Trp Arg Gly Gly 50
55 60Gln Val Ser Leu Lys Val Ser Asn Asp
Gly Pro Thr Leu Ile Gly Ala65 70 75
80Asn Ala Ser Phe Ser Ile Ala Leu Asn Phe Pro Gly Ser Gln
Lys Val 85 90 95Leu Pro
Asp Gly Gln Val Ile Trp Val Asn Asn Thr Ile Ile Asn Gly 100
105 110Ser Gln Val Trp Gly Gly Gln Pro Val
Tyr Pro Gln Glu Thr Asp Asp 115 120
125Ala Cys Ile Phe Pro Asp Gly Gly Pro Cys Pro Ser Gly Ser Trp Ser
130 135 140Gln Lys Arg Ser Phe Val Tyr
Val Trp Lys Thr Trp Gly Gln Tyr Trp145 150
155 160Gln Val Leu Gly Gly Pro Val Ser Gly Leu Ser Ile
Gly Thr Gly Arg 165 170
175Ala Met Leu Gly Thr His Thr Met Glu Val Thr Val Tyr His Arg Arg
180 185 190Gly Ser Arg Ser Tyr Val
Pro Leu Ala His Ser Ser Ser Ala Phe Thr 195 200
205Ile Thr Asp Gln Val Pro Phe Ser Val Ser Val Ser Gln Leu
Arg Ala 210 215 220Leu Asp Gly Gly Asn
Lys His Phe Leu Arg Asn Gln Pro Leu Thr Phe225 230
235 240Ala Leu Gln Leu His Asp Pro Ser Gly Tyr
Leu Ala Glu Ala Asp Leu 245 250
255Ser Tyr Thr Trp Asp Phe Gly Asp Ser Ser Gly Thr Leu Ile Ser Arg
260 265 270Ala Leu Val Val Thr
His Thr Tyr Leu Glu Pro Gly Pro Val Thr Ala 275
280 285Gln Val Val Leu Gln Ala Ala Ile Pro Leu Thr Ser
Cys Gly Ser Ser 290 295 300Pro Val Pro
Gly Thr Thr Asp Gly His Arg Pro Thr Ala Glu Ala Pro305
310 315 320Asn Thr Thr Ala Gly Gln Val
Pro Thr Thr Glu Val Val Gly Thr Thr 325
330 335Pro Gly Gln Ala Pro Thr Ala Glu Pro Ser Gly Thr
Thr Ser Val Gln 340 345 350Val
Pro Thr Thr Glu Val Ile Ser Thr Ala Pro Val Gln Met Pro Thr 355
360 365Ala Glu Ser Thr Gly Met Thr Pro Glu
Lys Val Pro Val Ser Glu Val 370 375
380Met Gly Thr Thr Leu Ala Glu Met Ser Thr Pro Glu Ala Thr Gly Met385
390 395 400Thr Pro Ala Glu
Val Ser Ile Val Val Leu Ser Gly Thr Thr Ala Ala 405
410 415Gln Val Thr Thr Thr Glu Trp Val Glu Thr
Thr Ala Arg Glu Leu Pro 420 425
430Ile Pro Glu Pro Glu Gly Pro Asp Ala Ser Ser Ile Met Ser Thr Glu
435 440 445Ser Ile Thr Gly Ser Leu Gly
Pro Leu Leu Asp Gly Thr Ala Thr Leu 450 455
460Arg Leu Val Lys Arg Gln Val Pro Leu Asp Cys Val Leu Tyr Arg
Tyr465 470 475 480Gly Ser
Phe Ser Val Thr Leu Asp Ile Val Gln Gly Ile Glu Ser Ala
485 490 495Glu Ile Leu Gln Ala Val Pro
Ser Gly Glu Gly Asp Ala Phe Glu Leu 500 505
510Thr Val Ser Cys Gln Gly Gly Leu Pro Lys Glu Ala Cys Met
Glu Ile 515 520 525Ser Ser Pro Gly
Cys Gln Pro Pro Ala Gln Arg Leu Cys Gln Pro Val 530
535 540Leu Pro Ser Pro Ala Cys Gln Leu Val Leu His Gln
Ile Leu Lys Gly545 550 555
560Gly Ser Gly Thr Tyr Cys Leu Asn Val Ser Leu Ala Asp Thr Asn Ser
565 570 575Leu Ala Val Val Ser
Thr Gln Leu Ile Met Pro Gly Gln Glu Ala Gly 580
