EA042507B1 - ANTIGEN-BINDING MOLECULE CONTAINING ANTIBODY VARIABLE REGION - Google Patents

Description

Technical field

The present invention provides an antigen-binding molecule comprising an antibody variable region that is capable of binding to two different antigens (a first antigen and a second antigen) but does not bind to these antigens simultaneously, and an antibody variable region that binds to a third antigen that is different from these antigens, it also provides a pharmaceutical composition containing an antigen-binding molecule and a method for producing an antigen-binding molecule.

BACKGROUND OF THE INVENTION

Antibodies have attracted attention as drugs because they are highly stable in plasma and have little adverse effects (Nat. Biotechnol. 23, 10731078 (2005) (non-patent literature list 1) and Eur. J. Pharm. Biopharm. 59 (3) , 389-396 (2005) (non-patent literature list 2)). Antibodies not only have antigen-binding and agonist or antagonist effects, but exhibit effector cell-mediated cytotoxic activity (also referred to as effector function), ADCC (antibody-dependent cytotoxicity), ADCP (antibody-dependent cellular phagocytosis), or CDC (complement-dependent cytotoxicity). First of all, antibodies of the IgG1 subclass exhibit effector functions in relation to tumor cells. Thus, many antibody-based drugs have been developed in the field of oncology.

For antibodies to exhibit ADCC, ADCP, or CDC activity, their Fc regions must bind to antibody receptors (FcyR) that are present on effector cells (such as NK natural killer cells or macrophages) and to various complement components. According to published data, there is a family of FcyR proteins in humans, including the isoforms FcyRIa, FcyRIIa, FcyRIIb, FcyRIIIa and FcyRIIIb, as well as their corresponding allotypes (Immunol. Lett. 82, 57-65 (2002) (non-patent literature list 3)). Of these isoforms, the FcyRIa, FcyRIIa and FcyRIIIa proteins contain in their intracellular domains an ITAM (immunoreceptor tyrosine activating motif) domain that transduces activating signals. Conversely, only FcyRIIb has an ITIM (immunoreceptor tyrosine inhibitory motif) domain in its intracellular domain that transduces inhibitory signals. All of these FcyR isoforms are known to convert signals by cross-linking (ie, cross-linking) with immunocomplexes or the like. (Nat. Rev. Immunol. 8, 34-47 (2008)) (non-patent literature list 4)). Indeed, when antibodies exhibit effector functions against tumor cells, FcyR molecules on the membranes of the effector cells form clusters due to the Fc fragments of many antibodies bound to the tumor cell membranes and thus transduce activation signals through the effector cells. The result is a cytotoxic effect. In this regard, the cross-linking of FcyR fragments is limited to effector cells localized near tumor cells, which indicates the activation of the immune system localized on tumor cells (Ann. Rev. Immunol. 6, 251-281 (1988) (non-patent literature list 5)) .

Natural immunoglobulins bind to antigens via their variable regions, and bind to receptors such as FcyR, FcRn, FcaR and FcεR or complement components via their conserved regions. Each FcRn molecule (a binding molecule that interacts with an IgG Fc fragment) binds to each antibody heavy chain in a one-to-one junction. Therefore, according to the literature, two FcRn molecules bind to one IgG antibody molecule. Unlike FcRn and the like, FcyR interacts with the antibody hinge and CH2 domains and only one FcyR molecule binds to one IgG type antibody molecule (J. Bio. Chem., 276, 16469-16477 (2001)). It was found that certain amino acid residues in the hinge region, CH2 domains of the antibody, as well as carbohydrate residues attached to the Asn 297 residue (EU numbering according to Kabat (Chem. Immunol. 65, 88-110 ( 1997) (non-patent literature list 6), Eur. J. Immunol. 23, 1098-1104 (1993) (non-patent literature list 7) and Immunol. 86, 319-324 (1995) (non-patent literature list 8)). Fc region variants with different FcyR binding properties have been investigated, focusing on this binding site in order to obtain Fc region variants with the highest binding activity for FcyR activation (WO2000/042072 (Patent Literature List 1) and WO2006/019447 (List patent literature 2)). , Ala 330 and Ile 332 (Kabat numbering) in the human IgG1 sequence for Asn, Leu and Glu residues, respectively (Proc. Natl. Acad. sci. U.S.A. 103, 4005-4010 (2006) (non-patent literature list 9) and WO2006/019447 (patent literature list 2). This modified form has approximately 9 times the binding activity compared to the binding activity of the wild-type immunoglobulin against FcyRIIIa/FcyIIb (A/I). In another case, the group of authors of Shinkawa et al. managed to significantly increase the binding activity against FcyRIIIa by approximately 100 times due to the deletion of fucose residues.

- 1 042507 in the carbohydrate chain attached to the residue Asn 297 (J. Biol. Chem. 278, 3466-3473 (2003) (non-patent literature list 10). These methods can significantly increase the ADCC activity of human IgG1 compared to natural IgG1 .

Natural antibodies of the IgG type usually recognize and bind to one epitope through its variable region (Fab) and therefore can only bind to one antigen. At the same time, it is known that many types of proteins are involved in the development of cancer or inflammation, and these proteins can interact with each other. For example, some inflammatory cytokines (TNF, IL1 and IL6) are known to be involved in the development of an immunological disease (Nat. Biotech., 28, 502510 (2011) (non-patent literature list 11)). It is also known that the activation of other receptors is one of the mechanisms underlying the development of cancer drug resistance (Endocr Relat Cancer 13, 45-51 (2006) (non-patent literature list 12)). In such a case, a conventional antibody that recognizes a single epitope cannot inhibit multiple proteins.

Antibodies that bind to two or more types of antigens and that are present in a single molecule (such antibodies are called bispecific antibodies) have been investigated as multi-target inhibitory molecules. Binding activity with two different antigens (first antigen and second antigen) can be achieved by modifying natural IgG type antibodies (mAbs. (2012) Mar 1, 4 (2)). Therefore, such a single molecule antibody not only has an effect of neutralizing these two or more types of antigens, but also an effect of increasing antitumor activity by crosslinking cells having cytotoxic activity against cancer cells. Previously, data were published on the molecular forms of bispecific antibodies, that is, on a molecule with an antigen-binding site attached to the N- or C-terminus of an antibody (DVDIg and scFv-IgG), on a molecule with different sequences of two Fab regions of an antibody (a bispecific antibody with a common L-chain and hybrid hybridoma), a molecule in which one Fab region recognizes two antigens (IgG two-in-one), and a molecule containing a loop of the CH3 domain as another antigen-binding site (Fcab) (Nat. Rev., 10, 301-316 (2010) (non-patent literature list 13) and Peds, 23 (4), 289-297 (2010) (non-patent literature list 14)). Since any of these bispecific antibodies interact with the FcyR receptor through their Fc regions, the effector functions of the antibody are preserved. Thus, a bispecific antibody binds to any antigen which is thereby recognized at the same time as binding to FcyR and exhibits ADCC activity against cells expressing the antigen.

Provided that all antigens recognized by the bispecific antibody are antigens specifically expressed in cancer, a bispecific antibody that binds to any of the antigens will exhibit cytotoxic activity against cancer cells and therefore such antibodies would be expected to have a stronger anticancer effect. effect than a conventional drug based on a single antigen. However, if any of the antigens recognized by the bispecific antibody is expressed in normal tissue, or is expressed on immunocytes, there will be destruction of normal tissue or release of cytokines by cross-linking with FcyR (J. Immunol., 163 (3), 1246- 1252 (1999) Aug 1 (non-patent literature list 15)). As a result, strong negative reactions are induced.

For example, catumaxomab is known to be a bispecific antibody that recognizes a protein expressed on T cells and a protein expressed on cancer cells (cancer antigen). Catumaxomab binds two Fab fragments to the cancer antigen (EpCAM) and to the CD3ε chain expressed on T cells, respectively. Catumaxomab induces T-cell mediated cytotoxic activity by binding to cancer antigen and CD3ε at the same time, and induces cytotoxic activity mediated by killer cells or antigen presenting cells (e.g. macrophage) by binding to the same time with cancer antigen and FcyR. Through the use of these two cytotoxic activities, catumaxomab has a high therapeutic effect on malignant ascites when administered intraperitoneally, and thus it has been approved for use in Europe (Cancer Treat Rev. 36 (6), 458-467 (2010) Oct (non-patent literature list 16 )). In addition, it is reported that the administration of catumaxomab in some cases leads to the production of antibodies reactive to cancer cells, indicating the induction of adaptive immunity (Future Oncol. 8 (1), 73-85 (2012) Jan (non-patent literature list 17)). In connection with these results, such antibodies exhibiting both T cell-mediated cytotoxic activity and an effect exerted by cells such as killer cells or macrophages with FcyR involvement (such antibodies are primarily referred to as trifunctional antibodies) have attracted attention, since a strong anti-cancer effect and induction of adaptive immunity were expected.

However, trifunctional antibodies bind at the same time to CD3ε and FcyR even in the absence of a cancer antigen and therefore cross-link the T-cell-expressed CD3ε fragment to FcyR-expressed cells even in a cancer-free environment, resulting in

- 2 042507 production of various cytokines in large quantities. Such cancer antigen-independent induction of production of various cytokines currently limits the introduction of trifunctional antibodies into the peritoneal cavity (Cancer Treat Rev. 36 (6), 458-467 (2010 Oct) (non-patent literature list 16)). The introduction of trifunctional antibodies systemically presents great difficulties due to severe negative reactions associated with strong cytokine influx (Cancer Immunol Immunother. 56 (9): 1397-1406 (2007 Sep) (non-patent literature list 18)).

A bispecific antibody produced by standard technology is capable of binding to both antibodies, i.e., the first cancer antibody (EpCAM) and the second CD3ε antigen, and simultaneously binding to FcyR, and therefore cannot eliminate, due to its molecular structure, such negative reactions caused by binding to FcyR and to the second CD3ε antigen at the same time.

A modified antibody has recently been prepared that elicits T-cell mediated cytotoxic activity while abolishing negative reactions, and such an antibody was prepared using an Fc region with reduced FcyR binding activity (WO2012/073985).

However, even such an antibody is unable to act on two immunoreceptors, ie CD3ε and FcyR, and at the same time bind to the cancer antigen due to its molecular structure.

So far, no antibody is known that exhibits both T cell-mediated cytotoxic activity and non-T cell-mediated cytotoxic activity in a cancer antigen-specific mechanism, and at the same time eliminates negative reactions.

List of cited literature

Patent Literature.

Patent Literature 1: WO2000/042072.

Patent Literature 2: WO2006/019447.

non-patent literature.

Non-Patent Literature 1: Nat. Biotechnol. 23, 1073-1078 (2005).

Non-Patent Literature 2: Eur J Pharm Biopharm. 59 (3), 389-396 (2005).

Non-Patent Literature 3: Immunol. Lett. 82, 57-65 (2002).

Non-Patent Literature 4: Nat. Rev. Immunol. 8, 34-47 (2008).

Non-Patent Literature 5: Ann. Rev. Immunol. 6. 251-81 (1988).

Non-Patent Literature 6: Chem. Immunol. 65, 88-110 (1997).

Non-Patent Literature 7: Eur. J. Immunol. 23, 1098-1104 (1993).

Non-Patent Literature 8: Immunol. 86, 319-324 (1995).

Non-Patent Literature 9: Proc. Natl. Acad. sci. U.S.A.) 103, 4005-4010 (2006).

Non-Patent Literature 10: J. Biol. Chem. 278, 3466-3473 (2003).

Non-Patent Literature 11: Nat. Biotech., 28, 502-10 (2011).

Non-Patent Literature 12: Endocr Relat Cancer 13, 45-51 (2006).

Non-Patent Literature 13: Nat. Rev. 10, 301-316 (2010).

Non-Patent Literature 14: Peds (2010), 23 (4), 289-297.

Non-Patent Literature 15: J. Immunol. 163(3), 1246-52 (1999) Aug 1.

Non-Patent Literature 16: Cancer Treat Rev. 36 (6), 458-67 (2010) Oct.

Non-Patent Literature 17: Future Oncol. (1), 73-85 (2012) Jan 8.

Non-Patent Literature 18: Cancer Immunol Immunother. 56 (9): 1397-406 2007 Sep.

Brief description of the essence of the present invention

Technical task.

The present invention has been made in light of these circumstances. In the object of the present invention, it is proposed to obtain an antigen-binding molecule, including: an antibody variable region that has binding activity with two different antigens (first antigen and second antigen), but does not bind to these antigens simultaneously, as well as a variable region with an antigen (third antigen) that differs from these antigens, and a pharmaceutical composition comprising an antigen-binding molecule and a method for producing an antigen-binding molecule is provided.

The solution of the problem.

The inventors of the present invention have carried out thorough research to achieve their goal. As a result, the authors successfully obtained an antigen-binding molecule, including: an antibody variable region that has binding activity with two different antigens (the first antigen and the second antigen), but does not bind to these antigens simultaneously, as well as a variable region that binds to the antigen (the third antigen) different from these antigens, and the increased activity carried out by this antigen-binding molecule due to the use of the binding activity of the antigen-binding molecule with three different antigens. Except

- 3 042507 In addition, the present inventors have successfully obtained an antigen-binding molecule capable of avoiding the cross-linking of different cells, which occurs when a standard multispecific antigen-binding molecule is bound to antigens expressed on various cells, which is believed to be responsible for the development of negative reactions when using a multispecific antigen-binding molecule as a drug.

In more detail, the present invention provides the following objects.

1. An antigen-binding molecule comprising an antibody variable region having binding activity with a first antigen and a second antigen different from the first antigen, but not simultaneously binding to the first antigen and the second antigen, and a variable region that binds to a third antigen different from the first antigen and second antigen.

2. An antigen-binding molecule comprising an antibody variable region with the heavy chain amino acid residue of the variable domain altered such that the variable region is capable of binding to a first antigen and a second antigen different from the first antigen, but not capable of binding to the first antigen and the second antigen simultaneously.

3. An antigen-binding molecule according to claim 1 or 2, wherein the variable region that does not bind both the first antigen and the second antigen simultaneously is the variable region that does not bind both the first antigen and the second antigen, each of which is expressed on different cells.

4. An antigen binding molecule according to any one of claims 1 to 3, further comprising an Fc region of an antibody.

5. An antigen-binding molecule according to claim 4, wherein the Fc region is an Fc region with reduced binding activity to the FcyR receptor compared to the Fc region of a native human IgG1 antibody.

6. An antigen binding molecule according to any one of claims 1 to 5, wherein the antigen binding molecule is a multispecific antibody.

7. An antigen-binding molecule according to any one of claims 1 to 6, wherein the variable region of the antibody capable of binding to the first antigen and the second antigen is a variable region with at least one amino acid residue altered.

8. An antigen binding molecule according to claim 7, wherein the change is a substitution or insertion of at least one amino acid residue.

9. The antigen-binding molecule according to claim 7 or 8, wherein the change is a substitution of a portion of the amino acid sequence of the variable region that binds to the first antigen with an amino acid sequence that binds to the second antigen, or insertion of an amino acid sequence that binds to the second antigen into the amino acid sequence of the variable region binding to the first antigen.

10. An antigen binding molecule according to claim 8 or 9, wherein the number of amino acid residues to be inserted is from 1 to 25.

11. An antigen binding molecule according to any one of claims 7-10, wherein the amino acid residue changed is an amino acid residue in the CDR1, CDR2, CDR3, or FR3 regions of the variable region of the antibody.

12. An antigen binding molecule according to any one of claims 7-11, wherein the altered amino acid residue is an amino acid residue in a loop.

13. An antigen-binding molecule according to any one of claims 7-11, wherein the altered amino acid residue is at least one amino acid residue selected from amino acid positions according to Kabat numbering 31-35, 50-65, 71-74 and 95-102 in severe the H chain of the variable domain, and according to Kabat numbering 24-34, 50-56 and 89-97 in the L light chain of the variable domain.

14. An antigen binding molecule according to any one of claims 1 to 13, wherein either of the first antigen and the second antigen is a molecule specifically expressed on the surface of a T cell and the other antigen is a molecule expressed on the surface of a T cell or other immunocyte.

15. The antigen-binding molecule of claim 14 wherein either the first antigen and the second antigen is CD3 and the other antigen is FcyR, TLR, lectin, IgA, immune checkpoint molecule, TNF subfamily molecule, TNFR subfamily molecule, or killer cell receptor molecule.

16. An antigen-binding molecule according to claim 14 or 15, wherein the third antigen is a molecule specifically expressed in cancer tissue.

17. A pharmaceutical composition comprising an antigen-binding molecule according to any one of claims 1-16 and a pharmaceutically acceptable carrier.

18. A method for producing an antigen-binding molecule according to any one of claims 1 to 16, wherein the method includes steps (i)-(iv):

(i) obtaining a library of antigen-binding molecules with at least one altered amino acid residue in the variable regions of the antibody, each of which binds to the first

- 4 042507 antigen or with a second antigen, and the altered variable regions differ from each other by at least one amino acid residue;

(ii) selecting from the resulting library an antigen-binding molecule comprising a variable region that exhibits binding activity to the first antigen and to the second antigen, but which does not simultaneously bind to the first antigen and to the second antigen;

(iii) culturing host cells comprising a nucleic acid encoding a variable region of an antigen-binding molecule selected in step (ii) and/or a nucleic acid encoding a variable region of an antigen-binding molecule that binds to a third antigen to express an antigen-binding molecule comprising the variable region of an antibody capable of binding to the first antigen and the second antigen, but not binding to the first antigen and the second antigen at the same time, and/or a variable region that binds to the third antigen; and (iv) isolating the antigen-binding molecule from the host cell culture.

19. The method of claim 18, wherein the variable region that does not bind both the first antigen and the second antigen at the same time contained in the antigen-binding molecule selected in step (ii) is the variable region that does not bind both the first antigen and the second antigen , each of which is expressed on different cells.

20. The method according to claim 18 or 19, wherein the host cells cultured in step (iii) further comprise a nucleic acid encoding the Fc region of the antibody.

21. The method of claim 20, wherein the Fc region is an Fc region with reduced binding activity for the FcyR receptor compared to the Fc region of a natural human IgG1 antibody.

22. The method of any one of claims 18-21, wherein the antigen-binding molecule produced is a multispecific antibody.

23. The method according to any one of claims 18-22, wherein at least one amino acid residue in the variable region in step (i) is a substituted or inserted amino acid residue.

24. The method of claim 23 wherein the number of amino acid residues inserted is between 1 and 25.

25. The method according to any one of claims 18-24, wherein the change is a change in an amino acid residue in a CDR1, CDR2, CDR3 or FR3 region in the variable region of the antibody.

26. The method of any one of claims 18-25, wherein the change is a change in the amino acid residue in the loop.

27. The method according to any one of claims 18-25, wherein the change is a change in at least one amino acid residue selected from amino acid positions according to Kabat numbering 31-35, 50-65, 71-74 and 95-102 in the heavy chain H variable domain, and according to Kabat numbering 24-34, 50-56 and 89-97 in the L light chain of the variable domain.

28. The method according to any one of claims 18-27, wherein either of the first antigen and the second antigen is a molecule specifically expressed on the surface of T cells, and the other antigen is a molecule expressed on the surface of T cells or any other immunocyte.

