阅读说明：本技术 异二聚体蛋白及其用途 (Heterodimeric proteins and uses thereof ) 是由 T·施赖伯 G·弗罗姆 S·达西瓦 于 2019-06-21 设计创作，主要内容包括：本发明尤其涉及包含可用于治疗疾病,例如针对癌症和自身免疫的免疫疗法的异二聚体蛋白的组合物和方法。(The invention relates, inter alia, to compositions and methods comprising heterodimeric proteins useful in the treatment of diseases, such as immunotherapy against cancer and autoimmunity.)

1. A heterodimeric protein comprising a first polypeptide chain and a second polypeptide chain, wherein

The first polypeptide chain comprises a first subunit of the first protein at the amino terminus linked to a first subunit of the second protein at the carboxy terminus by a first charge polarizing core domain; and is

A second polypeptide chain comprising a second subunit of the first protein at the amino terminus linked to a second subunit of the second protein at the carboxy terminus by a second charge polarizing core domain; and is

Wherein the first polypeptide chain and the second polypeptide chain form a heterodimer via electrostatic interaction between positively charged amino acid residues and negatively charged amino acid residues on the first and second charge polarizing core domains.

2. A heterodimeric protein according to claim 1 wherein said first and/or second charge polarized core domain comprises a polypeptide linker, optionally selected from a flexible amino acid sequence, an IgG hinge region or an antibody sequence.

3. A heterodimeric protein according to claim 2 wherein said linker is a synthetic linker, optionally PEG.

4. A heterodimeric protein according to claim 2 wherein said linker comprises a hinge-CH 2-CH3 Fc domain derived from IgG1, optionally human IgG 1.

5. A heterodimeric protein according to claim 2 wherein said linker comprises a hinge-CH 2-CH3 Fc domain derived from IgG4, optionally human IgG 4.

6. A heterodimeric protein according to any of the preceding claims wherein said first and/or second charge polarizing core domain further comprises a peptide having positively and/or negatively charged amino acid residues at the amino and carboxy terminus of said charge polarizing core domain.

7. A heterodimeric protein according to claim 6 wherein said peptide comprising positively charged amino acid residues may comprise one or more amino acids selected from His, Lys and Arg.

8. A heterodimeric protein according to claim 7 wherein said peptide comprising a positively charged amino acid residue comprises a sequence selected from: y is_nX_nY_nX_nY_n(wherein X is a positively charged amino acid such as arginine, histidine or lysine and Y is a spacer amino acid such as serine or glycine) (SEQ ID NO: 1), YY_nXX_nYY_nXX_nYY_n(wherein X is a positively charged amino acid such as arginine, histidine or lysine and Y is a spacer amino acid such as serine or glycine) (SEQ ID NO: 3) and Y_nX_nCY_nX_nY_n(wherein X is a positively charged amino acid such as arginine, histidine or lysine and Y is a spacer amino acid such as serine or glycine) (SEQ ID NO: 5).

9. A heterodimeric protein according to claim 8 wherein said peptide comprising a positively charged amino acid residue comprises the sequence RKGGKR (SEQ ID NO: 11) or GSGSRKGGKRGS (SEQ ID NO: 12).

10. A heterodimeric protein according to claim 6 wherein said peptide comprising negatively charged amino acid residues may comprise one or more amino acids selected from Asp and Glu.

11. A heterodimeric protein according to claim 9 wherein said peptide comprising negatively charged amino acid residues comprises a sequence selected from: y is_nZ_nY_nZ_nY_n(wherein Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a spacer amino acid such as serine or glycine) (SEQ ID NO: 2), YY_nZZ_nYY_nZZ_nYY_n(wherein Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a spacer amino acid such as serine or glycine) (SEQ ID NO: 4) and Y_nZ_nCY_nZ_nY_n(wherein Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a spacer amino acid such as serine or glycine) (SEQ ID NO: 6).

12. A heterodimeric protein according to claim 8 wherein said peptide comprising a positively charged amino acid residue comprises the sequence DEGGED (SEQ ID NO: 13) or GSGSDEGGEDGS (SEQ ID NO: 14).

13. A heterodimeric protein according to any of the preceding claims wherein said first and/or second charge polarizing core domain comprises one or more amino acid changes to promote heterodimerization via increased hydrogen bonding and/or van der waals forces.

14. A heterodimeric protein according to claim 13 wherein said one or more amino acid changes result in a knob in the hole motif.

15. A heterodimeric protein according to claim 14 wherein the knob in the hole motif is formed by one or more amino acid changes that replace one or more tyrosine (Y) residues with one or more threonine (T) residues in the first charge polarized core domain, comprises and/or is formed by one or more amino acid changes that replace one or more threonine (T) residues with one or more tyrosine (Y) residues in the second charge polarized core domain.

16. A heterodimeric protein according to claim 14 or 15, wherein the knob in the hole motif is formed by one or more amino acid changes replacing one or more tyrosine (Y) residues with one or more threonine (T) residues in the second charge polarized core domain, comprises and/or is formed by one or more amino acid changes replacing one or more threonine (T) residues with one or more tyrosine (Y) residues in the first charge polarized core domain.

17. The heterodimeric protein of any one of claims 13 to 16, wherein one or both of the charge polarizing core domains comprise one or more effector and complement silent substitutions selected from L234A and L235A (LALA), and optionally P329G.

18. The heterodimeric protein of any one of claims 13 to 17, wherein one or both of the charge polarizing core domains comprise one or more half-life extending mutations selected from M252Y, S254T, and T256E.

19. A heterodimeric protein according to any of the preceding claims wherein said first protein is selected from table 1.

20. A heterodimeric protein according to any of the preceding claims wherein said second protein is selected from table 1.

21. A heterodimeric protein according to any of claims 1 to 20 wherein said first and/or second protein is selected from a cytokine, a growth factor and/or a hormone.

22. A heterodimeric protein according to claim 21 wherein said first and/or second protein is an interleukin.

23. The heterodimeric protein of claim 22, wherein the first and/or second protein is IL-35 comprising IL12a and IL27 β subunits.

24. A heterodimeric protein according to any of the preceding claims wherein said first and/or second protein is selected from receptors for cytokines, growth factors and/or hormones.

25. A heterodimeric protein according to claim 24 wherein said first and/or second protein is a receptor for an interleukin.

26. The heterodimeric protein of claim 25, wherein the first and/or second protein is an IL 6receptor comprising IL6R a and gp130 subunit.

27. The heterodimeric protein of claim 25, wherein the first and/or second protein is an IL21 receptor comprising IL21r and IL2rg subunits.

28. The heterodimeric protein of claim 25, wherein the first and/or second protein is an IL21 receptor comprising IFNgR and IFNgR2 subunits.

29. A heterodimeric protein according to any of the preceding claims wherein said heterodimeric protein is capable of (i) reducing or eliminating an immunosuppressive signal and (ii) increasing or activating an immunostimulatory signal.

30. A heterodimeric protein according to any of the previous claims wherein said heterodimeric protein is capable of increasing the ratio of effector T cells to regulatory T cells.

31. A heterodimeric protein according to any of the preceding claims wherein said heterodimeric protein is capable of increasing and/or preventing the decrease in a subpopulation of: cytotoxic T cells; effector memory T cells; central memory T cells; CD8⁺Stem cell memory effector cells; TH1 effector T cells; TH2 effector T cells; TH9 effector T cells; TH17 effector T cells; and/or effector T cells secreting IL-2, IL-4 and/or IFN- γ.

32. A pharmaceutical composition comprising a heterodimeric protein according to any of the preceding claims.

33. An expression vector comprising a nucleic acid encoding the first and/or second polypeptide chain of a heterodimeric protein according to any of the preceding claims.

34. The expression vector of claim 33, wherein the expression vector is a mammalian expression vector.

35. The expression vector of claim 34, wherein the expression vector comprises DNA or RNA.

36. A host cell comprising the expression vector of any one of claims 33 to 35.

37. A method of treating cancer, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 32.

38. A method of treating an autoimmune disease or disorder, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 32.

39. A method of modulating an immune response in a patient comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 32.

40. The method of any one of claims 37 to 39, wherein the patient's T cells are activated by an extracellular domain with an immunostimulatory signal.

41. The method of any one of claims 37 to 39, wherein the patient has a tumor and one or more tumor cells are prevented from transmitting immunosuppressive signals activated by the extracellular domain having immunosuppressive signals.

42. The method of claim 38, wherein the autoimmune disease or disorder is selected from rheumatoid arthritis, systemic lupus erythematosus, diabetes, ankylosing spondylitis, sjogren's syndrome, inflammatory bowel disease (e.g., ulcerative colitis, crohn's disease), multiple sclerosis, sarcoidosis, psoriasis, graves 'disease, hashimoto's thyroiditis, psoriasis, hypersensitivity reactions (e.g., hypersensitivity, hay fever, asthma, and acute edema-induced type I hypersensitivity reactions) and vasculitis.

Technical Field

The present invention relates to heterodimeric proteins useful in the treatment of diseases, such as immunotherapy against cancer and autoimmunity.

Priority

The present application claims benefit of U.S. provisional application No. 62/688,167 filed on 21/6/2018 and U.S. provisional application No. 62/703,248 filed on 25/7/2018. The contents of which are incorporated herein by reference in their entirety.

Description of an electronically submitted text file

The contents of a text file submitted electronically together are incorporated herein by reference in their entirety: a computer-readable version copy of the Sequence Listing (filename: SHK-004PC-Sequence _ Listing _ ST 25; creation date: 2019, 6, month 21; file size: 112 KB).

Background

Protein-protein interactions are critical to the normal physiological function of cells and multicellular organisms. For example, cytokines act as ligands that bind to their associated receptors to regulate essential biological processes, such as inflammation and immunity. In this regard, there are many natural cytokines, cytokine receptors, integrins and other proteins that exist or function as multimeric protein complexes. Some multimers (such as those in the tumor necrosis factor superfamily) function as homotrimers, and other ligands include heterodimers formed by the IL-12 family of cytokines (such as IL12, IL23, IL27, or IL-35). Similarly, cytokine receptors may also function as heterodimeric complexes. For example, many interleukin receptors form heterodimers for signal transduction.

Modulation of protein-protein interactions is a useful mechanism for therapeutic intervention in a variety of diseases and pathologies. Soluble binding proteins that interact with ligands can potentially isolate the ligand from the receptor, thereby reducing activation of specific receptor pathways. Alternatively, sequestering a ligand may delay its elimination or degradation, thereby increasing the time of its action and biological activity. In addition, soluble ligands can be used to activate or inhibit specific receptors. However, synthesis and production of soluble proteins may be hindered when production of heterodimeric proteins is desired. In particular, the efficiency of the synthesis is greatly reduced by the formation of a mixture of homodimers and heterodimers.

Thus, there remains a need for new methods for efficiently synthesizing and producing heterodimeric proteins for therapeutic use.

Summary of The Invention

In various embodiments, the present invention provides heterodimeric proteins comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first subunit of the first protein at the amino terminus linked to a first subunit of the second protein at the carboxy terminus by a first charge polarizing core domain; and the second polypeptide chain comprises a second subunit of the first protein at the amino terminus linked to a second subunit of the second protein at the carboxy terminus through a second charge polarizing core domain.

In various embodiments, the first polypeptide chain and the second polypeptide chain heterodimer are through electrostatic interactions between positively charged amino acid residues and negatively charged amino acid residues on the first and second charge polarizing core domains. In some embodiments, the positively charged amino acid residue may comprise one or more amino acids selected from His, Lys and Arg. In some embodiments, the negatively charged amino acid residue can include one or more amino acids selected from Asp and Glu.

Thus, in various embodiments, the first and/or second charge polarizing core domain each comprises a peptide having positively or negatively charged amino acid residues located at the amino and carboxy termini of the core domain. In an exemplary embodiment, the first charge polarizing core domain may comprise a peptide having a positively charged amino acid at the amino terminus that is adjoined to a peptide having a negatively charged amino acid residue at the carboxy terminus (adjoin to) by a linker, such as a stabilizing domain. In such embodiments, the second charge polarizing core domain may comprise a peptide having negatively charged amino acids at the amino terminus, which is adjoined to a peptide having positively charged amino acid residues at the carboxy terminus by a linker (e.g., a stabilizing domain). In another exemplary embodiment, the first charge polarizing core domain may comprise a peptide having negatively charged amino acids at the amino terminus, which is adjoined by a linker (e.g., a stabilizing domain) to a peptide having positively charged amino acid residues at the carboxy terminus. In such embodiments, the second charge polarizing core domain may comprise a peptide having a positively charged amino acid at the amino terminus, which is adjoined by a linker (e.g., a stabilizing domain) to a peptide having a negatively charged amino acid residue at the carboxy terminus.

In various embodiments, each of the first and/or second charge polarizing core domains further comprises a linker (e.g., a stabilizing domain) adjacent to the peptide having a positively or negatively charged amino acid. In some embodiments, the linker (e.g., stabilizing domain) is optionally selected from a flexible amino acid sequence, an IgG hinge region, or an antibody sequence. In one embodiment, the linker (e.g., stabilizing domain) comprises a hinge-CH 2-CH3 Fc domain derived from IgG1, optionally human IgG 1. In another embodiment, the linker (e.g., stabilizing domain) comprises a hinge-CH 2-CH3 Fc domain derived from IgG4, optionally human IgG 4.

In some embodiments, the first and/or second protein is selected from a cytokine, a growth factor, and/or a hormone. In some embodiments, the first and/or second protein is selected from receptors for cytokines, growth factors, and/or hormones.

In some embodiments, in the heterodimeric protein, the first protein is selected from table 1 and/or the second protein is selected from table 1.

In some embodiments, the first and/or second protein is an interleukin. In some embodiments, the first and/or second protein is IL-35 comprising subunits of IL12a and IL27 β.

In some embodiments, the first and/or second protein is selected from receptors for cytokines, growth factors, and/or hormones. In some embodiments, the first and/or second protein is a receptor for an interleukin.

In some embodiments, the first and/or second protein is an IL 6receptor comprising IL6R α and gp130 subunit.

In some embodiments, the first and/or second protein is an IL21 receptor comprising IL21r and an IL2rg subunit.

In some embodiments, the first and/or second protein is an IL21 receptor comprising IFNgR and IFNgR2 subunits.

In some embodiments, the protein on the amino or carboxy terminus is a native heterodimer, and wherein the protein on the opposite terminus is not a native heterodimer.

Also in various aspects, the heterodimeric proteins of the invention are used in methods of treating autoimmune diseases comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising a heterodimeric protein. In a further aspect, the heterodimeric proteins of the invention are used in methods of treating infection (including but not limited to viral infection or other intracellular pathogen). In a still further aspect, the heterodimeric proteins of the invention are used in methods of treating cancer.

Any aspect or embodiment disclosed herein may be combined with any other aspect or embodiment disclosed herein.

Brief description of the drawings

FIG. 1 provides a schematic representation of an engineered embodiment of the proteins of the invention, showing exemplary heterodimeric proteins of the invention comprising the IL 6receptor (which includes IL6R α and gp130 subunits) and the IL-35 cytokine (which includes IL12 α and IL27 β subunits). Heterodimeric proteins are preferably formed by electrostatic interactions between charge-polarized core domains.

FIG. 2 provides a Western blot analysis of products of gp130-Fc-IL12a and IL6RA-Fc-IL27 β electrophoresis from transient transfection cultures. The secreted protein was captured using affinity chromatography and eluted from the column to obtain a protein containing a domain recognized by a human Fc-specific antibody. Since the protein was electrophoresed under denaturing conditions, individual bands for the alpha and beta chains were visible.

FIG. 3 provides a Western blot analysis of purified gp130-Fc-IL12a and IL6RA-Fc-IL27 β heterodimer proteins. SDS-PAGE gels (two left gels) indicated the presence of a single band at about 300kDa under non-denaturing conditions (left lane except molecular weight standards in each blot). This band separates into the components alpha and beta chains (middle lane in each gel) after incubation with β -mercaptoethanol, which electrophoresed at higher apparent molecular weights than expected, indicating that possible post-translational modifications include glycosylation. This is confirmed in the right-most lane of each gel, which shows that the molecular weight of the alpha and beta chains is reduced to the expected molecular weight after removal of the N-linked and O-linked glycosylation. For the right gel, a native PAGE gel (native PAGE gel) was used to further investigate the proportion of purified protein present as an alpha/beta heterodimer compared to the alpha/alpha or beta/beta homodimer forms. This gel indicated that the alpha/beta heterodimer was enriched to about 60% of the total protein in the preparation, compared to about 30% for the alpha/alpha homodimer and about 10% for the beta/beta homodimer.

FIG. 4 depicts the quantification of captured heterodimeric IL-6R-Fc-IL-35 protein using spectrophotometry.

FIG. 5 provides size-exclusion chromatography (SEC) chromatograms of the IL-6R-Fc-IL-35 construct after double transfection of gp130-alpha-IL12A and IL6RA-beta-IL27B constructs in CHO cells and subsequent purification of the secreted protein using protein A.

FIG. 6 provides a schematic representation of an ELISA assay developed to demonstrate that IL-6R-Fc-IL-35 protein is capable of binding to immobilized human IL-6. Only the substance of interest (shown in the upper middle panel) is expected to bind to IL-6 in this assay, which can be specifically detected with antibodies directed against the IL-27a (EBI3) domain of the heterodimer.

FIG. 7 provides another schematic of an ELISA assay developed to demonstrate that IL-6R-Fc-IL-35 protein is capable of binding to immobilized human IL-6. The IL-6RA domain is used to detect the bound protein.

FIG. 8 provides a schematic of an ELISA assay developed using anti-human gp130 antibody to specifically capture exemplary heterodimeric proteins of the invention and anti-human IL-27a (EBI3) antibody to detect the bound protein.

FIG. 9 provides a schematic of an ELISA assay developed using anti-human gp130 antibody to specifically capture exemplary heterodimeric proteins of the invention and IL-6RA domain to detect the bound protein.

FIG. 10 provides a schematic of an ELISA assay developed using IL-6RA domain specific capture of exemplary heterodimeric proteins of the invention and detection of bound protein using anti-human IL-27a (EBI3) antibody.

FIG. 11 provides a schematic of an ELISA assay developed using the IL-6RA domain to specifically capture exemplary heterodimeric proteins of the invention and the IL-6RA domain to detect the bound protein.

FIG. 12 provides a schematic of an ELISA assay developed using IL-12a p35 to specifically capture exemplary heterodimeric proteins of the invention and anti-human IL-27a (EBI3) antibody to detect the bound protein.

FIG. 13 provides a schematic of an ELISA assay developed using the IL-12a p35 domain to specifically capture exemplary heterodimeric proteins of the invention and the IL-6RA domain to detect the bound protein.

FIG. 14 provides a schematic of an ELISA assay developed using anti-human IL-27a (EBI3) antibody to specifically capture an exemplary heterodimeric protein of the invention and anti-human IL-27a (EBI3) antibody to detect the bound protein.

FIG. 15 provides a schematic of an ELISA assay developed using anti-human IL-27a (EBI3) antibody to specifically capture an exemplary heterodimeric protein of the invention and IL-6RA domain to detect the bound protein.

FIGS. 16A and 16B provide Size Exclusion Chromatography (SEC) chromatograms of IL-6R-Fc-IL-35 heterodimeric proteins. In FIG. 16A, the absorption wavelength is 210nm, and in FIG. 16B, the absorption wavelength is 280 nm.

FIG. 17 shows a graph of the results of an IL-6SINK assay using IL-6R-Fc-IL-35 heterodimeric protein.

FIG. 18 includes graphs showing the ability of IL-6R-Fc-IL-35 heterodimer protein (identified as HdA' 00) to induce at least IL-35. The condition "halocon" refers to treatment with a control chimeric protein.

FIG. 19A shows a schematic of an IL-21R-Fc-IL-35 heterodimer protein comprising IL-21R-Fc (alpha) -IL12a chain and IL2rg-Fc (beta) -IL27B chain. FIG. 19B shows an SDS-PAGE gel indicating the presence of two single bands at about 84.4kDa and 78.1kDa under reduced deglycosylation conditions (rightmost lane).

FIG. 20 provides a Size Exclusion Chromatography (SEC) chromatogram of IL-21R-Fc-IL-35IFN γ R-Fc-IL-35 heterodimer protein after double transfection of the L-21R-Fc (alpha) -IL12a and IL2rg-Fc (beta) -IL27B constructs in CHO cells and subsequent purification of the secreted protein using protein A.

FIG. 21 is a Western blot analysis of an IFN γ R-Fc-IL-35 heterodimeric protein comprising the IFNgR-Alpha-IL12a chain and the IFNGR2-Beta-IL27B chain, detected by the antibody shown below each blot. Proteins were electrophoresed under non-denaturing conditions (left lane in each blot except for molecular weight standards), under denaturing conditions with β -mercaptoethanol treatment (middle lane in each gel), and denaturing and deglycosylation treatments.

FIG. 22 provides Size Exclusion Chromatography (SEC) chromatograms of IFN γ R-Fc-IL-35 heterodimer proteins following double transfection of IFNgR-Alpha-IL12a and IFNGR2-Beta-IL27B chain constructs in CHO cells and subsequent purification of the secreted proteins using protein A.

Detailed Description

The present invention relates to protein engineering platforms for the synthesis and production of heterodimeric proteins. The methods of the invention can be effective in generating heterodimeric proteins useful for modulating immune signals to treat a variety of diseases, including but not limited to autoimmune diseases.

Charge Polarized Core Domain

In one aspect, the invention relates to heterodimeric proteins. In various embodiments, the heterodimeric proteins of the invention comprise two polypeptide chains. The first polypeptide chain comprises a first subunit of a first protein at the amino terminus linked to a first subunit of a second protein at the carboxy terminus by a first charge polarizing core domain. The second polypeptide chain comprises a second subunit of the first protein at the amino terminus linked to a second subunit of the second protein at the carboxy terminus by a second charge polarizing core domain. In various embodiments, the first polypeptide chain and the second polypeptide chain form heterodimers via electrostatic interactions between positively charged amino acid residues and negatively charged amino acid residues on the first and second polarized core domains.

In various embodiments, each of the first and second charge polarized core domains comprises a peptide having positively or negatively charged amino acid residues at the amino and carboxy termini of the core domain. In an exemplary embodiment, the first charge polarizing core domain may comprise a peptide having positively charged amino acids at the amino terminus, which is adjoined to a peptide having negatively charged amino acid residues at the carboxy terminus by a linker (e.g., a stabilizing domain). The second charge polarizing core domain may comprise a peptide having negatively charged amino acids at the amino terminus, which is adjoined to a peptide having positively charged amino acid residues at the carboxy terminus by a linker (e.g., a stabilizing domain).

In another exemplary embodiment, the first charge polarizing core domain may comprise a peptide having negatively charged amino acids at the amino terminus, which is adjoined to a peptide having positively charged amino acid residues at the carboxy terminus by a linker (e.g., a stabilizing domain). The second charge polarizing core domain may comprise a peptide with positively charged amino acids at the amino terminus, which is adjoined to a peptide with negatively charged amino acid residues at the carboxy terminus by a linker (e.g., a stabilizing domain).

In various embodiments, formation of heterodimeric proteins is driven by electrostatic interactions between positively and negatively charged amino acid residues located at the amino and carboxy termini of the first and second charge polarizing core domains. In addition, the formation of homodimeric proteins is prevented by repulsion between positively or negatively charged amino acid residues located at the amino and carboxy termini of the first and second charge polarized core domains.

In various embodiments, the peptide comprising positively and/or negatively charged amino acid residues located at the amino or carboxy terminus of the charge polarizing core domain is about 2 to about 50 amino acids in length. For example, peptides comprising positively and/or negatively charged amino acid residues located at either end of a charge polarizing core domain can be about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids in length.

In various embodiments, the peptide comprising positively charged amino acid residues may comprise one or more amino acids selected from the group consisting of His, Lys and Arg. In various embodiments, a peptide comprising negatively charged amino acid residues may include one or more amino acids selected from Asp and Glu.

In various embodiments, each of the first and/or second charge polarizing core domains may comprise a peptide comprising an amino acid sequence provided in the table below or an amino acid sequence at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identical thereto.

For example, in one embodiment, each of the first and second charge polarizing core domains may comprise a peptide comprising the sequence YYnXXnYnXXnYnYnYn (where X is a positively charged amino acid such as arginine, histidine or lysine and Y is a spacer amino acid such as serine or glycine; SEQ ID NO: 3). Exemplary peptide sequences include, but are not limited to, RKGGKR (SEQ ID NO: 11) or GSGSRKGGKRGS (SEQ ID NO: 12).

In another exemplary embodiment, each of the first and second charge polarizing core domains may comprise a peptide comprising the sequence yynzzny (where Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a spacer amino acid such as serine or glycine). Exemplary peptide sequences include, but are not limited to, DEGGED (SEQ ID NO: 13) or GSGSDEGGEDGS (SEQ ID NO: 14).

In some embodiments, the charge polarizing core domain (negative-positive, also referred to herein as "alpha core domain") is provided below:

in some embodiments, the heterodimeric protein comprises a variant alpha core domain. As an example, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In some embodiments, a charge polarizing core domain (positive-negative, also referred to herein as "beta core domain") is provided as follows:

in some embodiments, the heterodimeric protein comprises a variant beta core domain. As an example, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In various embodiments, peptides comprising charged amino acid residues may further comprise one or more cysteine residues to promote disulfide bonding between electrostatically charged (electrostatically charged) core domains as an alternative method of stabilizing heterodimers.

In various embodiments, each of the first and second charge-polarized core domains comprises a linker sequence that may optionally function as a stabilizing domain. In various embodiments, the linker may be derived from a naturally occurring multidomain protein, or may be an empirical linker, such as described in Chichil et al, (2013), protein Sci.22(2): 153-. In some embodiments, linkers can be designed using a linker design database and computer programs such as those described in Chen et al, (2013), Adv Drug Deliv Rev.65(10): 1357-.

In some embodiments, the linker (e.g., stabilizing domain) is a synthetic linker, such as PEG.

In other embodiments, the linker (e.g., stabilizing domain) is a polypeptide. In some embodiments, the linker (e.g., stabilizing domain) is less than about 500 amino acids, about 450 amino acids, about 400 amino acids, about 350 amino acids, about 300 amino acids, about 250 amino acids, about 200 amino acids, about 150 amino acids, or about 100 amino acids in length. For example, a linker (e.g., a stabilizing domain) can be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids in length.

In various embodiments, the linker (e.g., stabilization domain) comprises predominantly glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% glycine and serine).

In various embodiments, the linker (e.g., stabilizing domain) is a hinge region of an antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses such as IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA 2). The hinge region found in IgG, IgA, IgD and IgE class antibodies serves as a flexible spacer, allowing the Fab portion to move freely in space. In contrast to the constant regions, hinge domains are structurally diverse, with sequences and lengths that vary between different immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies between different IgG subclasses. The hinge region of IgG1 encompasses amino acids 216 and 231 and, since it is freely flexible, the Fab fragment can rotate about its axis of symmetry and move within the sphere centered on the first of the two inter-heavy chain disulfide bridges. The hinge of IgG2 is shorter than IgG1, which has 12 amino acid residues and 4 disulfide bridges. The hinge region of IgG2 lacks glycine residues, is relatively short, and contains a rigid polyproline double helix stabilized by additional inter-heavy chain disulfide bridges. These properties limit the flexibility of the IgG2 molecule. IgG3 differs from the other subclasses in its unique extended hinge region (approximately four times the IgG1 hinge) containing 62 amino acids (including 21 prolines and 11 cysteines) forming an inflexible polyproline double helix. In IgG3, the Fab fragment is relatively distant from the Fc fragment, which gives the molecule greater flexibility. The lengthened hinge in IgG3 is also responsible for its higher molecular weight compared to other subclasses. The hinge region of IgG4 is shorter than that of IgG1, and its flexibility is intermediate between IgG1 and IgG 2. It was reported that the flexibility of the hinge region decreased in the order of IgG3> IgG1> IgG4> IgG 2. In other embodiments, the linker may be derived from human IgG4 and contain one or more mutations to enhance dimerization (including S228P) or FcRn binding.