585 590Leu Gly Gln Val Pro Leu Ile Val Gly Ile Leu Leu
Val Leu Met Ala 595 600 605Val Val
Leu Ala Ser Leu Ile Tyr Arg Arg Arg Leu Met Lys Gln Asp 610
615 620Phe Ser Val Pro Gln Leu Pro His Ser Ser Ser
His Trp Leu Arg Leu625 630 635
640Pro Arg Ile Phe Cys Ser Cys Pro Ile Gly Glu Asn Ser Pro Leu Leu
645 650 655Ser Gly Gln Gln
Val 660269PRTArtificial SequenceSynthetic peptide. 26Lys Thr
Trp Gly Gln Tyr Trp Gln Val1 5279PRTArtificial
SequenceSynthetic peptide. 27Ile Met Asp Gln Val Pro Phe Ser Val1
5289PRTArtificialChemically synthesized peptide 28Ile Thr Asp Gln
Val Pro Phe Ser Val1 5299PRTArtificial SequenceSynthetic
peptide. 29Tyr Leu Glu Pro Gly Pro Val Thr Val1
5309PRTArtificialChemically synthesized peptide 30Tyr Leu Glu Pro Gly Pro
Val Thr Ala1 531204PRTArtificial SequenceSynthetic peptide.
31Met Asp Leu Asp Ala Val Arg Ile Lys Val Asp Thr Val Asn Ala Lys1
5 10 15Pro Gly Asp Thr Val Asn
Ile Pro Val Arg Phe Ser Gly Ile Pro Ser 20 25
30Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp
Pro Asn Val 35 40 45Leu Glu Ile
Ile Glu Ile Lys Pro Gly Glu Leu Ile Val Asp Pro Asn 50
55 60Pro Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro Asp
Arg Lys Met Ile65 70 75
80Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly Ala Tyr Ala Ile Thr
85 90 95Lys Asp Gly Val Phe Ala
Thr Ile Val Ala Lys Val Lys Glu Gly Ala 100
105 110Pro Asn Gly Leu Ser Val Ile Lys Phe Val Glu Val
Gly Gly Phe Ala 115 120 125Asn Asn
Asp Leu Val Glu Gln Lys Thr Gln Phe Phe Asp Gly Gly Val 130
135 140Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro
Thr Thr Pro Val Thr145 150 155
160Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala Ala Arg Ser Ala Phe Thr
165 170 175Ile Met Asp Gln
Val Pro Phe Ser Val Ser Val Ser Ala Ser Arg Lys 180
185 190Gly Ala Ala Ala Leu Glu His His His His His
His 195 20032118PRTArtificial SequenceSynthetic
peptide. 32Met Pro Arg Glu Asp Ala His Phe Ile Tyr Gly Tyr Pro Lys Lys
Gly1 5 10 15His Gly His
Ser Tyr Thr Thr Ala Glu Glu Ala Ala Gly Ile Gly Ile 20
25 30Leu Thr Val Ile Leu Gly Val Leu Leu Leu
Ile Gly Cys Trp Tyr Cys 35 40
45Arg Arg Arg Asn Gly Tyr Arg Ala Leu Met Asp Lys Ser Leu His Val 50
55 60Gly Thr Gln Cys Ala Leu Thr Arg Arg
Cys Pro Gln Glu Gly Phe Asp65 70 75
80His Arg Asp Ser Lys Val Ser Leu Gln Glu Lys Asn Cys Glu
Pro Val 85 90 95Val Pro
Asn Ala Pro Pro Ala Tyr Glu Lys Leu Ser Ala Glu Gln Ser 100
105 110Pro Pro Pro Tyr Ser Pro
115339PRTArtificial SequenceSynthetic peptide. 33Ala Ala Gly Ile Gly Ile
Leu Thr Val1 53410PRTArtificial SequenceSynthetic peptide.