29. The method of claim 28 wherein either first antigen and second antigen is CD3 and the other antigen is FcyR, TLR, lectin, IgA, immune checkpoint molecule, TNF subfamily molecule, TNFR subfamily molecule, or killer cell receptor molecule.

30. The method according to claim 28 or 29, wherein the third antigen is a molecule specifically expressed in cancer tissue.

31. A method of treating cancer, comprising administering an antigen-binding molecule according to any one of claims 1-16.

32. An antigen binding molecule according to any one of claims 1 to 16 for use in the treatment of cancer.

33. The use of an antigen-binding molecule according to any one of claims 1 to 16 for the preparation of a therapeutic agent for the treatment of cancer.

34. A method for producing a therapeutic agent for cancer, comprising the step of using an antigen-binding molecule according to any one of claims 1-16.

35. Those skilled in the art will appreciate that any combination of two or more of the objects described above is also included within the scope of the present invention, unless conflicts arise based on general technical principles known to those skilled in the art.

Brief description of the figures

In FIG. 1 is a schematic diagram of an antibody that binds to a first antigen and a second antigen, but does not bind to those antigens at the same time.

In FIG. 2 is a schematic diagram of an antibody that does not crosslink due to the fact that the antibody does not bind to two antigens at the same time.

In FIG. 3 is a schematic diagram of an antibody that binds to two antigens at the same time, but does not connect two cells at the same time.

In FIG. 4 is a schematic diagram of an antibody that cross-links a cancer cell with a T-5042507 cell expressing the first receptor.

In FIG. 5 is a schematic diagram of an antibody that cross-links a cancer cell with a cell expressing a second receptor.

In FIG. 6 is a schematic diagram of an antibody that crosslinks a cancer cell to an immunocyte but does not crosslink the immunocyte.

In FIG. 7 is a graph showing the results of the CE115 assay for CD3ε by enzyme-linked immunosorbent assay (ELISA).

In FIG. 8 is a diagram of the molecular form of EGFR_ERY22_CE115.

In FIG. 9 is a graph showing the results of the TDCC analysis (SK-pca13a) for the EGFR_ERY22_CE115 form.

In FIG. 10 is a graph showing the binding activity of humanized CE115 antibodies to the CD3ε receptor.

In FIG. 11 is a graph showing the results of an electroluminescent enzyme-linked immunosorbent assay (ECL-ELISA) for detecting integrin binding of CE115 antibodies with an RGD insert.

In FIG. 12 is a graph showing the results of an ECL-ELISA assay to detect CD3ε binding of CE115 antibodies with an RGD insert.

In FIG. 13 is a graph showing the results of an ECL-ELISA assay to detect co-binding to CD3ε and integrin of CE115 antibodies with an RGD insert. The results of simultaneous binding of altered forms to the indicated proteins are shown.

In FIG. 14 is a graph showing the results of an ECL-ELISA assay to detect co-antigen binding of CE115 antibodies with an RGD insert. The results of binding of altered forms that do not bind to antigens at the same time are shown.

In FIG. 15 is a graph showing the results of an ECL-ELISA for detecting TLR2 binding of CE115 antibodies with a TLR2 binding peptide insert.

In FIG. 16 is a graph showing the results of an ECL-ELISA for detecting CD3ε binding of CE115 antibodies with a TLR2 binding peptide insert.

In FIG. 17 is a graph showing the results of an ECL-ELISA assay to detect simultaneous binding to TLR2 and CD3 of CE115 antibodies with a TLR2 binding peptide insert.

In FIG. 18 shows a typical sensogram of an antibody, with a ratio of bound components less than 0.8. The ordinate shows the value in arbitrary units (response signal). Time is indicated on the abscissa.

In FIG. 19 is a diagram of the binding of the phage-displayed Fab domain to CD3ε and IL6R.

In FIG. 20 is a diagram of the binding of the Fab domain displayed by the phage to CD3ε and human IgA (hIgA). NC denotes its binding on the plate, which did not immobilize the antigen.

In FIG. 21 is a diagram of the binding of an IgG-converted clone to CD3ε and human IgA (hIgA).

In FIG. 22 is a graph showing that the binding of an IgG-converted clone to human IgA is inhibited by the CD3ε receptor, such that the clone is unable to simultaneously bind to human IgA (hIgA) and CD3ε.

In FIG. 23 is a diagram of the binding of the Fab domain displayed by the phage to CD3ε and to human CD154. NC denotes its binding on the plate, which did not immobilize the antigen.

In FIG. 24 is a diagram of the binding of an IgG-converted clone to CD3ε and to human CD154.

In FIG. 25 is a graph showing that binding of an IgG-converted clone to human CD154 is inhibited by the CD3ε receptor such that the clone is unable to bind to human CD154 (hIgA) and CD3ε simultaneously.

Description of Embodiments of the Present Invention

As used herein, the term antibody variable region generally means a region comprising a domain consisting of four framework regions (FRs) and three complementarity determining regions (CDRs) flanking both ends of the region, and also comprising a partial sequence of these regions of sufficient length for expression binding activity with part of the antigen or the whole antigen. First of all, preferably the region includes the light chain of the variable domain (VL) of the antibody and the heavy chain of the variable domain (VH). The variable region of an antibody of the present invention may contain an arbitrary sequence, and may contain a variable region derived from any antibody, such as a mouse antibody, a rat antibody, a rabbit antibody, a goat antibody, a camel antibody, and a humanized antibody obtained by humanizing any of these antibodies without human and human antibodies. The term humanized antibody, also called reshaped antibody, is derived by grafting non-human CDR regions from mammalian antibodies, such as a mouse antibody to

- 6 042507

human CDR. Methods for detecting CDR regions are known in the art (Kabat et al.,

Sequence of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md. (1987), and Chothia et al., Nature 342: 877 (1989)). Basic genetic engineering techniques are also known in the art (see European Patent Application Publication No. EP 125023 and WO 96/02576).

The term variable region of an antibody of the present invention that does not bind both the first antigen and the second antigen means that the variable region of the antibody of the present invention cannot bind to the second antigen if it is already bound to the first antigen, and at the same time, the variable region cannot bind to the first antigen if it is already bound to the second antigen. In this context, the phrase does not simultaneously bind to the first antigen and the second antigen also includes the absence of crosslinking of the cell expressing the first antigen with the cell expressing the second antigen, or the absence of simultaneous binding to the first antigen and the second antigen, each of which is expressed on different cells. This phrase also includes the case where the variable region is capable of binding to both the first antigen and the second antigen at the same time, if the first antigen and the second antigen are not expressed on the cell membrane, as with soluble proteins, or when both antigens are deposited on the same cell, but cannot bind simultaneously to the first antigen and the second antigen, each of which is expressed on different cells. Such an antibody variable region is not particularly limited as long as the antibody variable region exhibits its functions. Examples include variable regions derived from the variable region of an IgG antibody by modifying a portion of its amino acid residues to bind to the desired antigen. The amino acids to be modified are selected, for example, from amino acids whose modifications do not affect antigen binding in the variable region of the antibody that binds to the first antigen or to the second antigen.

In this context, the phrase expressed on different cells simply means that the antigens are expressed on individual cells. The combination of such cells may include, for example, cells of the same type, such as a T cell and another T cell, or may include cells of a different type, such as a T cell and a killer cell.

In the present invention, a change in a single amino acid residue or a change in multiple amino acid residues in combination can be used.

In the case of using multiple amino acid residue changes in combination, the number of changes to be combined is not particularly limited, and may accordingly be in a range that achieves the object of the present invention. The number of changes to be combined is, for example, 2 or more and 30 or less, preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or more and 20 or less, 2 or more and 15 or less. less than, 2 or more and 10 or less, 2 or more and 5 or less, or 2 or more and 3 or less.

The plurality of amino acid residue changes to be combined may be added to the antibody variable heavy chain or antibody variable light chain alone, or may be respectively distributed in both the heavy and light chains of the variable domain.

It is acceptable to change one or more amino acid residues in the variable region, provided that antigen-binding activity is maintained. In the case of an amino acid residue change in the variable region, the resulting variable region preferably retains the binding activity of the corresponding unchanged antibody, and preferably exhibits 50% or more, more preferably 80% or more, even more preferably 100% or more of the binding activity before the change, although the variable region of the present invention is not limited to them. Binding activity can be increased by changing amino acid residues, and, for example, can be increased by 2-fold, 5-fold, or 10-fold compared to the binding activity before the changes.

Examples of a preferred region for amino acid residue changes include solvent-exposed regions and loops in the variable region. Among others, CDR1, CDR2, CDR3, FR3 and loop regions are preferred. More particularly preferred are positions according to Kabat numbering 31-35, 50-65, 71-74 and 95-102 in the variable domain of the H chain and positions according to Kabat numbering 24-34, 50-56 and 89-97 in the variable domain L- chains. During amino acid residue changes, additional amino acid residues can also be included that increase antigen-binding activity.

The term loop according to the present invention means the region containing residues that do not take part in maintaining the β-sheet structure of the immunoglobulin.

In the present invention, a change in amino acid residues means a substitution, deletion, addition, insertion or modification, or a combination thereof. In the present invention, changing amino acid residues can be used interchangeably with amino acid mutation and still used in the same sense.

The replacement of an amino acid residue is carried out by replacing with another amino acid residue with

- 7 042507 for the purpose of change, for example, according to any of the following paragraphs (a)-(c):

(a) a polypeptide chain of a region with a folded sheet or helical structure;

(b) the electrical charge or hydrophobicity of the target site; and (c) side chain size.

Amino acid residues are classified into the following groups based on the properties of their side chains:

(1) hydrophobic residues: norleucine, Met, Ala, Val, Leu and Ile, (2) neutral hydrophilic residues: Cys, Ser, Thr, Asn and Gln, (3) acidic residues: Asp and Glu, (4) basic residues : His, Lys and Arg, (5) residues affecting chain orientation: Gly and Pro; and (6) aromatic residues: Trp, Tyr and Phe.

Substitutions of an amino acid residue with an amino acid residue from the same specified group are called conservative substitutions, while substitutions of an amino acid residue from one of these groups with an amino acid residue from another group are called non-conservative substitutions.

The substitution of the present invention may be a conservative substitution or may be a non-conservative substitution. Alternatively, a conservative substitution and a non-conservative substitution may be combined.

Altering an amino acid residue also includes: selecting a variable region capable of binding to the first antigen and the second antigen, but not able to simultaneously bind to these antigens, from regions obtained by randomly changing amino acid residues, the change of which does not abolish antigen-binding activity, in the variable region of the antibody binding to the first antigen or to the second antigen, and altered by inserting a peptide previously known to have binding activity to the desired antigen in the region indicated above. Examples of known peptides that have binding activity with the desired antigen include the peptides listed in table. 1.

Table 1

Binding component/test protein Literature VEGFR J Bici Chern. 2002 Nov 8;277(45):43137-42. Epub 2002 Aug 14., EMBO J. 2000 Apr 3; 19(7):1525-33, J Med Chern. 2010 Jun 1O;53(11):4428-40. TNFR Mol Immunol. 2004 Jul;41(8):741-9., Eur J Pharmacol. 2011 Apr 10;656(1-3):119-24. TLR5 J Immunol 2010; 185; 1744-1754 TLR4 PLoS ONE February 2012 | Volume 7 | Issue 2 | e30839 TLR2 WO2006/083706A2, T cell/VLA receptor Int Immunopharmacol. 2003 Mar;3(3):435-43. PDGFR Biochemical Pharmacology (2003), 66(7), 1307-1317, FEBS Lett. 1997 Dec 15;419(2-3):166-70. Naip5(NLR) NATURE IMMUNOLOGY VOLUME 9 NUMBER 10 OCTOBER 2008 1171- Integrin WO 95/14714, WO 97/08203, WO 98/10795, WO 99/24462, J. Biol. Chem. 274: 1979-1985 FcgRIla J Biol Chem. 2009 Jan 9;284(2):1126-35 EGFR Journal of Biotechnology(2005), 116(3) 211-219 DR5 agonist Journal of Biotechnology(2006), 361(3) 522-536 CXCR4 Science 330, 1066 (2010); Vol. 330 no. 6007 pp. 1066-1071 CD40 Eur J Biochem. 2003 May;270(10):2287-94. CD154 J Mol Med (Berl). 2009 Feb;87(2):181-97.

In one aspect of the present invention, an antigen-binding molecule is provided, comprising an antibody variable region with an amino acid residue in the heavy chain of the variable domain altered such that the variable region is capable of binding to a first antigen and a second antigen different from the first antigen, but not simultaneously binding to the first antigen. and a second antigen. A variable region capable of binding to the first antigen and a second antigen different from the first antigen, but which does not simultaneously bind to the first antigen and the second antigen, can be obtained, for example, by changing the amino acid residue (substitution, deletion, insertion, addition or modification, or their combination), as described above, in the heavy chain of the variable domain. The position of the amino acid residue to be changed is preferably in the heavy chain of the variable domain. Examples of a more preferred region include solvent-exposed regions and putles in the variable domain. Among

- 8 042507 CDR1, CDR2, CDR3, FR3 and loops are preferred. In more detail, positions according to Kabat numbering 31-35, 50-65, 71-74 and 95-102 in the variable domain of the H chain are preferred, and most preferably positions according to Kabat numbering 31, 52a-61, 71-74 and 97- 101 in the variable domain of the H chain. During amino acid residue changes, additional amino acid residues can also be included that increase antigen-binding activity.

In the variable region of an antibody of the present invention, the change above can be combined with a change known in the art. For example, a modification known in the art is to modify the N-terminal glutamine in the variable region by cyclizing glutamine to form pyroglutaminin. Thus, an antibody of the present invention containing glutamine at the N-terminal position of the heavy chain may contain a variable region with the N-terminal glutamine replaced by pyroglutamic acid.

Such an antibody variable region may further include altering an amino acid residue to improve, for example, antigen binding, pharmacokinetics, stability, or antigenicity. The variable region of an antibody of the present invention can be altered in such a way as to provide a pH-dependent binding activity to an antigen and thus to obtain the ability to re-bind to an antigen (WO2009/125825).

You can also change the amino acid residues to change the antigen-binding activity in accordance with the concentration of the compound specific to the target tissue, for example, in the variable region of the antibody that binds to the third antigen (WO2013/180200).

The variable region can be further modified to e.g. increase binding activity, improve specificity, decrease IEP value, confer pH dependent antigen binding properties, improve binding thermal stability, improve solubility, improve chemical modification stability, improve carbohydrate chain heterogeneity, eliminate T an epitope identified by in silico prediction or in vitro T cell based assay to reduce immunogenicity, or to include a T cell epitope to activate regulatory T cells (mAbs 3: 243-247, 2011).

The ability of the variable region of the present invention to bind to the first antigen and the second antigen can be assessed by a method known in the art.

Such a method includes, for example, the electroluminescent method (ECL method) (BMC Research Notes 2011, 4: 281).

In more detail, for example, a small molecule antibody comprising a region capable of binding to a first antigen and a second antigen, such as a Fab region, a biotin labeled antigen-binding molecule to be analyzed, or a monovalent antibody (an antibody that does not contain one of the two Fab regions , which are contained in a conventional antibody), are mixed with the first antigen or the second antigen containing the sulfo-label (Ru complex), and the mixture is added to the plate with immobilized streptavidin. At this stage, the biotin labeled antigen-binding molecule to be analyzed is bound to the streptavidin on the plate. The radiation observed from the sulfo tag and the luminescent signal can be measured using a Sector Imager 600 or 2400 (MSD K.K.) or similar device, thereby confirming the binding of the above region of the antigen-binding molecule to be analyzed with the first antigen or the second antigen.

Alternatively, such an analysis can be carried out by ELISA, FACS (fluorescence-activated cell sorting), ALPHAScreen (homogeneous proximity-enhanced luminescence), BIACORE (a method based on the surface plasmon resonance (SPR) phenomenon, etc. ( Proc Natl Acad Sci USA 103 (11), 4005-4010 (2006)).

In more detail, the analysis can be performed using, for example, a Biacore interaction analyzer (GE Healthcare Japan Corp.) based on the phenomenon of surface plasmon resonance (SPR). The Biacore analyzer includes any model such as Biacore T100, T200, X100, A100, 4000, 3000, 2000, 1000, or C. Any sensor chip can be used as the sensor chip for the Biacore analyzer, such as CM7, CM5, CM4, CM3, C1, SA, NTA, L1, HPA or Au. For immobilizing on the sensor chip the antigen-binding molecule capture proteins of the present invention, such as protein A, protein G, protein L, anti-human IgG antibodies, anti-human IgG-Fab antibodies, anti-human light chain antibodies, anti-human Fc antibodies, antigenic proteins or antigenic peptides, a condensation method such as amino condensation, disulfide condensation, or aldehyde condensation can be used. To obtain a sensogram, the first antigen or the second antigen is applied to the chip as the analyzed compound and the interaction is measured. In this operation, the concentration of the first antigen or the second antigen can be selected in the range from a few μm to a few pM in accordance with the strength of the interaction (eg, KD) of the analyzed sample.

Alternatively, instead of immobilizing the antigen-binding molecule on the sensor chip, one can immobilize the first antigen or the second antigen and evaluate the interaction with the antibody sample to be analyzed. The ability of the variable region of the antigen-binding molecule of the present invention to bind to the first antigen and the second antigen can be confirmed using the dissociation constant (KD) value calculated from the interaction sensorgram or by the degree of increase in the signal on the sensorogram after exposure to the antigen-binding molecule sample compared to the level before interaction.

The ALPHAScreen assay is carried out using ALPHA technology using two types of beads (donor and acceptor) based on the following principle: a luminescent signal is detected only when these two types of beads approach each other in the biological interaction of the molecule associated with the donor bead and the molecule associated with the acceptor bead . The laser-activated photosensitizer in the donor granule converts atmospheric oxygen into excited singlet oxygen. Singlet oxygen diffuses around the donor bead and reaches a nearby acceptor bead, thereby inducing a chemiluminescent reaction in the bead, which eventually emits light. In the absence of interaction between the molecule bound to the donor bead and the molecule bound to the acceptor bead, the singlet oxygen generated in the donor bead does not reach the acceptor bead. As a result, the chemiluminescent reaction does not occur.

One of the substances (ligand), between which interaction should be observed, is immobilized on a thin gold film of the sensor chip. The sensor chip is irradiated from below with light in such a way that total reflection occurs at the interface between the thin gold film and the glass. As a result, in part of the reflected light, a section with a decrease in the reflection intensity (SPR signal) is formed. The other of the substances (analyte) between which an interaction is to be observed is introduced onto the surface of the sensor chip. As the analyte binds to the ligand, the mass of the immobilized ligand molecule increases, changing the refractive index of the solvent on the surface of the sensor chip. Such a change in the refractive index shifts the position of the SPR signal (and vice versa, the dissociation of bound molecules shifts the signal to its original position). In the Biacore system, the y-axis plots the amount of shift, that is, the change in mass on the surface of the sensor chip, and displays the time-dependent change in mass as analysis data (sensogram). The amount of ligand-bound analyte captured on the surface of the sensor chip (quantitative change in signal response on the sensorgram between before and after the interaction of the analyte) can be determined using the data on the sensorgram. However, since the amount of bound substance also depends on the amount of ligand, the comparison should be made under conditions in which substantially equal amounts of ligand are used. The kinetics, ie the association rate constant (k _a ) and the dissociation rate constant (k _d ), can be determined from the curve on the sensogram, while the affinity constant (K _D ) can be determined from the ratio of these constants. The inhibition assay is also preferably carried out by the Biacore method. Examples of inhibition assays are described in Proc. Natl. Acad. sci. USA 103 (11), 4005-4010 (2006).