According to crystallographic studies, immunoglobulin hinge regions can be further functionally subdivided into three regions: an upper hinge region, a core region, and a lower hinge region. See Shin et al, 1992Immunological Reviews 130: 87. The upper hinge region includes a hinge from C_H1To the first movement-limiting residue in the hinge (typically the first cysteine residue forming an interchain disulfide bond between the two heavy chains). The length of the upper hinge region is related to the flexibility of the segment of the antibody. The core hinge region contains heavy interchain disulfide bridges and the lower hinge region connects C_H2Amino terminal to the domain and comprising C_H2The residue of (1). As above. The core hinge region of wild-type human IgG1 contains the sequence Cys-Pro-Cys, which when dimerized by forming disulfide bonds forms a cyclic octapeptide that is thought to act as a pivot (pivot) to impart flexibility. In various embodiments, the linkers of the invention (e.g., stabilizing domains) comprise one or two or three of the upper, core and lower hinge regions of any antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses such as IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA 2). The hinge region may also contain one or more glycosylation sites, which include a number of structurally diverse sites for carbohydrate binding. For example, IgA1 contains five glycosylation sites within a 17 amino acid segment of the hinge region, conferring resistance to enteroproteases to hinge region polypeptides, which is considered to be an advantageous property of secretory immunoglobulins. In various embodiments, linkers of the invention (e.g., stabilization)A glycosylation domain) comprises one or more glycosylation sites.

In various embodiments, the linker (e.g., stabilizing domain) comprises an Fc domain of an antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses such as IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA 2). In various embodiments, the linker (e.g., stabilizing domain) comprises a hinge-CH 2-CH3 Fc domain derived from a human IgG4 antibody. In various embodiments, the linker (e.g., stabilizing domain) comprises a hinge-CH 2-CH3 Fc domain derived from a human IgG1 antibody. In some embodiments, the Fc domain exhibits increased affinity for and enhanced binding to a neonatal Fc receptor (FcRn). In some embodiments, the Fc domain comprises one or more mutations that increase affinity for and enhance binding to FcRn. Without wishing to be bound by theory, it is believed that the increased affinity for and enhanced binding to FcRn increases the in vivo half-life of the heterodimeric proteins of the invention.

In some embodiments, the Fc domain contains one or more amino acid substitutions at amino acid residues 250, 252, 254, 256, 308, 309, 311, 428, 433, or 434 (numbering according to Kabat), or an equivalent thereof. In one embodiment, the amino acid substitution at amino acid residue 250 is a substitution with glutamine. In one embodiment, the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, phenylalanine, tryptophan, or threonine. In one embodiment, the amino acid substitution at amino acid residue 254 is a substitution with threonine. In one embodiment, the amino acid substitution at amino acid residue 256 is a substitution with serine, arginine, glutamine, glutamic acid, aspartic acid, or threonine. In one embodiment, the amino acid substitution at amino acid residue 308 is a substitution with threonine. In one embodiment, the amino acid substitution at amino acid residue 309 is a substitution with proline. In one embodiment, the amino acid substitution at amino acid residue 311 is a substitution with serine. In one embodiment, the amino acid substitution at amino acid residue 385 is with arginine, aspartic acid, serine, threonine, histidine, lysine, alanine, or glycine. In one embodiment, the amino acid substitution at amino acid residue 386 is a substitution with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine. In one embodiment, the amino acid substitution at amino acid residue 387 is a substitution with arginine, proline, histidine, serine, threonine, or alanine. In one embodiment, the amino acid substitution at amino acid residue 389 is a substitution with proline, serine, or asparagine. In one embodiment, the amino acid substitution at amino acid residue 428 is a substitution with leucine. In one embodiment, the amino acid substitution at amino acid residue 433 is a substitution with arginine, serine, isoleucine, proline or glutamine. In one embodiment, the amino acid substitution at amino acid residue 434 is a substitution with histidine, phenylalanine or tyrosine.

In some embodiments, the Fc domain (e.g., comprising an IgG constant region) comprises one or more mutations, e.g., substitutions, at amino acid residues 252, 254, 256, 433, 434, or 436 (numbering according to Kabat). In one embodiment, the IgG constant region includes three M252Y/S254T/T256E mutations or YTE mutations. In another embodiment, the IgG constant region comprises a triple H433K/N434F/Y436H mutation or KFH mutation. In further embodiments, the IgG constant region comprises a combination of YTE and KFH mutations.

In some embodiments, the modified humanized antibodies of the invention comprise an IgG constant region comprising one or more mutations at amino acid residues 250, 253, 307, 310, 380, 428, 433, 434 and 435. Exemplary mutations include T250Q, M428L, T307A, E380A, I253A, H310A, M428L, H433K, N434A, N434F, N434S, and H435A. In one embodiment, the IgG constant region comprises a M428L/N434S mutation or a LS mutation. In another embodiment, the IgG constant region comprises a T250Q/M428L mutation or a QL mutation. In another embodiment, the IgG constant region comprises the N434A mutation. In another embodiment, the IgG constant region comprises a T307A/E380A/N434A mutation or an AAA mutation. In another embodiment, the IgG constant region comprises the I253A/H310A/H435A mutation or IHH mutation. In another embodiment, the IgG constant region comprises the H433K/N434F mutation. In another embodiment, the IgG constant region comprises a combination of M252Y/S254T/T256E and H433K/N434F mutations.

In various embodiments, mutations are introduced to increase the stability and/or half-life of the Fc domain. An exemplary Fc stabilizing mutant is S228P. Other exemplary Fc half-life extending mutants are T250Q, M428L, V308T, L309P, and Q311S, and the linkers (e.g., stabilizing domains) of the invention may comprise 1, or 2, or 3, or 4, or 5 of these mutants.

In some embodiments, the core domain lacking charge polarization has the following sequence:

other exemplary mutations in IgG constant regions are described, for example, in Robbie, et al, Antichronobiological Agents and Chemotherapy (2013),57(12), 6147-.

In various embodiments, the joint may be flexible, including but not limited to being highly flexible. In various embodiments, the linker may be rigid, including but not limited to a rigid alpha helix.

In various embodiments, the linker may be functional. For example, but not limited to, the linker may serve to improve the folding and/or stability, improve expression, improve pharmacokinetics, and/or improve biological activity of the heterodimeric proteins of the invention. In another example, the linker can function to target the heterodimeric protein to a particular cell type or location.

In some embodiments, the core domain comprises one or more "knob-hole" amino acid changes. The "knob" amino acid change is a rational design strategy previously used in antibody engineering for heterodimerization of its heavy chains. See, for example, Ridgway, J.B. et al, "Knobs-into-holes' engineering of antibodiy CH3 domains for latent chain catalysis," Protein Eng.9(7):617-2(1996) and Carter, "Bispecific human IgG by design," immunological methods,248(1-2):7-15(2001), the contents of which are incorporated herein by reference in their entirety. Here, the amino acid changes were engineered to create a "knob" in the CH3 domain of the "alpha" heavy chain and a "hole" in the CH3 of the "beta" heavy chain; alternatively, here, the amino acid changes are engineered to create a "knob" in the CH3 domain of the "beta" heavy chain and a "hole" in the CH3 of the "alpha" heavy chain. In one example, a "knob" is represented by tyrosine (Y) in the amino acid type where the IMGT volume is "very large", while a "hole" is represented by (T) in the threonine type where the IMGT volume is "small". Characterization of the IMGT class of amino acids is described in Pommi, c. et al, "IMGT stabilized criteria for statistical analysis of immunoglobulin V-REGION amino acid properties," j.mol.recognit.,17,17-32(2004), the contents of which are incorporated herein by reference in their entirety. In the interface between the two CH3 domains on separate heavy chains, threonine (T) T22 in the beta heavy chain is within the hydrogen bonding distance of tyrosine (Y) Y86 in the alpha heavy chain. Y86 is the main interdomain contact of T22, and these amino acids participate in hydrogen bonding. However, Y86 also has many van der waals contacts with Y86 and lysine (K) K88 on its opposite heavy chain.

The following are exemplary hinges-CH 2-CH3 that contain "knob-and-hole" amino acid changes and can be used in the present invention. The following exemplary sequences are based on IgG1, and further comprise other effector and complement silencing substitutions (effectors and complementary silencing substitutions): L234A and L235A (LALA) and optionally P329G; and half-life extension mutations: M252Y, S254T, T256E.

An exemplary human IGHG1 knob and hole "alpha core domain" (T22Y) is shown below:

exemplary human IGHG1 mortar and pestle "beta core Domain" (Y86T)

Any core domain useful in the present invention may comprise one or more "knob" mutations.

Protein subunits

In various embodiments, the heterodimeric proteins of the invention comprise two polypeptide chains. In various embodiments, each polypeptide chain comprises a subunit of the first protein linked to a subunit of the second protein by a charge polarizing core domain. Under electrostatic interaction between the charge-polarized core domains, the subunits are heterodimerized to form a functional dimeric first protein and a functional dimeric second protein. In some embodiments, the polypeptide chains form a functional bilateral heterodimeric protein connected via a charge-polarized core domain, which optionally comprises a linker (e.g., a stabilizing domain) such as an Fc region.

In various embodiments, the first and second proteins can be any multimeric protein having two or more subunits. In some embodiments, the first protein and the second protein are selected from cytokines, growth factors, and/or hormones. Illustrative examples of such cytokines, growth factors and hormones include, but are not limited to, lymphokines, monokines, traditional polypeptide hormones, including, but not limited to, Colony Stimulating Factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL), such as but not limited to IL-18, IL-27 and IL-35; interleukin receptors such as, but not limited to, IL-2R, IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-9R, IL-10R, IL-11R, IL-12R, IL-13R, IL-15R, IL-17R, IL-18R, IL-20R, IL-21R, IL-22R, IL-23R, IL-27R, IL-35R; and other polypeptide factors including, but not limited to, EGFR, integrin, neurofibrillarin, and somatostatin receptors. As used herein, cytokines, growth factors, and hormones include proteins obtained from natural sources or produced from recombinant bacterial, eukaryotic, or mammalian cell culture systems as well as biologically active equivalents of the native sequence cytokines.

In some embodiments, the first and/or second protein is an immunomodulator, e.g., one or more of an interleukin and an interferon.

In some embodiments, the first and/or second protein is an interleukin, including, for example, IL-18, IL-27, and IL-35, or a fragment, variant, analog, or family member thereof. Interleukins are a group of multifunctional cytokines synthesized by lymphocytes, monocytes and macrophages. Known functions include stimulation of immune cell (e.g., T helper, B cell, eosinophil, and lymphocyte) proliferation, chemotaxis of neutrophils and T lymphocytes, and/or inhibition of interferon. Assays known in the art can be used to determine interleukin activity: matthews et al in Lymphokines and interferences A Practical Approach, Clemens et al eds, IRL Press, Washington, D.C.1987, pp.221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20.

In some embodiments, the first and/or second protein is a hormone, such as somatostatin.

In various embodiments, the first and/or second protein is a receptor for a cytokine, a growth factor, and/or a hormone. In some embodiments, the first protein and/or the second protein is a type I cytokine receptor, a type II cytokine receptor, a chemokine receptor, a TGF- β receptor, a receptor in the immunoglobulin (Ig) superfamily, and/or a receptor in the tyrosine kinase superfamily.

In some embodiments, the first and/or second protein is a type I cytokine receptor. Type I cytokine receptors are known in the art and include, but are not limited to, receptors for IL2(β subunit), IL3, IL4, IL5, IL6, IL7, IL9, IL11, IL12, GM-CSF, G-CSF, LIF, CNTF, and receptors for Thrombopoietin (TPO), prolactin (prolactin), and growth hormone. Exemplary type I cytokine receptors include, but are not limited to, GM-CSF receptor, G-CSF receptor, LIF receptor, CNTF receptor, TPO receptor, and type I IL receptor.

In some embodiments, the first and/or second protein is a type II cytokine receptor. Type II cytokine receptors are multimeric receptors comprising heterologous subunits and are primarily receptors for interferons. This family of receptors includes, but is not limited to, receptors for interferon- α, interferon- β and interferon- γ, IL10, IL22, and tissue factor. Exemplary type II cytokine receptors include but are not limited to IFN-alpha receptors (e.g., IFNAR1 and IFNAR2), IFN-beta receptors, IFN-gamma receptors (e.g., IFNGR1 and IFNGR2), and type II IL receptors.

In some embodiments, the first and/or second protein is a G protein-coupled receptor. Chemokine receptors are G protein-coupled receptors with seven transmembrane structures and are coupled to G proteins for signal transduction. Chemokine receptors include, but are not limited to, the CC chemokine receptor, the CXC chemokine receptor, the CX3C chemokine receptor, and the XC chemokine receptor (XCR 1). Exemplary chemokine receptors include, but are not limited to, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5, CSCR6, CXCR7, XCR1, and CX3CR 1.

In some embodiments, the first and/or second protein is a TGF- β receptor. TGF- β receptors are single transmembrane (single pass) serine/threonine kinase receptors. TGF- β receptors include, but are not limited to, TGFBR1, TGFBR2, and TGFBR 3.

In some embodiments, the first and/or second protein is an Ig superfamily receptor. Receptors in the immunoglobulin (Ig) superfamily share structural homology with immunoglobulins. Receptors in the Ig superfamily include, but are not limited to, interleukin-1 receptor, CSF-1R, PDGFR (e.g., PDGFRA and PDGFRB), and SCFR.

In some embodiments, the first and/or second protein is a member of the B7 superfamily. Members of the B7 superfamily share structural homology with each other. Members of this family include, but are not limited to, CD28, CD80, CD86, ICOS, ICOSL, B7-H3, B7-H4, PD-1, PD-L1, PD-L2, and the like.

In some embodiments, the first and/or second protein is a tyrosine kinase superfamily receptor. Receptors in the tyrosine kinase superfamily are well known in the art. There are approximately 58 known Receptor Tyrosine Kinases (RTKs), divided into 20 subfamilies. Receptors in the tyrosine kinase superfamily include, but are not limited to, FGF receptors and their various isoforms, such as FGFR1, FGFR2, FGFR3, FGFR4, and FGFR 5.

In an exemplary embodiment, the first and/or second protein is an IFN- α/β receptor (IFNAR) comprising subunits of IFNAR1 and/or IFNAR 2.

In an exemplary embodiment, the first and/or second protein is an interferon-gamma receptor (IFNGR) comprising IFNGR1 (also known as IFNGR) and IFNGR2 subunit.

In an exemplary embodiment, the first and/or second proteins are VEGF receptors, including VEGFR-1, VEGFR-2, and VEGFR-3.

In any of the heterodimeric proteins disclosed herein, the amino or carboxy terminus is native heterodimeric, and wherein the protein on the opposite terminus is not native heterodimeric.

In an exemplary embodiment, the first and/or second protein is a receptor for IL-1, such as IL-1R1 and/or IL-1 RAcP.

In an exemplary embodiment, the first and/or second protein is a receptor for IL-2, such as IL-2R α or IL-2R β or IL-2R γ.

In an exemplary embodiment, the first and/or second protein is a receptor for IL-3, which is a heterodimer with a unique alpha chain paired with a common beta (β c or CD131) subunit.

In an exemplary embodiment, the first and/or second protein is a receptor for IL-4, such as a type 1 or type 2 IL-4 receptor.

In an exemplary embodiment, the first and/or second protein is a receptor for IL-6, which is a cell surface type I cytokine receptor complex comprising a ligand-binding IL-6R chain (CD126 or IL-6R α) and a signal transduction component gp 130.

In an exemplary embodiment, the first and/or second protein is a receptor for IL-10, such as IL-10 receptor-1 and IL-10 receptor-2.

In an exemplary embodiment, the first and/or second protein is a receptor for IL-11, such as IL-11R α or IL-11R β or gp 130.

In an exemplary embodiment, the first and/or second protein is IL-12 receptor, such as IL-12R beta 1 and/or IL-12R beta 2.

In an exemplary embodiment, the first and/or second protein is a receptor for IL-13, such as IL-4 receptor (IL-4R α) or IL-13R α 1.

In an exemplary embodiment, the first and/or second protein is IL-18. In another exemplary embodiment, the first and/or second protein is a receptor for IL-18, such as IL-18R α and/or IL-18R β.

In an exemplary embodiment, the first and/or second protein is a receptor for IL-21, which is a cell surface type I cytokine receptor complex comprising a ligand-binding IL-21R chain comprising IL-21R and IL-2 rg.

In an exemplary embodiment, the first and/or second protein is a receptor for IL-33, such as the ST-2 receptor or IL-1 RAcP.

In an exemplary embodiment, the first and/or second protein is IL-35 (e.g., comprising subunits of IL12a and IL27 β). In another exemplary embodiment, the first and/or second protein is a receptor for IL-35, such as an IL-35 receptor comprising IL6R α and gp130 subunits.

In an exemplary embodiment, the first and/or second protein is a receptor for EGP, e.g., EGFR (ErbB1), ErbB2, ErbB3, and ErbB 4.

In an exemplary embodiment, the first and/or second protein is a receptor for insulin or an insulin analogue, such as an insulin receptor and/or an IGF1 or IGF2 receptor.

In an exemplary embodiment, the first and/or second protein is a receptor for EPO, such as EPO receptor (EPOR) receptor and/or ephrin receptor (EphR).

In various embodiments, the first and second proteins may comprise domains of soluble (e.g., non-membrane bound) proteins. In various embodiments, the first and second proteins can comprise fragments of a soluble protein involved in signaling (e.g., a portion of a soluble protein that interacts with a receptor).

In various embodiments, the first and second proteins may comprise the extracellular domain of a transmembrane protein. In various embodiments, one of the extracellular domains transduces an immunosuppressive signal and one of the extracellular domains transduces an immunostimulatory signal.

In some embodiments, an extracellular domain refers to a portion of a transmembrane protein that is capable of interacting with an extracellular environment. In various embodiments, an extracellular domain refers to a portion of a transmembrane protein sufficient to bind to a ligand or receptor and effectively transmit a signal to a cell. In various embodiments, the extracellular domain is the entire amino acid sequence of a transmembrane protein that is outside of the cell or cell membrane. In various embodiments, the extracellular domain is a portion of the amino acid sequence of a transmembrane protein that is external to the cell or cell membrane and is required for signal transduction and/or ligand binding, as can be determined using methods known in the art (e.g., in vitro ligand binding and/or cell activation assays).

In some embodiments, an immunosuppressive signal refers to a signal that reduces or eliminates an immune response. For example, in the field of oncology, such signals may attenuate or eliminate anti-tumor immunity. Under normal physiological conditions, inhibitory signals can be used to maintain self-tolerance (e.g., prevent autoimmunity) and also to protect tissues from damage when the immune system responds to pathogen infection. For example, but not limited to, immunosuppressive signals can be recognized by detecting an increase in cell proliferation, cytokine production, cell killing activity, or phagocytic activity when such inhibitory signals are blocked.

In some embodiments, an immunostimulatory signal refers to a signal that enhances an immune response. For example, in the field of oncology, such signals may enhance anti-tumor immunity. For example, but not limited to, immunostimulatory signals may be recognized by direct stimulation of leukocyte proliferation, cytokine production, killing activity, or phagocytic activity. Specific examples include direct stimulation of cytokine receptors such as IL-2R, IL-7R, IL-15R, IL-17R or IL-21R using fusion proteins encoding ligands for these receptors (IL-2, IL-7, IL-15, IL-17 or IL-21, respectively). Stimulation from any of these receptors can directly stimulate proliferation and cytokine production of a single subpopulation of T cells.

In some embodiments, the extracellular domain can be used to produce a soluble protein to competitively inhibit signaling by a ligand of that receptor. For example, but not limited to, competitive inhibition of PD-L1 or PD-L2 may be achieved using PD-1, or competitive inhibition of PVR may be achieved using TIGIT. In some embodiments, the extracellular domain can be used to provide artificial signaling.

In some embodiments, the heterodimeric proteins of the invention deliver or mask immunosuppressive signals. In some embodiments, the heterodimeric proteins of the invention deliver or mask an immunostimulatory signal.

In various embodiments, the heterodimeric proteins of the invention comprise two independent binding domains, each from one subunit of a heterodimeric human protein. Table 1 provides exemplary proteins that may form part of the heterodimeric proteins of the present invention. In various embodiments, the heterodimeric proteins of the invention have one of the exemplary proteins provided in table 1. In various embodiments, the heterodimeric proteins of the invention have two exemplary proteins provided in table 1.

TABLE 1

Exemplary proteins that can be incorporated into the compositions and methods of the invention include the following (as used herein, "entry" refers to a protein entry in the Uniprot database, and "entry name" refers to a protein entry in the Uniprot database):

in various embodiments, the heterodimeric proteins of the invention can be engineered to target one or more molecules residing on human leukocytes, including but not limited to the following extracellular domains (if applicable): SLAMF, IL-2R alpha, IL-2R beta, ALCAM, B-1, IL-4-H, BLAME/SLAMF, CEACAM, IL-6-7R alpha, IL-10R alpha, IL-l 0R beta, IL-12R beta 1, IL-12R beta 2, CD, IL-13R alpha 1, IL-13, CD, ILT/CDS 5, lutegrin alpha 4/CD49, CDS, integrin alpha E/CD103, CD, integrin alpha M/CD 11B, CDS, integrin alpha X/CD11, integrin beta 2/CDlS, KIR/CD15, KIR2DL, CD2, KIR2 DL/CD 15, CD/PECAM-1, KIR2DS, CD-3, CD, LAG, LAR, CDS, LAR-B, CDS, IL-12R beta 1, IL-13R alpha 1, IL-13, CD, CDS4/SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, common gamma chain/IL-2 Rgamma, osteopontin, CRACC/SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, CX3CR1, CX3CL1, L-selectin, SIRP beta 1, SLAM, TCCR/WSX-1, DNAM-1, Thymopoietin (Thymoietin), EMIN/CD 147, CX-1, EphB6, TIM-462, TIM-3, TIM-4, gamma Fc RIII/46CD 45, nu-6, Lysozyme (TSICAM), TSICAM/CD 147, IFN-24, IFN-R1, IFN-R102, CD 573R, CD11, CD-R, CD-5, IFN-R24, and CD-R.

In some embodiments, the heterodimeric proteins of the invention can be engineered to target one or more molecules involved in immunosuppression, including, for example: CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA/VSIG8, KIR, 2B4, TIGIT, CD160 (also known as BY55), CHK1 and CHK2 kinases, A2aR, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), and various B-7 family ligands (including but not limited to B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7).

In some embodiments, the heterodimeric proteins of the invention comprise an extracellular domain of an immunosuppressive agent.

In some embodiments, the heterodimeric proteins of the invention comprise an extracellular domain of an immunosuppressive soluble protein or membrane protein.

In some embodiments, the heterodimeric proteins of the invention mimic the binding of an inhibitory signaling ligand to its cognate receptor, but inhibit the transmission of inhibitory signals to immune cells (e.g., T cells, macrophages, or other leukocytes).

In various embodiments, the heterodimeric protein comprises an immunosuppressive receptor extracellular domain and an immunostimulatory ligand extracellular domain that can, but are not limited to, deliver immune stimulation to T cells while masking the tumor cells' immunosuppressive signals. In various embodiments, the heterodimeric protein delivers a signal with the end result of T cell activation (net result).

In some embodiments, the heterodimeric proteins of the invention comprise an extracellular domain of a soluble protein or a membrane protein having immunostimulatory properties.

In some embodiments, the heterodimeric proteins useful in the invention comprise the extracellular domain of Gp 130. Gp130 (also known as interleukin-6receptor subunit beta, IL-6R-beta, IL-6RB, and IL-6ST) is a signaling molecule. Receptor systems for IL6, LIF, OSM, CNTF, IL11, CTF1 and BSF3 may utilize Gp130 to initiate signaling. Binding of IL6 to IL6R induces homodimerization of IL6ST and forms a high affinity receptor complex that activates Janus kinase. This results in phosphorylation of Gp130 tyrosine residue, which in turn activates STAT 3. Gp130 mediates signals (by similarity) that regulate immune responses, hematopoiesis, pain control, and bone metabolism.

In some embodiments, the heterodimeric proteins useful in the invention comprise variants of the extracellular domain of Gp 130. As an example, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In some embodiments, the extracellular domain of Gp130 has the following amino acid sequence:

in some embodiments, the heterodimeric protein comprises a variant of the extracellular domain of Gp 130. As an example, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In some embodiments, one strand of the heterodimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 18.

Variants of the known amino acid sequence of Gp130 can be selected by the skilled worker by reference, for example, by Hibi et al, "Molecular cloning and expression of an IL-6signal transducer, Gp 130" Cell 63(6),1149-1157 (1990); wattzig et al, "N-linked catalysis for the stability but not the signaling function of the interactive-6 signal converter 130", J.biol.chem.285(3),1781-1789 (2010); schutt et al, "gp 130 activation is regulated by D2-D3 Interactive connectivity", biochem. J.450(3),487- "496 (2013); bravo et al, "Crystal structure of a cytokine-binding region of gp 130", EMBO J.17(6), 1665-; chow et al, "Structure of an extracellular gp130 cytokine receptor signaling complex", Science 291(5511),2150-2155 (2001); boulanger et al, "Hexameric structure and establishment of the interferometric in-6/IL-6alpha-receptor/gp130 complex", Science 300(5628),2101-2104 (2003); xu et al, "Crystal structure of the incident immune of gp130: instruments into the molecular assembly of the target cytokine receptors", J.biol.chem.285(28),21214 and 21218(2010), which are incorporated herein by reference in their entirety.

In some embodiments, heterodimeric proteins useful in the invention comprise the extracellular domain of IL-6 RA. IL-6RA (also known as interleukin-6receptor subunit alpha, IL-6R subunit alpha, and IL-6R-alpha) is a part of the interleukin 6receptor that binds IL6 with low affinity but does not transduce a signal. Signaling activation requires binding to gp 130. Activation can lead to modulation of immune responses, acute phase responses, and hematopoiesis. The soluble form of the IL 6receptor at low concentrations acts as an agonist of IL6 activity. Dysregulated production of IL6 and this receptor has been implicated in the pathogenesis of a number of diseases such as multiple myeloma, autoimmune diseases and prostate cancer.

In some embodiments, heterodimeric proteins useful in the invention comprise variants of the extracellular domain of IL-6 RA. By way of example, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In some embodiments, the extracellular domain of IL-6RA has the following amino acid sequence:

in some embodiments, the heterodimeric protein comprises a variant of the extracellular domain of IL-6 RA. As an example, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In some embodiments, one strand of the heterodimeric protein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID No. 19.

Variants of known amino acid sequences of IL-6RA may be selected by the skilled artisan by reference, for example, to Yamasaki et al, "Cloning and expression of the human interleukin-6(BSF-2/IFN beta 2) receptor" Science 241(4867),825-828 (1988); buk et al, "incorporated association with specific membranes/lipid columns of exogenous target mutants of the endogenous target gp80," Eur. J. cell biol.84(10), 819. 831 (2005); yawata et al, Structure-function analysis of human IL-6 receiver: association of amino acid residues for IL-6-binding and for IL-6signal transduction through gp130, "EMBO J.12(4), 1705-; horiuchi et al, "simple interlayer cleaned from T cell or gradient cell and human intrinsic biological cells area genetic tissue" Eur.J.Immunol.24(8), 1945-; boulanger et al, "Hexameric structure and assembly of the interactive in-6/IL-6 alpha-receiver/gp 130 complex", Science 300(5628),2101-2104(2003), which is incorporated herein by reference in its entirety.

In some embodiments, can be used in the invention of the heterodimeric proteins comprising IL-12A extracellular domain. IL-12A (also known as interleukin-12 subunit alpha and IL-12 subunit p35) is a cytokine that acts as a growth factor for activated T and NK cells, enhances the lytic activity of NK/lymphokine-activated killer cells, and stimulates IFN- γ production by resting PBMCs. The cytokine is a disulfide-linked heterodimer consisting of a 35kD subunit encoded by the gene and a 40kD subunit of a cytokine receptor family member. This cytokine is required for T-cell dependent induction of interferon gamma (INF- γ) and is important for differentiation of both Th1 and Th2 cells. The response of lymphocytes to this cytokine is mediated by the activator of the transcription protein STAT 4.

In some embodiments, the heterodimeric proteins useful in the invention comprise variants of the extracellular domain of IL-12A. By way of example, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In some embodiments, the extracellular domain of IL-12A has the following amino acid sequence:

in some embodiments, the heterodimeric protein comprises a variant of the extracellular domain of IL-12A. As an example, the variant may be identical to SEQ ID NO: 20 has a molecular weight of at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In some embodiments, one chain of the heterodimeric protein comprises a sequence identical to SEQ ID NO: 20 is at least 95% identical to the amino acid sequence of seq id no.