34Glu Ala Ala Gly Ile Gly Ile Leu Thr Val1 5
103510PRTArtificial SequenceSynthetic peptide. 35Glu Leu Ala Gly Ile
Gly Ile Leu Thr Val1 5
1036204PRTArtificial SequenceSynthetic peptide. 36Met Asp Leu Asp Ala Val
Arg Ile Lys Val Asp Thr Val Asn Ala Lys1 5
10 15Pro Gly Asp Thr Val Asn Ile Pro Val Arg Phe Ser
Gly Ile Pro Ser 20 25 30Lys
Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn Val 35
40 45Leu Glu Ile Ile Glu Ile Lys Pro Gly
Glu Leu Ile Val Asp Pro Asn 50 55
60Pro Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro Asp Arg Lys Met Ile65
70 75 80Val Phe Leu Phe Ala
Glu Asp Ser Gly Thr Gly Ala Tyr Ala Ile Thr 85
90 95Lys Asp Gly Val Phe Ala Thr Ile Val Ala Lys
Val Lys Glu Gly Ala 100 105
110Pro Asn Gly Leu Ser Val Ile Lys Phe Val Glu Val Gly Gly Phe Ala
115 120 125Asn Asn Asp Leu Val Glu Gln
Lys Thr Gln Phe Phe Asp Gly Gly Val 130 135
140Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro Val
Thr145 150 155 160Thr Pro
Thr Thr Thr Asp Asp Leu Asp Ala Ala Arg Thr Ala Glu Glu
165 170 175Leu Ala Gly Ile Gly Ile Leu
Thr Val Ile Leu Gly Ala Ser Arg Lys 180 185
190Gly Ala Ala Ala Leu Glu His His His His His His
195 20037241PRTArtificial SequenceSynthetic peptide.
37Met Asp Leu Asp Ala Val Arg Ile Lys Val Asp Thr Val Asn Ala Lys1
5 10 15Pro Gly Asp Thr Val Asn
Ile Pro Val Arg Phe Ser Gly Ile Pro Ser 20 25
30Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp
Pro Asn Val 35 40 45Leu Glu Ile
Ile Glu Ile Lys Pro Gly Glu Leu Ile Val Asp Pro Asn 50
55 60Pro Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro Asp
Arg Lys Met Ile65 70 75
80Val Phe Leu Phe Ala Glu Asp Ser Gly Thr Gly Ala Tyr Ala Ile Thr
85 90 95Lys Asp Gly Val Phe Ala
Thr Ile Val Ala Lys Val Lys Glu Gly Ala 100
105 110Pro Asn Gly Leu Ser Val Ile Lys Phe Val Glu Val
Gly Gly Phe Ala 115 120 125Asn Asn
Asp Leu Val Glu Gln Lys Thr Gln Phe Phe Asp Gly Gly Val 130
135 140Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro
Thr Thr Pro Val Thr145 150 155
160Thr Pro Thr Thr Thr Asp Asp Leu Asp Ala Ala Ser Asp Thr Thr Glu
165 170 175Ala Arg His Pro
His Pro Pro Val Thr Thr Pro Thr Thr Asp Arg Lys 180
185 190Gly Thr Thr Ala Glu Glu Leu Ala Gly Ile Gly
Ile Leu Thr Val Ile 195 200 205Leu
Gly Gly Lys Arg Thr Asn Asn Ser Thr Pro Thr Lys Gly Glu Phe 210
215 220Cys Arg Tyr Pro Ser His Trp Arg Pro Leu
Glu His His His His His225 230 235
240His3866PRTArtificial SequenceSynthetic peptide. 38Ala Ser Asp
Thr Thr Glu Ala Arg His Pro His Pro Pro Val Thr Thr1 5
10 15Pro Thr Thr Thr Asp Arg Lys Gly Thr
Thr Ala Glu Glu Leu Ala Gly 20 25
30Ile Gly Ile Leu Thr Val Ile Leu Gly Gly Lys Arg Thr Asn Asn Ser
35 40 45Thr Pro Thr Lys Gly Glu Phe
Cys Arg Tyr Pro Ser His Trp Arg Pro 50 55
60Arg Leu653957DNAArtificial 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 Lys1 5
10 15Pro Gly Asp Thr Val Asn Ile Pro Val Arg Phe Ser
Gly Ile Pro Ser 20 25 30Lys
Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn Val 35
40 45Leu Glu Ile Ile Glu Ile Lys Pro Gly
Glu Leu Ile Val Asp Pro Asn 50 55
60Pro Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro Asp Arg Lys Met Ile65
70 75 80Val Phe Leu Phe Ala
Glu Asp Ser Gly Thr Gly Ala Tyr Ala Ile Thr 85
90 95Lys Asp Gly Val Phe Ala Thr Ile Val Ala Lys