The ability of the antigen-binding molecule of the present invention not to bind simultaneously to the first antigen and the second antigen can be confirmed as follows: confirm the binding activity with the first antigen and the second antigen, and then ensure that the first antigen or the second antigen has previously bound to the antigen-binding molecule, including the variable region, which has such binding activity, and then determine the presence or absence of its binding activity with another antigen by the method described above.

Alternatively, this ability can also be confirmed by determining the inhibition of binding of an antigen-binding molecule to a first antigen or a second antigen immobilized on an ELISA plate or sensor chip when another antigen is added to the solution.

In more detail, in the case of using the ECL method, a biotin-labeled antigen-binding molecule of interest, a sulfo-labelled first antigen (Ru complex), and an unlabeled second antigen are prepared. If the tested antigen-binding molecule is able to bind to the first antigen and the second antigen, but does not bind simultaneously to the first antigen and the second antigen, then the luminescent signal from the sulfo-label is detected in the absence of unlabeled second antigen when a mixture of the tested antigen-binding molecule and the first antigen is added to the wells of the plate with immobilized streptavidin followed by light irradiation. Conversely, the luminescent signal will decrease in the presence of the second antigen. This reduction in luminescent signal can be used to quantify binding activity.

In the case of the ALPHAScreen assay, the antigen-binding molecule of interest interacts with the first antigen in the absence of a competing second antigen, generating signals at 520-620 nm. The unlabeled second antigen competes with the first antigen for binding to the antigen-binding molecule of interest. The decrease in fluorescence caused by competition can be quantified and thereby determine the relative binding activity. Biotinylation of polypeptides using sulfo-NHS-biotin or the like is known in the art. A GST (glutathione-S-transferase) tag can be incorporated into the first antigen using a specially optimized method, which includes, for example, melt fusion of the polynucleotide encoding the first antigen with the polynucleotide encoding the GST and expressing the resulting fusion gene. in cells or similar carrier vectors capable of expressing them, and then purification on a column with immobilized glutathione. The resulting signals are preferably analyzed using, for example, GRAPHPAD PRISM software (GraphPad Software, Inc., San Diego) optimized for a single site competition model based on non-linear regression analysis. Such an assay can be carried out in a similar manner using a labeled second antigen and an unlabeled first antigen.

Alternatively, the fluorescence resonance energy transfer (FRET) method can be used. FRET means a phenomenon in which excitation energy is transferred directly from one fluorescent molecule to another fluorescent molecule located close to each other by means of electronic resonance. When the FRET phenomenon occurs, the excitation energy of the donor (a fluorescent molecule in an excited state) is transferred to an acceptor (another fluorescent molecule located near the donor) so that the fluorescence emitted by the donor disappears (more precisely, the fluorescence emission time decreases), and the acceptor emits fluorescence . Using this phenomenon, the presence or absence of a double Fab fragment can be analyzed. For example, if the first antigen carries a fluorescent donor and the second antigen carries a fluorescent acceptor bound to an antigen-binding molecule to be analyzed at the same time, then the donor's fluorescence disappears while the acceptor emits fluorescence. Therefore, a change in the fluorescence wavelength is observed. Thus, it is confirmed that the antibody does not contain a double Fab. On the other hand, if the mixing of the first antigen, the second antigen and the antigen-binding molecule to be analyzed does not change the fluorescence wavelength of the donor bound to the first antigen, then the antigen-binding molecule to be analyzed can be considered to contain a double Fab.

For example, a biotin-labeled antigen-binding molecule to be analyzed is incubated to bind to streptavidin on a donor bead, while a first GST-labeled antigen is incubated to bind to an acceptor bead. The antigen-binding molecule to be analyzed interacts with the first antigen in the absence of a competing second antigen, generating signals in the range of 520 to 620 nm. The unlabeled second antigen competes with the first antigen for interaction with the antigen-binding molecule to be analyzed. The decrease in fluorescence caused by competition can be quantified, thereby determining the relative binding activity. Biotinylation of polypeptides using sulfo-NHS-biotin or the like is known in the art. A GST (glutathione-S-transferase) tag can be incorporated into the first antigen using a specially optimized method, which includes, for example, fusing a polynucleotide encoding the first antigen in a melt with a polynucleotide encoding a GST, and expressing the resulting hybrid gene in cells or similar carrier vectors capable of expressing them, and then purification on a column with immobilized glutathione. The resulting signals are preferably analyzed using, for example, GRAPHPAD PRISM software (GraphPad Software, Inc., San Diego) optimized for a single site competition model based on non-linear regression analysis.

Inclusion of a tag is not limited to a GST tag, and any tag can be included, such as, but not limited to, a histidine tag, MBP, CBP, Flag, HA, V5, or c-myc tags. Binding of the antigen-binding molecule to be assayed to the donor bead is not limited to binding using the biotin-streptavidin reaction. First of all, if the antigen-binding molecule comprises an Fc, then an exemplary method involves incubating the antigen-binding molecule to be assayed to bind through an Fc-recognizing protein such as protein A or protein G on a donor bead.

And also in the case when the variable region is able to bind to the first antigen and the second antigen simultaneously, if the first antigen and the second antigen are not expressed on cell membranes, as well as with soluble proteins, or if both are deposited on the same cell, but do not bind with a first antigen and a second antigen, each of which is expressed on different cells, can be analyzed simultaneously by a method known in the art.

In more detail, the antigen-binding molecule to be analyzed and confirmed to bind both the first antigen and the second antigen by ECL-ELISA is also mixed with the cell expressing the first antigen and the cell expressing the second antigen. The inability of the analyzed antigen-binding molecule to bind to the first antigen and the second antigen expressed on different cells at the same time can be detected, provided that the antigen-binding molecule and these cells do not simultaneously bind to each other. This analysis can be carried out, for example, by cell-based ECL-ELISA. The cell expressing the first antigen is preliminarily immobilized on the plate. After the analyzed antigen-binding molecule has bound to the first antigen, a cell expressing the second antigen is added to the plate. Another antigen expressed only on the cell expressing the second antigen

- 11 042507 are detected using sulfo-labeled antibodies against this antigen. The signal is recorded when the antigen-binding molecule binds simultaneously to two antigens respectively expressed on two cells. There is no signal if the antigen-binding molecule does not simultaneously bind to these antigens.

Alternatively, this analysis can be performed by the ALPHAScreen method. The antigen-binding molecule to be analyzed is mixed with a cell expressing a first antigen associated with a donor bead and a cell expressing a second antigen associated with an acceptor bead. A signal is recorded if the antigen-binding molecule simultaneously binds to two antigens expressed on two cells, respectively. The signal is not registered if the antigen-binding molecule does not simultaneously bind to these antigens.

Alternatively, this analysis can be performed using the octet interaction method. First, a cell expressing the peptide-labeled first antigen is incubated to bind to a biosensor that recognizes the peptide label. The cell expressing the second antigen and the antigen-binding molecule to be analyzed are placed in the wells and the interaction is analyzed. A significant wavelength shift caused by the binding of the analyzed antigen-binding molecule and the cell expressing the second antigen to the biosensor is observed when the antigen-binding molecule simultaneously binds to two antigens expressed on two cells, respectively. A slight wavelength shift caused by the binding of only the analyzed antigen-binding molecule to the biosensor is observed when the antigen-binding molecule does not bind simultaneously to these antigens.

Instead of these methods based on binding activity, you can use the analysis based on biological activity. For example, a cell expressing a first antigen and a cell expressing a second antigen are mixed with the antigen-binding molecule to be analyzed and cultured. Two antigens expressed by two cells, respectively, are co-activated in the presence of the analyzed antigen-binding molecule when the antigen-binding molecule binds to the two antigens simultaneously. Therefore, a change in the activation signal, such as increased levels of subsequent antigen phosphorylation, can be detected. In another embodiment, the activation induces the production of cytokines. Therefore, it is possible to measure the amount of cytokines produced, thereby confirming the presence or absence of simultaneous binding to two cells.

Used in this context, the term Fc region means the region including the hinge region or part of it and the CH2 and CH3 domains in an antibody molecule. The Fc region of the IgG class means, but is not limited to, a fragment from cysteine 226 (Kabat numbering or EU index used in this context) to the C-terminus or from proline 230 to the C-terminus. The Fc region can preferably be obtained by partially digesting, for example, IgG1, IgG2, IgG3, or IgG4 monoclonal antibodies with a proteolytic enzyme such as pepsin, and then eluting the protein A or protein G immobilized column fraction. Such a proteolytic enzyme is not particularly limited, provided that the enzyme is capable of cleaving a full-length antibody to form Fab or F(ab')2 fragments under appropriate conditions for the enzyme (eg, pH). Examples of such enzymes may include pepsin and papain.

As used herein, the term antigen-binding molecule is not particularly limited as long as the molecule includes the variable region of an antibody of the present invention. The antigen binding molecule may further include a peptide or protein of approximately 5 or more amino acid residues in length. The peptide or protein is not limited to a peptide or protein derived from an organism, and may mean, for example, a polypeptide containing an artificial sequence. A natural polypeptide, a synthetic polypeptide, a recombinant polypeptide or the like may also be used.

Preferred examples of an antigen-binding molecule of the present invention may include an antigen-binding molecule containing the Fc region of an antibody.

As the Fc region of the present invention, natural IgG, for example, can be used. In this context, natural IgG means a polypeptide containing an amino acid sequence identical to natural IgG, and refers to a class of antibodies that are mainly encoded by the gamma-immunoglobulin gene. Natural human IgG means, for example, natural human IgG1, natural human IgG2, natural human IgG3 or natural human IgG4. Natural IgGs also include variants or similar compounds spontaneously derived from them. Many allotypic sequences based on gene polymorphism are described as conserved regions of human IgG1, human IgG2, human IgG3 and human IgG4 in the list of immunological proteins, see NIH National Health Institute Publication No. 91-3242, and any of these immunoglobulins can be used in the present invention. First of all, the sequence of human IgG1 may contain the amino acid sequences DEL or EEM, according to Kabat numbering fragment 356-358.

The Fc region of an antibody was found to be the Fc region of immunoglobulins of the type

- 12 042507

IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 or IgM. As the Fc region of the antibody of the present invention, for example, the Fc region from natural human IgG can be used. As the Fc region of the antibody of the present invention, the Fc region from the conserved region of natural IgG can be used, in more detail, the conserved region (SEQ ID NO: 1) obtained from natural human IgG1, the conserved region (SEQ ID NO: 2) obtained from natural human IgG2, a conserved region (SEQ ID NO: 3) derived from natural human IgG3, or a conserved region (SEQ ID NO: 4) derived from natural human IgG4. The conserved region of natural IgG also includes variants or similar compounds spontaneously derived from them.

The Fc region of the present invention is preferably and primarily an Fc region having reduced binding activity to the Fcy receptor. In this context, an Fcy receptor (herein referred to as FcyR) means a receptor capable of binding to the Fc region of IgG1, IgG2, IgG3, or IgG4, and means any member of the family of proteins primarily encoded by Fcy receptor genes. In humans, this family includes, but is not limited to: FcyRI (CD64), including the FcyRIa, FcyRIb or FcyRIc isoforms; FcyRII (CD32), including isoforms FcyRIIa (including allotypes H131 (H type) and R131 (R type)), FcyRIIb (including FcyRIIb-1 and FcyRIIb-2), and FcyRIIc; and FcyRIII (CD16), including the isoforms FcyRIIIa (including allotypes V158 and F158) and FcyRIIIb (including allotypes FcyRIIIb-NA1 and FcyRIIIb-NA2); and any not yet discovered human FcyR or isoform or allotype of human FcyR. FcyR includes forms isolated from humans, mice, rats, rabbits and monkeys. FcyR is not limited to these molecules and can be obtained from any organism. Mouse FcyRs include, but are not limited to, FcyRI (CD64), FcyRII (CD32), FcyRIII (CD16), and FcyRIII-2 (CD16-2), and any yet-to-be-discovered mouse FcyR or FcyR isoform or allotype. Preferred examples of such Fcy receptors include human FcyRI (CD64), FcyRIIa (CD32), FcyRIIb (CD32), FcyRIIIa (CD16) and/or FcyRIIIb (CD16).

FcyR is found in the forms of an activating receptor containing ITAM (activated tyrosine immunoreceptor motif) and an inhibitory receptor containing ITIM (inhibitory tyrosine immunoreceptor motif). FcyRs are classified into FcyR activating (FcyRI, FcyRIIa R, FcyRIIa H, FcyRIIIa and FcyRIIIb) FcyR inhibitory (FcyRIIb).

The polynucleotide sequence and amino acid sequence of FcyR are listed in NM_000566.3 and NP_000557.1, respectively; the polynucleotide sequence and amino acid sequence of FcyRIIa are listed in BC020823.1 and AAH20823.1, respectively; the polynucleotide sequence and amino acid sequence of FcyRIIb are listed in BC146678.1 and AAI46679.1, respectively; the polynucleotide sequence and amino acid sequence of FcyRIIIa are listed in BC033678.1 and AAH33678.1, respectively; and the polynucleotide sequence and amino acid sequence of FcyRIIIb are listed in BC128562.1 and AAI28563.1, respectively (RefSeq registration numbers). FcyRIIa is characterized by two types of gene polymorphism, where the 131st amino acid residue in the FcyRIIa sequence is replaced by a histidine (H type) or arginine (R type) residue (J. Exp. Med, 172, 19-25, 1990). FcyRIIb is characterized by two types of gene polymorphism, where the 232nd amino acid residue in the FcyRIIb sequence is replaced by an isoleucine (type I) or threonine (type T) residue (Arthritis. Rheum. 46: 1242-1254 (2002)). FcyRIIIa is characterized by two types of gene polymorphism, where the 158th amino acid residue in the FcyRIIIa sequence is replaced by a valine (V type) or phenylalanine (F type) residue (J. Clin. Invest. 100 (5): 1059-1070 (1997)). FcyRIIIb is characterized by two types of gene polymorphism (NA1 type and NA2 type) (J. Clin. Invest. 85: 1287-1295 (1990)).

Reduced binding activity to the Fcy receptor can be confirmed by a widely used method such as FACS, ELISA, ALPHAScreen (homogeneous proximity enhanced luminescence assay) or the BIACORE method based on the surface plasmon resonance (SPR) phenomenon (Proc. Natl. Acad. Sci. USA 103 (11), 4005-4010 (2006)).

The ALPHAScreen assay is carried out using ALPHA technology using two types of beads (donor and acceptor) based on the following principle: a luminescent signal is detected only when these two types of beads approach each other in the biological interaction of the molecule associated with the donor bead and the molecule associated with the acceptor bead . The laser-activated photosensitizer in the donor granule converts atmospheric oxygen into excited singlet oxygen. Singlet oxygen diffuses around the donor bead and reaches a nearby acceptor bead, thereby inducing a chemiluminescent reaction in the bead, which eventually emits light. In the absence of interaction between the molecule bound to the donor bead and the molecule bound to the acceptor bead, the singlet oxygen generated in the donor bead does not reach the acceptor bead. As a result, the chemiluminescent reaction does not occur.

For example, a biotin-labeled antigen-binding molecule intended for analysis, in

- 13 042507 are cubated to bind to the donor bead while the Fcy receptor labeled with GST (glutathione S-transferase) is incubated to bind to the acceptor bead. In the absence of a competing antigen-binding molecule containing a mutated Fc region, an antigen-binding molecule containing a wild-type Fc region interacts with the Fcy receptor, generating signals in the range of 520 to 620 nm. An unlabeled antigen-binding molecule containing a mutated Fc region competes with an antigen-binding molecule containing a wild-type Fc region for interaction with the Fcy receptor. The decrease in fluorescence caused by competition can be quantified, thereby determining the relative binding activity. Biotinylation of an antigen-binding molecule (eg, antibody) using sulfo-NHS-biotin or the like is known in the art. A GST (glutathione-S-transferase) tag can be incorporated into the Fcy receptor using a specially optimized method, which includes, for example, fusing a polynucleotide encoding an Fcy receptor in a melt with a polynucleotide encoding a GST, and expressing the resulting hybrid gene in cells or similar vectors. carriers capable of expressing them, and then purification on a column with immobilized glutathione. The resulting signals are preferably analyzed using, for example, GRAPHPAD PRISM software (GraphPad Software, Inc., San Diego) optimized for a single site competition model based on non-linear regression analysis.

One of the substances (ligand), between which interaction should be observed, is immobilized on a thin gold film of the sensor chip. The sensor chip is irradiated from below with light in such a way that total reflection occurs at the interface between the thin gold film and the glass. As a result, in part of the reflected light, a section with a decrease in the reflection intensity (SPR signal) is formed. One of the other substances (the analyte) between which an interaction is to be observed is introduced onto the surface of the sensor chip. As the analyte binds to the ligand, the mass of the immobilized ligand molecule increases, changing the refractive index of the solvent on the surface of the sensor chip. Such a change in the refractive index shifts the position of the SPR signal (and vice versa, the dissociation of bound molecules shifts the signal to its original position). In the Biacore system, the y-axis plots the amount of shift, that is, the change in mass on the surface of the sensor chip, and displays the time-dependent change in mass as analysis data (sensogram). The kinetics, ie the association rate constant (k _a ) and the dissociation rate constant (kd) can be determined from the curve on the sensogram, while the affinity constant (K _D ) can be determined from the ratio of these constants. The inhibition assay is also preferably carried out by the Biacore method. Examples of inhibition assays are described in Proc. Natl. Acad. sci. USA 103 (11), 40054010 (2006).

As used herein, reduced Fcy receptor binding activity means that the antigen-binding molecule being analyzed exhibits binding activity, e.g., 50% activity or less, preferably 45% or less, 40% or less, 35% or less, 30% or less, 20% or less, or 15% or less, especially preferably 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less than, 2% or less, or 1% or less, as compared to the binding activity of a control antigen-binding molecule containing an Fc region, as determined by the assay described above.

As a control antigen-binding molecule, an antigen-binding molecule containing the Fc region of an IgG1, IgG2, IgG3 or IgG4 monoclonal antibody can suitably be used. The structure of the Fc region is described in the sequence SEQ ID NO: 1 (RefSeq No. AAC82527.1 with A added at the N-terminus), SEQ ID NO: 2 (RefSeq No. AAB59393.1 with A added at the N-terminus), SEQ ID NO: 3 (RefSeq Registration No. CAA27268.1 with an A residue added at the N-terminus), or SEQ ID NO: 4 (RefSeq Registration No. AAB59394.1 with an A residue, added at the N-terminus). In the case of using an antigen-binding molecule containing a variant Fc region of an antibody of a particular isotype as an analyte, an antigen-binding molecule containing an Fc region of an antibody of that particular isotype can be used as a control substance to study the effect of mutation of this variant on binding activity to the Fcy receptor. Thus, it was confirmed that a corresponding antigen-binding molecule containing a variant Fc region with reduced binding activity to the Fcy receptor was obtained.