The ordinarily skilled artisan can select variants of the known amino acid sequence of IL-12A by reference, for example, Wolf et al, "Cloning of cDNA for natural killer cell catalytic factor, a heterologous cytokine with multiple biological effects on T and natural killer cells", J.Immunol.146(9),3074-3081 (1991); devergne et al, "Epstein-Barr virus-induced gene 3and the p35 subBunit of interleukin 12for a novel optoelectronic peptide", Proc. Natl.Acad.Sci.U.S.A.94(22),12041-12046 (1997); yoon et al, "Charged residues doped a unique interlocking grafting in the heterologous cytokine intercalation-12", EMBO J.19(14), 3530-.

In some embodiments, heterodimeric proteins useful in the invention comprise the extracellular domain of IL-27B. IL-27B (also known as interleukin-27 subunit beta, IL-27 subunit beta, and IL-27B) forms interleukin 35(IL-35) with IL-12 a. IL-35 is a dimeric protein consisting of IL-12 α and IL-27 β chains, encoded by two separate genes, termed IL12A and EBI3, respectively. IL-27 has pro-inflammatory and anti-inflammatory properties, it regulates T helper cell development, inhibits T cell proliferation, stimulates cytotoxic T cell activity, induces isotype switching in B cells, and has multiple effects on innate immune cells. The IL-27 gene was identified by its induced expression in B lymphocytes in response to Epstein-Barr virus infection. The IL-27 moiety modulates T cell and inflammatory responses by activating the Jak/STAT pathway of CD4+ T cells.

In some embodiments, heterodimeric proteins useful in the invention comprise variants of the extracellular domain of IL-27B. By way of example, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In some embodiments, the extracellular domain of IL-27B has the following amino acid sequence:

in some embodiments, the heterodimeric protein comprises a variant of the extracellular domain of IL-27B. As an example, the variant may be identical to SEQ ID NO: 21 has a molecular weight of at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In some embodiments, one chain of the heterodimeric protein comprises a sequence identical to SEQ ID NO: 21, an amino acid sequence that is at least 95% identical to the amino acid sequence of seq id no.

Variants of the known amino acid sequence of IL-27B may be selected by the skilled artisan by reference, for example Devergne "Epstein-Barr virus-induced gene 3and the p35 subbunit of interleukin 12for a novel heterologous peptide, Proc. Natl.Acad.Sci.U.S. A.94(22),12041-12046 (1997); pflanz et al, "IL-27, a heterologous cytokine composition of EBI 3and p28 protein, indeces promotion of negative CD4+ T cells", Immunity 16(6), 779-; batten and Ghiardi "The biological and therapeutic potential of interleukin 27", J.mol.Med.85(7),661-672(2007), incorporated herein by reference in its entirety.

In some embodiments, the alpha chain of a heterodimeric chimeric protein useful in the invention comprises: (1) comprises the amino acid sequence of SEQ ID NO:18, (b) a first domain comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof, and (c) an alpha core domain or a variant thereof, comprising an amino acid sequence substantially identical to SEQ ID NO:16 or SEQ ID NO: 24 amino acid sequence that is at least 95% identical. Such an Alpha chain may be referred to as "Gp 130-Alpha-IL 12A".

In embodiments, the Gp130-Alpha-IL12A chain used in the present invention has the following amino acid sequence:

in some embodiments, the beta chain of a heterodimeric chimeric protein useful in the invention comprises: (1) comprises the amino acid sequence of SEQ ID NO:19, (b) a first domain comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof, and (c) an alpha core domain or a variant thereof, comprising an amino acid sequence substantially identical to SEQ ID NO:17 or SEQ ID NO: 25 amino acid sequences at least 95% identical. Such an alpha chain may be referred to as "IL 6RA-Beta-IL 27B". In some embodiments, the IL6RA-Beta-IL27B chain used in the present invention has the following amino acid sequence:

when Gp130-Alpha-IL12A chain and IL6RA-Beta-IL27B chain are combined (in cells or in vitro), they form a heterodimeric protein, referred to herein as IL-6R-Fc-IL-35.

In some embodiments, heterodimeric proteins useful in the invention comprise the extracellular domain of IL-21 r. The interleukin-21receptor (also known as the IL-21receptor and IL-21R) is a receptor for interleukin-21, belongs to the class I cytokine receptor, and has been shown to form heterodimeric receptor complexes with a common gamma chain (also a receptor subunit common to the receptors for interleukins 2,4, 7, 9 and 15). This receptor transduces the growth-promoting signal of IL21 and is important for the proliferation and differentiation of T cells, B cells, and Natural Killer (NK) cells. Ligand binding of the receptor results in activation of a number of downstream signaling molecules, including JAK1, JAK3, STAT1, and STAT 3. Knockout studies of similar genes in mice indicate a role for this gene in regulating immunoglobulin production.

In some embodiments, heterodimeric proteins useful in the invention comprise variants of the extracellular domain of IL-21 r. By way of example, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In some embodiments, the extracellular domain of IL-21r has the following amino acid sequence:

in some embodiments, the heterodimeric protein comprises a variant of the extracellular domain of IL-21 r. As an example, the variant may be identical to SEQ ID NO: 26 has a molecular weight of at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In some embodiments, one chain of the heterodimeric protein comprises a sequence identical to SEQ ID NO: 26, an amino acid sequence that is at least 95% identical to the amino acid sequence of seq id no.

Variants of known amino acid sequences of IL-21r can be selected by the skilled artisan by reference, for example, Ozaki et al, "Cloning of a type I cytokine receptor motor related to the IL-2receptor beta chain", Proc. Natl. Acad. Sci. U.S.A.97(21),11439-11444 (2000); kotlarz et al, "Loss-of-function details in the IL-21 receiver gene cause a primary immunological syndrome" J.Exp.Med.210(3),433-443 (2013); hamming et al, "Crystal Structure of Interactive-21 receiver (IL-21R) bound to IL-21 improvements of this molecular chain interaction with WSXWS motif is integral part of IL-21R" J.biol.chem.287(12),9454-9460(2012), which is incorporated herein by reference in its entirety.

In some embodiments, heterodimeric proteins useful in the invention comprise the extracellular domain of IL2 RG. Interleukin-2 receptor subunit γ (also known as cytokine receptor common subunit γ, IL-2receptor subunit γ, IL-2R subunit γ, and IL-2RG) is a common subunit of multiple receptors for interleukins (including receptors for interleukins-2, -4, -7, and-21) and is therefore referred to as the common γ chain. Mutations in this gene can lead to X-linked severe combined immunodeficiency (XSCID), and X-linked combined immunodeficiency (XCID) (less severe immunodeficiency disorder).

In some embodiments, the heterodimeric proteins useful in the invention comprise variants of the extracellular domain of IL2 RG. By way of example, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In some embodiments, the extracellular domain of IL2RG has the amino acid sequence:

in some embodiments, the heterodimeric protein comprises a variant of the extracellular domain of IL2 RG. As an example, the variant may be identical to SEQ ID NO: 27 has a molecular weight of at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In some embodiments, one chain of the heterodimeric protein comprises a sequence identical to SEQ ID NO: 27 is at least 95% identical to the amino acid sequence of seq id no.

Variants of the known amino acid sequence of IL2RG may be selected by the skilled artisan by reference, for example, Takeshita et al, "Cloning of the gamma chain of the human IL-2 receiver", Science 257(5068),379-382 (1992); ratthe et al, "Interleukin-15 enzymes human neutrophile proteins by a Syk-dependent mechanism of the IL-15Ralpha chain", J.Leukoc.biol.76(1),162-168 (2004); bamborough et al, "The interactive-2 and interactive-4 receptors by molecular modification", Structure 2(9), 839-; wang et al, "Structure of the quantitative complex of interfukin-2 with its alpha, beta, and gamma receptors" Science 310(5751),1159, 1163 (2005); stauber et al, "Crystal Structure of the IL-2signaling complex," front for a terrestrial cytokine receiver, "Proc. Natl. Acad. Sci. U.S.A.103(8),2788-2793(2006), which are incorporated herein by reference in their entirety.

In some embodiments, the alpha chain of a heterodimeric chimeric protein useful in the invention comprises: (1) comprises the amino acid sequence of SEQ ID NO: 26, (b) a first domain comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof, and (c) an alpha core domain or a variant thereof, comprising an amino acid sequence substantially identical to SEQ ID NO:16 or SEQ ID NO: 24 amino acid sequence that is at least 95% identical. Such an Alpha chain may be referred to as "IL 21r-Alpha-IL12 a".

In some embodiments, the IL21r-Alpha-IL12a chain used in the present invention has the following amino acid sequence:

in some embodiments, the beta chain of a heterodimeric chimeric protein useful in the invention comprises: (1) comprises the amino acid sequence of SEQ ID NO: 27, (b) a first domain comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof, and (c) an alpha core domain or a variant thereof, comprising an amino acid sequence substantially identical to SEQ ID NO:17 or SEQ ID NO: 25 amino acid sequences at least 95% identical. Such Beta chains may be referred to as "IL 2rg-Beta-IL 27B".

In some embodiments, the IL2rg-Beta-IL27B chain used in the present invention has the following amino acid sequence:

when IL21R-Alpha-IL12a chain and IL2rg-Beta-IL27B chain are combined (in cells or in vitro), they form a heterodimeric protein, referred to herein as IL-21R-Fc-IL-35.

In some embodiments, the alpha chain of a heterodimeric chimeric protein useful in the invention comprises: (1) comprises the amino acid sequence of SEQ ID NO: 26, (b) a first domain comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof, and (c) an alpha core domain or a variant thereof, comprising an amino acid sequence substantially identical to SEQ ID NO:17 or SEQ ID NO: 25 amino acid sequences at least 95% identical. Such an alpha chain may be referred to as "IL 21r-Beta-IL12 a".

In some embodiments, the IL21r-Beta-IL12a chain used in the present invention has the following amino acid sequence:

in some embodiments, the beta chain of a heterodimeric chimeric protein useful in the invention comprises: (1) comprises the amino acid sequence of SEQ ID NO: 27, (b) a first domain comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof, and (c) an alpha core domain or a variant thereof, comprising an amino acid sequence substantially identical to SEQ ID NO:16 or SEQ ID NO: 24 amino acid sequence that is at least 95% identical. Such beta chains may be referred to as "IL 2rg-Alpha-IL 27B".

In some embodiments, the chain of IL2rg-Alpha-IL27B used in the present invention has the following amino acid sequence:

when IL21R-Beta-IL12a chain and IL2rg-Alpha-IL27B chain are combined (in cells or in vitro), they form a heterodimeric protein, which may also be referred to herein as IL-21R-Fc-IL-35.

In some embodiments, heterodimeric proteins useful in the invention comprise an extracellular domain of IFNgR. IFNgR (also known as interferon gamma receptor 1, IFN-gamma-R1, IFN-gamma-R-alpha, IFN gamma R, and IFNgR1) binds to IFNgR2 to form a receptor for the cytokine interferon gamma (IFNG). Ligand binding stimulates the activation of the JAK/STAT signaling pathway. It plays a crucial role in the IFN- γ pathway required for cellular response to infectious agents. Genetic variation in IFNGR1 is associated with susceptibility to helicobacter pylori infection. In addition, defects in IFNGR1 are responsible for mendelian genetic susceptibility to mycobacteriosis (also known as familial disseminated atypical mycobacterial infection).

In some embodiments, the heterodimeric proteins useful in the invention comprise a variant of the extracellular domain of IFNgR. As an example, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In embodiments, the extracellular domain of IFNgR has the following amino acid sequence:

in some embodiments, the heterodimeric protein comprises a variant of the extracellular domain of IFNgR. As an example, the variant may be identical to SEQ ID NO: 30 has a molecular weight of at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In some embodiments, one chain of the heterodimeric protein comprises a sequence identical to SEQ ID NO: 30 is at least 95% identical to the amino acid sequence of seq id no.

Variants of the known amino acid sequence of IFNgR may be selected by the skilled person by reference, for example to Aguet et al, "Molecular cloning and expression of the human interference-gamma receptor" Cell 55(2),273-280 (1988); stuber et al, "Alignment of discrete bases of the extracellular domain of the interference gamma receiver and inhibition of the control in biological activity", Biochemistry 32(9),2423 and 2430 (1993); sakatsume et al, "The Jak enzymes differential association with The alpha and beta (access factor) channels of The interference gamma receptor to The functional receptor units of activating STAT transcription factors", J.biol.chem.270(29),17528-17534 (1995); walter et al, "Crystal Structure of a complex between interference-gamma and its soluble high-affinity receiver", Nature 376(6537), 230-; sogabe et al, "neutral suspensions on the extracellular interface gamma receiver (IFNgamma) alpha-chain characterized by homolog scanning mutagenesis and X-chain crystal structure of the A6fab-IFNgamma 1-108 complex", J.Mol.biol.273(4), 882. gamma. 897 (1997); thiel et al, "administration of an unexpected third receiver module in the crystal Structure of human interface-gamma receiver module", Structure 8(9),927 (2000); van de Wetering et al, "Functional analysis of natural curing amino acid substistention in human IFN-gamma 1," mol.Immunol.47:1023-1030(2010), which is incorporated herein by reference in its entirety.

In some embodiments, heterodimeric proteins useful in the invention comprise the extracellular domain of IFNGR 2. IFNGR2 (also known as interferon gamma receptor 2, also known as IFN-gamma receptor 2and IFN-gamma-R2) is the non-ligand binding beta chain of the gamma interferon receptor. Human interferon-gamma receptors are heterodimers of IFNGR1 and IFNGR 2. Ligand binding stimulates the activation of the JAK/STAT signaling pathway. IFNGR2 is essential for signal transduction relative to the other receptor subunits responsible for ligand binding. Defects in IFNGR2 are responsible for mendelian genetic susceptibility to mycobacteriosis (MSMD), also known as familial disseminated atypical mycobacterial infection. MSMD is a genetically heterogeneous disease with autosomal recessive inheritance, autosomal dominant inheritance, or X-linked inheritance.

In some embodiments, the heterodimeric proteins useful in the invention comprise variants of the extracellular domain of IFNGR 2. As an example, the variant may have at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In some embodiments, the extracellular domain of IFNGR2 has the following amino acid sequence:

in some embodiments, the heterodimeric protein comprises a variant of the extracellular domain of IFNGR 2. As an example, the variant may be identical to SEQ ID NO: 31 have a molecular weight of at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In some embodiments, one chain of the heterodimeric protein comprises a sequence identical to SEQ ID NO: 31, an amino acid sequence that is at least 95% identical to the amino acid sequence of seq id no.

Variants of the known amino acid sequence of IFNGR2 may be selected by the skilled artisan by reference, for example, Soh et al, "Identification and sequence of an access factor required for activation of the human interference gamma receiver", Cell 76(5), 793-; sakatsume et al, "The Jak enzymes differential association with The alpha and beta (access factor) channels of The interference gamma receptor to The functional receptor units of activating STAT transcription factors", J.biol.chem.270(29),17528-17534 (1995); rosenzweig et al, "Characterization of a peptide movement regulating IFN-gamma receptor 2plasma membrane accumulation and IFN-gamma resistance", J.Immunol.173(6), 3991-; mikulecky et al, "Crystal Structure of human interface-gamma receiver 2 derivatives the Structure base for receiver specificity", Acta Crystalloger.D 75, 1017-; kotenko et al, "Interaction between the components of the interference gamma receiver complex," J.biol.chem.270:20915-20921(1995), which is incorporated herein by reference in its entirety.

In some embodiments, the alpha chain of a heterodimeric chimeric protein useful in the invention comprises: (1) comprises the amino acid sequence of SEQ ID NO: 30, (b) a first domain comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof, and (c) an alpha core domain or a variant thereof, comprising an amino acid sequence substantially identical to SEQ ID NO:16 or SEQ ID NO: 24 amino acid sequence that is at least 95% identical. Such an Alpha chain may be referred to as "IFNgR-Alpha-IL 12 a".

In some embodiments, the IFNgR-Alpha-IL12a chain used in the invention has the following amino acid sequence:

in some embodiments, the beta chain of a heterodimeric chimeric protein useful in the invention comprises: (1) comprises the amino acid sequence of SEQ ID NO: 31, (b) a first domain comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof, and (c) an alpha core domain or a variant thereof, comprising an amino acid sequence substantially identical to SEQ ID NO:17 or SEQ ID NO: 25 amino acid sequences at least 95% identical. Such Beta chains may be referred to as "IFNGR 2-Beta-IL 27B".

In some embodiments, the IFNGR2-Beta-IL27B chain used in the present invention has the following amino acid sequence:

when IFNgR-Alpha-IL12a chain and IFNGR2-Beta-IL27B chain combined (in cells or in vitro), they formed a heterodimeric protein, referred to herein as IFN gamma R-Fc-IL-35.

In some embodiments, the alpha chain of a heterodimeric chimeric protein useful in the invention comprises: (1) comprises the amino acid sequence of SEQ ID NO: 30, (b) a first domain comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof, and (c) an alpha core domain or a variant thereof, comprising an amino acid sequence substantially identical to SEQ ID NO:17 or SEQ ID NO: 25 amino acid sequences at least 95% identical. Such an alpha chain may be referred to as "IFNgR-Beta-IL 12 a".

In some embodiments, the IFNgR-Beta-IL12a chain used in the present invention has the following amino acid sequence:

in some embodiments, the beta chain of a heterodimeric chimeric protein useful in the invention comprises: (1) comprises the amino acid sequence of SEQ ID NO: 31, (b) a first domain comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof, and (c) an alpha core domain or a variant thereof, comprising an amino acid sequence substantially identical to SEQ ID NO:16 or SEQ ID NO: 24 amino acid sequence that is at least 95% identical. Such beta chains may be referred to as "IFNGR 2-Alpha-IL 27B".

In some embodiments, the IFNGR2-Alpha-IL27B chain used in the present invention has the following amino acid sequence:

when IFNgR-Beta-IL12a chain and IFNGR2-Alpha-IL27B chain are combined (in cells or in vitro), they form a heterodimeric protein, which may also be referred to herein as IFN γ R-Fc-IL-35.

One embodiment of the IL-6R-Fc-IL-35 heterodimeric protein, comprising the Gp130-Alpha-IL12A chain and the IL6RA-Beta-IL27B chain, is disclosed above. In an alternative embodiment, the IL-6R-Fc-IL-35 heterodimeric protein may comprise IL6RA-Alpha-IL12a chain and Gp130-Beta-IL27 b.

In some embodiments, the alpha chain of a heterodimeric chimeric protein useful in the invention comprises: (1) comprises the amino acid sequence of SEQ ID NO:19, (b) a first domain comprising the amino acid sequence of SEQ ID NO: 20, or a variant thereof, and (c) an alpha core domain or a variant thereof, comprising an amino acid sequence substantially identical to SEQ ID NO:16 or SEQ ID NO: 24 amino acid sequence that is at least 95% identical. Such an Alpha chain may be referred to as "IL 6RA-Alpha-IL12 a".

In some embodiments, the chain of IL6RA-Alpha-IL12a used in the present invention has the following amino acid sequence:

in some embodiments, the beta chain of a heterodimeric chimeric protein useful in the invention comprises: (1) comprises the amino acid sequence of SEQ ID NO:18, (b) a first domain comprising the amino acid sequence of SEQ ID NO: 21, or a variant thereof, and (c) an alpha core domain or a variant thereof, comprising an amino acid sequence substantially identical to SEQ ID NO:17 or SEQ ID NO: 25 amino acid sequences at least 95% identical. Such Beta chain may be referred to as "Gp 130-Beta-IL27 b".

In some embodiments, the Gp130-Beta-IL27b chain used in the present invention has the following amino acid sequence:

when IL6RA-Alpha-IL12a chain and Gp130-Beta-IL27b chain are combined (in cells or in vitro), they form a heterodimeric protein, which may also be referred to herein as IL-6R-Fc-IL-35.

In various embodiments, the heterodimeric proteins of the invention can comprise variants of any known cytokine, growth factor, and/or hormone. In various embodiments, the heterodimeric proteins of the invention can comprise variants of any known receptor for cytokines, growth factors, and/or hormones. In various embodiments, a heterodimeric protein of the invention may comprise variants of any known extracellular domain, e.g., having at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity.

In various embodiments, the heterodimeric proteins of the invention can comprise an amino acid sequence having one or more amino acid mutations relative to any known protein sequence. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.

In some embodiments, the amino acid mutation is an amino acid substitution, and may include conservative and/or non-conservative substitutions.

"conservative substitutions" may be made, for example, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be divided into the following six standard amino acid groups: (1) hydrophobic: met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: cys, Ser, Thr, Asn, Gln; (3) acidic: asp and Glu; (4) basic: his, Lys, Arg; (5) chain orientation affecting residues: gly, Pro; and (6) aromatic: trp, Tyr, Phe.

As used herein, "conservative substitutions" are defined as exchanges of one amino acid for another amino acid listed in the same group of the six standard amino acid groups shown above. For example, exchange of Asp for Glu retains one negative charge in the polypeptide so modified. In addition, glycine and proline may be substituted for each other based on their ability to disrupt the alpha-helix.

As used herein, "non-conservative substitutions" are defined as exchanges of one amino acid for another amino acid listed in a different one of the six standard amino acid groups (1) to (6) shown above.

In various embodiments, substitutions may also include non-classical amino acids in general (e.g., selenocysteine, pyrrolysine, N-formylmethionine beta-alanine, GABA and delta-aminolevulinic acid, 4-aminobenzoic acid (PABA), the D-isomer of common amino acids, 2, 4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminocaproic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteine, t-butylglycine (t-butylglycine), t-butylalanine (t-butyllanine), phenylglycine, cyclohexylalanine, Beta-alanine, fluoroamino acids, designer amino acids (designer amino acids) such as beta methyl amino acids, C alpha methyl amino acids, N alpha methyl amino acids, and amino acid analogs).

The nucleotide sequence of the heterodimeric protein may also be mutated with reference to the genetic code, including taking into account codon degeneracy.

In various embodiments, the heterodimeric proteins of the invention are capable of and can be used in methods that include promoting immune activation (e.g., against a tumor). In various embodiments, the heterodimeric proteins of the invention are capable of and can be used in methods that include suppressing immunosuppression (e.g., which allows tumor survival). In various embodiments, the heterodimeric proteins of the invention provide improved immune activation and/or improved suppression of immunosuppression.

In various embodiments, the heterodimeric proteins of the invention can be or can be used in methods comprising modulating the magnitude of an immune response, e.g., modulating effector output levels. In some embodiments, for example, when used to treat cancer, the heterodimeric proteins of the invention alter the extent of immune stimulation compared to immunosuppression to increase the magnitude of T cell responses, including but not limited to stimulating increased levels of cytokine production, proliferation, or targeted lethality.

In various embodiments, the heterodimeric proteins of the invention can, in some embodiments, be or find use in methods involving masking an inhibitory ligand on the surface of a tumor cell and replacing the immunosuppressive ligand with an immunostimulatory ligand. Thus, the heterodimeric proteins of the invention can, in some embodiments, or find use in methods involving reducing or eliminating inhibitory immune signals and/or increasing or activating immunostimulatory signals. For example, a tumor cell carrying an inhibitory signal (and thus evading the immune response) may be replaced by a positive signal that binds to a T cell (which may then attack the tumor cell). Thus, in some embodiments, the heterodimeric proteins of the invention mask inhibitory immune signals and activate stimulatory immune signals. These beneficial properties are enhanced by the single construction method (single construct propaach) of the heterodimeric proteins of the present invention. For example, signal substitutions can be effected at approximately the same time, and tailored to be localized at a site of clinical importance (e.g., a tumor microenvironment).

In various embodiments, the heterodimeric proteins of the invention can be or find use in methods comprising stimulating or enhancing binding of an immunostimulatory receptor/ligand pair.

In other embodiments, the heterodimeric proteins of the invention can be or find use in methods involving enhancing, restoring, promoting and/or stimulating immune modulation. In some embodiments, the inventive heterodimeric proteins described herein restore, promote, and/or stimulate the activity or activation of one or more immune cells against tumor cells, including but not limited to: t cells, cytotoxic T lymphocytes, T helper cells, Natural Killer (NK) cells, natural killer T (nkt) cells, anti-tumor macrophages (e.g., M1 macrophages), B cells, and dendritic cells. In some embodiments, the heterodimeric proteins of the invention enhance, restore, promote, and/or stimulate the activity and/or activation of T cells, including by way of non-limiting example, activating and/or stimulating one or more T cell endogenous signals, including a survival-promoting signal; autocrine or paracrine growth signals; p38 MAPK-, ERK-, STAT-, JAK-, AKT-, or PI 3K-mediated signals; anti-apoptotic signals; and/or facilitating one or more of the following and/or signals necessary therefor: pro-inflammatory cytokine production or T cell migration or T cell tumor infiltration.

In some embodiments, the heterodimeric proteins of the invention can be used or found to be useful in relation to causing an increase in one or more of the following cells in a tumor or tumor microenvironment: t cells (including but not limited to cytotoxic T lymphocytes, T helper cells, natural killer T (nkt) cells), B cells, Natural Killer (NK) cells, natural killer T (nkt) cells, dendritic cells, monocytes, and macrophages (e.g., one or more of M1 and M2). In some embodiments, the heterodimeric proteins of the invention can be used or found to be useful in relation to inhibiting and/or causing the recruitment of immunosuppressive cells (e.g., myeloid-derived suppressor cells (MDSCs), regulatory T cells (tregs), tumor-associated neutrophils (TAN), M2 macrophages, and tumor-associated macrophages (TAMs)) to a tumor or reduction of the tumor microenvironment. In some embodiments, the therapies of the invention can alter the ratio of M1 to M2 macrophages in the tumor site and/or TME to favor M1 macrophages.

In various embodiments, the heterodimeric proteins of the present invention are capable of and can be used in methods comprising inhibiting and/or reducing T cell inactivation and/or immune tolerance to a tumor, comprising administering to a subject an effective amount of a heterodimeric protein described herein. In some embodiments, the heterodimeric proteins of the invention are capable of increasing serum levels of various cytokines including, but not limited to, one or more of the following: IFN gamma, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F and IL-22. In some embodiments, the heterodimeric proteins of the invention are capable of enhancing IL-2, IL-4, IL-5, IL-10, IL-13, IL-17A, IL-22, or IFN γ in the serum of a treated subject.

In various embodiments, the heterodimeric proteins of the invention inhibit, block and/or reduce cell death of anti-tumor CD8+ and/or CD4+ T cells; or stimulating, inducing and/or increasing cell death of pro-tumor T cells (pro-tumor T cells). T cell failure is a state of T cell dysfunction characterized by progressive loss of proliferation and effector functions culminating in clonal deletion. Thus, a pro-tumor T cell refers to a state of T cell dysfunction that occurs during many chronic infections and cancers. This dysfunction is defined as poor proliferation and/or effector function, sustained expression of inhibitory receptors, and transcriptional state distinct from functional effector or memory T cell states. Failure prevents optimal control of infection and tumors. In addition, anti-tumor CD8+ and/or CD4+ T cells refer to T cells that can immunoreact with a tumor. Exemplary tumor promoting T cells include, but are not limited to, tregs, CD4+ and/or CD8+ T cells expressing one or more checkpoint inhibitory receptors, Th2 cells, and Th17 cells. Checkpoint inhibitory receptors refer to receptors expressed on immune cells that can prevent or suppress uncontrolled immune responses (e.g., CTLA-4, B7-H3, B7-H4, TIM-3).