Val Lys Glu Gly Ala 100 105
110Pro Asn Gly Leu Ser Val Ile Lys Phe Val Glu Val Gly Gly Phe Ala
115 120 125Asn Asn Asp Leu Val Glu Gln
Lys Thr Gln Phe Phe Asp Gly Gly Val 130 135
140Asn Val Gly Asp Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro Val
Thr145 150 155 160Thr Pro
Thr Thr Thr Asp Asp Leu Asp Ala Ala Ser Glu Gly Gly Ser
165 170 175Leu Ala Ala Leu Thr Ala His
Gln Ala Cys His Leu Pro Leu Glu Thr 180 185
190Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu
Gln Cys 195 200 205Gly Tyr Pro Val
Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu 210
215 220Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala
Leu Ala Ser Pro225 230 235
240Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln
245 250 255Ala Arg Leu Ala Leu
Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val 260
265 270Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala
Asn Gly Pro Ala 275 280 285Asp Ser
Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu 290
295 300Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser
Thr Arg Gly Thr Gln305 310 315
320Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu
325 330 335Arg Gly Tyr Val
Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala 340
345 350Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg
Ser Gln Asp Leu Asp 355 360 365Ala
Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr 370
375 380Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala
Arg Gly Arg Ile Arg Asn385 390 395
400Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly
Phe 405 410 415Tyr Arg Thr
Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val 420
425 430Glu Arg Leu Ile Gly His Pro Leu Pro Leu
Arg Leu Asp Ala Ile Thr 435 440
445Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro 450
455 460Leu Ala Glu Arg Thr Val Val Ile
Pro Ser Ala Ile Pro Thr Asp Pro465 470
475 480Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile
Pro Asp Lys Glu 485 490
495Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro
500 505 510Pro Arg Glu Asp Leu Lys
51542614PRTArtificial SequenceSynthetic peptide. 42Gln Ile Gln Leu
Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5
10 15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr Ser Phe Thr Asn Tyr 20 25
30Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met
35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly
Glu Ser Thr Tyr Ala Asp Asp Phe 50 55
60Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr65
70 75 80Leu Gln Ile Ser Asn
Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe Cys 85
90 95Ala Arg Gly Asp Phe Arg Tyr Tyr Tyr Phe Asp
Tyr Trp Gly Gln Gly 100 105
110Thr Thr Leu Thr Gly Ser Ser Ala Lys Thr Lys Gly Pro Ser Val Phe
115 120 125Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135
140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp145 150 155 160Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
Lys Pro 195 200 205Ser Asn Thr Lys
Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210
215 220Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly
Pro Ser Val Phe225 230 235
240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
245 250 255Glu Val Thr Cys Val