For example, as such a variant, the prior art describes variants with the following changes: deletion 231A-238S (WO 2009/011941), C226S, C229S, P238S, (C220S) (J. Rheumatol (2007) 34, 11), C226S, C229S (Hum. Antibod. Hybridomas (1990) 1 (1), 47-54), C226S, C229S, E233P, L234V or L235A (Blood (2007) 109, 1185-1192) (numbering of these amino acid residues is given according to Kabat numbering) .

Preferred examples of such variants include antigen-binding molecules containing an Fc region derived from the Fc region of an antibody of a particular isotype by substituting any of the following constituent amino acid residues at positions 220, 226, 229, 231, 232, 233, 234, 235,

- 14 042507

236, 237, 238, 239, 240, 264, 265, 266, 267, 269, 270, 295, 296, 297, 298, 299, 300, 325, 327, 328, 329, 330,

331 and 332, according to Kabat numbering. The isotype of the antibody from which the Fc region is derived is not particularly limited, and the Fc region of an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody can equally be used. It is preferable to use the Fc region from a natural human IgG1 antibody.

For example, one can equally use an antigen-binding molecule derived from the Fc region of an IgG1 antibody by substitutions of the following groups of constituent amino acids (the number corresponds to the position of the amino acid residue determined according to Kabat numbering, the one-letter amino acid code before the number indicates the amino acid residue before the substitution, and the one-letter amino acid code indicated after the number means the amino acid residue after the substitution):

(a) L234F, L235E and P331S, (b) C226S, C229S and P238S, (c) C226S and C229S and (d) C226S, C229S, E233P, L234V and L235A, or by deleting the amino acid sequence at positions 231-239 according to numbering Kabat.

One can equally use an antigen-binding molecule derived from the Fc region of an IgG2 antibody by substitutions of the following groups of constituent amino acids (the number corresponds to the position of the amino acid residue determined according to Kabat numbering, the one-letter amino acid code before the number indicates the amino acid residue before the substitution, and the one-letter code amino acids indicated after the number means the amino acid residue after the replacement):

(e) H268Q, V309L, A330S and P331S, (f) V234A, (g) G237A, (h) V234A and G237A, (i) A235E and G237A and (j) V234A, A235E and G237A.

One can equally use an antigen-binding molecule derived from the Fc region of an IgG3 antibody by substitutions of the following groups of constituent amino acids (the number corresponds to the position of the amino acid residue determined according to Kabat numbering, the one-letter amino acid code before the number indicates the amino acid residue before the substitution, and the one-letter code the amino acid indicated after the number means the amino acid residue after substitution: (k) F241A, (m) D265A and (n) V264A.

One can equally use an antigen-binding molecule derived from the Fc region of an IgG4 antibody by substitutions of the following groups of constituent amino acids (the number corresponds to the position of the amino acid residue determined according to Kabat numbering, the one-letter amino acid code before the number indicates the amino acid residue before the substitution, and the one-letter code the amino acid indicated after the number means the amino acid residue after substitution: (o) L235A, G237A and E318A, (n) L235E and (p) F234A and L235A.

Other preferred examples of such substitutions include an antigen-binding molecule derived from the Fc region of natural human IgG1 by substituting the following constituent amino acids: 233, 234, 235, 236, 237, 327, 330 and 331, determined according to Kabat numbering, with the amino acid residue of the Fc region in corresponding position according to Kabat numbering from the same IgG2 or IgG4 sequence.

Other preferred examples of such substitutions include an antigen-binding molecule derived from the Fc region of natural human IgG1 by replacing any one or more of the following constituent amino acids: 234, 235 and 297, defined according to Kabat numbering, with a different amino acid residue. The type of amino acid residue to which the substitution has been made is not particularly limited. First of all, an antigen-binding molecule containing an Fc region with any one or more substitutions of amino acid residues at positions 234, 235 and 297 with an alanine residue is preferred.

Other preferred examples of such substitutions include an antigen-binding molecule derived from the Fc region of natural human IgG1 by substituting a constituent amino acid residue at position 265, defined according to Kabat numbering, with a different amino acid residue. The type of amino acid residue to which the substitution has been made is not particularly limited. First of all, an antigen-binding molecule containing an Fc region with the amino acid residue at positions 265 replaced by an alanine residue is preferred.

A preferred form of the antigen-binding molecule of the present invention may be, for example, a multispecific antibody comprising the variable region of the present invention.

To associate a multispecific antibody, the method of suppressing undesirable association between H heavy chains by introducing a repulsive electric charge at the interface between the second conserved domains (CH2) and the third conserved domains (CH3) in the antibody heavy chains can be used (WO2006/106905 ).

In a method for suppressing unwanted association between H heavy chains by introducing a repulsive electrical charge at the CH2 or CH3 interface, examples of amino acid residues contacting each other at the interface between conserved H heavy chain domains may include residue 356 according to Kabat numbering, residue 439 according to Kabat numbering, residue 357 according to Kabat numbering, residue 370 according to Kabat numbering, residue 399 according to Kabat numbering, and residue 409 according to Kabat numbering in one CH3 domain, and their respective residues in another CH3 domain.

In more detail, for example, an antibody comprising two heavy chain CH3 domains can be prepared in the form of an antibody in which one to three amino acid residue pairs selected from the following amino acid residue pairs (1)-(3) in the first heavy chain, domain CH3 carries the same electrical charge:

(1) amino acid residues 356 and 439 according to Kabat numbering contained in the heavy chain CH3 domain, (2) amino acid residues 357 and 370 according to Kabat numbering contained in the heavy chain CH3 domain, and (3) amino acid residues 399 and 409 contained in CH3 heavy chain domain.

In addition, the antibody can be obtained in the form of an antibody in which one to three pairs of amino acid residues selected from the following pairs of amino acid residues (1) to (3) in the second heavy chain, the CH3 domain is different from the CH3 domain of the first heavy chain, in such a way that that it carries the same electrical charge as the CH3 domain in the first heavy chain, but the opposite charge to the corresponding amino acid residues of the CH3 domain in the first heavy chain.

Each amino acid residue described in pairs (1)-(3) is located near its component in the associated H heavy chains. One skilled in the art will be able to select the appropriate amino acid residues described in each pair (1)-(3) in the required domains. CH3 of the heavy chain or conserved heavy chain domains using homologous modeling or similar methods and appropriate commercial software, and modify the amino acid residues at the selected positions accordingly.

In the antibody described above, each of the residues carrying an electrical charge is preferably selected from, for example, amino acid residues included in any of the following groups (a) and (b):

(a) glutamic acid (E) and aspartic acid (D), and (b) lysine (K), arginine (R) and histidine (H).

In an antibody described above, the expression carrying the same electrical charge means that, for example, all of two or more amino acid residues are amino acid residues included in any of groups (a) and (b). The expression carrying opposite electrical charge means that, for example, one amino acid residue of two or more amino acid residues may refer to any of groups (a) and (b), while the other amino acid residue(s) may refer to the amino acid residue ( residues) included in another group.

In a preferred embodiment of the present invention, the antibody may comprise a first heavy chain CH3 domain and a second heavy chain CH3 domain linked by a disulfide bond.

The variable amino acid residue of the present invention is not limited to the amino acid residue in the variable region of an antibody or the conserved region of an antibody as described above. One skilled in the art can select the amino acid residues constituting the interface in the polypeptide variant or heteromultimer by homologous modeling or the like using commercial software and change the amino acid residue to control association.

Association for the formation of multispecific antibodies of the present invention can also be carried out according to an alternative method known in the art. The side chain of an amino acid residue present in the variable region of one heavy chain of the antibody is replaced with a longer side chain (protrusion), and the side chain of its component present in the variable domain of another heavy chain is replaced with a shorter side chain (depression). The protrusion can be located in a recess that effectively promotes association of Fc domain polypeptides (WO1996/027011; Ridgway JB et al., Protein Engineering 9, 617-621 (1996), and Merchant AM et al., Nature Biotechnology 16, 677-681 ( 1998)).

In addition to this method, another method known in the art can be used to generate the multispecific antibody of the present invention. The CH3 portion of one heavy chain of an antibody is converted into an IgA-derived component sequence, and the complementary portion in the CH3 of the other heavy chain is converted into an IgA-derived component sequence. The use of a genetically engineered (strand exchange) CH3 domain can induce efficient association between polypeptides of different sequences by complementary CH3 association (Protein Engineering Design & Selection, 23; 195-202, (2010)). Using this

- 16 042507 method known in the art, you can also effectively get the investigated multispecific antibody.

In another embodiment, a multispecific antibody can be obtained, for example, by the technology of generating antibodies using the association of antibodies CH1-CL and VH-VL, as described in application WO2011/028952, by the technology of producing a bispecific antibody using single monoclonal antibodies (Fab arm exchange), as described in WO2008/119353 and WO2011/131746, in the technology for controlling the association of CH3 domains of antibody heavy chains, as described in WO2012/058768 and WO2013/063702, in the technology for producing a bispecific antibody consisting of two types of light chains and one type of heavy chains as described in WO2012/023053 or a bispecific antibody technique using two bacterial cell lines each expressing half an antibody molecule consisting of one H heavy chain and one L light chain as described by Christoph et al. (Nature Biotechnology Vol. 31, p. 753-758 (2013)). In addition to these association methods, the CrossMab technology, known as the heteroassociation technology of a light chain forming a variable region associated with a first epitope and a light chain forming a variable region associated with a second epitope, can also be used to obtain a multispecific or multiparatopic antigen-binding molecule of the present invention, with a heavy chain forming a variable region associated with a first epitope and a heavy chain forming a variable region associated with a second epitope, respectively (Scaefer et al., Proc. Natl. Acad. Sci. U.S.A. 108, 11187-11192 (2011)) . Examples of technology for producing a bispecific antibody using separately prepared monoclonal antibodies may include a method that includes promoting antibody heterodimerization by incorporating monoclonal antibodies with a specific amino acid substitution in the CH3 domain of the heavy chain under reducing conditions to produce the desired bispecific antibody. Examples of amino acid substitution positions preferred by this method may include residue 392 according to Kabat numbering and residue 397 according to EU numbering in the CH3 domain. Moreover, a bispecific antibody can be made by using an antibody in which one to three pairs of amino acid residues selected from the following pairs of amino acid residues (1)-(3) in the first heavy chain, the CH3 domain carries the same electrical charge:

(1) EU amino acid residues 356 and 439 contained in the heavy chain CH3 domain, (2) EU amino acid residues 357 and 370 contained in the heavy chain CH3 domain, and (3) amino acid residues 399 and 409 contained in CH3 heavy chain domain.

A bespecifically antibody can also be prepared by using an antibody in which one to three amino acid residue pairs selected from the following amino acid residue pairs (1) to (3) in the second heavy chain have a CH3 domain different from the first heavy chain's CH3 domain such that it carries the same electrical charge as the CH3 domain in the first heavy chain, but carries the opposite charge to the corresponding amino acid residues of the CH3 domain in the first heavy chain.

Even if a multispecific antibody of interest cannot be efficiently generated, it can be obtained by isolating and purifying the antibody of interest from a plurality of produced antibodies. For example, the previously described method involves introducing an amino acid substitution into the variable domains of two types of heavy chains of interest in order to change the isoelectric points (IEP) of these chains in such a way as to allow purification of the two types of antibody homodimers and heterodimers, each separately, by ion exchange chromatography. (WO2007114325). Previously, as a method for purifying heterodimers, a method involving the use of protein A to purify the heterodimer of an antibody comprising a heavy chain H of a mouse IgG2a antibody capable of binding to protein A and a heavy chain H of a rat IgG2 antibody incapable of binding to protein A was published (WO98050431 and WO95033844). Alternatively, amino acid residues 435 and 436 according to Kabat numbering, which are contained in the binding site of protein A in IgG, can be replaced with amino acids such as Tyr and His, which provide different binding strength to protein A, and the resulting heavy chain H can be used for each H strand to interact with protein A. As a result, only the antibody heterodimer can be efficiently purified using a protein A immobilized column.

A plurality of two or more such methods, for example, may be used in combination. Such methods can also be applied separately to the two heavy chains intended for association. Based on the antigen-binding molecule of the present invention, but separately from the thus modified form, it is possible to obtain an antigen-binding molecule with an identical amino acid sequence to these forms.

Changing the amino acid sequence can be done by various methods known in the art. Examples of such methods that can be used include, but are not limited to, site-directed mutagenesis (Hashimoto-Gotoh T, Mizuno T, Ogasahara Y.

- 17 042507 and Nakagawa M., An oligodeoxyribonucleotide-directed dual amber method for site-directed mutagenesis, Gene 152, 271-275 (1995), Zoller MJ and Smith M., Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors, Methods Enzymol. 100, 468-500 (1983), Kramer W, Drutsa V, Jansen HW, Kramer B, Pflugfelder M. and Fritz HJ, The gapped duplex DNA approach to oligonucleotide-directed mutation construction, Nucleic Acids Res. 12, 9441-9456 (1984), Kramer W and Fritz HJ, Oligonucleotide-directed construction of mutations via gapped duplex DNA, Methods. Enzymol. 154, 350-367 (1987) and Kunkel TA, Rapid and efficient sitespecific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA. 82, 488-492 (1985)), PCR mutagenesis and cassette mutagenesis.

The antigen-binding molecule of the present invention may be an antibody fragment that includes both the heavy chain and the light chain constituting the variable region of the antibody of the present invention in a single polypeptide chain, but does not contain a conserved region. Such an antibody fragment may be, for example, a diabody (Db), a single chain antibody or sc(Fab') ₂ .

Db is a dimer consisting of two polypeptide chains (eg Holliger P et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), EP404097 and WO93/11161). These polypeptide chains are connected via a short linker, for example about 5 amino acid residues long, such that an L chain variable domain (VL) and an H chain variable domain (VH) within the same polypeptide chain cannot associate with each other.

Due to such a short linker, VH and VL encoded on the same polypeptide chain cannot form a single-stranded Fv, but rather dimerize with VH and VL, respectively, on the other polypeptide chain, forming two antigen-binding sites.

Examples of single chain antibodies include sc(Fv)2. The sc(Fv)2 fragment is a single chain antibody containing a single chain consisting of four variable domains, i.e. two VL and two VH connected via linkers such as peptide linkers (J Immunol. Methods 231 (1-2), 177-189 (1999)). These two VL and VH can be obtained from various monoclonal antibodies. Preferred examples of such fragments include bispecific sc(Fv) ₂ that recognize two types of epitopes present on the same antigen, as described in Journal of Immunology 152 (11), 5368-5374 (1994). Fragment sc(Fv) ₂ can be obtained known in the art. For example, an sc(Fv) ₂ fragment can be obtained by joining two sc(Fv) through a linker, such as a peptide linker.

Examples of the configuration of antigen-binding domains consisting of sc(Fv) ₂ described in this context include an antibody in which two VH chains and two VL chains are in the following order: VH, VL, VH and VL (i.e. [VH] -linker-[VL]-linker-[VH]-linker-[VL]), starting from the N-terminus of the single chain polypeptide. The arrangement order of the two VHs and the two VLs is not particularly limited by configuration, and they may be arranged in any order. Examples of such an arrangement include the following configurations:

[VL] -linker-[VH] -linker-[VH] -linker-[VL],

[VH]-linker-[VL]-linker-[VL]-linker-[VH],

[VH] -linker-[VH] -linker - [VL] -linker-[VL],

[VL] -linker-[VL] -linker-[VH] -linker-[VH] and

[VL]-linker-[VH]-linker-[VL]-linker-[VH].

The molecular form of sc(Fv) ₂ is also detailed in WO2006/132352. Based on the present description, a person skilled in the art can appropriately obtain the required sc(Fv)2 fragment to obtain the antigen-binding molecule described in the present context.

The antigen-binding molecule of the present invention can be conjugated to a polymeric carrier such as PEG or an organic compound such as an anticancer agent. Preferably, a carbohydrate chain may also be included in the antigen-binding molecule of the present invention when inserting a glycosylated sequence to confer the desired effect.

For example, an arbitrary peptide linker, which can be incorporated by genetic engineering, or a synthetic linker compound (eg, the compound described in Protein Engineering, 9 (3), 299-305 (1996)) can be used to attach antibody variable domains. In the present invention, a peptide linker is preferred. The length of the peptide linker is not particularly limited, and the person skilled in the art can appropriately select it according to the purpose. The length is preferably 5 or more amino acid residues (the upper limit is not particularly limited and is usually 30 amino acids or less, preferably 20 amino acids or less, especially 15 amino acids. If sc(Fv) ₂ contains three peptide linkers, all of these peptide linkers used are characterized the same length or different lengths.

Examples of peptide linkers may include the following sequences:

Ser, Gly-Ser, Gly-Gly-Ser, Ser-Gly-Gly, Gly-Gly-Gly-Ser (SEQ ID NO: 5),

Ser-Gly-Gly-Gly (SEQ ID NO: 6),

Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 7),

Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 8),

- 18 042507

Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 9),

Ser-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 10),

Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 11),

Ser-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 12), (Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 7)) _n and (Ser-Gly-Gly-Gly- Gly (SEQ ID NO: 8))n, where n is an integer 1 or more.

However, the length or sequence of the peptide linker can be chosen by one of skill in the art according to the intended use.

Synthetic linker compound (chemical crosslinker) is a crosslinker commonly used for crosslinking peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidyl propionate) (DSP) , dithiobis(sulfosuccinimidyl)propionate (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfoEGS), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES) or bis [2-(Sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (Sulfo-BSOCOES). These crosslinkers are commercially available.

Three linkers are typically used to connect the four variable domains of an antibody. All of these linkers used may be the same or different.

The F(ab') ₂ fragment includes two light chains and two heavy chains containing a conserved region (CH1 domains and part of CH2 domains), thus forming a disulfide bond between these two heavy chains.

The F(ab')2 fragment constituting the polypeptide associate described herein can preferably be obtained under partial cleavage conditions, for example, by cleaving a full-length monoclonal antibody containing the desired antigen-binding domains with a proteolytic enzyme such as pepsin, followed by removal of the Fc fragment adsorbed on the protein A immobilized column. Such a proteolytic enzyme is not particularly limited as long as the enzyme is capable of cleaving a full-length antibody to form a restricted form of F(ab') ₂ under appropriate enzyme conditions (eg, pH). Examples of such enzymes include pepsin and ficin.

The antigen-binding molecule of the present invention may further comprise other changes in addition to the amino acid residue changes mentioned above. Additional changes can be selected from, for example, substitution, deletion or modification of an amino acid, and combinations thereof.

For example, the antigen-binding molecule of the present invention can be further modified in an arbitrary manner without substantially changing the desired functions of the molecule. Such a mutation can be carried out, for example, by substitution of conservative amino acid residues. In another embodiment, even a change to change the desired functions of the antigen-binding molecule of the present invention can be carried out, provided that the functions changed as a result of such a change are included in the scope of the subject of the present invention.