In various embodiments, the heterodimeric proteins of the invention are capable of and can be used in methods comprising increasing the ratio of effector T cells to regulatory T cells. Exemplary effector T cells include ICOS⁺Effector T cells; cytotoxic T cells (e.g., α β TCR, CD 3)⁺、CD8⁺、CD45RO⁺)；CD4⁺Effector T cells (e.g., α β TCR, CD3⁺、CD4⁺、CCR7⁺、CD62Lhi、IL-7R/CD127⁺)；CD8⁺Effector T cells (e.g., α β TCR, CD3⁺、CD8⁺、CCR7⁺、CD62Lhi、IL-7R/CD127⁺) (ii) a Effector memory T cells (e.g. CD62L low, CD44⁺、TCR、CD3⁺、IL-7R/CD127⁺、IL-15R⁺CCR7 low); central memory T cells (e.g., CCR 7)⁺、CD62L⁺、CD27⁺(ii) a Or CCR7hi, CD44⁺、CD62Lhi、TCR、CD3⁺、IL-7R/CD127⁺、IL-15R⁺)；CD62L⁺Effector T cells; CD8⁺Effector memory T cells (TEM), including early effector memory T cells (CD27⁺CD62L^-) And late effector memory T cells (CD 27)^-CD62L^-) (TemE and TemL, respectively); CD127(⁺) CD25 (low /) effector T cells; CD127(^-)CD25(^-) Effector T cells; CD8⁺Stem cell memory effector cells (TSCMs) (e.g., CD44 (Low) CD62L (high) CD122 (high) sca: (⁺) ); TH1 effector T cells (e.g. CXCR 3)⁺、CXCR6⁺And CCR5⁺(ii) a Or α β TCR, CD3⁺、CD4⁺、IL-12R⁺、IFNγR⁺、CXCR3⁺) TH2 effector T cells (e.g. CCR 3)⁺、CCR4⁺And CCR8⁺(ii) a Or α β TCR, CD3⁺、CD4⁺、IL-4R⁺、IL-33R⁺、CCR4⁺、IL-17RB⁺、CRTH2⁺) (ii) a TH9 effector T cells (e.g., α β TCR, CD 3)⁺、CD4⁺) (ii) a TH17 effector T cells (e.g., α β TCR, CD 3)⁺、CD4⁺、IL-23R⁺、CCR6⁺、IL-1R⁺)；CD4⁺CD45RO⁺CCR7⁺Effector T cells, CD4⁺CD45RO⁺CCR7(^-) Effector T cells; and IL-2, IL-4 and/or IFN-gamma secreting effector T cells. Exemplary regulatory T cells include ICOS⁺Regulatory T cells, CD4⁺CD25⁺FOXP3⁺Regulatory T cells, CD4⁺CD25⁺Regulatory T cells, CD4⁺CD25^-Regulatory T cells, CD4⁺CD25 high regulatory T cells, TIM-3⁺PD-1⁺Regulatory T cell, lymphocyte activation gene 3(LAG-3)⁺Regulatory T cells, CTLA-4/CD152⁺Regulatory T cells, neuropilin 1(Nrp-1)⁺Regulatory T cells, CCR4⁺CCR8⁺Regulatory T cells, CD62L (L-selectin)⁺Regulatory T cells, CD45RB low regulatory T cells, CD127 low regulatory T cells, LRRC32/GARP⁺Regulatory T cells, CD39⁺Regulatory T cells, GITR⁺Regulatory T cells, LAP⁺Regulatory T cells, 1B11⁺Regulatory T cell, BTLA⁺Regulatory T cells, type 1 regulatory T cells (Tr1 cells), and T helper type 3 (Th3) cellsCell, natural killer T cell phenotype regulatory cell (NKTreg), CD8⁺Regulatory T cells, CD8⁺CD28^-Regulatory T cells and/or regulatory T cells secreting IL-10, IL-35, TGF-beta, TNF-alpha, galectin-1, IFN-gamma and/or MCP 1.

In various embodiments, the heterodimeric proteins of the invention are capable of and can be used in methods that include transiently stimulating effector T cells for no more than about 12 hours, about 24 hours, about 48 hours, about 72 hours, or about 96 hours, or about 1 week or about 2 weeks. In various embodiments, the heterodimeric proteins of the invention are capable of and can be used in methods comprising transiently depleting or inhibiting regulatory T cells for no more than about 12 hours, about 24 hours, about 48 hours, about 72 hours, or about 96 hours, or about 1 week or about 2 weeks. In various embodiments, transient stimulation of effector T cells and/or transient depletion or inhibition of regulatory T cells occurs substantially in the blood or in specific tissues/sites of a patient, including lymphoid tissues such as bone marrow, lymph nodes, spleen, thymus, mucosa-associated lymphoid tissue (MALT), non-lymphoid tissues or tumor microenvironment.

In various embodiments, the heterodimeric proteins of the present invention provide advantages including, but not limited to, ease of use and ease of production. This is because combining two different immunotherapeutic agents into a single product allows for a single manufacturing process rather than two separate manufacturing processes. In addition, administering a single agent rather than two separate agents allows for easier administration and greater patient compliance. Moreover, the heterodimeric proteins of the invention are easier and more cost-effective to manufacture than, for example, monoclonal antibodies (large multimeric proteins containing many disulfide bonds and post-translational modifications such as glycosylation).

In various embodiments, the heterodimeric proteins of the invention provide synergistic therapeutic effects as they allow for improved site-specific interaction of two immunotherapeutic agents. In some embodiments, the heterodimeric proteins of the invention provide the potential to reduce off-site and/or systemic toxicity.

Diseases; treatment methods and patient selection

In various embodiments, the invention relates to the use of heterodimeric proteins for the treatment of one or more autoimmune diseases or disorders. In various embodiments, treating an autoimmune disease or disorder can involve modulating the immune system with a heterodimeric protein of the invention to favor immunosuppression over immunostimulation. Exemplary autoimmune diseases or disorders treatable with the heterodimeric proteins of the invention include those in which body autoantigens are the target of an immune response, such as rheumatoid arthritis, systemic lupus erythematosus, diabetes (diabetes mellitis), ankylosing spondylitis, sjogren's syndrome, inflammatory bowel diseases (e.g., ulcerative colitis, crohn's disease), multiple sclerosis, sarcoidosis, psoriasis, Grave's disease, Hashimoto's thyroiditis, psoriasis, hypersensitivity reactions (e.g., type I hypersensitivity reactions caused by allergy, hay fever, asthma and acute edema), and vasculitis.

Exemplary autoimmune diseases or disorders that can be treated or prevented using the heterodimeric proteins of the invention include, but are not limited to, multiple sclerosis, diabetes, lupus, celiac disease, crohn's disease, ulcerative colitis, Guillain-Barre syndrome (Guillain-Barre syndrome), scleroderma (scleroderma), Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, laslemen's encephalitis (Rasmussen's encephalitis), primary biliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, Addison's disease, hashimoto's thyroiditis, Fibromyalgia (Fibromyalgia), Menier's syndrome; transplant rejection (e.g., prevention of allograft rejection), pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, graves ' disease, and other autoimmune diseases.

In various embodiments, the present invention is applicable to cancers and/or tumors; for example, treating or preventing cancer and/or tumors. As described elsewhere herein, in various embodiments, treating cancer may involve modulating the immune system with the heterodimeric proteins of the invention to favor immune stimulation over immunosuppression.

Cancer or tumor refers to an uncontrolled growth of cells and/or an abnormally increased survival and/or apoptosis of cells that is inhibited, which interferes with the normal function of body organs and systems. Including benign and malignant cancers, polyps, hyperplasia, and dormant tumors or micrometastases. Also included are cells with abnormal proliferation that are not impeded by the immune system (e.g., virus-infected cells). The cancer may be a primary cancer or a metastatic cancer. A primary cancer may be a region of cancer cells at a clinically detectable site of origin, and may be a primary tumor. In contrast, metastatic cancer can be the spread of disease from one organ or portion to another non-adjacent organ or portion. Metastatic cancer can result from cancer cells acquiring the ability to penetrate and infiltrate the surrounding normal tissue of a local area, forming a new tumor that can be a local metastasis. Cancer may also be caused by cancer cells that acquire the ability to penetrate the lymphatic and/or blood vessel walls, after which the cancer cells are able to circulate through the blood (and thus become circulating tumor cells) to other sites and tissues in the body. Cancer may be caused by processes such as lymphatic spread or spreading of blood origin. Cancer can also be caused by tumor cells that reside at another site, re-penetrate the blood vessel or wall, continue to proliferate, and eventually form another clinically detectable tumor. The cancer may be a new tumor (which may be a metastatic (or secondary) tumor).

Cancer may be caused by metastasized tumor cells, which may be secondary or metastatic tumors. The cells of the tumor may be similar to the cells in the original tumor. For example, if breast or colon cancer metastasizes to the liver, the secondary tumor, while present in the liver, is composed of abnormal breast or colon cells rather than abnormal liver cells. The tumor in the liver may thus be metastatic breast cancer or metastatic colon cancer, but not liver cancer.

Cancer may originate in any tissue. Cancer may originate from melanoma, colon, breast or prostate and may therefore consist of cells of origin in skin, colon, breast or prostate respectively. The cancer may also be a hematological malignancy, which may be leukemia or lymphoma. Cancer can invade tissues such as the liver, lung, bladder or intestine.

Representative cancers and/or tumors of the present invention include, but are not limited to, basal cell carcinoma, biliary tract carcinoma; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancers of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver cancer (hepatic carcinosoma); hepatoma (hepatoma); intraepithelial neoplasms (intra-epithelial neoplasms); kidney cancer (kidney or renal cancer); laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous carcinoma); melanoma; a myeloma cell; neuroblastoma; oral cancer (lip, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; a sarcoma; skin cancer; squamous cell carcinoma; gastric cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphomas, including hodgkin lymphoma and non-hodgkin lymphoma as well as B-cell lymphoma (including low grade/follicular non-hodgkin lymphoma (NHL); small Lymphocyte (SL) NHL; moderate/follicular NHL; moderate diffuse NHL; hyperimmune blast NHL; high lymphoblastoid NHL; highly small non-lysed cell NHL; giant mass nhl (bulk disease nhl); mantle cell lymphoma; AIDS-related lymphomas; and Macroglobulinemia fahrenheit (Waldenstrom's macrolobalinemia); chronic Lymphocytic Leukemia (CLL); acute Lymphocytic Leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), and abnormal vascular proliferation associated with maternal plaque disease (phakomatose), edema (e.g., associated with brain tumors), and Meigs' syndrome.

In some embodiments, a subject having a refractory cancer is treated with a heterodimeric protein. In some embodiments, the heterodimeric protein is used to treat a subject refractory to one or more immunomodulators. For example, in some embodiments, a subject that does not respond to treatment or even has progressed after about 12 weeks of treatment is treated with a heterodimeric protein. For example, in some embodiments, the subject is a PD-1 and/or PD-L1 and/or PD-L2 agent-refractory patient, including, for example, nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL myrs squid bb), pembrolizumab (pembrolizumab) (keyrun, MERCK), pidilizumab (pidilizumab) (CT-011, CURE TECH), MK-3475(MERCK), BMS936559 (brostol myrs suibb), Ibrutinib (Ibrutinib) (PHARMACYCLICS/abie), aleuzumab (atezolizumab) (TECENTRIQ, GENENTECH), and/or MPDL328OA (he) roc-refractory patient. For example, in some embodiments, the subject is refractory to an anti-CTLA-4 agent, e.g., an ipilimumab (yerveoy) refractory patient (e.g., a melanoma patient). Accordingly, in various embodiments, the present invention provides cancer treatment methods to save patients who are non-responsive to various therapies, including monotherapy with one or more immunomodulators.

In various embodiments, the present invention provides heterodimeric proteins that target cells or tissues within the tumor microenvironment. In some embodiments, cells or tissues within the tumor microenvironment express one or more targets or binding partners for the heterodimeric proteins. Tumor microenvironment refers to the cellular environment (cellular milieu) including cells, secreted proteins, small physiological molecules, and blood vessels in which tumors reside. In some embodiments, the cell or tissue within the tumor microenvironment is one or more of: tumor vasculature; tumor infiltrating lymphocytes; fibroblast reticulocytes; endothelial Progenitor Cells (EPC); cancer-associated fibroblasts; a pericyte; other stromal cells; a component of the extracellular matrix (ECM); a dendritic cell; an antigen presenting cell; a T cell; regulatory T cells; macrophages; neutrophils; and other immune cells located proximal to the tumor. In various embodiments, the heterodimeric proteins of the invention target cancer cells. In some embodiments, the cancer cell expresses one or more targets or binding partners of the heterodimeric protein.

In various embodiments, the heterodimeric proteins of the invention can be targeted to cells (e.g., cancer cells or immune cells) expressing any of the receptors described herein. For example, the heterodimeric proteins of the invention can target cells expressing receptors for any of the cytokines, growth factors, and/or hormones described herein.

In some embodiments, the present methods provide for treating patients refractory to other agents with heterodimeric proteins, the "other agents" being described elsewhere herein, including but not limited to the various chemotherapeutic agents described herein.

In some aspects, the chimeric agents of the invention are used to eliminate intracellular pathogens. In some aspects, one or more infections are treated using chimeric agents of the invention. In some embodiments, the heterodimeric proteins of the invention are used in methods of treating viral infections (including, e.g., HIV and HCV), parasitic infections (including, e.g., malaria), and bacterial infections. In various embodiments, the infection induces immunosuppression. For example, HIV infection often results in immunosuppression in infected subjects. Thus, as described elsewhere herein, in various embodiments, treating these infections may comprise modulating the immune system with the heterodimeric proteins of the invention to favor immune stimulation over immune suppression. Alternatively, the invention provides methods for treating an infection that induces immune activation. For example, intestinal helminth infections are associated with chronic immune activation. In these embodiments, treating these infections may include modulating the immune system with the heterodimeric proteins of the invention to favor immunosuppression over immunostimulation.

In various embodiments, the present invention provides methods of treating viral infections, including but not limited to acute or chronic viral infections, such as respiratory, papilloma virus, Herpes Simplex Virus (HSV), Human Immunodeficiency Virus (HIV), and viral infections of internal organs, such as hepatitis virus. In some embodiments, the viral infection is caused by a virus of the flaviviridae family. In some embodiments, the virus of the flaviviridae family is selected from the group consisting of yellow fever virus, west nile virus, dengue virus, japanese encephalitis virus, st. In other embodiments, the viral infection is caused by a virus of the picornaviridae family, such as poliovirus, rhinovirus, coxsackievirus. In other embodiments, the viral infection is caused by a member of the orthomyxoviridae family, such as an influenza virus. In other embodiments, the viral infection is caused by a member of the retroviral family, such as a lentivirus. In other embodiments, the viral infection is caused by a member of the paramyxoviridae family, such as respiratory syncytial virus, human parainfluenza virus, rubella virus (e.g., mumps virus), measles virus, and human metapneumovirus (human metapneumovirus). In other embodiments, the viral infection is caused by a member of the bunyaviridae family, such as hantavirus. In other embodiments, the viral infection is caused by a member of the reoviridae family, such as a rotavirus.

In various embodiments, the present invention provides methods of treating parasitic infections such as protozoan (protozoa) or helminth infections. In some embodiments, the parasitic infection is caused by a protozoan parasite (protozoan parasite). In some embodiments, the oritiziab parasite is selected from an intestinal protozoan, a tissue protozoan, or a blood protozoan. Exemplary pairs of protozoan parasites include, but are not limited to, Entamoeba hystolytica, Giardia lamblia (Giardia lamblia), Cryptosporidium murinus (Cryptosporidium muris), Trypanosoma gambiae (Trypanosomatida gamniense), Trypanosoma rhodanense (Trypanosomatida rhodesiense), Trypanosoma proteorum (Trypanosomatida crusi), Leishmania mexicana (Leishmania mexicana), Leishmania brasiliensis (Leishmania braziensis), Leishmania tropicalis (Leishmania tropicalis), Leishmania donovani (Leishmania donovani), Toxoplasma gondii (Toplasmia ndii), Plasmodium vivax (Plasmodium vivax), Plasmodium vivax (Plasmodium malariae), Plasmodium vivax (Plasmodium Trichomonas), Plasmodium vaginalis (Plasmodium). In some embodiments, the parasitic infection is caused by a helminthic parasite, such as the phylum nematoda (Adenophorea). In some embodiments, the parasite is selected from the group consisting of the phylum caucasianoides (secemenea) (e.g., Trichuris trichoderma), Ascaris hominis (Ascaris lumbricoides), enterobiasis (enterobiasis), ancylostomus duodenalis (ancylostome), ancylostomus americanus (subcaterocanus), Strongyloides stercoralis, Wuchereria brueckii (Wuchereria subcortica), medecinopharynus makinsonii (dracculus medius medinensis)). In some embodiments, the parasite is selected from the group consisting of flukes (e.g., schistosomes, liver flukes, intestinal flukes, and lung flukes). In some embodiments, the parasite is selected from the group consisting of: schistosoma mansoni (Schistosoma mansoni), Schistosoma japonicum (Schistosoma haematbium), Schistosoma japonicum (Schistosoma japonicum), Fasciola hepatica (Fasciola hepatica), Fasciola gigantica (Fasciola gigantica), Heterophaera heteroptera (Heterophaera heteroptera), and Euonymus westernum (Paragonimus westernani). In some embodiments, the parasite is selected from the group consisting of tapeworms (e.g., Taenia solium), beef tapeworms (Taenia saginata), hymenotheca teniae (Hymenolepis nana), Echinococcus granulosus (Echinococcus grandis).

In various embodiments, the present invention provides methods of treating bacterial infections. In various embodiments, the bacterial infection is caused by gram-positive bacteria, gram-negative bacteria, aerobic bacteria, and/or anaerobic bacteria. In various embodiments, the bacteria are selected from, but not limited to, Staphylococcus (Staphylococcus), Lactobacillus (Lactobacillus), Streptococcus (Streptococcus), Sarcina (Sarcina), Escherichia (Escherichia), Enterobacter (Enterobacter), Klebsiella (Klebsiella), Pseudomonas (Pseudomonas), Acinetobacter (Acinetobacter), Mycobacterium (Mycobacterium), Proteus (Proteus), Campylobacter (Campylobacter), Citrobacter (Citrobacter), Neisseria (neisia), Bacillus (Bacillus), Bacteroides (Bacteroides), Peptococcus (Peptococcus), Clostridium (Clostridium), Salmonella (Salmonella), Shigella (Shigella), Serratia (Serratia), Haemophilus (Haemophilus), and other organisms. In some embodiments, the bacteria are selected from, but not limited to, Pseudomonas aeruginosa (Pseudomonas aeruginosa), Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas acidovorax (Pseudomonas acidifera), Pseudomonas alcaligenes (Pseudomonas alcaligenes), Pseudomonas putida (Pseudomonas putida), Stenotrophomonas maltophilia (Stenotrophomonas maltophilia), Burkholderia cepacia (Burkholderia cepacia), Aeromonas hydrophila (Aeromonas hydrophylla), Escherichia coli (Escherichia coli), Citrobacter freundii (Citrobacter freundii), Salmonella typhimurium (Salmonella typhimurium), Salmonella paratyphi (Salmonella parayphi), Salmonella typhimurium (Shigella dysenteriae), Salmonella typhi (Shigella Enterobacter coli), Salmonella typhi (Shigella pneumoniae (Shigella Enterobacter coli), Salmonella paratyphi (Shigella dysenteriae), Escherichia coli (Shigella Enterobacter coli (Shigella) Klebsiella oxytoca (Klebsiella oxytoca), Serratia marcescens (Serratia marcescens), Francisella tularensis (Francisella tularensis), Morganella morganii (Morganella morgananii), Proteus mirabilis (Proteus mirabilis), Proteus vulgaris (Proteus vulgaris), Providella alcaligenes (Providelia alcaliensis), Providencia retta (Providelia Retentii), Providencia stuartii (Providelia nitili), Acinetobacter baumannii (Acinetobacter baumannii), Acinetobacter calcoaceticus (Acinetobacter caoecacicus), Acinetobacter haemolyticus (Acinetobacter haemolyticus) Haitellus, Klebsiella oxytoca (Klebsiella oxytoca), Yersinia pestis (Bordetella), Yersinia parahaemophila (Borteus flavipes), Bordetella parahaemophilus (Bordetella), Bordetella parahaemophilus influenzae (Yersinia), Bordetella pertussis parahaemophilus (Yersinia), Bordetella parahaemophilus (Yersinia), Bordetella) bacteria (Yersinia), Bordetella parahaemophilus pertussis parahaemophilus (Yersis), Bordetella) and Bordetella Haemophilus infection (Yersinia), Bordetella) A strain (Bordetella) and Bordetella Haemophilus, Haemophilus haemolyticus (Haemophilus haemolyticus), Haemophilus parahaemolyticus (Haemophilus parahaemolyticus), Haemophilus ducreyi (Haemophilus ducreyi), Pasteurella multocida (Pasteurella multocida), Pasteurella haemolyticus (Pasteurella haemolytica), Branhamella catarrhalis (Branhamella catarrhalis), Helicobacter pylori (Helicobacter pylori), Campylobacter foetidulus (Campylobacter fetalis), Campylobacter jejuni (Campylobacter jejuni), Campylobacter coli (Campylobacter coli), Bordetella Borrelia (Bordetella burgdorferi), Vibrio cholerae (Vibrio cholerae), Vibrio parahaemolyticus (Vibrio parahaemolyticus), Salmonella parahaemolyticus (Legionella), Salmonella meningitidis (Legionlla), Salmonella meningitidis), Salmonella cholerae (Legionlla meningitidis), Salmonella cholera meningitidis (Legionlla), Salmonella cholera (Salmonella), Salmonella (Salmonella), Salmonella, Salmon, Bacteroides 3452A homologous group (Bacteroides 3452A homology group), Bacteroides vulgatus (Bacteroides vulgatus), Bacteroides ovatus (Bacteroides ovatus), Bacteroides thetaiotaomicron (Bacteroides thetaiotaomicron), Bacteroides monoides (Bacteroides uniflora), Bacteroides exxoides (Bacteroides eggchoii), Bacteroides visceral Bacteroides (Bacteroides sphaericus), Clostridium difficile (Clostridium difficile), Mycobacterium tuberculosis (Mycobacterium tuberculosis), Mycobacterium avium (Mycobacterium avium), Mycobacterium intracellulare (Mycobacterium intracellulare), Mycobacterium leprae (Mycobacterium tuberculosis), Corynebacterium diphtheriae (Corynebacterium diphtheriae), Streptococcus ulceroides (Streptococcus), Streptococcus faecalis (Streptococcus faecalis), Streptococcus faecalis (Streptococcus), Streptococcus faecalis), Streptococcus (Streptococcus), Streptococcus faecalis (Streptococcus), Streptococcus (Streptococcus), Streptococcus faecalis (Streptococcus), Streptococcus (Streptococcus faecalis (Streptococcus) and Streptococcus (Streptococcus) are strain (Streptococcus), Streptococcus (Streptococcus faecalis (Streptococcus), Streptococcus (Streptococcus, Staphylococcus intermedius (Staphylococcus intermedius), Staphylococcus suis subsp. hyosus (Staphylococcus hyicus), Staphylococcus haemolyticus (Staphylococcus haemolyticus), Staphylococcus hominis (Staphylococcus hominis), or Staphylococcus saccharolyticus (Staphylococcus saccharolyticus).

In another further aspect, the invention relates to methods of treating and preventing T cell mediated diseases and disorders, such as, but not limited to, diseases or disorders described elsewhere herein, as well as inflammatory diseases or disorders, Graft Versus Host Disease (GVHD), transplant rejection, and T cell proliferative disorders.

In some aspects, the chimeric agents of the invention are used in methods of activating T cells, e.g., via an extracellular domain with an immunostimulatory signal.

In some aspects, the chimeric agents of the invention are used in methods of preventing cellular transmission of immunosuppressive signals. Combination therapy and conjugation

In some embodiments, the present invention provides heterodimeric proteins and methods further comprising administering to a subject an additional agent. In some embodiments, the invention relates to co-administration and/or co-formulation. Any of the compositions described herein can be co-formulated and/or co-administered.

In some embodiments, any of the heterodimeric proteins described herein act synergistically when co-administered with another agent, and are administered at doses lower than those typically employed when such agent is used as monotherapy. In various embodiments, any of the agents mentioned herein can be used in combination with any of the heterodimeric proteins described herein.

In various embodiments, any of the heterodimeric proteins disclosed herein can be co-administered with another heterodimeric protein disclosed herein. Without wishing to be bound by theory, it is believed that a combination regimen involving administration of one or more heterodimeric proteins that induce an innate immune response and one or more heterodimeric proteins that induce an adaptive immune response may result in a synergistic effect (e.g., a synergistic anti-tumor effect).

In various embodiments, any heterodimeric protein that induces an innate immune response can be used in the present invention. In various embodiments, any heterodimeric protein that induces an adaptive immune response can be used in the present invention.

In some embodiments, including but not limited to cancer applications, the invention relates to chemotherapeutic agents as other agents. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents, such as thiotepa and CYTOXAN cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzotepa, carboquone, meturedpa and uredpa; ethyleneimine and methylmelamine including altretamine, triethylenemelamine, triethylenephosphoramide (triethylenephosphoramide), triethylenethiophosphoramide (triethylenethiophosphoramide), and trimethylolmelamine (trimetylomelamine); polyacetylens (acetogenin) (e.g., bullatacin and bullatacin); camptothecin (including the synthetic analogue topotecan); bryostatin; a calicheatin (cally statin); CC-1065 (including its adozelesin (adozelesin), carzelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); cryptophycin (e.g., cryptophycin 1 and cryptophycin 8); dolastatin (dolastatin); duocarmycins (duocarmycins) (including synthetic analogs, KW-2189 and CB 1-TM 1); (ii) an elutherobin; coprinus annuus alkali (pancratistatin); sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlorophosphamide (cholphosphamide), estramustine (estramustine), ifosfamide (ifosfamide), dichloromethyl diethylamine (mechlerothiamine), chlorambucil oxide, melphalan (melphalan), neomustard (novembichin), benzene mustard (phenesterine), prednimustine (trofosfamide), uracil mustard; nitrosoureas (nitrosureas), such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ranimustine (ranirnustine); antibiotics, such as enediynes antibiotics (e.g., calicheamicin (calicheamicin), especially calicheamicin gamma and calicheamicin omega (see, e.g., Agnew, chem. int. ed. Engl.,33:183-186(1994)), daptomycin (dynemicin), including daptomycin A; bisphosphonates, such as clodronate; esperamicin (esperamicin), and neocarzinostain chromophores (neocarzinostatin chromophoromophores) and related chromoproteins enediyne antibiotic chromophores (related chromophoric protein diynes)), aclacinomycin (lacinystatin), actinomycins (actinomycins), actinomycins (leucomycins), actinomycins (monocarbomycins) (monocrotamycins) (6-5), monochromycins (monochromycins) (leucomycins), actinomycins (monochromycin-6-D-monochromycin (monochromycin), monochromycin (monochromycin), monochromycin (monochromycin A-D-monochromycin, monochromycin (monochromycin), monochromycin (monochromycin, monochromycin (monochromycin), monochromycin, monochro L-norleucine), ADRIAMYCIN doxorubicin (doxorubicin) (including morpholino-doxorubicin), cyanomorpholino doxorubicin, 2-pyrrole-doxorubicin and deoxydoxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), mariomycin (marcellomycin), mitomycin (mitomycin) such as mitomycin c, mycophenolic acid (mycophenolic acid), norramycin (nogalamycin), olivomycin (olivomycin), pelomycin (polyplomycin), pofiomycin (potfiromycin), puromycin (puromycin), griseofibrinomycin (lamycin), robinicin (urorubicin), streptozocin (streptozocin), streptozotocin (streptozotocin), tubercidin (zotocin), tubercidin (tubercidin), tubercidin (zotocin); antimetabolites such as methotrexate (methotrexate) and 5-fluorouracil (5-fluorouricil, 5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine (fludarabine), 6-mercaptopurine, thiamiprine (thiamiprine), thioguanine; pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine (6-azauridine), carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine, deoxyfluorouridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); androgens such as testosterone Caroterone (calusterone), drostandrosterone propionate (dromostanolone propionate), epithioandrostanol (epithioandrostane), mepiquat (mepiquitazone), lactonone (telectalactone); anti-adrenalines such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trostane (trilostane); folic acid supplements such as folinic acid; acetoglucuronolactone (acegultone); (ii) an aldophosphamide glycoside; aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); bestrabuucil; bisantrene; edatrexate (edatraxate); colchicine (demecolcine); diazaquinone (diaziqutone); eflornithine (elformithine); ammonium etilate (ellitinium acetate); epothilone (epothilone); etoglut (etoglucid); gallium nitrate; a hydroxyurea; mushroom polysaccharides (lentinan); lonidamine (lonidainine); maytansinoids such as maytansine (maytansinoid) and ansamitocins (ansamitocins); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidanol (mopidanmol); nicergoline (nitrarine); pentostatin (pentostatin); phenamett; pirarubicin (pirarubicin); losoxantrone (losoxantrone); podophyllinic acid (podophyllic acid); 2-ethyl hydrazide; procarbazine, PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane (rizoxane); rhizomycin (rhizoxin); azofurans (sizofurans); germanium spiroamines (spirogyranium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2' -trichlorotriethylamine; trichothecenes (trichothecenes) (e.g., T-2 toxin, verrucin a (verrucarin a), tuberculin a (roridin a), and serpentine (anguidine)); urethane (urethane); vindesine (vindesine); dacarbazine (dacarbazine); mannomustine (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); (iii) a parthenosine; cytarabine (Ara-C), cyclophosphamide; thiotepa; taxanes (taxoids), such as TAXOL paclitaxel (paclitaxel) (Bristol-Myers Squibb Oncology, Princeton, n.j.), albumin engineered paclitaxel nanoparticle formulations of ABRAXANE without hydrogenated castor oil (ABRAXANE Cremophor-free, albumin-engineered nanoparticles of paclitaxel, American Pharmaceutical ligands, Schaumberg,111.) and TAXOL docetaxel (taxotexel) (Rhone-Poulenc Rorer, Antony, nce); nitrogen mustard phenylbutyric acid; GeMZAR gemcitabine (gemcitabine), 6-thioguanine, mercaptopurine, methotrexate; platinum analogs such as cisplatin (cissplatin), oxaliplatin (oxaliplatin), and carboplatin (carboplatin); vinblastine (vinblastine); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (vincristine); navelbine vinorelbine (vinorelbine); nuantro (novantrone); teniposide (teniposide); edatrexate (edatrexate); daunomycin (daunomycin); aminopterin (aminopterin); (xiloda); ibandronate (ibandronate); irinotecan (irinotecan) (Camptosar, CPT-11) (treatment regimens that include irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids (retinoids), such as retinoic acid (retinoic acid); capecitabine (capecitabine); combretastatin (combretastatin); leucovorin (LV); oxaliplatin (oxaliplatin), including oxaliplatin treatment regimen (FOLFOX); lapatinib (lapatinib) (TYKERB); an inhibitor of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)), and VEGF-A that reduces cell proliferation, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above. Additionally, the method of treatment may further comprise the use of radiation. In addition, the method of treatment may further comprise the use of photodynamic therapy.