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260
265 270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr 275 280 285Lys Pro
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290
295 300Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys305 310 315
320Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser
325 330 335Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340
345 350Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val 355 360 365Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370
375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp385 390 395
400Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
Trp 405 410 415Gln Glu Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420
425 430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Leu Gly Lys Ala Ser 435 440
445Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro Val Thr Thr Pro Thr Thr 450
455 460Thr Asp Asp Leu Asp Ala Val Arg
Ile Lys Val Asp Thr Val Asn Ala465 470
475 480Lys Pro Gly Asp Thr Val Asn Ile Pro Val Arg Phe
Ser Gly Ile Pro 485 490
495Ser Lys Gly Ile Ala Asn Cys Asp Phe Val Tyr Ser Tyr Asp Pro Asn
500 505 510Val Leu Glu Ile Ile Glu
Ile Lys Pro Gly Glu Leu Ile Val Asp Pro 515 520
525Asn Pro Thr Lys Ser Phe Asp Thr Ala Val Tyr Pro Asp Arg
Lys Met 530 535 540Ile Val Phe Leu Phe
Ala Glu Asp Ser Gly Thr Gly Ala Tyr Ala Ile545 550
555 560Thr Lys Asp Gly Val Phe Ala Thr Ile Val
Ala Lys Val Lys Glu Gly 565 570
575Ala Pro Asn Gly Leu Ser Val Ile Lys Phe Val Glu Val Gly Gly Phe
580 585 590Ala Asn Asn Asp Leu
Val Glu Gln Lys Thr Gln Phe Phe Asp Gly Gly 595
600 605Val Asn Val Gly Asp Thr 61043308PRTArtificial
SequenceSynthetic peptide. 43Leu Asp Asp Leu Asp Ala Val Arg Ile Lys Val
Asp Thr Val Asn Ala1 5 10
15Lys Pro Gly Asp Thr Val Arg Ile Pro Val Arg Phe Ser Gly Ile Pro
20 25 30Ser Lys Gly Ile Ala Asn Cys
Asp Phe Val Tyr Ser Tyr Asp Pro Asn 35 40
45Val Leu Glu Ile Ile Glu Ile Glu Pro Gly Asp Ile Ile Val Asp
Pro 50 55 60Asn Pro Asp Lys Ser Phe
Asp Thr Ala Val Tyr Pro Asp Arg Lys Ile65 70
75 80Ile Val Phe Leu Phe Ala Glu Asp Ser Gly Thr
Gly Ala Tyr Ala Ile 85 90
95Thr Lys Asp Gly Val Phe Ala Thr Ile Val Ala Lys Val Lys Glu Gly
100 105 110Ala Pro Asn Gly Leu Ser
Val Ile Lys Phe Val Glu Val Gly Gly Phe 115 120
125Ala Asn Asn Asp Leu Val Glu Gln Lys Thr Gln Phe Phe Asp
Gly Gly 130 135 140Val Asn Val Gly Asp
Thr Thr Glu Pro Ala Thr Pro Thr Thr Pro Val145 150
155 160Thr Thr Pro Thr Thr Thr Asp Asp Leu Asp
Ala Leu Glu Ala Asp Gln 165 170
175Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val
180 185 190Asp Gln Leu Lys Asn
Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro 195
200 205Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser
Ala Phe Ser Cys 210 215 220Phe Gln Lys
Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg225
230 235 240Ile Ile Asn Val Ser Ile Lys
Lys Leu Lys Arg Lys Pro Pro Ser Thr 245
250 255Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys
Pro Ser Cys Asp 260 265 270Ser
Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser 275
280 285Leu Leu Gln Lys Met Ile His Gln His
Leu Ser Ser Arg Thr His Gly 290 295
300Ser Glu Asp Ser305
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