The change in the amino acid sequence of the present invention also includes post-translational modification. In more detail, post-translational modification may mean the insertion or deletion of a carbohydrate chain. An antigen-binding molecule of the present invention, for example, containing a conserved region of the IgG1 type, may contain an amino acid residue at position 297 according to EU numbering with a modified carbohydrate chain. The structure of the carbohydrate chain to be used in the modification is not limited. In general, antibodies expressed by eukaryotic cells include modification of the carbohydrate chain in their conserved regions. Thus, antibodies expressed by the following cells typically involve modification of some carbohydrate chains: antibody-producing mammalian cells, and eukaryotic cells transformed with expression vectors comprising DNA that encodes antibodies.

In this context, eukaryotic cells include yeast and animal cells. For example, typical animal cells to be transformed with expression vectors containing DNA that encode antibodies are CHO or HEK293H cells. On the other hand, the antibodies of the present invention also include antibodies that lack modified carbohydrate chains at this position. Antibodies with unmodified carbohydrate chains within conserved regions can be generated by expression of the genes encoding these antibodies in prokaryotic cells such as E. coli.

In more detail, an additional change according to the present invention can be carried out, for example, by including sialic acid in the carbohydrate chain in the Fc region (mAbs. 2 (5): 519527 (2010 Sep-Oct)).

If the antigen-binding molecule of the present invention contains an Fc region, amino acid residue substitutions can be further made to improve binding activity to FcRn

- 19 042507 (J Immunol. 176 (1): 346-356 (2006 Jan 1), J Biol Chem. 281 (33): 23514-23524 (2006 Aug 18), Int Immunol.

(12): 1759-1769 (2006 Dec), Nat Biotechnol. 28 (2): 157-159 (2010 Feb), WO2006/019447,

WO2006/053301 and WO2009/086320) or substitution of amino acid residues to improve antibody heterogeneity or stability (WO2009/041613).

The term antibody is used in the present invention in its broadest sense and also includes any antibody such as monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody variants, antibody fragments, multispecific antibodies (e.g., bispecific antibodies), hybrid antibodies, and humanized antibodies. provided that the antibody exhibits the desired biological activity.

The antibody of the present invention is not limited to its type of antigen, its source, and the like, and may be any antibody. Examples of antibody sources may include, but are not limited to, human antibodies, mouse antibodies, rat antibodies, and rabbit antibodies.

Antibodies can be obtained by a method known in the art. For example, monoclonal antibodies can be produced by the hybridoma method (Kohler and Milstein, Nature 256: 495 (1975)) or by the recombinant method (U.S. Patent No. 4816567). Alternatively, monoclonal antibodies can be isolated from antibody phage display libraries (Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991)). Monoclonal antibodies can also be isolated from whole B cell clones (N. Biotechnol. 28 (5): 253-457 (2011)).

Humanized antibodies are also referred to as reshaped human antibodies. In more detail, for example, a humanized antibody is known in the art, comprising a human antibody CDR-grafted with a non-human animal (eg, mouse) antibody. Approaches based on the recombination of essential genes are also known for the production of humanized antibodies. In more detail, for example, overlapping primer PCR is known in the art as a method for grafting mouse antibodies to human FR regions.

DNA encoding antibody variable domains, each containing three CDRs and four FR regions connected, and DNA encoding human antibody conserved domains can be inserted into expression vectors so as to form a hybrid product of variable domain DNA and conserved domain DNA in frame readouts and obtain vectors for expression of the humanized antibody. These insert vectors are transformed into host organisms to produce recombinant cells. The recombinant cells are then cultured to express DNA encoding humanized antibodies, and humanized antibodies are obtained in cultured cells (see European Patent Publication No. EP 239400 and International Publication No. WO1996/002576).

If necessary, the amino acid residue(s) in the FR fragment can be changed so that the CDR regions of the reshaped antibody will form the corresponding antigen-binding site. For example, the amino acid sequence of an FR fragment can be changed by mutating the PCR method that is used to graft mouse CDRs onto human FR fragments.

The desired human antibody can be obtained by DNA immunization using transgenic animals that have the entire set of human antibody genes (see International Publication No. WO1993/012227, WO1992/003918, WO1994/002602, WO1994/025585, WO1996/034096 and WO 1996/033735) as in immunized animals.

In addition, a method for obtaining human antibodies, the so-called panning method (fractionation and cell sorting), using an antibody library is known. For example, the V region of a human antibody is expressed as a single chain antibody (scFv) on the surface of phage by phage display. The phage expressing the antigen-binding scFv can then be selected. The gene of the selected phage can be analyzed to determine the DNA sequence encoding the V region of the human antigen binding antibody. Once the DNA sequence of an antigen-binding scFv has been determined, the V region sequence can be fused with the desired human antibody C region sequence and then incorporated into appropriate expression vectors to produce expression vectors. Expression vectors are transferred to the preferred expression cells listed above for expression of genes encoding human antibodies, and human antibodies are obtained. These methods are already known in the art (see International Publication No. WO1992/001047, WO1992/020791, WO1993/006213, WO1993/011236, WO1993/019172,

WO1995/001438 and WO1995/015388).

The variable regions constituting an antibody of the present invention may be variable regions that recognize an arbitrary antigen.

Used in the present description, the term antigen is not particularly limited, may be any antigen. Examples of antigens include 17-IA, 4-1 BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, active, active A, active AB, B active, C active, RIA active, RIA active ALK-2, RIB ALK-4 active, RIIA active, RIIB active, ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5 , addresses,

- 20 042507 adiponectin, ADP-ribosylcyclase-1, aFGF, AGE, ALCAM, ALK, ALK-1, ALK-7, allergen, alpha-1 antichymotrypsin, alpha-1 antitrypsin, alpha-synuclein, alpha-V/beta-1 antagonist, aminine , amylin, amyloid beta, amyloid immunoglobulin heavy chain variable region, amyloid immunoglobulin light chain variable region, androgen, ANG, angiotensinogen, angiopoietin ligand-2, anti-Id, antithrombin III, anthrax, APAF-1, APE, APJ, apo A1 , serum apo A amyloid, Apo-SAA, APP, APRIL, AR, ARC, ART, artemin, ASPARTIC, atrial natriuretic factor, atrial natriuretic peptide, atrial natriuretic peptides A, atrial natriuretic peptides B, atrial natriuretic peptides C, av/b3 integrin, Axl, B7-1, B7-2, B7-H, BACE, BACE-1, protective anthrax antigen, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, BcI , BCMA, BDNF, b-ECGF, beta-2-microglobulin, beta-lactamase, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, B-lymphocyte stimulator ( BIyS), BMP, BMP-2 (BMP-2a), BMP-3 (osteogenin), BMP-4 (BMP-2b), BMP-5, BMP-6 (Vgr-1), BMP-7 (OP-1 ), BMP-8 (BMP-8a), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BMPR-II (BRK-3), BMPs, BOK, bombesin, bone neurotrophic factor , bovine growth hormone, BPDE, BPDE-DNA, BRK-2, BTS, T cell adhesion molecule, C10, C1 inhibitor, C1q, C3, C3a, C4, C5, C5a (complement 5a), CA125, CAD -8, cadherin-3, calcitonin, cAMP, carbonic anhydrase-IX, carcinoembryonic antigen (CEA), carcinoma antigen, cardiotrophin-1, cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D, cathepsin E, cathepsin H, cathepsin L , cathepsin O, cathepsin S, cathepsin V, cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1/I-309, CCLU/eotaxin, CCL12/MCP-5, CCL13/MCP-4, CCL14/HCC -1, CCL15/HCC-2, CCL16/HCC-4, CCL17/TARC, CCL18/PARC, CCL19/ELC, CCL2/MCP-1, CCL20/MIP-3-a, CCL21/SLC, CCL22/MDC, CCL23 /MPIF-1, CCL24/eotaxin-2, CCL25/TECK, CCL26/eotaxin-3, CCL27/CTACK, CCL28/MEC, CCL3/M1P-1-a, CCL3L1/LD-78-p, CCL4/MI P-1-e, CCL5/RANTES, CCL6/C10, CCL7/MCP-3, CCL8/MCP2, CCL9/10/MTP-1-y, CCR, CCR1, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD10, CD105, CD11a, CD11b, CD11c, CD123, CD13, CD137, CD138, CD14, CD140a, CD146, CD147, CD148, CD15, CD152, CD16, CD164, CD18, CD19, CD2, CD20, CD21, CD22, CD23, CD25, CD26, CD27L, CD28, CD29, CD3, CD30, CD30L, CD32, CD33 (p67 proteins), CD34, CD37, CD38, CD3E, CD4, CD40, CD40L, CD44, CD45, CD46, CD49a, CD49b, CD5, CD51, CD52, CD54, CD55, CD56, CD6, CD61, CD64, CD66e, CD7, CD70, CD74, CD8, CD80 (B7-1), CD89, CD95, CD105, CD158a, CEA , CEACAM5, CFTR, cGMP, CGRP receptor, CINC, CKb8-1, claudin 18, CLC, clostridium botulinum toxin, clostridium difficile toxin, clostridium perfringens toxin, c-Met, CMV, CMV UL, CNTF, CNTN-1, complement factor 3 (C3), complement factor D, corticosteroid-binding globulin, colony stimulating factor receptor-1, COX, C-Ret, CRG-2, CRTH2, CT-1, STACK, CTGF, CTLA-4, CX3CL1/fractalkine, CX3CR1, CXCL, CXCL1/Gro-a, CXC L10, CXCL11/I-TAC, CXCL12/SDF-1-a/e, CXCL13/BCA-1, CXCL14/BRAK, CXCL15/Lungkine, CXCL16, CXCL16, CXCL2/Gro-e, CXCL3/Gro-y , CXCL3, CXCL4/PF4, CXCL5/ENA-78, CXCL6/GCP-2, CXCL7/NAP-2, CXCL8/IL-8, CXCL9/Mig, CXCL10/IP-10, CXCR, CXCR1, CXCR2, CXCR3, CXCR4 , CXCR5, CXCR6, cystatin C, cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, decay accelerator factor, delta-like protein ligand 4, des(1-3)-IGF-1 (brain IGF-1), Dhh , DHICA oxidase, Dickkopf-1, digoxin, dipeptidyl peptidase IV, DK1, DNAM-1, DNase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1 ), EGF-like domain containing protein 7, elastase, elastin, EMA, EMMPRIN, ENA, ENA-78, endosialin, endothelin receptor, endotaxin, enkephalinase, eNOS, Eot, eotaxin, eotaxin-2, eotaxin, EpCAM, ephrin B2 /EphB4, Epha2 tyrosine kinase receptor, epidermal growth factor receptor (EGFR), ErbB2 receptor, ErbB3 tyrosine kinase receptor, ERCC, EREG, erythropoietin (EPO), erythropoietin receptor, E-c selectin, ET-1, Exodus-2, F-protein RSV, F10, F11, F12, F13, F5, F9, factor Ia, factor IX, factor Xa, factor VII, factor VIII, factor VIIIc, Fas, FcaR, FcεRI , FcyIIb, FcyRI, FcyRIIa, FcyRIIIa, FcyRIIIb, FcRn, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF-2 receptor, FGF-3, FGF-8, acidic FGF, basic FGF, FGFR, FGFR-3, fibrin, fibroblast activation protein (FAP), fibroblast growth factor, fibroblast growth factor 10, fibronectin, FL, FLIP, Flt-3, FLT3 ligand, folate receptor, follicle-stimulating hormone (FSH), fractalkine (CX3C), free heavy chain, free light chain, FZD1, FZD10, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, G250, Gas 6, GCP-2, GCSF, G-CSF, G-CSF receptor, GD2, GD3 , GDF, GDF-1, GDF-15 (MIC-1), GDF-3 (Vgr-2), GDF5 (BMP-14/CDMP-1), GDF-6 (BMP-13/CDMP-2), GDF -7 (BMP-12/CDMP-3), GDF-8 (myostatin), GDF-9, GDNF, gelsolin, GFAP, GF-CSF, GFR-a1, GFR-a2, GFR-a3, GF-βΓ envelope glycoprotein gH, GITR, glucagon, glucagon receptor, glucagon-like peptide-1 receptor, Glut 4, glutamate carboxypeptidase II, glycoprotein hormone receptors, glycoprotein IIb/IIIa (GPIIb/IIIa), glypican-3, GMCSF, GM-CSF receptor, gp130, gp140, gp72, granulocyte-CSF (G-CSF), GRO/MGSA, growth hormone releasing factor, GRO-β, GRO-y, H. pylori, hapten (NP-sar or NIP-sar), HB-EGF, HCC, HCC 1, HCMV envelope glycoprotein gB, HCMV UL, hematopoietic growth factor ( HGF), Hep B gp120, heparanase, heparin cofactor II, hepatic growth factor, anthrax protective antigen, hepatitis C, hepatitis E glycoprotein E2, hepcidin, Her1, Her2/neu (ErbB-2), Her3 (ErbB-3) , Her4 (ErbB-4), herpes simplex virus (HSV) glycoprotein gB, HGF, HGFA, high molecular weight melanoma

- 21 042507 associated antigen (HMW-MAA), HIV envelope proteins such as GP120, HIV MIB loop gp 120 V3, HLA, HLA-DR, HM1.24, HMFG PEM, HMGB-1, HRG, Hrk, HSP47, Hsp90 , HSV glycoprotein gD, human cardiac myosin, human cytomegalovirus (HCMV), human growth hormone (hGH), human serum albumin, human tissue plasminogen activator (t-PA), huntingtin, HVEM, IAP, ICAM, ICAM-1, ICAM- 3, ICE, ICOS, IFN-α, IFN-β, IFN-γ, IgA, IgA receptor, IgE, IGF, IGF-binding proteins, IGF-1, IGF-1 R, IGF-2, IGFBP, IGFR, IL , IL-1, IL-10, IL-10 receptors, IL-11, IL-11 receptors, IL12, IL-12 receptors, IL-13, IL-13 receptors, IL-15, IL-15 receptors, IL- 16, IL-16 receptors, IL-17 receptors, IL-17 receptors, IL-18 (IGIF), IL-18 receptors, IL-1a, IL-1e, IL-1 receptors, IL-2 receptors, IL-2 receptors, IL-20, IL-20 receptors, IL-21, IL-21 receptors, IL-23, IL-23 receptors, IL-2 receptors, IL-3, IL-3 receptors, IL-31, IL-31 receptors, IL-3 receptors, IL-4 receptors, IL-4 receptors, IL-5 receptors, IL-5 receptors, IL-6 receptors, IL-6 receptors, IL-7, IL-7 receptors, IL-8, IL-8 receptors, IL-9 receptors, IL-9 receptors, immunoglobulin immune complex, immunoglobulins, IFN-a, IFN-a receptors, INF-β, INF-β receptors , IFN-γ, IFN-γ receptors, IFN type I, IFN type I receptor, influenza virus, inhibin, inhibin a, inhibin β, iNOS, insulin, insulin A-chain, insulin B-chain, insulin-like growth factor 1, insulin-like growth factor 2, insulin-like growth factor binding proteins, integrin, integrin a2, integrin a3, integrin a4, integrin α4/β1, integrin α-Υ/β-3, integrin a-V/β-6, integrin α4/β7, integrin α5/ β1, integrin α5/β3, integrin α5/β6, integrin α σ (αΥ), integrin α θ, integrin β 1, integrin β2, integrin β3(GPIIb-IIIa), IP-10, I-TAC, JE, kallikrein, kallikrein 11, kallikrein 12, kallikrein 14, kallikrein 15, kallikrein 2, kallikrein 5, kallikrein 6, kallikrein L1, kallikrein L2, kallikrein L3, kallikrein L4, kallistatin, KC, KDR, keratinocyte growth factor (KGF), keratinocyte growth factor- 2 (KGF-2), KGF, immunoglobulin-like rec killer cell eptor, kit ligand (KL), tyrosine kinase Kit, laminin 5, LAMP, LAPP (amylin, islet amyloid polypeptide), LAP (TGF-1), latent-associated peptide, latent TGF-1, latent TGF-1 bp1 , LBP, LDGF, LDL, LDL receptor, LECT2, Lefty, leptin, luteinizing hormone (LH), Lewis Y antigen, Lewis Y related antigen, LFA-1, LFA-3, LFA-3 receptors, Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotactin, lymphotaxin-β receptor, lysosphingolipid receptor, Mac-1, macrophage-CSF ( M-CSF), MAdCAM, MAG, MAP2, MARC, maspin, MSAM, MSC-2, MCP, MCP-1, MCP-2, MCP-3, MCP-4, MCP-I (MCAF), M-CSF, MDC, MDC (67 a.a.o.), MDC (69 a.a.o.), megzin, Mer, MET tyrosine kinase receptor family, metalloproteases, OX2 membrane glycoprotein, mesothelin, MGDF receptor, MGMT, MHC (HLA- DR), proteins of microorganisms, MIF, MIG, MIP, MIP-1α, MIP-1β, MIP-3a, MIP-3β, MIP-4, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMR-12 , MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, monocytic chemotactic protein, monocytic colony-inhibiting factor, mouse gonadotropin-associated peptide , MPIF, Mpo, MSK, MSP, MUC-16, MUC18, Mucin (Mud), Müllerian inhibitory substance, Mug, MuSK, myelin-associated glycoprotein, myeloid inhibitory factor-1 precursor (MPIF-I), NAIP, nanobody, NAP , NAP-2, NCA 90, NCAD, N-cadherin, NCAM, neprilysin, neuronal cell adhesion molecule, neuroserpin, neuronal growth factor (NGF), neurotrophin-3, neurotrophin-4, neurotrophin-6, neuropilin 1, neuroturin, NGF -β, NGFR, NKG20, N-methionyl human growth hormone, nNOS, NO, Nogo-A, Nogo receptor, non-structural protein type 3 (NS3) of hepatitis C virus, NOS, Npn, NRG-3, NT, NT-3, NT -4, NTN, OB, OGG1, oncostatin M, OP-2, OPG, OPN, OSM, OSM receptors, osteoinductive factors, osteopontin, OX40L, OX40R, oxidized LDL, p150, p95, PADPr, parathyroid hormone, PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PCSK9, PDGF, PDGF receptor, PDGF-AA, PDGF-AB, PDGF-BB, PDGF-D, PDK-1, PECAM, PEDF, PEM, PF-4, PGE, PGF, PGI2, PGJ2, PIGF, PIN , PLA2, placental growth factor, placental alkaline phosphatase (PLAP), placental lactogen, plasminogen activator-1 inhibitor, platelet growth factor, plgR, PLP, various lengths of polyethylene glycol chains (eg, PEG-20, PEG-30, PEG40), PP14 , prekallikrein, prion protein, procalcitonin, programmed cell death protein 1, proinsulin, prolactin, PC9 proprotein convertase, prorelaxin, prostate-specific membrane antigen (PSMA), protein A, protein C, protein D, protein S, protein Z, PS, PSA, PSCA, PsmAr, PTEN, PTHrp, Ptk, PTN, P-selectin glycoprotein ligand-1, R51, RAGE, RANK, RANKL, RANTES, relaxin, relaxin A-chain, relaxin B-chain, renin, respiratory syncytial virus (RSV) F, Ret, reticulon 4, rheumatoid factors, RLI P76, RPA2, RPK-1, RSK, RSV Fgp, S100, RON-8, SCF/KL, SCGF, sclerostin, SDF-1, SDF1a, SDF1β, antigen SERINE, serum pure amyloid P, serum albumin, sFRP-3, Shh, Shiga-like toxin II, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, sphingosine 1-phosphate receptor 1, staphylococcal lipoteichoic acid, Stat, STEAP, STEAP-II, stem cell factor (SCF), streptokinase, superoxide dismutase, syndecan-1, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TB, TCA-3, α/β T receptors -cells, TdT, TECK, TEM1, TEM5, TEM7, TEM8, tenascin, TERT, testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-α, TGF-β, pan-specific TGF-β, TGF-β RII, TGF-βa RIIb, TGF-β RIIL TGF-β R1 (ALK-5), TGFβ1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, TGF-I, thrombin, thrombopoietin (TPO), thymus receptor stromal lipoprotein, thymic Ck-1, thyroid-stimulating hormone (TSH), thyroxine, thyroxin-binding