In various embodiments, including but not limited to cancer applications, other agents of the invention are one or more immunomodulatory agents selected from the group consisting of: agents that block, reduce and/or inhibit binding of PD-1 and PD-L1 or PD-L2 and/or PD-1 to PD-L1 or PD-L2 (as non-limiting examples, nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, Merck), MK-3475(MERCK), BMS936559(BRISTOL MYERS SQUIBB), Atuzumab (TECENTRIQ, GENENTECH), MPDL328OA (ROCHE)), agents that increase and/or stimulate binding of CD137(4-1BB) and/or CD137(4-1BB) to one or more 4-1BB ligands (as non-limiting examples, Uramumab (BMS) (BMS-663513 and anti-4-1 BB antibodies), and agents that block, reduce and/or inhibit CTLA-4 activity and/or CTLA-4-AP-CTLA activity and CTLA-1 to PD-L1 or PD-L2) Agents that bind to one or more of CD80, CD86, SHP-2, and PPP2R5A and/or to bind OX40 to OX40L (by way of non-limiting example GBR830(GLENMARK), MEDI6469 (medimmone).

In some embodiments, including but not limited to infectious disease applications, the invention relates to anti-infective agents as other agents. In some embodiments, the anti-infective agent is an antiviral agent, including, but not limited to, Abacavir (Abacavir), Acyclovir (Acyclovir), Adefovir (Adefovir), Amprenavir (Amprenavir), Atazanavir (Atazanavir), Cidofovir (Cidofovir), Darunavir (daunarvir), Delavirdine (Delavirdine), Didanosine (Didanosine), behenyl (Docosanol), efavir (Efavirenz), Elvitegravir (Elvitegravir), Emtricitabine (Emtricitabine), envivirtide (enfurovirtide), Etravirine (Etravirine), Famciclovir (Famciclovir) and Foscarnet (foscarat net). In some embodiments, the anti-infective agent is an antibacterial agent, including, but not limited to, cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and cefbiprole); fluoroquinolones (ciprofloxacin (cipro), levofloxacin (Levaquin), floxuridine (floxin), gatifloxacin (tequin), moxifloxacin (avelox) and norfloxacin (norflox)); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin (vancomycin), and methicillin); monobactam antibiotic (aztreonam); and carbapenem antibiotics (ertapenem), doripenem (doripenem), imipenem (imipenem)/cilastatin (cilastatin), and meropenem (meropenem)). In some embodiments, the anti-infective agent comprises an antimalarial agent (e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether/lumefantrine, atovaquone/proguanil, and sulfadoxine/pyrimethamine), metronidazole, tinidazole, ivermectin, pyrantel pamoate (pyrantel pamoate), and albendazole (albendazole).

In some embodiments, including but not limited to autoimmune applications, the other agent is an immunosuppressive agent. In some embodiments, the immunosuppressive agent is an anti-inflammatory agent, such as a steroidal anti-inflammatory agent or a non-steroidal anti-inflammatory agent (NSAID). Steroids, particularly adrenocortical steroids and their synthetic analogs, are well known in the art. Examples of corticosteroids that may be used in the present invention include, but are not limited to, triamcinolone (hydroxytriamcinolone), alpha-methyl dexamethasone (alpha-methyl dexamethasone), beta-methyl betamethasone (beta-methyl betamethasone), beclomethasone dipropionate (beclomethasone dipropionate), betamethasone benzoate (betamethasone benzoate), betamethasone dipropionate (betamethasone dipropionate), betamethasone valerate (betamethasone valerate), clobetasone valerate (clobetasol valerate), desonide (desonide), desoximetasone (desoxymethasone), dexamethasone, diflunisone diacetate (difluorideone diacetate), diflunisone (diflunisone valerate), flutolamine (fludioxolone), fluocinonide (fluocinolone acetonide), fluocinonide (fluketoflunisolone), fluocinonide (flunisolone acetate), fluocinonide (flunisolone), fluocinonide (flunisolone), fluocinonide (flunisolone, fluocinonide (fluocinonide), fludroxysone (flurandrenone), halcinonide (halcinonide), hydrocortisone acetate (hydrocortisone acetate), hydrocortisone butyrate (hydrocortisone butyrate), methylprednisolone (methylprednisolone), triamcinolone acetonide (triamcinolone acetonide), cortisone (cortisone), cotolosone (cortixolone), fluocinolone (corticonone), fluocinonide (fludrolone), fludrocortisone (fludrocortisone), diflorasone (diflorolone diacetate), fludaronone acetate, fluocinolone acetonide, medrysone (medrysone), amcinafelone (amfenasone), amcinolone (amcinamide), betamethasone (betasone ester), prednisolone (chlorodesosone), clocortolone (clolone), thilone (methalone), triamcinolone (flunisolone), triamcinolone (fluprednillone (flunisolone), triamcinolone acetonide (flunisolone), triamcinolone acetonide (flunisolone), triamcinolone (flunisolone) and the balance of the ester of the balance of the mixture of, Beclomethasone dipropionate (beclomethasone dipropionate). Examples of (NSAIDS) that may be used in the present invention include, but are not limited to, salicylic acid, acetylsalicylic acid, methyl salicylate, glycol salicylate (glycol salicylate), salicylamide (salicylate), benzyl-2,5-diacetoxybenzoic acid (benzyl-2,5-diacetoxybenzoic acid), ibuprofen, fulvindac (fulvindac), naproxen (naproxen), ketoprofen, etofenamate (etofenamate), phenylbutazone (phenylbutazone), and indomethacin (indomethacin). In some embodiments, the immunosuppressive agent can be a cytostatic agent such as an alkylating agent, an antimetabolite (e.g., azathioprine, methotrexate), a cytotoxic antibiotic, an antibody (e.g., basiliximab, daclizumab, and muromab), an anti-immunophilin (anti-immunophilin) (e.g., cyclosporine, tacrolimus, sirolimus), an interferon, an opioid, a TNF binding protein, mycophenolate mofetil, and a small biological agent (e.g., fingolimod, myriocin).

In some embodiments, the heterodimeric proteins (and/or other agents) described herein include derivatives that are modified, i.e., by covalently binding any type of molecule to the composition such that the covalent binding does not interfere with the activity of the composition. For example, but not limited to, derivatives include compositions that have been modified by, for example: glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a number of chemical modifications may be made by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin (turicamycin), and the like. In addition, the derivative may contain one or more non-canonical amino acids. In other embodiments, the heterodimeric proteins (and/or other agents) described herein further comprise a cytotoxic agent, including in exemplary embodiments a toxin, a chemotherapeutic agent, a radioisotope, and an agent that causes apoptosis or cell death. These agents may be coupled to the compositions described herein (conjugated).

The heterodimeric proteins (and/or other agents) described herein can thus be post-translationally modified to add effector moieties such as chemical linkers, detectable moieties such as fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as streptavidin, avidin, biotin, cytotoxins, cytotoxic agents, and radioactive materials.

Preparation of

The heterodimeric proteins (and/or other agents) described herein can have a functional group that is sufficiently basic to be reactive with an inorganic or organic acid, or a carboxyl group that is reactive with an inorganic or organic base, to form a pharmaceutically acceptable salt. As is well known in the art, pharmaceutically acceptable acid addition salts are formed from pharmaceutically acceptable acids. Such salts include, for example, the pharmaceutically acceptable salts listed below: journal of Pharmaceutical Science,66,2-19(1977) and The Handbook of Pharmaceutical Salts; properties, Selection, and use, p.h.stahl and c.g.wermuth (eds.), Verlag, zurich (switzerland)2002, which is incorporated herein by reference in its entirety.

In some embodiments, the compositions described herein are in the form of a pharmaceutically acceptable salt.

In addition, any of the heterodimeric proteins (and/or other agents) described herein can be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier or vehicle. Such compositions may optionally comprise a suitable amount of a pharmaceutically acceptable excipient to provide a form for suitable administration. Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipient may be, for example, saline, gum arabic (gum acacia), gelatin, starch paste, talc (talc), keratin, colloidal silica, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants may be used. In one embodiment, the pharmaceutically acceptable excipient is sterile when administered to a subject. Water is a useful excipient when any of the agents described herein are administered intravenously. Saline solutions, as well as aqueous dextrose and glycerol solutions, may also be employed as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk (chalk), silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any of the agents described herein may also contain minor amounts of wetting or emulsifying agents or pH buffering agents, as desired.

In some embodiments, the compositions described herein are resuspended in a saline buffer (including but not limited to TBS, PBS, and the like).

In various embodiments, the heterodimeric protein may be conjugated and/or fused with another agent to increase half-life or improve pharmacodynamic and pharmacokinetic properties. In some embodiments, the heterodimeric protein may be fused or conjugated to one or more of the following: PEG, XTEN (e.g., as rPEG), polysialic acid (POLYXEN), albumin (e.g., human serum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP, transferrin, and the like. In various embodiments, each individual heterodimeric protein is fused to one or more agents described in BioDrugs (2015)29: 215-.

Administration/dosing, dosing and treatment regimen

The invention includes such heterodimeric proteins (and/or other agents) in various formulations. Any of the heterodimeric proteins (and/or other agents) described herein can take the form of a solution, suspension, emulsion, drop, tablet, pill(s), pellet(s), capsule, liquid-containing capsule, powder, sustained release formulation, suppository, emulsion, aerosol, spray, suspension, or any other suitable form for use. DNA or RNA constructs encoding protein sequences may also be used. In one embodiment, the composition is in the form of a capsule (see, e.g., U.S. Pat. No. 5,698,155). Other examples of suitable Pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-.

If desired, the formulation comprising the heterodimeric protein (and/or other agent) may further comprise a solubilizing agent. The agents may also be delivered using suitable vehicles or delivery devices known in the art. The combination therapeutics outlined herein can be co-delivered in a single delivery vehicle or delivery device. Compositions for administration may optionally include a local anesthetic such as lidocaine (lignocaine) to reduce pain at the site of injection.

Formulations comprising the heterodimeric proteins (and/or other agents) of the invention can be conveniently presented in unit dosage form and can be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing into association the therapeutic agent with the carrier which constitutes one or more accessory ingredients. Generally, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulation dosage form (e.g., wet or dry granulation, powder mixtures, and the like, followed by compression by conventional methods known in the art).

In one embodiment, any of the heterodimeric proteins (and/or other agents) described herein are formulated in accordance with conventional methods into compositions suitable for the modes of administration described herein.

Routes of administration include, for example, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, by inhalation or topically, especially to the ear, nose, eye or skin. In some embodiments, administration is by oral or parenteral injection. In most cases, administration results in the release of any of the agents described herein into the bloodstream.

Any of the heterodimeric proteins (and/or other agents) described herein can be administered orally. These heterodimeric proteins (and/or other agents) may also be administered by any other convenient route, such as by intravenous infusion or bolus injection (bolus injection), absorbed through epithelial or mucocutaneous layers (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered with other biologically active agents. Administration may be systemic or local. Various delivery systems are known, e.g., encapsulated in liposomes, microparticles, microcapsules, capsules, etc., and can be used for administration.

In particular embodiments, it may be desirable to apply topically to the area in need of treatment. In one embodiment, the heterodimeric protein (and/or other agent) is administered, for example, in the treatment of cancer, in a tumor microenvironment (e.g., in cells, molecules, extracellular matrix, and/or blood vessels surrounding tumor cells and/or feeder tumor cells, including, for example, tumor vasculature, tumor infiltrating lymphocytes, fibroblast reticulocytes, Endothelial Progenitor Cells (EPCs), cancer-associated fibroblasts, pericytes, other stromal cells, components of extracellular matrix (ECM), dendritic cells, antigen presenting cells, T cells, regulatory T cells, macrophages, neutrophils, and other immune cells located proximal to the tumor) or lymph nodes and/or targeted to the tumor microenvironment or lymph nodes. In various embodiments, the heterodimeric protein (and/or other agent) is administered intratumorally, for example in the treatment of cancer.

In various embodiments, the heterodimeric proteins of the invention allow for a dual effect that provides fewer side effects than seen with conventional immunotherapy (e.g., treatment with one or more of OPDIVO, KEYTRUDA, YERVOY, and TECENTRIQ). For example, the heterodimeric proteins of the invention reduce or prevent commonly observed immune-related adverse events affecting various tissues and organs, including skin, gastrointestinal tract, kidney, peripheral and central nervous system, liver, lymph nodes, eye, pancreas, and endocrine system; such as hypophysitis, colitis, hepatitis, pneumonia, rashes and rheumatism. In addition, the local administration (e.g., intratumoral) of the invention eliminates adverse events seen with conventional systemic administration (e.g., IV infusion) and is used in conjunction with conventional immunotherapy (e.g., treatment with one or more of OPDIVO, keytrda, YERVOY, and TECENTRIQ).

Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions (e.g., lyophilized compositions), which may be dissolved or suspended in a sterile injectable medium immediately prior to use. They may contain, for example, suspending or dispersing agents as known in the art.

The dosage and schedule of administration of any of the heterodimeric proteins (and/or other agents) described herein can depend on various parameters, including but not limited to the disease being treated, the general health of the subject, and the discretion of the attending physician. Any of the heterodimeric proteins described herein can be administered to a subject in need thereof prior to, concurrently with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) administration of other agents (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks). In various embodiments, any of the heterodimeric proteins and other agents described herein are administered at1 minute intervals, 10 minutes intervals, 30 minutes intervals, less than 1 hour intervals, 1 hour to 2 hours intervals, 2 hours to 3 hours intervals, 3 hours to 4 hours intervals, 4 hours to 5 hours intervals, 5 hours to 6 hours intervals, 6 hours to 7 hours intervals, 7 hours to 8 hours intervals, 8 hours to 9 hours intervals, 9 hours to 10 hours intervals, 10 hours to 11 hours intervals, 11 hours to 12 hours intervals, 1 day intervals, 2 days intervals, 3 days intervals, 4 days intervals, 5 days intervals, 6 days intervals, 1 week intervals, 2 weeks intervals, 3 weeks intervals, or 4 weeks intervals.

In various embodiments, the present invention relates to the co-administration of a heterodimeric protein that induces an innate immune response and another heterodimeric protein that induces an adaptive immune response. In such embodiments, the heterodimeric protein that induces an innate immune response can be administered prior to, concurrently with, or subsequent to the administration of the heterodimeric protein that induces an adaptive immune response. For example, the heterodimeric protein can be administered at1 minute intervals, 10 minute intervals, 30 minute intervals, less than 1 hour intervals, 1 hour to 2 hour intervals, 2 hour to 3 hour intervals, 3 hour to 4 hour intervals, 4 hour to 5 hour intervals, 5 hour to 6 hour intervals, 6 hour to 7 hour intervals, 7 hour to 8 hour intervals, 8 hour to 9 hour intervals, 9 hour to 10 hour intervals, 10 hour intervals to 11 hour intervals, 11 hour to 12 hour intervals, 1 day intervals, 2 days intervals, 3 days intervals, 4 days intervals, 5 days intervals, 6 days intervals, 1 week intervals, 2 weeks intervals, 3 weeks intervals, or 4 weeks intervals. In an exemplary embodiment, the heterodimeric protein that induces the innate immune response and the heterodimeric protein that induces the adaptive response are administered 1 week apart, or are administered alternately weekly (i.e., the heterodimeric protein that induces the adaptive immune response is administered one week after the heterodimeric protein that induces the innate immune response is administered, and so on).

The dosage of any of the heterodimeric proteins (and/or other agents) described herein can depend on several factors, including the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the subject to be treated. In addition, pharmacogenomic (the effect of genotype on the pharmacokinetics, pharmacodynamics, or efficacy profile of a therapeutic) information about a particular subject can affect the dosage used. In addition, the exact individual dosage may be adjusted somewhat depending on a variety of factors including the particular combination of agents administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the condition, and the anatomical location of the condition. Some variation in dosage is contemplated.

For administration of any of the heterodimeric proteins (and/or other agents) described herein by parenteral injection, the dose can be from about 0.1mg to about 250mg per day, from about 1mg to about 20mg per day, or from about 3mg to about 5mg per day. Generally, when administered orally or parenterally, the dose of any of the agents described herein can be from about 0.1mg to about 1500mg per day, or from about 0.5mg to about 10mg per day, or from about 0.5mg to about 5mg per day, or from about 200 to about 1,200mg per day (e.g., about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1,000mg, about 1,100mg, about 1,200mg per day).

In some embodiments, the heterodimeric proteins (and/or other agents) described herein are administered by parenteral injection at the following doses: from about 0.1mg to about 1500mg per treatment, or from about 0.5mg to about 10mg per treatment, or from about 0.5mg to about 5mg per treatment, or from about 200 to about 1,200mg per treatment (e.g., from about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1,000mg, about 1,100mg, about 1,200mg per treatment).

In some embodiments, a suitable dose of heterodimeric protein (and/or other agent) ranges from about 0.01mg/kg to about 100mg/kg of subject's body weight, or from about 0.01mg/kg to about 10mg/kg of subject's body weight, e.g., about 0.01mg/kg, about 0.02mg/kg, about 0.03mg/kg, about 0.04mg/kg, about 0.05mg/kg, about 0.06mg/kg, about 0.07mg/kg, about 0.08mg/kg, about 0.09mg/kg, about 0.1mg/kg, about 0.2mg/kg, about 0.3mg/kg, about 0.4mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.7mg/kg, about 0.8mg/kg, about 0.9mg/kg, about 1mg/kg, about 1.1mg/kg, about 1.2mg/kg, about 0.6mg/kg, about 0.7mg/kg, About 1.3mg/kg, about 1.4mg/kg, about 1.5mg/kg, about 1.6mg/kg, about 1.7mg/kg, about 1.8mg/kg, 1.9mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, including all values and ranges therebetween.

In another embodiment, delivery may be in vesicles, particularly Liposomes (see Langer,1990, Science 249: 1527-1533; Treat et al, in lipids in the Therapy of infection diseases and Cancer, Lopez-beer and Fidler (eds.), Liss, New York, pp.353-365 (1989)).

Any of the heterodimeric proteins (and/or other agents) described herein can be administered by controlled or sustained release means or by delivery devices known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Pat. nos. 3,845,770; 3,916,899; 3,536,809, respectively; 3,598,123, respectively; 4,008,719, respectively; 5,674,533, respectively; 5,059,595, respectively; 5,591,767, respectively; 5,120,548, respectively; 5,073,543, respectively; 5,639,476, respectively; 5,354,556, respectively; and 5,733,556, all of which are incorporated herein by reference in their entirety. Such dosage forms may be used in varying proportions to produce a desired release profile, for example, of hydroxypropylmethylcellulose, other polymeric matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof, for providing controlled or sustained release of one or more active ingredients. Controlled or sustained release of the active ingredient may be stimulated by a variety of conditions, including but not limited to changes in pH, changes in temperature, stimulation by light of the appropriate wavelength, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.

In another embodiment, polymeric materials may be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres, Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas,1983, J.Macromol. Sci.Rev. Macromol. chem.23: 61; see also Levy et al, 1985, Science 228: 190; During et al, 1989, Ann.Neurol.25: 351; Howard et al, 1989, J.Neurog.71: 105).

In another embodiment, a Controlled Release system may be placed near the target area to be treated, whereby only a fraction of the systemic dose is required (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol.2, pp.115-138 (1984)). Other controlled release systems discussed in the reviews by Langer,1990, Science 249: 1527-.

The administration of any of the heterodimeric proteins (and/or other agents) described herein can independently be 1 to 4 times per day or 1 to 4 times per month or 1 to 6 times per year or once every two, three, four or five years. Administration may last one day or one month, two months, three months, six months, one year, two years, three years, and may even be the lifetime of the subject.

The dosage regimen for any of the heterodimeric proteins (and/or other agents) described herein can be selected based on a variety of factors including the type, species, age, weight, sex, and medical condition of the subject; the severity of the condition to be treated; the route of administration; kidney or liver function of the subject; pharmacogenomic composition of individuals; and the specific compounds of the invention used. Any of the heterodimeric proteins (and/or other agents) described herein can be administered in a single daily dose, or the total daily dose can be administered in divided doses of two, three, or four times daily. In addition, any of the heterodimeric proteins (and/or other agents) described herein can be administered continuously, rather than intermittently, during a dosing regimen.

Cells and nucleic acids

In various embodiments, the invention provides an expression vector comprising a nucleic acid encoding a heterodimeric protein described herein (e.g., a heterodimeric protein comprising a first and a second polypeptide chain). In various embodiments, the expression vector comprises DNA or RNA. In various embodiments, the expression vector is a mammalian expression vector.

Both prokaryotic and eukaryotic vectors can be used to express heterodimeric proteins. Prokaryotic vectors include constructs based on E.coli (E.coli) sequences (see, e.g., Makrides, Microbiol Rev 1996, 60: 512-. Non-limiting examples of regulatory regions that can be used for expression in E.coli include lac, trp, lpp, phoA, recA, tac, T3, T7, and λ P_L. Non-limiting examples of prokaryotic expression vectors may include the lambda gt vector series such as lambda gt11(Huynh et al, in "DNA Cloning technologies, Vol.I: A Practical Approach," 1984, (D.Glover, ed.), pp.49-78, IRL Press, Oxford) and pET vector series (Studier et al, Methods Enzymol 1990,185: 60-89). However, prokaryotic host-vector systems are not capable of performing most of the post-translational processing of mammalian cells. Thus, eukaryotic-host vector systems may be particularly useful. Various regulatory regions can be used to express heterodimeric proteins in mammalian host cells. For example, the SV40 early and late promoters, the Cytomegalovirus (CMV) early promoter and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter may be used. Inducible promoters that can be used in mammalian cells include, but are not limited to, promoters associated with the metallothionein II gene, the glucocorticoid-responsive long terminal repeat (MMTV-LTR) of mouse mammary tumor virus, the interferon-beta gene, and the hsp70 gene (see Williams et al, Cancer Res 1989,49: 2735-42; and Taylor et al, Mol Cell Biol 1990,10: 165-75). A heat shock promoter or stress promoter may also be advantageous for driving expression of the fusion protein in a recombinant host cell.

In some embodiments, the expression vectors of the invention comprise a nucleic acid encoding at least a first and/or second polypeptide chain of a heterodimeric protein (and/or other agent), or a complement thereof, operably linked to an expression control region functional in a mammalian cell, or a complement thereof. The expression control region is capable of driving expression of an operably linked nucleic acid encoding a blocking and/or stimulatory agent such that the blocking and/or stimulatory agent is produced in a human cell transformed with the expression vector.

Expression control regions are regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, that affect the expression of an operably linked nucleic acid. The expression control region of the expression vectors of the invention is capable of expressing the operably linked coding nucleic acids in human cells. In one embodiment, the cell is a tumor cell. In another embodiment, the cell is a non-tumor cell. In one embodiment, the expression control region provides regulatable expression to the operably linked nucleic acid. The signal (sometimes referred to as a stimulus) can increase or decrease the expression of a nucleic acid operably linked to such an expression control region. Such expression control regions that increase expression in response to a signal are often referred to as inducible. Such expression control regions that decrease expression in response to a signal are often referred to as repressible. Generally, the amount of increase or decrease provided by these elements is directly proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.

In one embodiment, the present invention contemplates the use of an inducible promoter capable of transiently producing high levels of expression in response to a signal (cue). For example, when in the vicinity of a tumor cell, cells transformed with an expression vector comprising such a heterodimeric protein (and/or other agent) of an expression control sequence are induced to transiently produce high levels of the agent by exposing the transformed cells to an appropriate signal. Exemplary inducible expression control regions include those regions that comprise an inducible promoter that is stimulated by a signal (e.g., a small molecule compound). Specific examples can be found, for example, in U.S. patent nos. 5,989,910, 5,935,934, 6,015,709, and 6,004,941, which are all incorporated herein by reference in their entirety.

Expression control regions and locus control regions include full-length promoter sequences, such as native promoter and enhancer elements, as well as subsequences or polynucleotide variants that retain all or part of full-length or non-variant function. As used herein, the term "functional" and grammatical variants thereof, when used in reference to a nucleic acid sequence, subsequence or fragment, refers to a sequence having one or more functions of a native nucleic acid sequence (e.g., a non-variant or unmodified sequence).

As used herein, "operatively linked" refers to the physical juxtaposition of components, described as allowing them to function in their intended manner. In examples where the expression control element is operably linked to the nucleic acid, the relationship is such that the control element modulates expression of the nucleic acid. Typically, an expression control region that regulates transcription is juxtaposed near the 5' end (i.e., "upstream") of the transcribed nucleic acid. The expression control region may also be located at the 3' end of the transcribed sequence (i.e., "downstream") or within the transcript (e.g., in an intron). The expression control element can be located at a distance from the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to 5000, or more nucleotides from the nucleic acid). A specific example of an expression control element is a promoter, which is typically located 5' to the transcribed sequence. Another example of an expression control element is an enhancer, which may be located 5 'or 3' to, or within, a transcribed sequence.

Expression systems that function in human cells are well known in the art and include viral systems. Generally, a promoter functional in human cells is any DNA sequence capable of binding to mammalian RNA polymerase and initiating transcription of the coding sequence downstream (3') into mRNA. A promoter will have a transcriptional initiation region usually located near the 5' end of the coding sequence, and usually will have a TATA box located 25-30 base pairs upstream of the transcriptional initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct position. Promoters will also typically contain upstream promoter elements (enhancer elements), typically located within 100 to 200 base pairs upstream of the TATA box. Upstream promoter elements determine the rate of transcription initiation and can function in either direction. Particularly useful promoters are those from mammalian viral genes, since viral genes are generally highly expressed and have a wide host range. Examples include the SV40 early promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter, the herpes simplex virus promoter, and the CMV promoter.

Typically, the transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and, thus, flank the coding sequence along with the promoter element. The 3' end of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation. Examples of transcription terminators and polyadenylation signals include those derived from SV 40. Introns may also be included in the expression construct.

There are a variety of techniques available for introducing nucleic acids into living cells. Suitable techniques for transferring nucleic acids into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, polymer-based systems, DEAE-dextran, viral transduction, calcium phosphate precipitation, and the like. For in vivo gene transfer, a variety of techniques and agents may also be used, including liposomes; natural polymer-based delivery vehicles, such as chitosan and gelatin; viral vectors are also suitable for in vivo transduction. In some cases, it is desirable to provide targeting agents, such as antibodies or ligands specific for tumor cell surface membrane proteins. Where liposomes are employed, proteins that bind to cell surface membrane proteins associated with endocytosis may be used to target and/or facilitate uptake, e.g., capsid proteins or fragments thereof that are compatible with a particular cell type, antibodies to proteins that undergo internalization in circulation, proteins that target intracellular localization and prolong cellular half-life. Techniques for receptor-mediated endocytosis are described, for example, by Wu et al, J.biol.chem.262,4429-4432 (1987); and Wagner et al, Proc.Natl.Acad.Sci.USA 87,3410-3414 (1990).

Gene delivery agents, such as integration sequences, may also be used where appropriate. Many integration sequences are known in the art (see, e.g., Nunes-Duby et al, Nucleic Acids Res.26:391-406, 1998; Sadwoski, J.Bacteriol.,165:341-357, 1986; Bestor, Cell,122(3):322-325, 2005; Plastk et al, TIG 15:326-332, 1999; Kootstra et al, Ann.Rev.pharm.Toxicol.,43:413-439, 2003). These include recombinases and transposases. Examples include Cre (Sternberg and Hamilton, J.Mol.biol.,150: 467-. In addition, direct and targeted genetic integration strategies including CRISPR/CAS9, zinc fingers, TALENs, and meganuclease gene editing techniques can be used to insert nucleic acid sequences encoding the chimeric fusion proteins.

In one aspect, the expression vectors provided herein for expressing the heterodimeric protein (and/or other agent) are viral vectors. Many viral vectors are known to be useful in gene therapy (see, e.g., Lundstrom, Trends Biotechnol.,21: 117,122,2003. exemplary viral vectors include those selected from the group consisting of anti-virus (LV), Retrovirus (RV), Adenovirus (AV), adeno-associated virus (AAV), and alphavirus, although other viral vectors may also be used. For example, alphaviruses and adenoviruses, exemplary types of alphaviruses include Sindbis virus (Sindbis virus), Venezuelan Equine Encephalitis (VEE) virus, and Semliki Forest Virus (SFV), for in vitro use, viral vectors integrated into the host genome are suitable, in one embodiment, the invention provides a method of transducing a human cell in vivo comprising contacting a solid tumor in vivo with a viral vector of the invention.

In various embodiments, the present invention provides a host cell comprising an expression vector comprising a heterodimeric protein as described herein.

The expression vector may be introduced into a host cell to produce the heterodimeric protein of the invention. For example, the cells may be cultured in vitro or genetically engineered. Useful mammalian host cells include, but are not limited to, cells derived from humans, monkeys, and rodents (see, e.g., Kriegler in "Gene Transfer and Expression: A Laboratory Manual," 1990, New York, Freeman & Co.). These include monkey kidney cell lines transformed by SV40 (e.g., COS-7, ATCC CRL 1651); human embryonic kidney cell lines (e.g., 293-EBNA or 293 cells subcloned for growth in suspension culture, Graham et al, J Gen Virol 1977,36: 59); baby hamster kidney cells (e.g., BHK, ATCC CCL 10); chinese hamster ovary-cell-DHFR (e.g., CHO, Urlaub and Chasin, Proc Natl Acad Sci USA 1980,77: 4216); DG44 CHO cells, CHO-K1 cells, mouse testicular support cells (mouse sertoli cell) (Mather, Biol Reprod 1980,23: 243-; mouse fibroblasts (e.g., NIH-3T3), monkey kidney cells (e.g., CV1 ATCC CCL 70); vero cells (e.g., VERO-76, ATCC CRL-1587); human cervical cancer cells (e.g., HELA, ATCC CCL 2); canine kidney cells (e.g., MDCK, ATCC CCL 34); buffalo rat hepatocytes (e.g., BRL 3A, ATCC CRL 1442); human lung cells (e.g., W138, ATCC CCL 75); human hepatocytes (e.g., Hep G2, HB 8065); and mouse breast tumor cells (e.g., MMT 060562, ATCC CCL 51). Exemplary cancer cell types for expressing the fusion proteins described herein include mouse fibroblast cell line, NIH3T3, mouse Lewis lung cancer cell line, LLC, mouse mast cell tumor cell line, P815, mouse lymphoma cell line, EL4 and its ovalbumin transfectants, e.g7, mouse melanoma cell line, B16F10, mouse fibrosarcoma cell line, MC57 and human mouse lung cancer cell lines, SCLC # 2and SCLC # 7.

Host cells can be obtained from normal or affected subjects (including healthy humans, cancer patients, and patients with infectious diseases), private laboratory collections, public Culture collections (e.g., American Type Culture Collection), or commercial suppliers.

Cells that can be used to produce the heterodimeric proteins of the invention in vitro, ex vivo, and/or in vivo include, but are not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, particularly hematopoietic stem or progenitor cells (e.g., cells obtained from bone marrow), cord blood, peripheral blood, fetal liver, and the like. The choice of cell type depends on the type of tumor or infectious disease being treated or prevented and can be determined by one skilled in the art.

The production and purification of Fc-containing macromolecules (e.g., Fc fusion proteins) has become a standardized process with little variation between products. For example, many Fc-containing macromolecules are produced by Human Embryonic Kidney (HEK) cells (or variants thereof) or Chinese Hamster Ovary (CHO) cells (or variants thereof), or in some cases, by bacterial or synthetic methods. After production, Fc-containing macromolecules secreted by HEK or CHO cells were purified by binding to a protein a chromatography column and then "stripped" using various methods. Generally, purified Fc-containing macromolecules are stored in liquid form for a period of time, frozen for an extended period of time, or in some cases lyophilized. In various embodiments, the production of heterodimeric proteins contemplated herein can have unique characteristics compared to traditional Fc-containing macromolecules. In certain examples, heterodimeric proteins can be purified using specific chromatography resins or using chromatography methods that do not rely on protein a capture. In other embodiments, the heterodimeric protein in the oligomeric state or states may be purified and enriched for a particular oligomeric state using a particular method. Without being bound by theory, these methods may include treatment with a particular buffer including a particular salt concentration, pH, and additive composition. In other examples, such methods may include treatments that favor one oligomerization state over another. The heterodimeric proteins obtained herein can be additionally "stripped" using methods specified in the art. In some embodiments, the heterodimeric proteins are highly stable and able to tolerate a wide range of pH exposures (between pH 3-12), able to tolerate a large amount of freeze/thaw stress (greater than 3 freeze/thaw cycles), and able to tolerate prolonged incubation at high temperatures (more than 2 weeks at 40 degrees celsius). In other embodiments, it is shown that heterodimeric proteins remain intact with no signs of degradation, deamidation, etc. under these stress conditions.

Subjects and/or animals

In some embodiments, the subject and/or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or a non-human primate, e.g., monkey, chimpanzee, or baboon. In other embodiments, the subject and/or animal is a non-mammal, such as zebrafish. In some embodiments, the subject and/or animal may comprise fluorescently labeled cells (e.g., labeled with GFP). In some embodiments, the subject and/or animal is a transgenic animal comprising fluorescent cells.

In some embodiments, the subject and/or animal is a human. In some embodiments, the human is a young child. In other embodiments, the human is an adult. In other embodiments, the human is an elderly human. In other embodiments, the human may be referred to as a patient.

In certain embodiments, the age range of the human is about 0 month to about 6 months, about 6 to about 12 months old, about 6 to about 18 months old, about 18 to about 36 months old, about 1 to about 5 years old, about 5 to about 10 years old, about 10 to about 15 years old, about 15 to about 20 years old, about 20 to about 25 years old, about 25 to about 30 years old, about 30 to about 35 years old, about 35 to about 40 years old, about 40 to about 45 years old, about 45 to about 50 years old, about 50 to about 55 years old, about 55 to about 60 years old, about 60 to about 65 years old, about 65 to about 70 years old, about 70 to about 75 years old, about 75 to about 80 years old, about 80 to about 85 years old, about 85 to about 90 years old, about 90 to about 95 years old, or about 95 to about 100 years old.

In other embodiments, the subject is a non-human animal, and thus the invention relates to veterinary uses. In a particular embodiment, the non-human animal is a domestic pet. In another specific embodiment, the non-human animal is a livestock animal.

Reagent kit

The present invention provides kits that can simplify the administration of any of the agents described herein. Exemplary kits of the invention comprise a unit dosage form of any of the compositions described herein. In one embodiment, the unit dosage form is a container, e.g., a pre-filled syringe, which may be sterile, containing any of the agents described herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle. The kit may further comprise a label or printed instructions indicating the use of any of the agents described herein. The kit may also include a lid speculum (lid speculum), a local anesthetic, and a cleanser for the administration site. The kit may further comprise one or more additional agents as described herein. In one embodiment, a kit comprises a container containing an effective amount of a composition of the invention and an effective amount of another composition (such as those described herein).

Definition of

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the term "or" is to be understood as being inclusive and encompassing both "or" and "unless specified otherwise or apparent from the context.

Unless specifically stated or otherwise apparent from the context, as used herein, the term "about" should be understood to be within the normal tolerance (normal tolerance) in the art, e.g., within 2 standard deviations of the mean. About is understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value. All numerical values provided herein are modified by the term "about," unless the context clearly dictates otherwise.

The specified ranges are to be understood as any value between the specified ranges and at the ends of the specified ranges. By way of example, a range between 1 and 5 includes 1,2, 3, 4, and 5; the range between 1 and 10 includes 1,2, 3, 4,5, 6, 7, 8, 9 and 10; ranges between 1 and 100 include 1,2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although other probes, compositions, methods, and kits similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Any aspect or embodiment described herein can be combined with any other aspect or embodiment disclosed herein.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

Examples

Example 1: construction and characterization of IL-6R-Fc-IL-35 heterodimer proteins

Heterodimeric proteins comprising IL 6receptor (IL6R) linked to IL-35 via a charge polarizing core domain were constructed (see, e.g., fig. 1). In particular, heterodimeric proteins comprise two polypeptide chains. The first polypeptide chain comprises IL6R subunit Gp130 linked to IL-35 subunit IL12 α through a charge polarizing core domain. The second polypeptide chain comprises IL6R subunit IL6R α linked to IL-35 subunit IL27 β through a charge polarizing core domain. IL-6R-Fc-IL-35 heterodimer proteins were expressed in mammalian cells by double transient transfection with IL6RA-Beta-IL27 Beta and gp130-Alpha-IL12 Alpha constructs. Coomassie blue staining indicated the presence of expressed proteins, which were confirmed using anti-human IgG western blots to include proteins corresponding to the approximate molecular weights of the Alpha and Beta constructs (see, e.g., figure 2).

Further analysis of the purified protein under non-reducing, reducing and deglycosylating conditions provided further evidence for the assembly of the heterodimeric constructs. Specifically, staining by Western blotting of anti-human Fc and anti-human IL-6R demonstrated the presence of a single high molecular weight band corresponding to the approximate molecular weight of the alpha/beta heterodimer comprising IL-6R-Fc-IL-35. The heterodimer can decompose under reducing conditions into constitutive alpha and beta chains that exhibit apparent molecular weights that are higher than predicted molecular weights based solely on amino acid content. This is expected since glycosylation sites are known to be present, and deglycosylation of the alpha and beta chains results in individual proteins that appear at their predicted molecular weights by protein analysis (see, e.g., FIG. 3).

In addition, the IL-6R-Fc-IL-35 construct was further analyzed by Blue Native PAGE since the presence of SDS disrupts any charge interactions that contribute to protein multimerization. These data indicate that most of the secreted proteins (estimated at 60%) represent alpha/beta heterodimers (FIG. 3). The concentration of purified IL-6R-Fc-IL-35 protein was confirmed by spectrophotometric analysis (see, e.g., FIG. 4).

IL-6R-Fc-IL-35 heterodimer protein was subjected to Size Exclusion Chromatography (SEC) after double transfection of gp130-Fc (alpha) -IL12A and IL6RA-Fc (beta) -IL27B constructs in CHO cells and subsequent purification of the secreted protein using protein A. One single peak appearing by SEC indicates that only a single species of heterodimeric protein may be present, as expected due to the use of charge-polarized linking domains (Fc-alpha and Fc-beta) in both constructs (FIG. 5).

To confirm that the assembled IL-6R-Fc-IL-35 heterodimer retains the ability to bind to a cognate ligand (e.g., IL-6) and be recognized by each of the protein components directed against the assembled heterodimer (i.e., IL-6RA, gp130, IL27 β/EBI3, and IL12 α), a series of ELISA assays were performed to demonstrate the specific presence of the IL-6R-Fc-IL-35 heterodimer. In fig. 6 to 15, a schematic of the ELISA assay is shown at the top of each figure. In the schematic, the capture and detection strategies are illustrated. In each case by capture with recombinant IL-6 and detection with anti-IL-27B/EBI 3 (FIG. 6), capture with recombinant IL-6 and detection with anti-IL-6 RA (FIG. 7), capture with anti-human gp130 and detection with anti-IL 27B/EBI3 (FIG. 8), capture with anti-human gp130 and detection with anti-human IL-6RA (FIG. 9), capture with anti-IL-6 RA and detection with anti-IL 27B/EBI3 (FIG. 10), capture with anti-IL-6 RA and detection with anti-IL-6 RA (FIG. 11), capture with anti-human p35 and detection with anti-IL-27B/EBI 3 (FIG. 12), capture with anti-human p35 and detection with anti-human IL-6RA (FIG. 13), capture with anti-human p35 and detection with anti-IL 27B/EBI3 (FIG. 14), and capture with anti-IL 27B/EBI 3and detection with anti-human IL-6RA (FIG. 15), to observe the presence of IL-6R-Fc-IL-35 heterodimers.

SEQ ID NO:16 provides the sequence of an exemplary charge polarizing core domain (negative-positive, i.e., "alpha core domain"), and SEQ ID NO: an exemplary alpha core domain comprising a knob and hole mutation is provided in 24.

SEQ ID NO: exemplary charge polarizing core domain (positive-negative, or "beta core domain") sequences are provided in fig. 17, and SEQ ID NO: an exemplary beta core domain comprising a knob and hole mutation is provided in 25.

The sequences of the components of exemplary polypeptide chains are shown below: SEQ ID NO:18 is Gp130ECD (type 1), SEQ ID NO:19 is IL-6RA ECD (type 1), SEQ ID NO: 20 is IL-12a (type 2, first part of IL-35), SEQ ID NO: 21 is IL-27b (type 2, second part of IL-35).

An exemplary Gp130-Alpha-IL12A chain has the amino acid sequence of SEQ ID NO: 22, and an exemplary IL6RA-Beta-IL27B chain has the sequence shown in SEQ ID NO: 23, or a sequence shown in seq id no.

In an alternative embodiment, the IL-6R-Fc-IL-35 heterodimeric protein may comprise IL6RA-Alpha-IL12a chain (SEQ ID NO: 34) and Gp130-Beta-IL27b chain (SEQ ID NO: 35).

Example 2: further characterization of IL-6R-Fc-IL-35 heterodimeric proteins

Size Exclusion Chromatography (SEC) was performed on IL-6R-Fc-IL-35 heterodimer protein. The single peak appearing by SEC at an absorption wavelength of 210nm indicates that there may be only a single species of protein, as expected due to the use of charge-polarized linker domains (Fc-alpha and Fc-beta) in both constructs (FIG. 16A). Interestingly, SEC with an absorption wavelength of 280nm showed a second, lower molecular weight band (FIG. 16B).

The IL-6R-Fc-IL-35 heterodimeric protein was then used in an IL-6SINK assay. Here, IL-6R-Fc-IL-35 heterodimeric proteins were tested for their ability to block IL 6. Cultures of DS-1 cells (B cell lines that survive dependent on exogenous IL 6) were incubated with IL-6R-Fc-IL-35 heterodimeric protein in the presence of exogenous IL 6. DS-1 cells, when not exposed to IL6, result in cell death. Therefore, these experiments were performed to determine whether IL-6R-Fc-IL-35 heterodimeric proteins could block IL6 and cause DS-1 cell death.

DS-1 cells were cultured for 24 hours in the presence of an increased molar ratio of IL-6R-Fc-IL-35 to IL-6. Cell death was measured by caspase 3/7 activity (by luciferase readout).

FIG. 17 shows that IL-6R-Fc-IL-35 heterodimer proteins (identified as Lot '00 and Lot' 48) are capable of inducing cell death in DS-1 cells. In fact, depending on the batch used, the heterodimeric protein showed an IL-6 blocking capacity 7 to 281 times higher than that of truzumab (Tocilizumab, an anti-human IL-6receptor monoclonal antibody that blocks DS-1 binding to IL 6), depending on the batch used.

IL-6R-Fc-IL-35 heterodimeric proteins were then tested for function. IL-35 has been reported to induce an atypical regulatory phenotype in CD4T cells characterized by little or no production of FoxP3 that is associated with IL-35 production. In addition, IL-35 is known to shut down the production of TGF-. beta.and IL-10.

Here, magnetically enriched human naive CD4T cells were isolated from a single donor and activated with α CD3/α CD28 beads and cultured for 5 days in the presence of indicated reagents (as shown in FIG. 18). Total mRNA was isolated and RT-qPCR was performed.

FIG. 18 shows that IL-6R-Fc-IL-35 heterodimer protein (identified as HdA' 00) induces production of IL-35 (a dimer of EB13 and IL 12A). Surprisingly, heterodimeric proteins also increased the production of FoxP 3. In addition, the IL-6R-Fc-IL-35 heterodimer protein allows cell proliferation, unlike other treated agents. Although IL-35 is known to shut down the production of TGF-. beta.and IL-10, the IL-6R-Fc-IL-35 heterodimer protein results in detectable levels of IL-10 production (2-fold higher than controls). Finally, the heterodimeric protein had no significant effect on IL-6 production.

Example 3: construction and characterization of IL-21R-Fc-IL-35 heterodimer proteins

Constructs encoding IL21r-Alpha-IL12a chain and IL2rg-Beta-IL27B chain were double transfected into CHO cells, and the secreted protein was purified using protein A. When IL21R-Alpha-IL12a chain and IL2rg-Beta-IL27B chain (in cells or in vitro) combined, they formed a heterodimeric protein, referred to herein as IL-21R-Fc-IL-35. (FIG. 19A).

Western blots were performed on the expressed heterodimeric proteins. These revealed bands corresponding to predicted molecular weights of IL21r-Alpha-IL12a chain and IL2rg-Beta-IL27B chain under denaturing and deglycosylation conditions (FIG. 19B).

Size Exclusion Chromatography (SEC) was performed with IL-21R-Fc-IL-35 heterodimer protein. The single peak appearing by SEC indicates that there may be only a single class of protein, as expected due to the use of charge-polarized linker domains (Fc-alpha and Fc-beta) in both constructs (FIG. 20).

In these experiments, SEQ ID NO:16 provides exemplary charge polarizing core domain (negative-positive, i.e., "alpha core domain") sequences, while SEQ ID NOs: exemplary alpha core domains including a knob and hole mutation are provided in fig. 9. SEQ ID NO: exemplary charge polarizing core domain (positive-negative, or "beta core domain") sequences are provided in fig. 17, while SEQ ID NO: an exemplary beta core domain containing a knob and hole mutation is provided in 25.

The sequences of the components of the exemplary polypeptide chains used in this example are shown below: SEQ ID NO: 26 is the extracellular domain of IL-21r, SEQ ID NO: 27 is the extracellular domain of IL2RG, SEQ ID NO: 20 is IL-12a, SEQ ID NO: 21 is IL-27 b. An exemplary IL21r-Alpha-IL12a chain has SEQ ID NO: 28, and an exemplary IL2rg-beta-IL27B chain has the sequence shown in SEQ ID NO: 29, or a sequence shown in seq id no.

In an alternative embodiment, the IL-21R-Fc-IL-35 heterodimeric protein may comprise IL2rg-Alpha-IL27B chain (SEQ ID NO: 36) and IL21R-Beta-IL12a chain (SEQ ID NO: 37).

Example 4: construction and characterization of IFN γ R-Fc-IL-35 heterodimer proteins

Constructs encoding IFNgR-Alpha-IL12a chain and IFNGR2-Beta-IL27B chain were double transfected into CHO cells, and then the secreted protein was purified using protein A. When IFNgR-Alpha-IL12a chain and IFNGR2-Beta-IL27B chain (in cells or in vitro) combined, they formed a heterodimeric protein, referred to herein as IFN gamma R-Fc-IL-35.

Western blots were performed on IFN γ R-Fc-IL-35 heterodimer proteins comprising IFNgR-Alpha-IL12a chain and hIFNGR2-Beta-IL27B chain, and the antibodies used for detection were as indicated below each blot. These revealed bands corresponding to predicted molecular weights of IFNgR-Alpha-IL12a and hIFNGR2-Beta-IL27B under denaturing and deglycosylation conditions (FIG. 21). Bands highlighted in yellow are nonspecific bands.

Size Exclusion Chromatography (SEC) was performed on the IFN γ R-Fc-IL-35 heterodimer protein. The single peak appearing by SEC indicates that there may be only a single class of protein, as expected due to the use of charge-polarized linker domains (Fc-alpha and Fc-beta) in both constructs (FIG. 22).

In these experiments, SEQ ID NO: exemplary charge polarizing core domain (negative-positive, i.e., "alpha core domain") sequences are provided in SEQ ID NO: an exemplary alpha core domain comprising a knob and hole mutation is provided in 24. SEQ ID NO: exemplary charge polarizing core domain (positive-negative, or "beta core domain") sequences are provided in fig. 17, and SEQ ID NO: an exemplary beta core domain containing a knob and hole mutation is provided in 25.

The sequences of the components of the exemplary polypeptide chains used in this example are shown below: SEQ ID NO: 30 is the extracellular domain of IFNgR, SEQ ID NO: 31 is the extracellular domain of IFNGR2, SEQ ID NO: 20 is IL-12a, and SEQ ID NO: 21 is IL-27 b. An exemplary IFNgR-Alpha-IL12a chain has the amino acid sequence of SEQ ID NO: 32, and an exemplary IFNGR2-Beta-IL27B chain has the sequence shown in SEQ ID NO: 33, or a sequence shown in seq id no.

In an alternative embodiment, the IFN γ R-Fc-IL-35 heterodimeric protein may comprise IFNGR2-Alpha-IL27B chain (SEQ ID NO: 39) and IFNgR-Beta-IL12a chain (SEQ ID NO: 38).

Equivalent scheme

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed by the scope of the appended claims.

Is incorporated by reference

All patents and publications cited herein are incorporated by reference in their entirety.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are for organizational purposes only and are not intended to limit the disclosure in any way. The contents of any individual chapter may be equally applicable to all chapters.

Sequence listing

<110> Santakg laboratory Co., Ltd (Shattuck Lab Inc.)

T.Schreiber (Schreiber, Taylor)

G From (Fromm, George)

S Darcy tile (De Silva, Suresh)

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<151> 2018-06-21

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Val Leu Thr Val Leu His Gln Asp Trp Leu Ser Gly Lys Glu Tyr Lys