- 22 042507 globulin, Tie, TIMP, TIQ, tissue factor, tissue factor protease inhibitor, tissue factor protein, TMEFF2, Tmpo, TMPRSS2, TNF I receptor, receptor

TNF II, TNF-a, TNF-β, ΤΝΡ-β2, TNFc, TNF-RI, TNF-RII, TNFRSF10A (TRAIL RI Apo-2/DR4), TNFRSF10B (TRAIL R2 DR5/KILLER/TRICK2A/TRICK-B) , TNFRSF10C (TRAIL R3 DcRl/LIT/TRID), TNFRSF10D (TRAIL R4 DcR2/TRUNDD), TNFRSF11A (RANK ODF R/TRANCE R), TNFRSF11B (OPG OCIF/TR1), TNFRSF12 (TWEAK R FN14), TNFRSF12A, TNFRSF13B ( TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR/HveA/LIGHT R/TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROY TAJ/TRADE), TNFRSF19L (RELT ), TNFRSF1A (TNF RI CD120a/p55-60), TNFRSF1B (TNF RII CD120b/p75-80), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRSF25 (DR3 Apo-3/LARD/TR3/TRAMP/WSL -l), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNF RIII/TNFC R), TNFRSF4 (0X40 ACT35/TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo1/APT1/CD95), TNFRSF6B (DcR3 M68/TR6 ), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9 (4-1 BB CD137/ILA), TNFRST23 (DcTRAIL RI TNFRH1), TNFSF10 (TRAIL (Apo-2 ligand)/TE2), TNFSF11 (TRANCE/RANK ODF ligand /OPG ligand), TNFSF12 (TWEAK (Apo-3 ligand)/ligand and DR3), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS/TALL1/THANK/TNFSF20), TNFSF14 (LIGHT HVEM/LTg ligand), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR ligand (AITR ligand)/TL6), TNFSF1A (conectin TNF-a/DIF/TNFSF2), TNFSF1B (TNF-β LTa/TNFSFl), TNFSF3 (LTb TNFC/p33), TNFSF4 (ligand 0X40 gp34/TXGPl), TNFSF5 (ligand CD40 CD154/gp39/HIGMl/IMD3 /TRAP), TNFSF6 (Fas ligand Apo-1 ligand/ΑΡΤΙ ligand), TNFSF7 (CD27 ligand (CD70)), TNFSF8 (CD30 ligand (CD153)), TNFSF9 (BB 4-1 ligand (CD137 ligand)), TNF- a, TNF-β, TNIL-I, toxic metabolite, TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, transforming growth factors (TGF) such as TGF-α and TGF-β, NMB transmembrane glycoprotein, transthyretin, TRF, Trk, TROP-2, trophoblastic glycoprotein, TSG, TSLP, tumor necrosis factor (TNF), CA 125 tumor-associated antigen, carbohydrate-expressing tumor-associated antigen , related to Lewis Y, TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VAP-1, endothelial factor vascular growth factor (VEGF), vaspin, VCAM, VCAM-1, VECAD, VEcadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEFGR-2, VEGF receptor (VEGFR), VEGFR-3 (flt- 4), VEGI, VIM, viral antigens, VitB12 receptor, vitronectin receptor, VLA, VLA-1, VLA-4, VNRintegrin, von Willebrand factor (vWF), WIF-1, WNT1, WNT10A, WNT10B, WNT11, WNT16, WNT2 , WNT2B/13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, XCL1, XCL2/SCM-1-3, XCL1/lymphotactin, XCR1, XEDAR, XIAP and XPD .

One of the two variable domains of an antibody constituting an antigen-binding molecule of the present invention is capable of binding to two different antigens, but is not capable of binding to said antigens simultaneously. The term first antigen or second antigen to which said variable domain binds preferably means, for example, a molecule on the surface of an immunocyte (for example, a molecule on the surface of T cells, a molecule on the surface of NK cells, a molecule on the surface of dendritic cells, a molecule on the surface of B cells , as well as a molecule on the surface of NKT cells, a molecule on the surface of MDSC cells and a molecule on the surface of macrophages), or an antigen that is expressed not only on tumor cells, tumor vessels, stem cells, etc., but also on healthy tissues ( integrin, cell factor, VEGFR, PDGFR, EGFR, IGFR, MET chemokine receptor, heparan sulfate proteoglycan, CD44, fibronectin, DR5, TNFRSF, etc.). Regarding the use of a combination of the first antigen and the second antigen, preferably either first antigen and second antigen is, for example, a molecule specifically expressed on a T cell, and the other antigen is a molecule expressed on the surface of a T cell or any other immunocyte. In another embodiment of the combination of the first antigen and the second antigen, preferably either the first antigen and the second antigen is, for example, a molecule specifically expressed on a T cell, and the other antigen is a molecule that is expressed on an immunocyte and is different from the preselected antigen. Specific examples of a molecule specifically expressed on a T cell include CD3 and T cell receptors. First of all, CD3 is preferred. For example, in the case of human CD3, the site within CD3 to which the antigen-binding molecule of the present invention binds can be any epitope present on the γ-chain, δ-chain or ε-chain constituting human CD3. First of all, an epitope present in the extracellular region of the ε-chain of the human CD3 complex is preferred. The polynucleotide sequences of the γ-chain, δ-chain and ε-chain structures constituting CD3 are shown in SEQ ID NOs: 83 (NM_000073.2), 85 (NM_000732.4) and 87 (NM_000733.3), and the corresponding polypeptide sequences are shown in SEQ ID nos: 84 (NP_000064.1), 86 (NP_000723.1), and 88 (NP_000724.1) (RefSeq accession numbers are shown in parentheses). Examples of another antigen include Fcy receptors, TLRs, lectin, IgA, immune checkpoint molecules, TNF superfamily molecules, TNFR superfamily molecules, and NK receptor molecules.

Of the two antibody variable domains included in the antigen-binding molecule of the present invention, the other variable domain binds to a third antigen that is different from the first antigen and second antigen mentioned above. The third antigen is preferably, for example, a tumor cell-specific antigen, and said antigen also includes an antigen expressed during a malignant change in cells, as well as when an abnormal carbohydrate sequence appears on the surface of cells or in a protein molecule during malignant transformation of cells. Specific examples of said antigen include ALK receptor (pleiotrophin receptor), pleiotrophin, pancreatic cancer antigen KS 1/4, ovarian cancer antigen (CA125), prostate acid phosphatase, prostate specific antigen (PSA), melanoma-associated p97 antigen, antigen gp75 melanoma, high molecular weight melanoma antigen (HMW-MAA), prostate-specific membrane antigen, carcinoembryonic antigen (CEA), polymorphic epithelial mucin antigen, human breast milk fat globule membrane antigen, colorectal cancer tumor-specific antigen (eg, CEA, TAG- 72, CO17-1A, GICA 19-9, CTA-1, and LEA), Burkitt's lymphoma antigen 38.13, CD19, human B-cell lymphoma antigen CD20, CD33, melanoma-specific antigen (eg, ganglioside GD2, ganglioside GD3, ganglioside GM2 and ganglioside GM3), tumor-specific transplantation antigen (TSTA), Tantigen, virus-induced tumor antigen (for example, oncogenic DNA virus capsular antigen and it cogenic RNA virus), colon CEA, oncofetal α-fetoprotein antigen (eg, oncofetal trophoblastic glycoprotein 5T4 and oncofetal bladder tumor antigen), differentiation antigen (eg, lung cancer antigens L6 and L20), fibrosarcoma antigen, Gp37 antigen, human T-cell leukemia-associated, neonatal glycoprotein, sphingolipid, breast cancer antigen (eg, EGFR (epithelial growth factor receptor)), NY-BR-16, NY-BR-16 and HER2 (p185HER2) antigen, polymorphic epithelial mucin (PEM), human lymphocyte malignant antigen APO-1, differentiation antigen such as antigen I found in fetal erythrocytes, primary endoderm antigen I found in mature erythrocytes, I (Ma) found in embryonic cells before transplantation or gastrointestinal cancer , M18 found in breast epithelium, M39, SSEA-1 found in bone marrow cells, VEP8, VEP9, Myl, VIM-D5, D156-22, found th in colorectal cancer cells, TRA-1-85 (blood type H), SCP-1 found in testicular cancer cells and ovarian cancer cells, C14 found in colon cancer cells, F3 found in lung cancer cells, AH6 , found in gastrointestinal cancer cells, hapten Y, Ley found in embryonic cancer cells, TL5 (blood type A), EGF receptor found in A431 cells, E1 series (blood type B), found in pancreatic cancer cells , FC10.2, found in embryonic cancer cells, gastrointestinal cancer antigen, CO-514 (blood type Lea), found in adenocarcinoma cells, NS-10, found in adenocarcinoma cells, CO-43 (blood type Leb), G49, found in the EGF receptor of A431 cells, MH2 (blood type ALeb/Ley), found in colon cancer cells, 19.9, found in colon cancer cells, gastrointestinal cancer mucin, T5A7, found in bone marrow cells, R24 found in chalk cells nomas, 4.2, GD3, D1.1, OFA-1, GM2, OFA-2, GD2, as well as M1:22:25:8, found in embryonic cancer cells, SSEA-3 and SSEA-4, found in 4- 8-cell embryos, skin T cell lymphoma associated antigen, MART-1 antigen, sialyl-Tn (STn) antigen, NY-CO-45 colon cancer cell antigen, NY-LU-12 lung cancer cell variant A antigen , adenocarcinoma antigen ART1, paraneoplastic antigen associated with brain-ovarian cancer (onconeuronal antigen MA2 and paraneoplastic neuronal antigen), neurooncological ventral antigen 2 (NOVA2), blood cancer antigen 520, tumor-associated antigen CO-029, tumor-associated antigen MAGE-C1 (CT7 cancer testicular antigen), MAGE-B1 (MAGE-XP antigen), MAGE-82 (DAM6), MAGE-2, MAGE-4a, MAGE-4b MAGE-X2, testicular cancer antigen (NY-EOS -1), YKL-40, as well as any fragment of these polypeptides and their modified structures (the above-mentioned modified phosphate groups, carbohydrate chains etc.), EpCAM, EREG, CA19-9, CA15-3, sialyl SSEA-1 (SLX), HER2, PSMA, CEA and CLEC12A.

- 24 042507

The antigen-binding molecule of the present invention can be obtained by known methods. For example, an antibody can be obtained by the following method, although the method for producing an antibody of the present invention is not limited to these methods. Many combinations of host cells and expression vectors are known in the art to generate antibodies by transferring isolated genes encoding polypeptides into appropriate host cells. All of these expression systems can be used to isolate the antigen-binding molecule of the present invention. In the case of using eukaryotic cells as host cells, animal cells, plant cells or fungal cells can be appropriately used. In more detail, examples of animal cells include the following cells:

(1) mammalian cells such as CHO (Chinese hamster ovary cell line), COS (monkey kidney cell line), myeloma cells (Sp2/0, NS0, etc.), BHK (newborn hamster kidney cell line), HEK293 (human embryonic kidney cell line with degraded adenovirus (Ad)5 DNA), PER.C6 cells (human embryonic retina cell line transformed with adenovirus type 5 (Ad5) E1A and E1B genes), Hela, and Vero (Current Protocols in Protein Science (May 2001, Section (Unit) 5.9, Table 5.9.1)), (2) amphibian cells such as Xenopus oocytes, and (3) insect cells such as sf9, sf21 and Tn.

The antibody can also be obtained using E. coli culture cells (mAbs, 4 (2): pp. 217-225 (2012 March-April)) or yeast (WO 2000/023579). The antibody generated using E. coli is non-glycosylated. On the other hand, an antibody produced using yeast is glycosylated.

DNA is expressed that encodes a heavy chain, including one or more amino acid residues in the variable domain, replaced with various desired amino acid residues, and DNA that encodes an antibody light chain. A DNA that encodes a heavy chain or a light chain comprising one or more amino acid residues in the variable domain substituted for various desired amino acid residues can be obtained, for example, by obtaining a DNA encoding the variable domain of an antibody prepared by a known technique against a particular antigen, and by substituting accordingly, such that codons that code for specific amino acid residues in the domain will code for the various desired amino acid residues.

In another embodiment, DNA encoding a protein in which one or more amino acid residues in the variable domain of an antibody produced by a known technique against a particular antigen are replaced with various desired amino acids can be pre-engineered and obtained by chemical synthesis and obtain a DNA that encodes a heavy chain , including one or more amino acid residues in the variable domain, which are replaced by various required amino acids. The amino acid substitution site and substitution type are not particularly limited. Examples of a region that is preferred for altering amino acid residues include solvent-exposed regions and loops in the variable domain. Among others, CDR1, CDR2, CDR3, FR3 and loops are preferred. First of all, the following positions in the H chain variable domain are preferred: 31-35, 50-65, 71-74 and 95-102 (according to Kabat numbering), as well as the following positions in the L chain variable domain: 24-34, 50-56 and 89-97 according to Kabat numbering. More preferred are the following positions in the H chain variable domain: 31, 52a-61, 71-74 and 97-101 according to Kabat numbering, as well as the following positions in the L chain variable domain: 24-34, 51-56 and 89-96 according to Kabat numbering.

The change in amino acid sequence is not limited to substitution and may be a deletion, addition, insertion or modification, or a combination of these operations.

DNA that encodes a heavy chain having one or more amino acid residues in the variable domain changed to the various desired amino acid residues can also be obtained as single DNA fragments. Examples of the combination of single DNAs include, but are not limited to, DNA encoding a variable domain and DNA encoding a conserved domain, as well as DNA encoding a Fab domain and DNA encoding an Fc domain. Similarly, the DNA encoding the light chain can also be obtained as single DNA fragments.

Such DNAs can be expressed in the following manner: for example, DNA encoding a heavy chain variable domain along with DNA encoding a conserved heavy chain domain is inserted into an expression vector to obtain a heavy chain expression vector. Similarly, the DNA encoding the light chain variable domain along with the DNA encoding the conserved light chain domain are inserted into an expression vector to obtain a light chain expression vector. These heavy chain and light chain genes can be inserted into one vector.

DNA encoding the desired antibody is inserted into expression vectors in such a way as to allow expression under conditions of control of certain expression regions, for example, using an enhancer and a promoter. The host cells are then transformed with the resulting expression vectors and antibodies are expressed. In this case, the appropriate host cells and expression vectors can be used in combination.

- 25 042507

Examples of vectors include the M13 series vectors, the pUC series, pBR322, pBluescript and pCRScript vectors. In addition to these vectors, for example, pGEM-T, pDIRECT or pT7 can also be used for subcloning and excising a cDNA segment.

First of all, expression vectors are suitable for use in the preparation of the antibody of the present invention. For example, if the host is an E. coli strain such as JM109, DH5a, HB101, or XL1-Blue, the expression vectors necessarily include a promoter that allows for efficient expression in E. coli, such as the lacZ promoter (Ward et al., Nature , 341, pp. 544-546 (1989) and Faseb J., 6, pp. 2422-2427 (1992), the contents of which are incorporated herein by reference in their entirety), the araB promoter (Better et al., Science, 240, 1041-1043 (1988), the contents of which are hereby incorporated by reference in their entirety), or the T7 promoter. Examples of these vectors include the vectors mentioned above, as well as the pGEX-5X-1 vector (manufactured by Pharmacia), the QIAexpress system vector set (manufactured by Qiagen N.V.), pEGFP and pET vectors (in this case, the host organism is preferably the BL21 strain expressing RNA -T7 polymerase).

The vectors may contain a signal sequence for the secretion of polypeptides. In the case of production in the periplasm of E. coli, the signal sequence pelB can be used as a signal sequence for the secretion of polypeptides (Lei S. P. et al., J. Bacteriol., 169, p. 4397 (1987), the contents of which are included in full to the present description by reference). Vectors can be transferred into host cells using, for example, a transfection system in the presence of lipofectin, calcium phosphate, or DEAE-dextran.

In addition to expression vectors for E. coli, examples of vectors for producing a polypeptide of the present invention include mammalian expression vectors (e.g., pcDNA3 vector (manufactured by Invitrogen Corp.), EGF-BOS vector (Nucleic Acids. Res., 18 (17), p. 5322 (1990, the contents of which are incorporated herein by reference in their entirety), pEF and pCDM8 vectors), insect expression vectors (e.g. Bac-to-BAC baculovirus expression system (manufactured by GIBCO BRL) and pBacPAK8), expression vectors plants (e.g., pMH1 and pMH2), animal expression vectors (e.g., pHSV, pMV, and pAdexLcw), retroviral expression vectors (e.g., pZIPneo), yeast expression vectors (e.g., Pichia Expression Kit (available from Invitrogen Corp.), pNV11 and SP-Q01), as well as Bacillus subtilis expression vectors (eg pPL608 and pKTH50).

For expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, or HEK293 cells, the vectors necessarily contain a promoter required for intracellular expression, such as the SV40 promoter (Mulligan et al., Nature, 277, p. 108 (1979 ), the content of which is incorporated herein by reference in its entirety), MMTV-LTR promoter, EF1a promoter (Mizushima et al., Nucleic Acids Res., 18, p. 5322 (1990), the content of which is incorporated herein in reference), a CAG promoter (Gene, 108, 193 (1991), the contents of which are incorporated herein by reference in their entirety) or a CMV promoter, and more preferably contain a gene for screening transformed cells (e.g., a gene for drug resistance). an agent that serves as a drug marker (neomycin, G418, etc.)). Examples of vectors with these properties include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV and pOP13. In addition, EBNA1 protein can be co-expressed to increase the number of gene copies. In this case, vectors containing the origin of replication OriP are used (articles Biotechnol. Bioeng., 75 (2), c. 197-203 (2001 Oct20) and Biotechnol. Bioeng., 91 (6), c. 670-677 (2005 Sep20)).

A typical method for stable gene expression and increase in intracellular gene copy number involves transforming CHO cells, in which the nucleic acid synthesis pathway is excluded, with vectors containing the DHFR gene serving as its complement (for example, pCHOI), and using methotrexate (MTX ) during gene amplification. A typical method for transient expression of a gene involves the use of COS cells containing the SV40 T antigen in their chromosomes, and the cells are transformed with vectors containing the SV40 origin of replication (pcD, etc.). As the origin of replication, a point derived from polyomavirus, adenovirus, bovine papillomavirus (BPV), and the like can be used. To increase the number of gene copies in the host cell system, expression vectors can include a selectable marker such as the aminoglycoside phosphotransferase (APH) gene, the thymidine kinase (TK) gene, the E. coli xanthineguanine phosphoribosyltransferase (Ecogpt) gene, or the dihydrofolate reductase (dhfr) gene.