100 105 110

Cys Lys Val Ser Ser Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

115 120 125

Ser Asn Ala Thr Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

130 135 140

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

145 150 155 160

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

165 170 175

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

180 185 190

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

195 200 205

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu

210 215 220

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Asp

225 230 235 240

Glu Gly Gly Glu Asp Gly Ser Gly Ser

245

<210> 17

<211> 249

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic sequence

<400> 17

Gly Ser Gly Ser Asp Glu Gly Gly Glu Asp Gly Ser Lys Tyr Gly Pro

1 5 10 15

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

20 25 30

Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr

35 40 45

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

50 55 60

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

65 70 75 80

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

85 90 95

Val Leu Thr Val Leu His Gln Asp Trp Leu Ser Gly Lys Glu Tyr Lys

100 105 110

Cys Lys Val Ser Ser Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

115 120 125

Ser Asn Ala Thr Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

130 135 140

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

145 150 155 160

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

165 170 175

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

180 185 190

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

195 200 205

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu

210 215 220

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Arg

225 230 235 240

Lys Gly Gly Lys Arg Gly Ser Gly Ser

245

<210> 18

<211> 597

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic sequence

<400> 18

Glu Leu Leu Asp Pro Cys Gly Tyr Ile Ser Pro Glu Ser Pro Val Val

1 5 10 15

Gln Leu His Ser Asn Phe Thr Ala Val Cys Val Leu Lys Glu Lys Cys

20 25 30

Met Asp Tyr Phe His Val Asn Ala Asn Tyr Ile Val Trp Lys Thr Asn

35 40 45

His Phe Thr Ile Pro Lys Glu Gln Tyr Thr Ile Ile Asn Arg Thr Ala

50 55 60

Ser Ser Val Thr Phe Thr Asp Ile Ala Ser Leu Asn Ile Gln Leu Thr

65 70 75 80

Cys Asn Ile Leu Thr Phe Gly Gln Leu Glu Gln Asn Val Tyr Gly Ile

85 90 95

Thr Ile Ile Ser Gly Leu Pro Pro Glu Lys Pro Lys Asn Leu Ser Cys

100 105 110

Ile Val Asn Glu Gly Lys Lys Met Arg Cys Glu Trp Asp Gly Gly Arg

115 120 125

Glu Thr His Leu Glu Thr Asn Phe Thr Leu Lys Ser Glu Trp Ala Thr

130 135 140

His Lys Phe Ala Asp Cys Lys Ala Lys Arg Asp Thr Pro Thr Ser Cys

145 150 155 160

Thr Val Asp Tyr Ser Thr Val Tyr Phe Val Asn Ile Glu Val Trp Val

165 170 175

Glu Ala Glu Asn Ala Leu Gly Lys Val Thr Ser Asp His Ile Asn Phe

180 185 190

Asp Pro Val Tyr Lys Val Lys Pro Asn Pro Pro His Asn Leu Ser Val

195 200 205

Ile Asn Ser Glu Glu Leu Ser Ser Ile Leu Lys Leu Thr Trp Thr Asn

210 215 220

Pro Ser Ile Lys Ser Val Ile Ile Leu Lys Tyr Asn Ile Gln Tyr Arg

225 230 235 240

Thr Lys Asp Ala Ser Thr Trp Ser Gln Ile Pro Pro Glu Asp Thr Ala

245 250 255

Ser Thr Arg Ser Ser Phe Thr Val Gln Asp Leu Lys Pro Phe Thr Glu

260 265 270

Tyr Val Phe Arg Ile Arg Cys Met Lys Glu Asp Gly Lys Gly Tyr Trp

275 280 285

Ser Asp Trp Ser Glu Glu Ala Ser Gly Ile Thr Tyr Glu Asp Arg Pro

290 295 300

Ser Lys Ala Pro Ser Phe Trp Tyr Lys Ile Asp Pro Ser His Thr Gln

305 310 315 320

Gly Tyr Arg Thr Val Gln Leu Val Trp Lys Thr Leu Pro Pro Phe Glu

325 330 335

Ala Asn Gly Lys Ile Leu Asp Tyr Glu Val Thr Leu Thr Arg Trp Lys

340 345 350

Ser His Leu Gln Asn Tyr Thr Val Asn Ala Thr Lys Leu Thr Val Asn

355 360 365

Leu Thr Asn Asp Arg Tyr Leu Ala Thr Leu Thr Val Arg Asn Leu Val

370 375 380

Gly Lys Ser Asp Ala Ala Val Leu Thr Ile Pro Ala Cys Asp Phe Gln

385 390 395 400

Ala Thr His Pro Val Met Asp Leu Lys Ala Phe Pro Lys Asp Asn Met

405 410 415

Leu Trp Val Glu Trp Thr Thr Pro Arg Glu Ser Val Lys Lys Tyr Ile

420 425 430

Leu Glu Trp Cys Val Leu Ser Asp Lys Ala Pro Cys Ile Thr Asp Trp

435 440 445

Gln Gln Glu Asp Gly Thr Val His Arg Thr Tyr Leu Arg Gly Asn Leu

450 455 460

Ala Glu Ser Lys Cys Tyr Leu Ile Thr Val Thr Pro Val Tyr Ala Asp

465 470 475 480

Gly Pro Gly Ser Pro Glu Ser Ile Lys Ala Tyr Leu Lys Gln Ala Pro

485 490 495

Pro Ser Lys Gly Pro Thr Val Arg Thr Lys Lys Val Gly Lys Asn Glu

500 505 510

Ala Val Leu Glu Trp Asp Gln Leu Pro Val Asp Val Gln Asn Gly Phe

515 520 525

Ile Arg Asn Tyr Thr Ile Phe Tyr Arg Thr Ile Ile Gly Asn Glu Thr

530 535 540

Ala Val Asn Val Asp Ser Ser His Thr Glu Tyr Thr Leu Ser Ser Leu

545 550 555 560

Thr Ser Asp Thr Leu Tyr Met Val Arg Met Ala Ala Tyr Thr Asp Glu

565 570 575

Gly Gly Lys Asp Gly Pro Glu Phe Thr Phe Thr Thr Pro Lys Phe Ala

580 585 590

Gln Gly Glu Ile Glu

595

<210> 19

<211> 346

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic sequence

<400> 19

Leu Ala Pro Arg Arg Cys Pro Ala Gln Glu Val Ala Arg Gly Val Leu

1 5 10 15

Thr Ser Leu Pro Gly Asp Ser Val Thr Leu Thr Cys Pro Gly Val Glu

20 25 30

Pro Glu Asp Asn Ala Thr Val His Trp Val Leu Arg Lys Pro Ala Ala

35 40 45

Gly Ser His Pro Ser Arg Trp Ala Gly Met Gly Arg Arg Leu Leu Leu

50 55 60

Arg Ser Val Gln Leu His Asp Ser Gly Asn Tyr Ser Cys Tyr Arg Ala

65 70 75 80

Gly Arg Pro Ala Gly Thr Val His Leu Leu Val Asp Val Pro Pro Glu

85 90 95

Glu Pro Gln Leu Ser Cys Phe Arg Lys Ser Pro Leu Ser Asn Val Val

100 105 110

Cys Glu Trp Gly Pro Arg Ser Thr Pro Ser Leu Thr Thr Lys Ala Val

115 120 125

Leu Leu Val Arg Lys Phe Gln Asn Ser Pro Ala Glu Asp Phe Gln Glu

130 135 140

Pro Cys Gln Tyr Ser Gln Glu Ser Gln Lys Phe Ser Cys Gln Leu Ala

145 150 155 160

Val Pro Glu Gly Asp Ser Ser Phe Tyr Ile Val Ser Met Cys Val Ala

165 170 175

Ser Ser Val Gly Ser Lys Phe Ser Lys Thr Gln Thr Phe Gln Gly Cys

180 185 190

Gly Ile Leu Gln Pro Asp Pro Pro Ala Asn Ile Thr Val Thr Ala Val

195 200 205

Ala Arg Asn Pro Arg Trp Leu Ser Val Thr Trp Gln Asp Pro His Ser

210 215 220

Trp Asn Ser Ser Phe Tyr Arg Leu Arg Phe Glu Leu Arg Tyr Arg Ala

225 230 235 240

Glu Arg Ser Lys Thr Phe Thr Thr Trp Met Val Lys Asp Leu Gln His

245 250 255

His Cys Val Ile His Asp Ala Trp Ser Gly Leu Arg His Val Val Gln

260 265 270

Leu Arg Ala Gln Glu Glu Phe Gly Gln Gly Glu Trp Ser Glu Trp Ser

275 280 285

Pro Glu Ala Met Gly Thr Pro Trp Thr Glu Ser Arg Ser Pro Pro Ala

290 295 300

Glu Asn Glu Val Ser Thr Pro Met Gln Ala Leu Thr Thr Asn Lys Asp

305 310 315 320

Asp Asp Asn Ile Leu Phe Arg Asp Ser Ala Asn Ala Thr Ser Leu Pro

325 330 335

Val Gln Asp Ser Ser Ser Val Pro Leu Pro

340 345

<210> 20

<211> 197

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic sequence

<400> 20

Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu

1 5 10 15

His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys

20 25 30

Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp

35 40 45

His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu

50 55 60

Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr

65 70 75 80

Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe

85 90 95

Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr

100 105 110

Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys

115 120 125

Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu

130 135 140

Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser

145 150 155 160

Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu

165 170 175

Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser

180 185 190

Tyr Leu Asn Ala Ser

195

<210> 21

<211> 209

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic sequence

<400> 21

Arg Lys Gly Pro Pro Ala Ala Leu Thr Leu Pro Arg Val Gln Cys Arg

1 5 10 15

Ala Ser Arg Tyr Pro Ile Ala Val Asp Cys Ser Trp Thr Leu Pro Pro

20 25 30

Ala Pro Asn Ser Thr Ser Pro Val Ser Phe Ile Ala Thr Tyr Arg Leu

35 40 45

Gly Met Ala Ala Arg Gly His Ser Trp Pro Cys Leu Gln Gln Thr Pro

50 55 60

Thr Ser Thr Ser Cys Thr Ile Thr Asp Val Gln Leu Phe Ser Met Ala

65 70 75 80

Pro Tyr Val Leu Asn Val Thr Ala Val His Pro Trp Gly Ser Ser Ser

85 90 95

Ser Phe Val Pro Phe Ile Thr Glu His Ile Ile Lys Pro Asp Pro Pro

100 105 110

Glu Gly Val Arg Leu Ser Pro Leu Ala Glu Arg Gln Leu Gln Val Gln

115 120 125

Trp Glu Pro Pro Gly Ser Trp Pro Phe Pro Glu Ile Phe Ser Leu Lys

130 135 140

Tyr Trp Ile Arg Tyr Lys Arg Gln Gly Ala Ala Arg Phe His Arg Val

145 150 155 160

Gly Pro Ile Glu Ala Thr Ser Phe Ile Leu Arg Ala Val Arg Pro Arg

165 170 175

Ala Arg Tyr Tyr Val Gln Val Ala Ala Gln Asp Leu Thr Asp Tyr Gly

180 185 190

Glu Leu Ser Asp Trp Ser Leu Pro Ala Thr Ala Thr Met Ser Leu Gly

195 200 205

Lys

<210> 22

<211> 1043

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic sequence

<400> 22

Glu Leu Leu Asp Pro Cys Gly Tyr Ile Ser Pro Glu Ser Pro Val Val

1 5 10 15

Gln Leu His Ser Asn Phe Thr Ala Val Cys Val Leu Lys Glu Lys Cys

20 25 30

Met Asp Tyr Phe His Val Asn Ala Asn Tyr Ile Val Trp Lys Thr Asn

35 40 45

His Phe Thr Ile Pro Lys Glu Gln Tyr Thr Ile Ile Asn Arg Thr Ala

50 55 60

Ser Ser Val Thr Phe Thr Asp Ile Ala Ser Leu Asn Ile Gln Leu Thr

65 70 75 80

Cys Asn Ile Leu Thr Phe Gly Gln Leu Glu Gln Asn Val Tyr Gly Ile

85 90 95

Thr Ile Ile Ser Gly Leu Pro Pro Glu Lys Pro Lys Asn Leu Ser Cys

100 105 110

Ile Val Asn Glu Gly Lys Lys Met Arg Cys Glu Trp Asp Gly Gly Arg

115 120 125

Glu Thr His Leu Glu Thr Asn Phe Thr Leu Lys Ser Glu Trp Ala Thr

130 135 140

His Lys Phe Ala Asp Cys Lys Ala Lys Arg Asp Thr Pro Thr Ser Cys

145 150 155 160

Thr Val Asp Tyr Ser Thr Val Tyr Phe Val Asn Ile Glu Val Trp Val

165 170 175

Glu Ala Glu Asn Ala Leu Gly Lys Val Thr Ser Asp His Ile Asn Phe

180 185 190

Asp Pro Val Tyr Lys Val Lys Pro Asn Pro Pro His Asn Leu Ser Val

195 200 205

Ile Asn Ser Glu Glu Leu Ser Ser Ile Leu Lys Leu Thr Trp Thr Asn

210 215 220

Pro Ser Ile Lys Ser Val Ile Ile Leu Lys Tyr Asn Ile Gln Tyr Arg

225 230 235 240

Thr Lys Asp Ala Ser Thr Trp Ser Gln Ile Pro Pro Glu Asp Thr Ala

245 250 255

Ser Thr Arg Ser Ser Phe Thr Val Gln Asp Leu Lys Pro Phe Thr Glu

260 265 270

Tyr Val Phe Arg Ile Arg Cys Met Lys Glu Asp Gly Lys Gly Tyr Trp

275 280 285

Ser Asp Trp Ser Glu Glu Ala Ser Gly Ile Thr Tyr Glu Asp Arg Pro

290 295 300

Ser Lys Ala Pro Ser Phe Trp Tyr Lys Ile Asp Pro Ser His Thr Gln

305 310 315 320

Gly Tyr Arg Thr Val Gln Leu Val Trp Lys Thr Leu Pro Pro Phe Glu

325 330 335

Ala Asn Gly Lys Ile Leu Asp Tyr Glu Val Thr Leu Thr Arg Trp Lys

340 345 350

Ser His Leu Gln Asn Tyr Thr Val Asn Ala Thr Lys Leu Thr Val Asn

355 360 365

Leu Thr Asn Asp Arg Tyr Leu Ala Thr Leu Thr Val Arg Asn Leu Val

370 375 380

Gly Lys Ser Asp Ala Ala Val Leu Thr Ile Pro Ala Cys Asp Phe Gln

385 390 395 400

Ala Thr His Pro Val Met Asp Leu Lys Ala Phe Pro Lys Asp Asn Met

405 410 415

Leu Trp Val Glu Trp Thr Thr Pro Arg Glu Ser Val Lys Lys Tyr Ile

420 425 430

Leu Glu Trp Cys Val Leu Ser Asp Lys Ala Pro Cys Ile Thr Asp Trp

435 440 445

Gln Gln Glu Asp Gly Thr Val His Arg Thr Tyr Leu Arg Gly Asn Leu

450 455 460

Ala Glu Ser Lys Cys Tyr Leu Ile Thr Val Thr Pro Val Tyr Ala Asp

465 470 475 480

Gly Pro Gly Ser Pro Glu Ser Ile Lys Ala Tyr Leu Lys Gln Ala Pro

485 490 495

Pro Ser Lys Gly Pro Thr Val Arg Thr Lys Lys Val Gly Lys Asn Glu

500 505 510

Ala Val Leu Glu Trp Asp Gln Leu Pro Val Asp Val Gln Asn Gly Phe

515 520 525

Ile Arg Asn Tyr Thr Ile Phe Tyr Arg Thr Ile Ile Gly Asn Glu Thr

530 535 540

Ala Val Asn Val Asp Ser Ser His Thr Glu Tyr Thr Leu Ser Ser Leu

545 550 555 560

Thr Ser Asp Thr Leu Tyr Met Val Arg Met Ala Ala Tyr Thr Asp Glu

565 570 575

Gly Gly Lys Asp Gly Pro Glu Phe Thr Phe Thr Thr Pro Lys Phe Ala

580 585 590

Gln Gly Glu Ile Glu Gly Ser Gly Ser Arg Lys Gly Gly Lys Arg Gly

595 600 605

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu

610 615 620

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu

625 630 635 640

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

645 650 655

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

660 665 670

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

675 680 685

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Ser

690 695 700

Gly Lys Glu Tyr Lys Cys Lys Val Ser Ser Lys Gly Leu Pro Ser Ser

705 710 715 720

Ile Glu Lys Thr Ile Ser Asn Ala Thr Gly Gln Pro Arg Glu Pro Gln

725 730 735

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

740 745 750

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

755 760 765

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

770 775 780

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

785 790 795 800

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

805 810 815

Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

820 825 830

Ser Leu Gly Lys Asp Glu Gly Gly Glu Asp Gly Ser Gly Ser Arg Asn

835 840 845

Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His His

850 855 860

Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg

865 870 875 880

Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu

885 890 895

Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu

900 905 910

Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe

915 920 925

Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met

930 935 940

Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val

945 950 955 960

Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln

965 970 975

Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln

980 985 990

Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu

995 1000 1005

Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu

1010 1015 1020

His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser

1025 1030 1035

Tyr Leu Asn Ala Ser

1040

<210> 23

<211> 804

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic sequence

<400> 23

Leu Ala Pro Arg Arg Cys Pro Ala Gln Glu Val Ala Arg Gly Val Leu

1 5 10 15

Thr Ser Leu Pro Gly Asp Ser Val Thr Leu Thr Cys Pro Gly Val Glu

20 25 30

Pro Glu Asp Asn Ala Thr Val His Trp Val Leu Arg Lys Pro Ala Ala

35 40 45

Gly Ser His Pro Ser Arg Trp Ala Gly Met Gly Arg Arg Leu Leu Leu

50 55 60

Arg Ser Val Gln Leu His Asp Ser Gly Asn Tyr Ser Cys Tyr Arg Ala

65 70 75 80

Gly Arg Pro Ala Gly Thr Val His Leu Leu Val Asp Val Pro Pro Glu

85 90 95

Glu Pro Gln Leu Ser Cys Phe Arg Lys Ser Pro Leu Ser Asn Val Val

100 105 110

Cys Glu Trp Gly Pro Arg Ser Thr Pro Ser Leu Thr Thr Lys Ala Val

115 120 125

Leu Leu Val Arg Lys Phe Gln Asn Ser Pro Ala Glu Asp Phe Gln Glu

130 135 140

Pro Cys Gln Tyr Ser Gln Glu Ser Gln Lys Phe Ser Cys Gln Leu Ala

145 150 155 160

Val Pro Glu Gly Asp Ser Ser Phe Tyr Ile Val Ser Met Cys Val Ala

165 170 175

Ser Ser Val Gly Ser Lys Phe Ser Lys Thr Gln Thr Phe Gln Gly Cys

180 185 190

Gly Ile Leu Gln Pro Asp Pro Pro Ala Asn Ile Thr Val Thr Ala Val

195 200 205

Ala Arg Asn Pro Arg Trp Leu Ser Val Thr Trp Gln Asp Pro His Ser

210 215 220

Trp Asn Ser Ser Phe Tyr Arg Leu Arg Phe Glu Leu Arg Tyr Arg Ala

225 230 235 240

Glu Arg Ser Lys Thr Phe Thr Thr Trp Met Val Lys Asp Leu Gln His

245 250 255

His Cys Val Ile His Asp Ala Trp Ser Gly Leu Arg His Val Val Gln

260 265 270

Leu Arg Ala Gln Glu Glu Phe Gly Gln Gly Glu Trp Ser Glu Trp Ser

275 280 285

Pro Glu Ala Met Gly Thr Pro Trp Thr Glu Ser Arg Ser Pro Pro Ala

290 295 300

Glu Asn Glu Val Ser Thr Pro Met Gln Ala Leu Thr Thr Asn Lys Asp

305 310 315 320

Asp Asp Asn Ile Leu Phe Arg Asp Ser Ala Asn Ala Thr Ser Leu Pro

325 330 335

Val Gln Asp Ser Ser Ser Val Pro Leu Pro Gly Ser Gly Ser Asp Glu

340 345 350

Gly Gly Glu Asp Gly Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

355 360 365

Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

370 375 380

Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

385 390 395 400

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

405 410 415

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

420 425 430

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

435 440 445

Gln Asp Trp Leu Ser Gly Lys Glu Tyr Lys Cys Lys Val Ser Ser Lys

450 455 460

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Asn Ala Thr Gly Gln

465 470 475 480

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met

485 490 495

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

500 505 510

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

515 520 525

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

530 535 540

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

545 550 555 560

Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln

565 570 575

Lys Ser Leu Ser Leu Ser Leu Gly Lys Arg Lys Gly Gly Lys Arg Gly

580 585 590

Ser Gly Ser Arg Lys Gly Pro Pro Ala Ala Leu Thr Leu Pro Arg Val

595 600 605

Gln Cys Arg Ala Ser Arg Tyr Pro Ile Ala Val Asp Cys Ser Trp Thr

610 615 620

Leu Pro Pro Ala Pro Asn Ser Thr Ser Pro Val Ser Phe Ile Ala Thr

625 630 635 640

Tyr Arg Leu Gly Met Ala Ala Arg Gly His Ser Trp Pro Cys Leu Gln

645 650 655

Gln Thr Pro Thr Ser Thr Ser Cys Thr Ile Thr Asp Val Gln Leu Phe

660 665 670

Ser Met Ala Pro Tyr Val Leu Asn Val Thr Ala Val His Pro Trp Gly

675 680 685

Ser Ser Ser Ser Phe Val Pro Phe Ile Thr Glu His Ile Ile Lys Pro

690 695 700

Asp Pro Pro Glu Gly Val Arg Leu Ser Pro Leu Ala Glu Arg Gln Leu

705 710 715 720

Gln Val Gln Trp Glu Pro Pro Gly Ser Trp Pro Phe Pro Glu Ile Phe

725 730 735

Ser Leu Lys Tyr Trp Ile Arg Tyr Lys Arg Gln Gly Ala Ala Arg Phe

740 745 750

His Arg Val Gly Pro Ile Glu Ala Thr Ser Phe Ile Leu Arg Ala Val

755 760 765

Arg Pro Arg Ala Arg Tyr Tyr Val Gln Val Ala Ala Gln Asp Leu Thr

770 775 780

Asp Tyr Gly Glu Leu Ser Asp Trp Ser Leu Pro Ala Thr Ala Thr Met

785 790 795 800

Ser Leu Gly Lys

<210> 24

<211> 232

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic peptide

<220>

<221> LALA

<222> (19)..(20)

<223> L234A and L235A

<220>

<221> M252Y

<222> (37)..(37)

<220>

<221> S254T

<222> (39)..(39)

<220>

<221> T256E

<222> (41)..(41)

<220>

<221> T-Y

<222> (151)..(151)

<223> pestle mutation

<220>

<221> T-Y

<222> (192)..(192)

<223> pestle mutation

<400> 24

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Tyr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 25

<211> 232

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic peptide

<220>

<221> LALA

<222> (19)..(20)

<223> L234A and L235A

<220>

<221> M252Y

<222> (37)..(37)

<220>

<221> S254T

<222> (39)..(39)

<220>

<221> T256E

<222> (41)..(41)

<220>

<221> Y-T

<222> (151)..(151)

<223> mutation of mortar

<220>

<221> Y-T

<222> (192)..(192)

<223> mutation of mortar

<400> 25

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 26

<211> 213

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic peptide

<400> 26

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp Pro Ala Phe

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile Ser Val Asp

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser Ser Tyr Gln

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu

210

<210> 27

<211> 240

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic peptide

<400> 27

Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly Asn Glu Asp Thr Thr Ala

1 5 10 15

Asp Phe Phe Leu Thr Thr Met Pro Thr Asp Ser Leu Ser Val Ser Thr

20 25 30

Leu Pro Leu Pro Glu Val Gln Cys Phe Val Phe Asn Val Glu Tyr Met

35 40 45

Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro Gln Pro Thr Asn Leu Thr

50 55 60

Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn Asp Lys Val Gln Lys Cys

65 70 75 80

Ser His Tyr Leu Phe Ser Glu Glu Ile Thr Ser Gly Cys Gln Leu Gln

85 90 95

Lys Lys Glu Ile His Leu Tyr Gln Thr Phe Val Val Gln Leu Gln Asp

100 105 110

Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln Met Leu Lys Leu Gln Asn

115 120 125

Leu Val Ile Pro Trp Ala Pro Glu Asn Leu Thr Leu His Lys Leu Ser

130 135 140

Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn Arg Phe Leu Asn His Cys

145 150 155 160

Leu Glu His Leu Val Gln Tyr Arg Thr Asp Trp Asp His Ser Trp Thr

165 170 175

Glu Gln Ser Val Asp Tyr Arg His Lys Phe Ser Leu Pro Ser Val Asp

180 185 190

Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg Ser Arg Phe Asn Pro Leu

195 200 205

Cys Gly Ser Ala Gln His Trp Ser Glu Trp Ser His Pro Ile His Trp

210 215 220

Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe Leu Phe Ala Leu Glu Ala

225 230 235 240

<210> 28

<211> 659

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic peptide

<400> 28

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp Pro Ala Phe

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile Ser Val Asp

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser Ser Tyr Gln

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu Gly Ser Gly Ser Arg Lys Gly Gly Lys Arg Gly

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Ser

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Ser Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Asn Ala Thr Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly Lys Asp Glu Gly Gly Glu Asp Gly Ser Gly Ser Arg Asn

450 455 460

Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His His

465 470 475 480

Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg

485 490 495

Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu

500 505 510

Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu

515 520 525

Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe

530 535 540

Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met

545 550 555 560

Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val

565 570 575

Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln

580 585 590

Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln

595 600 605

Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu

610 615 620

Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His

625 630 635 640

Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser Tyr Leu

645 650 655

Asn Ala Ser

<210> 29

<211> 698

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic peptide

<400> 29

Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly Asn Glu Asp Thr Thr Ala

1 5 10 15

Asp Phe Phe Leu Thr Thr Met Pro Thr Asp Ser Leu Ser Val Ser Thr

20 25 30

Leu Pro Leu Pro Glu Val Gln Cys Phe Val Phe Asn Val Glu Tyr Met

35 40 45

Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro Gln Pro Thr Asn Leu Thr

50 55 60

Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn Asp Lys Val Gln Lys Cys

65 70 75 80

Ser His Tyr Leu Phe Ser Glu Glu Ile Thr Ser Gly Cys Gln Leu Gln

85 90 95

Lys Lys Glu Ile His Leu Tyr Gln Thr Phe Val Val Gln Leu Gln Asp

100 105 110

Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln Met Leu Lys Leu Gln Asn

115 120 125

Leu Val Ile Pro Trp Ala Pro Glu Asn Leu Thr Leu His Lys Leu Ser

130 135 140

Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn Arg Phe Leu Asn His Cys

145 150 155 160

Leu Glu His Leu Val Gln Tyr Arg Thr Asp Trp Asp His Ser Trp Thr

165 170 175

Glu Gln Ser Val Asp Tyr Arg His Lys Phe Ser Leu Pro Ser Val Asp

180 185 190

Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg Ser Arg Phe Asn Pro Leu

195 200 205

Cys Gly Ser Ala Gln His Trp Ser Glu Trp Ser His Pro Ile His Trp

210 215 220

Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe Leu Phe Ala Leu Glu Ala

225 230 235 240

Gly Ser Gly Ser Asp Glu Gly Gly Glu Asp Gly Ser Lys Tyr Gly Pro

245 250 255

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

260 265 270

Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr

275 280 285

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

290 295 300

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

305 310 315 320

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

325 330 335

Val Leu Thr Val Leu His Gln Asp Trp Leu Ser Gly Lys Glu Tyr Lys

340 345 350

Cys Lys Val Ser Ser Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

355 360 365

Ser Asn Ala Thr Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

370 375 380

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

385 390 395 400

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

405 410 415

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

420 425 430

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

435 440 445

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu

450 455 460

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Arg

465 470 475 480

Lys Gly Gly Lys Arg Gly Ser Gly Ser Arg Lys Gly Pro Pro Ala Ala

485 490 495

Leu Thr Leu Pro Arg Val Gln Cys Arg Ala Ser Arg Tyr Pro Ile Ala

500 505 510

Val Asp Cys Ser Trp Thr Leu Pro Pro Ala Pro Asn Ser Thr Ser Pro

515 520 525

Val Ser Phe Ile Ala Thr Tyr Arg Leu Gly Met Ala Ala Arg Gly His

530 535 540

Ser Trp Pro Cys Leu Gln Gln Thr Pro Thr Ser Thr Ser Cys Thr Ile

545 550 555 560

Thr Asp Val Gln Leu Phe Ser Met Ala Pro Tyr Val Leu Asn Val Thr

565 570 575

Ala Val His Pro Trp Gly Ser Ser Ser Ser Phe Val Pro Phe Ile Thr

580 585 590

Glu His Ile Ile Lys Pro Asp Pro Pro Glu Gly Val Arg Leu Ser Pro

595 600 605

Leu Ala Glu Arg Gln Leu Gln Val Gln Trp Glu Pro Pro Gly Ser Trp

610 615 620

Pro Phe Pro Glu Ile Phe Ser Leu Lys Tyr Trp Ile Arg Tyr Lys Arg

625 630 635 640

Gln Gly Ala Ala Arg Phe His Arg Val Gly Pro Ile Glu Ala Thr Ser

645 650 655

Phe Ile Leu Arg Ala Val Arg Pro Arg Ala Arg Tyr Tyr Val Gln Val

660 665 670

Ala Ala Gln Asp Leu Thr Asp Tyr Gly Glu Leu Ser Asp Trp Ser Leu

675 680 685

Pro Ala Thr Ala Thr Met Ser Leu Gly Lys

690 695

<210> 30

<211> 228

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic peptide

<400> 30

Glu Met Gly Thr Ala Asp Leu Gly Pro Ser Ser Val Pro Thr Pro Thr

1 5 10 15

Asn Val Thr Ile Glu Ser Tyr Asn Met Asn Pro Ile Val Tyr Trp Glu

20 25 30

Tyr Gln Ile Met Pro Gln Val Pro Val Phe Thr Val Glu Val Lys Asn

35 40 45

Tyr Gly Val Lys Asn Ser Glu Trp Ile Asp Ala Cys Ile Asn Ile Ser

50 55 60

His His Tyr Cys Asn Ile Ser Asp His Val Gly Asp Pro Ser Asn Ser

65 70 75 80

Leu Trp Val Arg Val Lys Ala Arg Val Gly Gln Lys Glu Ser Ala Tyr

85 90 95

Ala Lys Ser Glu Glu Phe Ala Val Cys Arg Asp Gly Lys Ile Gly Pro

100 105 110

Pro Lys Leu Asp Ile Arg Lys Glu Glu Lys Gln Ile Met Ile Asp Ile

115 120 125

Phe His Pro Ser Val Phe Val Asn Gly Asp Glu Gln Glu Val Asp Tyr

130 135 140

Asp Pro Glu Thr Thr Cys Tyr Ile Arg Val Tyr Asn Val Tyr Val Arg

145 150 155 160

Met Asn Gly Ser Glu Ile Gln Tyr Lys Ile Leu Thr Gln Lys Glu Asp

165 170 175

Asp Cys Asp Glu Ile Gln Cys Gln Leu Ala Ile Pro Val Ser Ser Leu

180 185 190

Asn Ser Gln Tyr Cys Val Ser Ala Glu Gly Val Leu His Val Trp Gly

195 200 205

Val Thr Thr Glu Lys Ser Lys Glu Val Cys Ile Thr Ile Phe Asn Ser

210 215 220

Ser Ile Lys Gly

225

<210> 31

<211> 220

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic peptide

<400> 31

Ser Gln Leu Pro Ala Pro Gln His Pro Lys Ile Arg Leu Tyr Asn Ala

1 5 10 15

Glu Gln Val Leu Ser Trp Glu Pro Val Ala Leu Ser Asn Ser Thr Arg

20 25 30

Pro Val Val Tyr Gln Val Gln Phe Lys Tyr Thr Asp Ser Lys Trp Phe

35 40 45

Thr Ala Asp Ile Met Ser Ile Gly Val Asn Cys Thr Gln Ile Thr Ala

50 55 60

Thr Glu Cys Asp Phe Thr Ala Ala Ser Pro Ser Ala Gly Phe Pro Met

65 70 75 80

Asp Phe Asn Val Thr Leu Arg Leu Arg Ala Glu Leu Gly Ala Leu His

85 90 95

Ser Ala Trp Val Thr Met Pro Trp Phe Gln His Tyr Arg Asn Val Thr

100 105 110

Val Gly Pro Pro Glu Asn Ile Glu Val Thr Pro Gly Glu Gly Ser Leu

115 120 125

Ile Ile Arg Phe Ser Ser Pro Phe Asp Ile Ala Asp Thr Ser Thr Ala

130 135 140

Phe Phe Cys Tyr Tyr Val His Tyr Trp Glu Lys Gly Gly Ile Gln Gln

145 150 155 160

Val Lys Gly Pro Phe Arg Ser Asn Ser Ile Ser Leu Asp Asn Leu Lys

165 170 175

Pro Ser Arg Val Tyr Cys Leu Gln Val Gln Ala Gln Leu Leu Trp Asn

180 185 190

Lys Ser Asn Ile Phe Arg Val Gly His Leu Ser Asn Ile Ser Cys Tyr

195 200 205

Glu Thr Met Ala Asp Ala Ser Thr Glu Leu Gln Gln

210 215 220

<210> 32

<211> 674

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic peptide

<400> 32

Glu Met Gly Thr Ala Asp Leu Gly Pro Ser Ser Val Pro Thr Pro Thr

1 5 10 15

Asn Val Thr Ile Glu Ser Tyr Asn Met Asn Pro Ile Val Tyr Trp Glu

20 25 30

Tyr Gln Ile Met Pro Gln Val Pro Val Phe Thr Val Glu Val Lys Asn

35 40 45

Tyr Gly Val Lys Asn Ser Glu Trp Ile Asp Ala Cys Ile Asn Ile Ser

50 55 60

His His Tyr Cys Asn Ile Ser Asp His Val Gly Asp Pro Ser Asn Ser

65 70 75 80

Leu Trp Val Arg Val Lys Ala Arg Val Gly Gln Lys Glu Ser Ala Tyr

85 90 95

Ala Lys Ser Glu Glu Phe Ala Val Cys Arg Asp Gly Lys Ile Gly Pro

100 105 110

Pro Lys Leu Asp Ile Arg Lys Glu Glu Lys Gln Ile Met Ile Asp Ile

115 120 125

Phe His Pro Ser Val Phe Val Asn Gly Asp Glu Gln Glu Val Asp Tyr

130 135 140

Asp Pro Glu Thr Thr Cys Tyr Ile Arg Val Tyr Asn Val Tyr Val Arg

145 150 155 160

Met Asn Gly Ser Glu Ile Gln Tyr Lys Ile Leu Thr Gln Lys Glu Asp

165 170 175

Asp Cys Asp Glu Ile Gln Cys Gln Leu Ala Ile Pro Val Ser Ser Leu

180 185 190

Asn Ser Gln Tyr Cys Val Ser Ala Glu Gly Val Leu His Val Trp Gly

195 200 205

Val Thr Thr Glu Lys Ser Lys Glu Val Cys Ile Thr Ile Phe Asn Ser

210 215 220

Ser Ile Lys Gly Gly Ser Gly Ser Arg Lys Gly Gly Lys Arg Gly Ser

225 230 235 240

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

275 280 285

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Ser Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Ser Lys Gly Leu Pro Ser Ser Ile