The antibody can be isolated, for example, by culturing the transformed cells and then separating the antibody from the molecule-transformed cells or from the culture medium. The antibody can then be isolated and purified, respectively, using combinations of methods such as centrifugation, fractionation at various concentrations of ammonium sulfate, salting out, ultrafiltration, column chromatography with immobilized C1q, FcRn, protein A and protein G, affinity chromatography, ion exchange chromatography and gel- filtration.

In order to efficiently produce a multispecific antibody, the method can use the technology mentioned above, such as the projection-in-recess technique (application WO 1996/027011, articles by Ridgway J. B. et al., Protein Engineering, 9, pp. 617-621 ( 1996) and Merchant A. M. et al., Nature Biotechnology, 16, pp. 677-681 (1998)) or technology for suppressing undesirable association of H-strands by introducing electrical repulsive charges (application WO 2006/106905).

The present invention further provides a method for producing an antigen-binding molecule of the present invention, and above all, a method for producing an antigen-binding molecule comprising: an antibody variable domain that is capable of binding to two different antigens (a first antigen and a second antigen) but does not bind with the first antigen and the second antigen at the same time (the specified variable domain is called the first variable domain), and the second variable domain that binds to the third antigen, which differs from the first antigen and the second antigen (the specified variable domain is called the second variable domain), and the method includes the step of obtaining libraries of antigen-binding molecules containing various amino acid sequences of the first variable domain.

Examples may include a production method comprising the following steps:

(i) obtaining a library of antigen-binding molecules containing at least one altered amino acid residue in the variable domains of the antibody, each of which binds to the first antigen or to the second antigen, and the altered variable domains differ from each other by at least one amino acid remainder;

(ii) selecting from the resulting library an antigen-binding molecule comprising a variable domain that binds to the first antigen and the second antigen, but does not bind to the first antigen and the second antigen simultaneously;

(iii) culturing host cells comprising a nucleic acid encoding the variable domain of the antigen-binding molecule selected in step (ii) and a nucleic acid encoding the variable domain of the antigen-binding molecule that binds to the third antigen, wherein the antigen-binding molecule comprising the variable domain of the antibody is expressed, which is able to bind to the first antigen and the second antigen, but does not bind to the first antigen and the second antigen at the same time, as well as a variable domain that binds to the third antigen; and (iv) isolating the antigen-binding molecule from host cell cultures.

In said production process, step (ii) may be the following selection step:

(v) selecting from the resulting library an antigen-binding molecule comprising a variable domain that binds to the first antigen and the second antigen, but does not bind simultaneously to the first antigen and the second antigen, each of which is expressed on different cells.

The antigen-binding molecules used in step (i) are not particularly limited as long as each of said molecules includes an antibody variable domain. Antigen binding molecules can be antibody fragments such as Fv, Fab or Fab', or antibodies containing an Fc region.

The amino acid to be changed is selected, for example, from amino acids whose change in the variable domain of the antibody that binds to the first antigen or to the second antigen does not affect binding to the antigen.

In the present invention, a change in a single amino acid residue may be used, or a change in multiple amino acid residues may be used in combination.

In the case of using a multiple amino acid change in combination, the number of changes to be combined is not particularly limited and is, for example, 2 or more and 30 or less, preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or more and 20 or less, 2 or more and 15 or less, 2 or more and 10 or less, 2 or more and 5 or less, or 2 or more and 3 or less.

The plurality of amino acid changes to be combined may be added either to the antibody heavy chain variable domain only or to the light chain variable domain, or respectively distributed to both the heavy chain variable domain and the light chain variable domain.

Examples of a region favored for altering the amino acid sequence include solvent-exposed regions and loops in the variable domain. Among others, CDR1, CDR2, CDR3, FR3 and loops are preferred. First of all, the following positions in the variable domain of the H chain are preferred: 31-35, 50-65, 71-74 and 95-102 according to Kabat numbering, as well as the following positions in the variable domain of the L chain: 24-34, 50- 56 and 89-97 according to the Kabat numbering. More preferred are the following positions in the H chain variable domain: 31, 52a-61, 71-74 and 97-101 according to Kabat numbering, as well as the following positions in the L chain variable domain: 24-34, 51-56 and 89- 96 according to Kabat numbering.

Changing the amino acid residue also includes randomly changing the amino acid residues in the region mentioned above in the variable domain of the antibody that binds to the first antigen or to the second antigen, and the insertion of a peptide of known binding activity with the desired

- 27 042507 antigen in the area mentioned above. The antigen-binding molecule of the present invention can be obtained by selecting, among the antigen-binding molecules thus modified, a variable domain that is capable of binding to the first antigen and the second antigen, but not capable of simultaneously binding to said antigens. Examples of a peptide with known binding activity to the desired antigen include those listed in Table 1 above. 1.

The ability of the variable domain to bind to the first antigen and the second antigen, and the lack of the ability to simultaneously bind to these antigens, and, in addition, the ability of the variable domain to simultaneously bind to both of these antigens - the first antigen and the second antigen, provided that any one of the first antigen and the second antigen is on the cell and the other antigen is present separately, both antigens are present separately, or both antigens are present on the same cell, and the lack of ability to simultaneously bind to these antigens when each antigen is expressed on different cells can also be confirmed by the indicated method.

The present invention further provides a method for producing an antigen-binding molecule of the present invention and, first of all, provides a method for producing an antigen-binding molecule comprising an antibody variable domain that is capable of binding to two different antigens (a first antigen and a second antigen) but does not bind to the first antigen and the second antigen simultaneously (the specified variable domain is called the first variable domain), while the method includes the step of obtaining a library of antigen-binding molecules containing different amino acid sequences of the first variable domain.

Examples of the production method for such an antigen-binding molecule include a production method comprising the following steps:

(i) obtaining a library of antigen-binding molecules containing in the variable domains of the antibody, at least one altered amino acid residue, each of which binds to the first antigen or to the second antigen, and the altered variable domains differ from each other by at least one amino acid remainder;

(ii) selecting from the resulting library an antigen-binding molecule comprising a variable domain that exhibits binding activity to the first antigen and the second antigen, but does not bind to the first antigen and the second antigen simultaneously;

(iii) culturing a host cell comprising a nucleic acid encoding the variable domain of the antigen-binding molecule selected in step (ii), wherein the expression of an antigen-binding molecule comprising an antibody variable domain that is capable of binding to the first antigen and the second antigen, but not binding to the first antigen and the second antigen at the same time; and (iv) isolating the antigen-binding molecule from host cell cultures.

Examples of a region favored for altering amino acids include the heavy chain variable domain. More preferred examples of this region include solvent-exposed regions and loops in the variable domain. Among others, CDR1, CDR2, CDR3, FR3 and loops are preferred. First of all, the following positions in the variable domain of the H chain are preferred: 31-35, 50-65, 71-74 and 95-102 according to Kabat numbering, as well as the following positions in the variable domain of the L chain: 24-34, 50- 56 and 89-97 according to the Kabat numbering. More preferred are the following positions in the H chain variable domain: 31, 52a-61, 71-74 and 97101 according to Kabat numbering, as well as the following positions in the L chain variable domain: 24-34, 51-56 and 89-96 according to Kabat numbering.

In said production method, step (ii) may be the following selection step:

(v) selecting from the resulting library an antigen-binding molecule comprising a variable domain that exhibits binding activity with the first antigen and the second antigen, but does not bind simultaneously with the first antigen and the second antigen when each antigen is expressed on different cells.

The antigen-binding molecules used in step (i) are not particularly limited as long as each of said molecules includes an antibody variable domain. The antigen binding molecules can be antibody fragments such as Fv, Fab or Fab', or antibodies containing an Fco region.

The amino acid residue to be modified is selected, for example, from amino acids whose modification does not affect the binding of the antigen to the antibody variable domain that binds to the first antigen or to the second antigen.

In the present invention, a change in a single amino acid residue may be used, or a change in multiple amino acid residues may be used in combination.

In the case of using a change in a plurality of amino acid residues in combination, the number of changes to be combined is not particularly limited and is, for example, 2 or more and 30 or less, preferably 2 or more and 25 or less, 2 or more and 22 or less, 2 or more and 20 or less, 2 or more and 15 or less, 2 or more and 10 or less, 2 or more and 5 or less, or 2

- 28 042507 or more and 3 or less.

The change in the plurality of amino acid residues to be combined may be carried out either in the heavy chain variable domain of the antibody only, or in the light chain variable domain, or may be distributed appropriately in both the heavy chain variable domain and the light chain variable domain.

Changing the amino acid sequence also includes: randomly changing amino acid residues in the above region in the variable domain of the antibody that binds to the first antigen or to the second antigen, and inserting in the above region a peptide with known binding activity to the desired antigen. The antigen-binding molecule of the present invention can be obtained by selecting, among the thus modified antigen-binding molecules, a variable domain that is capable of binding to the first antigen and the second antigen, but not capable of binding to said antigens simultaneously. Examples of a peptide with known binding activity to the desired antigen include those listed in Table 1. 1 above.

The ability of the variable domain to bind to the first antigen and the second antigen and the lack of the ability to simultaneously bind to these antigens, and, in addition, the ability of the variable domain to simultaneously bind to both of these antigens - the first antigen and the second antigen, provided that any one of the first antigen and the second antigen is on the cell and the other antigen is present separately, both antigens are present separately, or both antigens are present on the same cell, and the lack of ability to simultaneously bind to these antigens when each antigen is expressed on different cells can also be confirmed according to with the method mentioned above.

The present invention also includes an antigen-binding molecule obtained by any of the above production methods.

The type or region of change of the amino acid residues incorporated by the method of the present invention is not particularly limited.

In a non-limiting embodiment of the present invention, examples of the library of the present invention include a library consisting of antigen-binding molecules that bind to CD3 (in the case of human CD3, the γ-chain, δ-chain or ε-chain within human CD3), selected as the first antigen, as well as with a randomly selected second antigen.

As used herein, the term library refers to a plurality of antigen-binding molecules, or a plurality of hybrid polypeptides comprising antigen-binding molecules, or nucleic acids or polynucleotides encoding said sequences. The plurality of antigen-binding molecules or the plurality of hybrid polypeptides comprising antigen-binding molecules included in the library are antigen-binding molecules that differ from each other in sequence but do not contain single sequences or hybrid polypeptides included in the antigen-binding molecules.

In one embodiment of the present invention, a hybrid polypeptide can be prepared comprising an antigen-binding molecule of the present invention and a heterologous polypeptide. In one embodiment, the fusion polypeptide may comprise an antigen-binding molecule of the present invention coupled to at least a portion of a viral surface protein selected from the group consisting of, for example, pIII, pVIII, pVII, pIX, Soc, Hoc, gpD, and pVI, as well as variants of these proteins.

In one embodiment of the present invention, the antigen-binding molecule of the present invention is a ScFv, Fab fragment, F(ab)2 or F(ab') ₂ . In another embodiment, the present invention provides a library consisting essentially of a plurality of fusion polypeptides differing from each other in sequence, with each of the fusion polypeptides constituting any of said antigen-binding molecules and a heterologous polypeptide. First of all, the present invention provides a library consisting essentially of a plurality of fusion polypeptides differing from one another in sequence, each of the fusion polypeptides constituting any of said antigen-binding molecules coupled to at least a portion of a virus surface protein selected from a group including, for example, the surface proteins of the virus pIII, pVIII, pVII, pIX, Soc, Hoc, gpD and pVI, as well as their variants. The antigen-binding molecule of the present invention may further comprise a dimerization domain. In one embodiment, the dimerization domain may be located between an antibody heavy chain variable domain and an antibody light chain variable domain and at least a portion of the surface protein of the virus. The dimerization domain may include at least one dimerization sequence and/or a sequence including one or more cysteine residues. The specified dimerization domain is preferably attached to the C-terminal fragment of the variable domain or conserved domain of the heavy chain. The dimerization domain can be characterized by different structures depending on whether the antibody variable domain is obtained as a hybrid polypeptide component of the surface protein of the virus (there is no amber stop codon following the dimerization domain), or depending on whether

- 29 042507 the variable domain of the antibody predominantly without the inclusion of a component of the surface protein of the virus (for example, there is an amber stop codon following the dimerization domain). If the variable domain of the antibody is obtained predominantly as a fusion polypeptide component with the surface protein of the virus, then a bivalent display is obtained using one or more disulfide bonds and/or a single dimerization sequence.

As used herein, the term differing in sequence in relation to a plurality of antigen-binding molecules differing from each other in sequence means that the individual antigen-binding molecules in the library are characterized by different sequences. First of all, the number of different sequences in a library reflects the number of independent clones in the library that differ in sequence, and this term can also be called the size of the library. The size of a typical phage display library is from 10 ⁶ to 10 ¹² and can be increased to 10 ¹⁴ using techniques known in the art such as the ribosome display method. However, the actual number of phage particles to be panned for a phage library is typically 10 to 10,000 times greater than the size of the library. Said excess set, the so-called number of library equivalents, is between 10 and 10,000 individual clones, which may include the same amino acid sequence. Accordingly, the term differing in sequence as used in the present invention means that the individual antigen-binding molecules in the library, excluding the number of library equivalents, have different sequences, and more specifically, the term means that the library comprises from 10 ⁶ to 10 ¹⁴ , preferably from 10 ⁷ to 10 ¹² , more preferably 10 ⁸ to 10 ¹¹ , especially preferably 10 ⁸ to 10 ¹⁰ antigen-binding molecules differing from one another in sequence.

As used herein, the term consisting primarily of a library consisting primarily of a plurality of antigen-binding molecules indicates the number of antigen-binding molecules that differ in their binding activity to the first and/or second antigen among the independent clones in the library that differ in sequence. First of all, the library preferably includes at least 10 ⁴ antigen-binding molecules that exhibit the specified binding activity. More preferably, the present invention provides a library comprising at least 10 ⁵ antigen-binding molecules that exhibit said binding activity. Even more preferably, the present invention provides a library that includes at least 10 ⁶ antigen-binding molecules that exhibit the specified binding activity. First of all, preferably, the present invention provides a library comprising at least 10 ⁷ antigen-binding molecules that exhibit the specified binding activity. Also preferably, the present invention provides a library that includes at least 10 ⁸ antigen-binding molecules that exhibit the specified binding activity. In other words, this term preferably means the ratio of the number of antigen-binding molecules that differ in binding activity to the first and/or second antigen and the number of independent clones in the library that differ in sequence. More specifically, the present invention provides a library comprising antigen-binding molecules that exhibit said binding activity, with a ratio of said molecules to the number of independent clones in the library that differ in sequence from 0.1 to 80%, preferably from 0.5 to 60%. , more preferably 1 to 40%, even more preferably 2 to 20%, especially preferably 4 to 10%. For hybrid polypeptides, polynucleotide molecules or vectors, the number of molecules or their ratio to the total number of molecules can also be indicated, as in the case above. Similarly, for viruses, it is also possible to indicate the number of individuals carrying viruses, or their ratio to the total number of individuals carrying viruses, as in the case above.

As used herein, the term phage display refers to a technique in which polypeptide variants are displayed as fusion proteins linked to at least a portion of surface proteins on the surface of phage particles, eg, filamentous phages. The use of phage display is due to the fact that it provides a fast and efficient screening of a large library of randomized protein variants to select a sequence that binds to a target antigen with high affinity. The display of peptide and protein libraries on phage is used to screen millions of polypeptides to select polypeptides with specific binding properties. The polyvalent phage display method is used to display small random peptides and small random proteins when hybridized to either gene III or gene VIII of a filamentous phage (Wells and Lowman, Curr. Opin. Struct. Biol., 3, c. 355362 (1992) and references cited in the referenced article). Monovalent phage display includes a library of fusion proteins or peptides with gene III or part of it and expression of fusion proteins at low levels in the presence of wild-type gene III protein, with each phage particle displaying one copy of the fusion protein or displaying no copies. Monovalent phage displays

- 30 042507 are characterized by a lower avidity effect compared to polyvalent phages and, therefore, provide screening based on endogenous ligand affinity using phagemid vectors that simplify the processing of resulting DNA (Lowman and Wells, Methods: A Companion to

Methods in Enzymology, 3, p. 205-216 (1991)).

The term phagemid refers to a plasmid vector that includes an origin of bacterial replication, eg ColE1, and a copy of the bacteriophage intergenic region. Accordingly, a phagemid derived from any bacteriophage known in the art, such as filamentous bacteriophage or lambdoid bacteriophage, can be used. Typically, the plasmid also contains a selectable marker for antibiotic resistance. DNA fragments cloned into these vectors can be cultured as plasmids. If the cells containing these vectors include all the genes necessary for the production of phage particles, the plasmid replication pattern changes to rolling ring replication, with copies of one strand of plasmid DNA being formed and packaged into phage particles. The phagemida can form infectious or non-infectious phage particles. This term includes a phagemid that contains a phage surface protein gene or a fragment thereof linked to a gene for a heterologous polypeptide as a result of gene hybridization, in which the heterologous polypeptide is displayed on the surface of the phage particle.

The term phage vector refers to a double-stranded replicative form of a bacteriophage that contains a heterologous gene and is capable of replication. The phage vector includes an origin of phage replication that allows for phage replication and generation of phage particles. The phage is preferably a filamentous bacteriophage, eg M13, f1, fd or Pf3 phage or derivatives thereof, or a lambdoid phage, eg lambda, 21, phi80, phi81, 82, 424, 434 phage or any other phage or derivative thereof.

The term oligonucleotide refers to a short-chain single- or double-stranded polydeoxynucleotide that is prepared by a chemical synthesis method known in the art (e.g., a solid-phase method based on phosphotriesters, phosphites, or phosphoramidites, such as the method described in EP 266032, or a method of preparation through intermediate derivatives H-phosphonate deoxynucleotide described in Froeshler et al., Nucl Acids Res 14 5399-5407 (1986)). Other methods for the synthesis of oligonucleotides include the polymerase chain reaction (PCR) described below and other autoprimer methods, as well as the synthesis of nucleotides on solid supports. All of these methods are described in the article Engels and others, Agnew. Chem. Int. Ed. Engl., 28, p. 716-734 (1989). These methods are used if the complete nucleotide sequence of the gene is known or if a nucleotide sequence is available that is complementary to the coding strand. Alternatively, if the desired nucleic acid sequence is known, then a candidate nucleotide sequence can be appropriately constructed using known and preferred residues encoding each amino acid residue. The oligonucleotide can be purified using polyacrylamide gel columns or molecular sieve columns or precipitation.

The terms fusion protein and fusion polypeptide refer to a polypeptide comprising two segments covalently linked to each other. These segments in a polypeptide differ in their properties. Said property may be, for example, a biological property such as in vitro or in vivo activity. Alternatively, said property may be a chemical or physical property, such as binding to a target antigen or catalyzing a reaction. These two segments can be linked either directly through a single peptide bond, or through a peptide linker containing one or more amino acid residues. Typically, these two segments and the linker are located in the same reading frame. Preferably, the two polypeptide segments are derived from heterologous or different polypeptides.