340 345 350

Glu Lys Thr Ile Ser Asn Ala Thr Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Leu Gly Lys Asp Glu Gly Gly Glu Asp Gly Ser Gly Ser Arg Asn Leu

465 470 475 480

Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His His Ser

485 490 495

Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln

500 505 510

Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp

515 520 525

Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu

530 535 540

Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile

545 550 555 560

Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala

565 570 575

Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu

580 585 590

Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile

595 600 605

Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala

610 615 620

Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu

625 630 635 640

Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala

645 650 655

Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn

660 665 670

Ala Ser

<210> 33

<211> 678

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic peptide

<400> 33

Ser Gln Leu Pro Ala Pro Gln His Pro Lys Ile Arg Leu Tyr Asn Ala

1 5 10 15

Glu Gln Val Leu Ser Trp Glu Pro Val Ala Leu Ser Asn Ser Thr Arg

20 25 30

Pro Val Val Tyr Gln Val Gln Phe Lys Tyr Thr Asp Ser Lys Trp Phe

35 40 45

Thr Ala Asp Ile Met Ser Ile Gly Val Asn Cys Thr Gln Ile Thr Ala

50 55 60

Thr Glu Cys Asp Phe Thr Ala Ala Ser Pro Ser Ala Gly Phe Pro Met

65 70 75 80

Asp Phe Asn Val Thr Leu Arg Leu Arg Ala Glu Leu Gly Ala Leu His

85 90 95

Ser Ala Trp Val Thr Met Pro Trp Phe Gln His Tyr Arg Asn Val Thr

100 105 110

Val Gly Pro Pro Glu Asn Ile Glu Val Thr Pro Gly Glu Gly Ser Leu

115 120 125

Ile Ile Arg Phe Ser Ser Pro Phe Asp Ile Ala Asp Thr Ser Thr Ala

130 135 140

Phe Phe Cys Tyr Tyr Val His Tyr Trp Glu Lys Gly Gly Ile Gln Gln

145 150 155 160

Val Lys Gly Pro Phe Arg Ser Asn Ser Ile Ser Leu Asp Asn Leu Lys

165 170 175

Pro Ser Arg Val Tyr Cys Leu Gln Val Gln Ala Gln Leu Leu Trp Asn

180 185 190

Lys Ser Asn Ile Phe Arg Val Gly His Leu Ser Asn Ile Ser Cys Tyr

195 200 205

Glu Thr Met Ala Asp Ala Ser Thr Glu Leu Gln Gln Gly Ser Gly Ser

210 215 220

Asp Glu Gly Gly Glu Asp Gly Ser Lys Tyr Gly Pro Pro Cys Pro Pro

225 230 235 240

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

245 250 255

Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr

260 265 270

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

275 280 285

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

290 295 300

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

305 310 315 320

Leu His Gln Asp Trp Leu Ser Gly Lys Glu Tyr Lys Cys Lys Val Ser

325 330 335

Ser Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Asn Ala Thr

340 345 350

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

355 360 365

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

370 375 380

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

385 390 395 400

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

405 410 415

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

420 425 430

Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr

435 440 445

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Arg Lys Gly Gly Lys

450 455 460

Arg Gly Ser Gly Ser Arg Lys Gly Pro Pro Ala Ala Leu Thr Leu Pro

465 470 475 480

Arg Val Gln Cys Arg Ala Ser Arg Tyr Pro Ile Ala Val Asp Cys Ser

485 490 495

Trp Thr Leu Pro Pro Ala Pro Asn Ser Thr Ser Pro Val Ser Phe Ile

500 505 510

Ala Thr Tyr Arg Leu Gly Met Ala Ala Arg Gly His Ser Trp Pro Cys

515 520 525

Leu Gln Gln Thr Pro Thr Ser Thr Ser Cys Thr Ile Thr Asp Val Gln

530 535 540

Leu Phe Ser Met Ala Pro Tyr Val Leu Asn Val Thr Ala Val His Pro

545 550 555 560

Trp Gly Ser Ser Ser Ser Phe Val Pro Phe Ile Thr Glu His Ile Ile

565 570 575

Lys Pro Asp Pro Pro Glu Gly Val Arg Leu Ser Pro Leu Ala Glu Arg

580 585 590

Gln Leu Gln Val Gln Trp Glu Pro Pro Gly Ser Trp Pro Phe Pro Glu

595 600 605

Ile Phe Ser Leu Lys Tyr Trp Ile Arg Tyr Lys Arg Gln Gly Ala Ala

610 615 620

Arg Phe His Arg Val Gly Pro Ile Glu Ala Thr Ser Phe Ile Leu Arg

625 630 635 640

Ala Val Arg Pro Arg Ala Arg Tyr Tyr Val Gln Val Ala Ala Gln Asp

645 650 655

Leu Thr Asp Tyr Gly Glu Leu Ser Asp Trp Ser Leu Pro Ala Thr Ala

660 665 670

Thr Met Ser Leu Gly Lys

675

<210> 34

<211> 792

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic peptide

<400> 34

Leu Ala Pro Arg Arg Cys Pro Ala Gln Glu Val Ala Arg Gly Val Leu

1 5 10 15

Thr Ser Leu Pro Gly Asp Ser Val Thr Leu Thr Cys Pro Gly Val Glu

20 25 30

Pro Glu Asp Asn Ala Thr Val His Trp Val Leu Arg Lys Pro Ala Ala

35 40 45

Gly Ser His Pro Ser Arg Trp Ala Gly Met Gly Arg Arg Leu Leu Leu

50 55 60

Arg Ser Val Gln Leu His Asp Ser Gly Asn Tyr Ser Cys Tyr Arg Ala

65 70 75 80

Gly Arg Pro Ala Gly Thr Val His Leu Leu Val Asp Val Pro Pro Glu

85 90 95

Glu Pro Gln Leu Ser Cys Phe Arg Lys Ser Pro Leu Ser Asn Val Val

100 105 110

Cys Glu Trp Gly Pro Arg Ser Thr Pro Ser Leu Thr Thr Lys Ala Val

115 120 125

Leu Leu Val Arg Lys Phe Gln Asn Ser Pro Ala Glu Asp Phe Gln Glu

130 135 140

Pro Cys Gln Tyr Ser Gln Glu Ser Gln Lys Phe Ser Cys Gln Leu Ala

145 150 155 160

Val Pro Glu Gly Asp Ser Ser Phe Tyr Ile Val Ser Met Cys Val Ala

165 170 175

Ser Ser Val Gly Ser Lys Phe Ser Lys Thr Gln Thr Phe Gln Gly Cys

180 185 190

Gly Ile Leu Gln Pro Asp Pro Pro Ala Asn Ile Thr Val Thr Ala Val

195 200 205

Ala Arg Asn Pro Arg Trp Leu Ser Val Thr Trp Gln Asp Pro His Ser

210 215 220

Trp Asn Ser Ser Phe Tyr Arg Leu Arg Phe Glu Leu Arg Tyr Arg Ala

225 230 235 240

Glu Arg Ser Lys Thr Phe Thr Thr Trp Met Val Lys Asp Leu Gln His

245 250 255

His Cys Val Ile His Asp Ala Trp Ser Gly Leu Arg His Val Val Gln

260 265 270

Leu Arg Ala Gln Glu Glu Phe Gly Gln Gly Glu Trp Ser Glu Trp Ser

275 280 285

Pro Glu Ala Met Gly Thr Pro Trp Thr Glu Ser Arg Ser Pro Pro Ala

290 295 300

Glu Asn Glu Val Ser Thr Pro Met Gln Ala Leu Thr Thr Asn Lys Asp

305 310 315 320

Asp Asp Asn Ile Leu Phe Arg Asp Ser Ala Asn Ala Thr Ser Leu Pro

325 330 335

Val Gln Asp Ser Ser Ser Val Pro Leu Pro Gly Ser Gly Ser Arg Lys

340 345 350

Gly Gly Lys Arg Gly Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

355 360 365

Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

370 375 380

Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

385 390 395 400

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

405 410 415

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

420 425 430

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

435 440 445

Gln Asp Trp Leu Ser Gly Lys Glu Tyr Lys Cys Lys Val Ser Ser Lys

450 455 460

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Asn Ala Thr Gly Gln

465 470 475 480

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met

485 490 495

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

500 505 510

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

515 520 525

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

530 535 540

Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

545 550 555 560

Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln

565 570 575

Lys Ser Leu Ser Leu Ser Leu Gly Lys Asp Glu Gly Gly Glu Asp Gly

580 585 590

Ser Gly Ser Arg Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe

595 600 605

Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met

610 615 620

Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu

625 630 635 640

Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu

645 650 655

Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser

660 665 670

Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys

675 680 685

Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu

690 695 700

Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met

705 710 715 720

Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile

725 730 735

Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln

740 745 750

Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu

755 760 765

Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg

770 775 780

Val Met Ser Tyr Leu Asn Ala Ser

785 790

<210> 35

<211> 597

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic peptide

<400> 35

Glu Leu Leu Asp Pro Cys Gly Tyr Ile Ser Pro Glu Ser Pro Val Val

1 5 10 15

Gln Leu His Ser Asn Phe Thr Ala Val Cys Val Leu Lys Glu Lys Cys

20 25 30

Met Asp Tyr Phe His Val Asn Ala Asn Tyr Ile Val Trp Lys Thr Asn

35 40 45

His Phe Thr Ile Pro Lys Glu Gln Tyr Thr Ile Ile Asn Arg Thr Ala

50 55 60

Ser Ser Val Thr Phe Thr Asp Ile Ala Ser Leu Asn Ile Gln Leu Thr

65 70 75 80

Cys Asn Ile Leu Thr Phe Gly Gln Leu Glu Gln Asn Val Tyr Gly Ile

85 90 95

Thr Ile Ile Ser Gly Leu Pro Pro Glu Lys Pro Lys Asn Leu Ser Cys

100 105 110

Ile Val Asn Glu Gly Lys Lys Met Arg Cys Glu Trp Asp Gly Gly Arg

115 120 125

Glu Thr His Leu Glu Thr Asn Phe Thr Leu Lys Ser Glu Trp Ala Thr

130 135 140

His Lys Phe Ala Asp Cys Lys Ala Lys Arg Asp Thr Pro Thr Ser Cys

145 150 155 160

Thr Val Asp Tyr Ser Thr Val Tyr Phe Val Asn Ile Glu Val Trp Val

165 170 175

Glu Ala Glu Asn Ala Leu Gly Lys Val Thr Ser Asp His Ile Asn Phe

180 185 190

Asp Pro Val Tyr Lys Val Lys Pro Asn Pro Pro His Asn Leu Ser Val

195 200 205

Ile Asn Ser Glu Glu Leu Ser Ser Ile Leu Lys Leu Thr Trp Thr Asn

210 215 220

Pro Ser Ile Lys Ser Val Ile Ile Leu Lys Tyr Asn Ile Gln Tyr Arg

225 230 235 240

Thr Lys Asp Ala Ser Thr Trp Ser Gln Ile Pro Pro Glu Asp Thr Ala

245 250 255

Ser Thr Arg Ser Ser Phe Thr Val Gln Asp Leu Lys Pro Phe Thr Glu

260 265 270

Tyr Val Phe Arg Ile Arg Cys Met Lys Glu Asp Gly Lys Gly Tyr Trp

275 280 285

Ser Asp Trp Ser Glu Glu Ala Ser Gly Ile Thr Tyr Glu Asp Arg Pro

290 295 300

Ser Lys Ala Pro Ser Phe Trp Tyr Lys Ile Asp Pro Ser His Thr Gln

305 310 315 320

Gly Tyr Arg Thr Val Gln Leu Val Trp Lys Thr Leu Pro Pro Phe Glu

325 330 335

Ala Asn Gly Lys Ile Leu Asp Tyr Glu Val Thr Leu Thr Arg Trp Lys

340 345 350

Ser His Leu Gln Asn Tyr Thr Val Asn Ala Thr Lys Leu Thr Val Asn

355 360 365

Leu Thr Asn Asp Arg Tyr Leu Ala Thr Leu Thr Val Arg Asn Leu Val

370 375 380

Gly Lys Ser Asp Ala Ala Val Leu Thr Ile Pro Ala Cys Asp Phe Gln

385 390 395 400

Ala Thr His Pro Val Met Asp Leu Lys Ala Phe Pro Lys Asp Asn Met

405 410 415

Leu Trp Val Glu Trp Thr Thr Pro Arg Glu Ser Val Lys Lys Tyr Ile

420 425 430

Leu Glu Trp Cys Val Leu Ser Asp Lys Ala Pro Cys Ile Thr Asp Trp

435 440 445

Gln Gln Glu Asp Gly Thr Val His Arg Thr Tyr Leu Arg Gly Asn Leu

450 455 460

Ala Glu Ser Lys Cys Tyr Leu Ile Thr Val Thr Pro Val Tyr Ala Asp

465 470 475 480

Gly Pro Gly Ser Pro Glu Ser Ile Lys Ala Tyr Leu Lys Gln Ala Pro

485 490 495

Pro Ser Lys Gly Pro Thr Val Arg Thr Lys Lys Val Gly Lys Asn Glu

500 505 510

Ala Val Leu Glu Trp Asp Gln Leu Pro Val Asp Val Gln Asn Gly Phe

515 520 525

Ile Arg Asn Tyr Thr Ile Phe Tyr Arg Thr Ile Ile Gly Asn Glu Thr

530 535 540

Ala Val Asn Val Asp Ser Ser His Thr Glu Tyr Thr Leu Ser Ser Leu

545 550 555 560

Thr Ser Asp Thr Leu Tyr Met Val Arg Met Ala Ala Tyr Thr Asp Glu

565 570 575

Gly Gly Lys Asp Gly Pro Glu Phe Thr Phe Thr Thr Pro Lys Phe Ala

580 585 590

Gln Gly Glu Ile Glu

595

<210> 36

<211> 698

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic peptide

<400> 36

Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly Asn Glu Asp Thr Thr Ala

1 5 10 15

Asp Phe Phe Leu Thr Thr Met Pro Thr Asp Ser Leu Ser Val Ser Thr

20 25 30

Leu Pro Leu Pro Glu Val Gln Cys Phe Val Phe Asn Val Glu Tyr Met

35 40 45

Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro Gln Pro Thr Asn Leu Thr

50 55 60

Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn Asp Lys Val Gln Lys Cys

65 70 75 80

Ser His Tyr Leu Phe Ser Glu Glu Ile Thr Ser Gly Cys Gln Leu Gln

85 90 95

Lys Lys Glu Ile His Leu Tyr Gln Thr Phe Val Val Gln Leu Gln Asp

100 105 110

Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln Met Leu Lys Leu Gln Asn

115 120 125

Leu Val Ile Pro Trp Ala Pro Glu Asn Leu Thr Leu His Lys Leu Ser

130 135 140

Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn Arg Phe Leu Asn His Cys

145 150 155 160

Leu Glu His Leu Val Gln Tyr Arg Thr Asp Trp Asp His Ser Trp Thr

165 170 175

Glu Gln Ser Val Asp Tyr Arg His Lys Phe Ser Leu Pro Ser Val Asp

180 185 190

Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg Ser Arg Phe Asn Pro Leu

195 200 205

Cys Gly Ser Ala Gln His Trp Ser Glu Trp Ser His Pro Ile His Trp

210 215 220

Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe Leu Phe Ala Leu Glu Ala

225 230 235 240

Gly Ser Gly Ser Arg Lys Gly Gly Lys Arg Gly Ser Lys Tyr Gly Pro

245 250 255

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

260 265 270

Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr

275 280 285

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

290 295 300

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

305 310 315 320

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

325 330 335

Val Leu Thr Val Leu His Gln Asp Trp Leu Ser Gly Lys Glu Tyr Lys

340 345 350

Cys Lys Val Ser Ser Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

355 360 365

Ser Asn Ala Thr Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

370 375 380

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

385 390 395 400

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

405 410 415

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

420 425 430

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

435 440 445

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu

450 455 460

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Asp

465 470 475 480

Glu Gly Gly Glu Asp Gly Ser Gly Ser Arg Lys Gly Pro Pro Ala Ala

485 490 495

Leu Thr Leu Pro Arg Val Gln Cys Arg Ala Ser Arg Tyr Pro Ile Ala

500 505 510

Val Asp Cys Ser Trp Thr Leu Pro Pro Ala Pro Asn Ser Thr Ser Pro

515 520 525

Val Ser Phe Ile Ala Thr Tyr Arg Leu Gly Met Ala Ala Arg Gly His

530 535 540

Ser Trp Pro Cys Leu Gln Gln Thr Pro Thr Ser Thr Ser Cys Thr Ile

545 550 555 560

Thr Asp Val Gln Leu Phe Ser Met Ala Pro Tyr Val Leu Asn Val Thr

565 570 575

Ala Val His Pro Trp Gly Ser Ser Ser Ser Phe Val Pro Phe Ile Thr

580 585 590

Glu His Ile Ile Lys Pro Asp Pro Pro Glu Gly Val Arg Leu Ser Pro

595 600 605

Leu Ala Glu Arg Gln Leu Gln Val Gln Trp Glu Pro Pro Gly Ser Trp

610 615 620

Pro Phe Pro Glu Ile Phe Ser Leu Lys Tyr Trp Ile Arg Tyr Lys Arg

625 630 635 640

Gln Gly Ala Ala Arg Phe His Arg Val Gly Pro Ile Glu Ala Thr Ser

645 650 655

Phe Ile Leu Arg Ala Val Arg Pro Arg Ala Arg Tyr Tyr Val Gln Val

660 665 670

Ala Ala Gln Asp Leu Thr Asp Tyr Gly Glu Leu Ser Asp Trp Ser Leu

675 680 685

Pro Ala Thr Ala Thr Met Ser Leu Gly Lys

690 695

<210> 37

<211> 659

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic peptide

<400> 37

Cys Pro Asp Leu Val Cys Tyr Thr Asp Tyr Leu Gln Thr Val Ile Cys

1 5 10 15

Ile Leu Glu Met Trp Asn Leu His Pro Ser Thr Leu Thr Leu Thr Trp

20 25 30

Gln Asp Gln Tyr Glu Glu Leu Lys Asp Glu Ala Thr Ser Cys Ser Leu

35 40 45

His Arg Ser Ala His Asn Ala Thr His Ala Thr Tyr Thr Cys His Met

50 55 60

Asp Val Phe His Phe Met Ala Asp Asp Ile Phe Ser Val Asn Ile Thr

65 70 75 80

Asp Gln Ser Gly Asn Tyr Ser Gln Glu Cys Gly Ser Phe Leu Leu Ala

85 90 95

Glu Ser Ile Lys Pro Ala Pro Pro Phe Asn Val Thr Val Thr Phe Ser

100 105 110

Gly Gln Tyr Asn Ile Ser Trp Arg Ser Asp Tyr Glu Asp Pro Ala Phe

115 120 125

Tyr Met Leu Lys Gly Lys Leu Gln Tyr Glu Leu Gln Tyr Arg Asn Arg

130 135 140

Gly Asp Pro Trp Ala Val Ser Pro Arg Arg Lys Leu Ile Ser Val Asp

145 150 155 160

Ser Arg Ser Val Ser Leu Leu Pro Leu Glu Phe Arg Lys Asp Ser Ser

165 170 175

Tyr Glu Leu Gln Val Arg Ala Gly Pro Met Pro Gly Ser Ser Tyr Gln

180 185 190

Gly Thr Trp Ser Glu Trp Ser Asp Pro Val Ile Phe Gln Thr Gln Ser

195 200 205

Glu Glu Leu Lys Glu Gly Ser Gly Ser Asp Glu Gly Gly Glu Asp Gly

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Ser

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Ser Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Asn Ala Thr Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly Lys Arg Lys Gly Gly Lys Arg Gly Ser Gly Ser Arg Asn

450 455 460

Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His His

465 470 475 480

Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg

485 490 495

Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu

500 505 510

Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu

515 520 525

Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe

530 535 540

Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met

545 550 555 560

Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val

565 570 575

Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln

580 585 590

Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln

595 600 605

Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu

610 615 620

Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His

625 630 635 640

Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser Tyr Leu

645 650 655

Asn Ala Ser

<210> 38

<211> 674

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic peptide

<400> 38

Glu Met Gly Thr Ala Asp Leu Gly Pro Ser Ser Val Pro Thr Pro Thr

1 5 10 15

Asn Val Thr Ile Glu Ser Tyr Asn Met Asn Pro Ile Val Tyr Trp Glu

20 25 30

Tyr Gln Ile Met Pro Gln Val Pro Val Phe Thr Val Glu Val Lys Asn

35 40 45

Tyr Gly Val Lys Asn Ser Glu Trp Ile Asp Ala Cys Ile Asn Ile Ser

50 55 60

His His Tyr Cys Asn Ile Ser Asp His Val Gly Asp Pro Ser Asn Ser

65 70 75 80

Leu Trp Val Arg Val Lys Ala Arg Val Gly Gln Lys Glu Ser Ala Tyr

85 90 95

Ala Lys Ser Glu Glu Phe Ala Val Cys Arg Asp Gly Lys Ile Gly Pro

100 105 110

Pro Lys Leu Asp Ile Arg Lys Glu Glu Lys Gln Ile Met Ile Asp Ile

115 120 125

Phe His Pro Ser Val Phe Val Asn Gly Asp Glu Gln Glu Val Asp Tyr

130 135 140

Asp Pro Glu Thr Thr Cys Tyr Ile Arg Val Tyr Asn Val Tyr Val Arg

145 150 155 160

Met Asn Gly Ser Glu Ile Gln Tyr Lys Ile Leu Thr Gln Lys Glu Asp

165 170 175

Asp Cys Asp Glu Ile Gln Cys Gln Leu Ala Ile Pro Val Ser Ser Leu

180 185 190

Asn Ser Gln Tyr Cys Val Ser Ala Glu Gly Val Leu His Val Trp Gly

195 200 205

Val Thr Thr Glu Lys Ser Lys Glu Val Cys Ile Thr Ile Phe Asn Ser

210 215 220

Ser Ile Lys Gly Gly Ser Gly Ser Asp Glu Gly Gly Glu Asp Gly Ser

225 230 235 240

Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

275 280 285

Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Ser Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Ser Lys Gly Leu Pro Ser Ser Ile

340 345 350

Glu Lys Thr Ile Ser Asn Ala Thr Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Leu Gly Lys Arg Lys Gly Gly Lys Arg Gly Ser Gly Ser Arg Asn Leu

465 470 475 480

Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His His Ser

485 490 495

Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln

500 505 510

Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp

515 520 525

Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu

530 535 540

Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile

545 550 555 560

Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala

565 570 575

Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu

580 585 590

Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile

595 600 605

Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala

610 615 620

Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu

625 630 635 640

Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala

645 650 655

Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn

660 665 670

Ala Ser

<210> 39

<211> 678

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> synthetic peptide

<400> 39

Ser Gln Leu Pro Ala Pro Gln His Pro Lys Ile Arg Leu Tyr Asn Ala

1 5 10 15

Glu Gln Val Leu Ser Trp Glu Pro Val Ala Leu Ser Asn Ser Thr Arg

20 25 30

Pro Val Val Tyr Gln Val Gln Phe Lys Tyr Thr Asp Ser Lys Trp Phe

35 40 45

Thr Ala Asp Ile Met Ser Ile Gly Val Asn Cys Thr Gln Ile Thr Ala

50 55 60

Thr Glu Cys Asp Phe Thr Ala Ala Ser Pro Ser Ala Gly Phe Pro Met

65 70 75 80

Asp Phe Asn Val Thr Leu Arg Leu Arg Ala Glu Leu Gly Ala Leu His

85 90 95

Ser Ala Trp Val Thr Met Pro Trp Phe Gln His Tyr Arg Asn Val Thr

100 105 110

Val Gly Pro Pro Glu Asn Ile Glu Val Thr Pro Gly Glu Gly Ser Leu

115 120 125

Ile Ile Arg Phe Ser Ser Pro Phe Asp Ile Ala Asp Thr Ser Thr Ala

130 135 140

Phe Phe Cys Tyr Tyr Val His Tyr Trp Glu Lys Gly Gly Ile Gln Gln

145 150 155 160

Val Lys Gly Pro Phe Arg Ser Asn Ser Ile Ser Leu Asp Asn Leu Lys

165 170 175

Pro Ser Arg Val Tyr Cys Leu Gln Val Gln Ala Gln Leu Leu Trp Asn

180 185 190

Lys Ser Asn Ile Phe Arg Val Gly His Leu Ser Asn Ile Ser Cys Tyr

195 200 205

Glu Thr Met Ala Asp Ala Ser Thr Glu Leu Gln Gln Gly Ser Gly Ser

210 215 220

Arg Lys Gly Gly Lys Arg Gly Ser Lys Tyr Gly Pro Pro Cys Pro Pro

225 230 235 240

Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

245 250 255

Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr

260 265 270

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

275 280 285

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

290 295 300

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

305 310 315 320

Leu His Gln Asp Trp Leu Ser Gly Lys Glu Tyr Lys Cys Lys Val Ser

325 330 335

Ser Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Asn Ala Thr

340 345 350

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

355 360 365

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

370 375 380

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

385 390 395 400

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

405 410 415

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

420 425 430

Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr

435 440 445

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Asp Glu Gly Gly Glu

450 455 460

Asp Gly Ser Gly Ser Arg Lys Gly Pro Pro Ala Ala Leu Thr Leu Pro

465 470 475 480

Arg Val Gln Cys Arg Ala Ser Arg Tyr Pro Ile Ala Val Asp Cys Ser

485 490 495

Trp Thr Leu Pro Pro Ala Pro Asn Ser Thr Ser Pro Val Ser Phe Ile

500 505 510

Ala Thr Tyr Arg Leu Gly Met Ala Ala Arg Gly His Ser Trp Pro Cys

515 520 525

Leu Gln Gln Thr Pro Thr Ser Thr Ser Cys Thr Ile Thr Asp Val Gln

530 535 540

Leu Phe Ser Met Ala Pro Tyr Val Leu Asn Val Thr Ala Val His Pro

545 550 555 560

Trp Gly Ser Ser Ser Ser Phe Val Pro Phe Ile Thr Glu His Ile Ile

565 570 575

Lys Pro Asp Pro Pro Glu Gly Val Arg Leu Ser Pro Leu Ala Glu Arg

580 585 590

Gln Leu Gln Val Gln Trp Glu Pro Pro Gly Ser Trp Pro Phe Pro Glu

595 600 605

Ile Phe Ser Leu Lys Tyr Trp Ile Arg Tyr Lys Arg Gln Gly Ala Ala

610 615 620

Arg Phe His Arg Val Gly Pro Ile Glu Ala Thr Ser Phe Ile Leu Arg

625 630 635 640

Ala Val Arg Pro Arg Ala Arg Tyr Tyr Val Gln Val Ala Ala Gln Asp

645 650 655

Leu Thr Asp Tyr Gly Glu Leu Ser Asp Trp Ser Leu Pro Ala Thr Ala

660 665 670

Thr Met Ser Leu Gly Lys

675