The term surface protein refers to a protein, at least part of which is present on the surface of a virus particle. Functionally, a surface protein is an arbitrary protein that binds to viral particles during the formation of viruses in host cells and remains bound there until the virus infects other host cells. The surface protein may be a major surface protein or a minor surface protein. A minor surface protein is usually a surface protein present in the viral capsule in an amount of preferably at least about 5, more preferably at least about 7, even more preferably at least about 10 or more protein copies per virion. . The basic surface protein may be present in tens, hundreds or thousands of copies per virion. Examples of the major surface protein include the p8 filamentous phage protein.

In a non-limiting embodiment of the present invention, examples of the library preparation method include the following 6 methods:

1) a method which consists in inserting a peptide (this term is used to include a polypeptide and a protein) that binds to the second antigen into antigen-binding molecules, each of which binds to the first antigen;

- 31 042507

2) a method that consists in obtaining a library in such a way that various amino acid residues are displayed in positions at which a change can be made, which allows lengthening (expanding) the changeable fragments of loops in antigen-binding molecules, and obtaining an antigen-binding molecule exhibiting binding activity with a second arbitrary antigen selected from a library using antigen binding activity as an index;

3) a method that consists in identifying amino acids that allow retaining binding activity with the first antigen, using an antibody obtained by site-directed mutagenesis from an antigen-binding molecule, which has previously been shown to bind to the first antigen, and an antigen-binding molecule exhibiting binding activity is obtained with a second arbitrary antigen selected from a library in which the identified amino acids are displayed using antigen-binding activity as an index;

4) the method according to claim 3, which further includes obtaining an antibody library so that various amino acid residues are displayed in positions that allow lengthening (expanding) of variable fragments of loops in antigen-binding molecules, and obtaining an antigen-binding molecule that exhibits binding activity with a second arbitrary antigen , selected from the library, using the binding activity with the antigen as an index;

5) the method according to claims 1, 2, 3 or claim 4, which further comprises changing the antigen-binding molecules to include carbohydrate sequences (for example, NxS and NxT, where x denotes an amino acid other than P), while inserting into the antigen-binding molecules carbohydrate chains that recognize carbohydrate chain receptors (for example, carbohydrate chains with a high mannose content are inserted into the composition of antigen-binding molecules, and it is known that carbohydrate chains with a high mannose content are obtained by adding kifunensin during antibody expression (mAbs, 4 (4), c 475-87 (2012 Jul-Aug)), and

6) the method according to claims 1, 2, 3 or claim 4, which additionally includes adding domains to the antigen-binding molecules, each of which binds to the second antigen through a covalent bond, inserting Cys, Lys or non-natural amino acid residues into loops or sites that have been found to be substituted with different amino acid residues, or substitution at said sites with Cys, Lys, or non-natural amino acid residues (this method is typed using antibody-drug conjugates, and this method is a Cys conjugation method , Lys or with a non-natural amino acid via a covalent bond (mAbs, 6, 1, c. 34-45 (2014 Jun-Feb), WO 2009/134891 A2 and Bioconjug. Chem., 19, 25 (2), c. 351-361 (2014 Feb)).

In the 6 library preparation methods listed above, a site in the Fab fragment or in the variable domain of the antigen binding molecule is preferably selected as the site for amino acid substitution in each antigen-binding molecule or as the site for inserting a peptide into the antigen-binding molecule. Examples of a preferred region include solvent-exposed regions and variable domain loops. Among others, CDR1, CDR2, CDR3, FR3 and loops are preferred. First of all, the following positions in the variable domain of the H chain are preferred: 31-35, 50-65, 71-74 and 95-102 according to Kabat numbering, as well as the following positions in the variable domain of the L chain: 24-34, 50- 56 and 89-97 according to the Kabat numbering. More preferred are the following positions in the H chain variable domain: 31, 52a-61, 71-74 and 97-101 according to Kabat numbering, as well as the following positions in the L chain variable domain: 24-34, 51-56 and 89-96 according to Kabat numbering.

In one embodiment, examples of Method 1, which includes inserting a second antigen-binding peptide into antigen-binding molecules each of which binds to the first antigen, may also include a G-CSF insertion method, as exemplarily described in Angew Chem., Int. . Ed., Engl., 52 (32), p. 8295-8298 (2013 Aug5). Alternatively, the peptide to be inserted can be obtained from a peptide library in a phage peptide display format. Alternatively, you can use a full-length natural protein or part of it.

In order to identify amino acids that allow retention of binding activity to the first CD3 antigen (in the case of human CD3, the γ-chain, δ-chain or ε-chain constituting human CD3), for example, antibodies are prepared comprising a change in one amino acid residue, when the amino acid residue in sites that are presumably involved in binding to the antigen, and analyze these antibodies. The binding of CD3 to antibodies comprising a change in one amino acid residue can be assessed by an appropriately selected method, well known to those skilled in the art, and can be determined, for example, by ELISA (enzymatic immunoassay), FACS (fluorescence activated cell sorting), ALPHAScreen (homogeneous analysis of proximity-enhanced luminescence) or the BIACORE method based on the phenomenon of surface plasmon resonance (SPR).

To identify amino acids that allow retention of CD3 binding activity,

- 32 042507 you can use data on the ratio of the amounts of antigens, for example, associated with various modified forms, and the amount of antigen associated with the corresponding unchanged antibody. In more detail, if the amount of the corresponding unaltered bound antibody is X and the amount of the form bound to one altered amino acid residue is Y, then Z (ratio of bound components)=Y/X can be used. An altered form may be considered to retain binding activity compared to the corresponding unaltered antibody if Z (ratio of bound components) is 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more, or 0 .9 or more, preferably 0.8 or more. An antibody library can be generated such that amino acids are displayed that allow retention of binding activity.

ECM (extracellular matrix) is an extracellular component and is present in vivo at various sites. In this regard, it is known that an antibody with high ECM binding activity is characterized by lower blood kinetics (shorter half-life) (application WO 2012/093704 A1). Thus, amino acids that do not increase binding to ECM are preferably selected as amino acids that are displayed in the antibody library.

To select amino acids that do not affect ECM binding, ECM binding is assessed, for example, by the procedure described in Comparative Example 2. The ECM binding value (electroluminescent reaction (ECL)) of each altered form is divided by the ECM binding value to the MRA antibody ( H-chain: SEQ ID NO: 57, L-chain: SEQ ID NO: 58) and use the resulting value. When evaluating the effect of increasing the binding of ECM to a variety of modified forms, the value obtained can be used as the indicated value up to 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold or 30x boost. Preferably, up to a 10-fold increase in binding is used to create the library. The library of antibodies can be obtained in such a way that the amino acids selected in this way are displayed.

In the case of insertion into CDR3 of a peptide comprising 6 amino acid residues, but not limited to this case, binding to ECM is increased if the extended loop of CDR3 is enriched in amino acids containing a positively charged side chain. Therefore, the loop should preferably not include three or more amino acid residues containing a positively charged side chain.

In the library of the present invention, a peptide to expand the diversity of the library can be inserted into each variable domain. Examples of the preferred region for a peptide insert include solvent-exposed regions and loops in the variable domain. Among others, CDR1, CDR2, CDR3, FR3 and loops are preferred. First of all, the following positions in the variable domain of the H chain are preferred: 31-35, 50-65, 71-74 and 95-102 according to Kabat numbering, as well as the following positions in the variable domain of the L chain: 24-34, 50- 56 and 89-97 according to the Kabat numbering. More preferred are the following positions in the H chain variable domain: 31, 52a-61, 71-74 and 97-101 according to Kabat numbering, as well as the following positions in the L chain variable domain: 24-34, 51-56 and 89- 96 according to Kabat numbering. Even more preferred are positions 99 and 100 in the variable domain of the H chain according to Kabat numbering. An amino acid that enhances antigen-binding activity can also be included at any time during amino acid residue changes.

In a non-limiting embodiment of the present invention, a typical length of the peptide to be inserted is 1 to 3 amino acid residues, 4 to 6 amino acid residues, 7 to 9 amino acid sequences, 10 to 12 amino acid residues, 13 to 15 amino acid residues, 15 to 20 amino acid residues; and 21 to 25 amino acid residues. The length of the peptide to be inserted is preferably 1 to 3 amino acid residues, 4 to 6 amino acid residues, or 7 to 9 amino acid residues.

Insertion site and peptide length to expand the diversity of the library can be investigated by obtaining molecules that include peptide inserts, and evaluating the binding of such molecules to CD3. Binding to CD3 can be assessed by a suitably chosen method well known to those skilled in the art and can be determined, for example, by ELISA (enzymatic immunoassay), FACS (fluorescence activated cell sorting), ALPHAScreen (homogeneous proximity enhanced luminescence assay). ) or by the BIACORE method based on the phenomenon of surface plasmon resonance (SPR).

In a non-limiting embodiment of the present invention, an antibody library for producing an antibody that binds to CD3 and a second antigen can be generated as follows:

step 1: selection of amino acid residues that retain the ability to bind to CD3 (to ensure binding to 80% or more of the unchanged CD3 antibody).

A library for producing an antibody that binds to CD3 and a second antigen can be generated, for example, such that the amino acids selected in step 1 are displayed.

- 33 042507

In a non-limiting embodiment of the present invention, an antibody library for producing an antibody that binds to CD3 and a second antigen can be generated as follows:

step 1: selection of amino acid residues that retain the ability to bind to CD3 (to ensure binding to 80% or more of the unchanged CD3 antibody), and step 2: insertion of an amino acid residue between positions 99 and 100 (according to Kabat numbering) in the H chain of CDR3 .

A library for producing an antibody that binds to CD3 and a second antigen can be created with an expanded library variety, for example, by performing step 1, as well as by inserting an amino acid residue into the CDR3 domain in step 2.

In a non-limiting embodiment of the present invention, an antibody library for producing an antibody that binds to CD3 and a second antigen can be generated as follows:

step 1: selection of amino acid residues that retain the ability to bind to CD3 (to ensure binding to 80% or more of the unchanged antibody to CD3), step 2: selection of amino acid residues that provide a 10-fold increase in binding to ECM compared to binding to MRA before change, and step 3: insertion of amino acids between positions 99 and 100 (according to Kabat numbering) in the H chain of CDR3.

Amino acid residues that do not increase ECM binding, for example in steps 1 and 3 as well as in step 2, can also be selected as amino acids displayed in the library, although the present invention is not limited to this approach. Even a library obtained without performing step 2 allows analysis of the antigen-binding molecule obtained from the library and evaluation of binding to ECM.

In a non-limiting embodiment of the present invention, the VH domain of CE115HA000 (SEQ ID NO: 52) can be used as a sequence template for an antibody that binds to CD3 (CD3ε). In this instance, exemplary amino acids for use in building the library include any one or more of the amino acids at positions 11, 31, 52a, 52b, 52c, 53, 54, 56, 57, 61, 72, 78, 98, 99, 100, 100a, 100b, 100c, 100d, 100e, 100f, 100g and 101 according to Kabat numbering contained in the heavy chain variable domain.

To create a library, it is preferable to change the V11L/L78I amino acid residues in the VH domain of CE115HA000 (SEQ ID NO: 52), although the library of the present invention is not limited to making this change. In addition, to create a library, it is preferable to change the amino acid residues V11L/D72A/L78I/D101Q in the VH domain of CE115HA000 (SEQ ID NO: 52), although the library of the present invention is not limited to this change.

In a non-limiting embodiment of the present invention, the VL domain of GLS3000 (SEQ ID NO: 53) can be used as a sequence template for an antibody that binds to CD3 (CD3ε). In this case, exemplary amino acids for use in building the library include any one or more amino acid residues at the following positions: 11, 24, 25, 26, 27, 27a, 27b, 27c, 27e, 30, 31, 33, 34, 51, 52 , 53, 54, 55, 56, 74, 77, 89, 90, 92, 93, 94, 96 and 107 according to Kabat numbering contained in the light chain variable domain.

Obtaining a library of the present invention includes, but not limited to, primarily, the creation of a library that includes antigen-binding domains, the amino acid residue in which in a particular site is replaced by the desired amino acid residue, or the creation of a library that includes a plurality of modified forms of antigen-binding molecules, including antigen-binding domains using art-known library building techniques, such as NNK or TRIM libraries (Gonzalez-Munoz A. et al., mAbs (2012), Lee C.V. et al., J. Mol. Biol. (2004), Knappik A. et al., J. Mol Biol (2000) and Tiller, T. et al., mAbs (2013)).

In the present invention, the term one or more amino acid residues is not limited to a specific number of amino acid residues, and may refer to 2 or more types of amino acid residues, 5 or more types of amino acid residues, 10 or more types of amino acid residues, 15 or more types of amino acids, or 20 types of amino acid residues.

In the case of fusion polypeptide display, the variable domain fusion polypeptide of the antigen-binding molecule can be displayed in various forms on the surface of cells, viruses and phagemid particles. These forms include single chain Fv fragments (scFvs), F(ab) fragments and multivalent forms of these fragments. Multivalent forms are preferably dimers ScFv, Fab and F(ab'), which are designated in this context as (ScFv) ₂ , F(ab) ₂ and F(ab') ₂ , respectively. The display of multivalent forms is preferred, probably in part because the display of multivalent forms usually allows the identification of clones with low affinity and/or containing many antigen-binding sites, which allows more efficient selection of rare clones during selection.

Methods for displaying hybrid polypeptides comprising antibody fragments on the surface of bacteriophages are known in the art and are described, for example, in WO 1992/001047 and also in

- 34 042507 of the present description. Other similar methods are described in WO 1992/020791, WO 1993/006213, WO 1993/011236 and WO 1993/019172. Those skilled in the art can appropriately use these methods. Other published documents (Hoogenboom H.R., Winter G., J. Mol. Biol., 227, pp. 381-388 (1992), applications WO 1993/006213 and WO 1993/011236) describe the identification of antibodies using a set of artificially rearranged variable domain genes for various antigens displayed on the surface of phages.

In the case of constructing a vector for displaying in the form of scFv, said vector includes nucleic acid sequences encoding the light chain variable domain and the heavy chain variable domain of the antigen-binding molecule. In general, a nucleic acid sequence encoding the heavy chain variable domain of an antigen-binding molecule is fused to a nucleic acid sequence encoding a virus surface protein component. The nucleic acid sequence encoding the light chain variable domain of the antigen binding molecule is attached to the nucleic acid of the heavy chain variable domain of the antigen binding molecule via a nucleic acid sequence encoding a peptide linker. The peptide linker usually contains 5 to 15 amino acid residues. Optionally, an additional sequence encoding, for example, a peptide tag suitable for purification or detection.

In the case of constructing a vector to display in the form F(ab), said vector includes nucleic acid sequences encoding the variable domains of the antigen-binding molecule and the conserved domains of the antigen-binding molecule. A nucleic acid sequence encoding a light chain variable domain of an antigen binding molecule is fused to a nucleic acid sequence encoding a conserved light chain domain. The nucleic acid sequence encoding the heavy chain variable domain of the antigen binding molecule is fused to the nucleic acid sequence encoding the conserved heavy chain CH1 domain. In general, a nucleic acid sequence encoding a heavy chain variable domain and a conserved domain is fused to a nucleic acid sequence encoding the entire surface protein of a virus or a portion thereof. The heavy chain variable domain and the conserved domain are preferably expressed as a fusion product with at least part of the surface protein of the virus, while the light chain variable domain and the conserved domain are expressed separately from the fusion protein comprising the heavy chain and the surface protein of the virus. The heavy chain and light chain may be linked to each other by a covalent bond or a non-covalent bond. Optionally, an additional sequence encoding, for example, a polypeptide tag suitable for for cleaning or detection.

With respect to the transfer of viruses into host cells, the vectors constructed as described above are transferred into host cells for amplification and/or expression. Vectors can be transferred into host cells by transformation methods known in the art, including electroporation, calcium phosphate precipitation, and the like. If the vectors are infectious particles such as viruses, the vectors themselves enter the host cells. Fusion proteins are displayed on the surface of phage particles upon transfection of host cells with replication expression vectors comprising inserts of polynucleotides encoding the fusion proteins and production of phage particles in a manner known in the art.

Replication expression vectors can be transferred into host cells using a variety of methods. In a non-limiting embodiment, vectors can be transferred into cells by electroporation as described in WO 2000/106717. The cells are cultured at 37°C, optionally for 6 hours to 48 hours (or until the optical density at 600 nm reaches a value of 0.6 to 0.8) in standard culture medium. The culture medium is then centrifuged and the cell supernatant is separated (eg by decantation). In the initial purification step, the cell pellet is preferably resuspended in a buffer solution (eg 1.0 mM HEPES (pH 7.4)). The suspension is then centrifuged again to separate the supernatant. The resulting cell pellet is resuspended in glycerol diluted to a concentration of, for example, 5 vol.% to 20 vol.%. The suspension is centrifuged again, whereby the supernatant is separated and a cell pellet is obtained. The cell pellet is resuspended in water or dilute glycerol. Based on the measured cell density of the resulting suspension, the final cell density is adjusted so as to obtain the required value using water or diluted glycerol.

Examples of preferred recipient cells include E. coli strain SS320 susceptible to

- 35 042507 electroporation (article Sidhu and others, Methods Enzymol., 328, c. 333-363 (2000)). E. coli strain SS320 is prepared by combining MC1061 cells with XL1-BLUE cells under conditions sufficient to transfer a fertile episome (F'-plasmid) or XL1-BLUE into MC1061 cells. E. coli strain SS320 is deposited with the American Culture Type Collection (ATCC, 10801 University Boulevard, Manassas, Virginia) under accession number 98795. Any F'episome capable of replicating phages in said strain can be used in the present invention. The corresponding episome can be obtained from strains deposited with ATCC or can be obtained as a commercial product (TG1, CJ236, CSH18, DHF', ER2738, JM101, JM103, JM105, JM107, JM109, JM110, KS1000, XL1-BLUE, 71- 18, etc.).

The use of higher concentrations of DNA (about 10 times higher) during electroporation allows you to increase the frequency of transformation and the number of DNA molecules that transform host cells. The use of high cell density also increases efficiency (approximately 10 times). An increased number of DNA transfers can provide a library with more diversity and more independent clones with different sequences. Transformed cells are usually selected based on the presence or absence of growth in the medium containing the antibiotic.

The present invention further provides a nucleic acid encoding an antigen-binding molecule of the present invention. The nucleic acid of the present invention may be present in any form such as DNA and RNA.

The present invention further provides a vector comprising the nucleic acid of the present invention. The type of vector can be appropriately selected by those skilled in the art according to the host cells into which the vector is transferred. For example, any of the vectors mentioned above can be used.

The present invention further relates to a host cell transformed with the vector of the present invention. The host cell can be suitably selected by those skilled in the art. For example, any of the host cells mentioned above can be used.

The present invention also provides a pharmaceutical composition comprising an antigen-binding molecule of the present invention and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention can be prepared according to a method known in the art by adding a pharmaceutically acceptable carrier to the antigen-binding molecule of the present invention. For example, the pharmaceutical composition may be used in the form of a sterile solution or suspension in water, or any other pharmaceutically acceptable solution for parenteral injection. For example, a pharmaceutical composition can be prepared by mixing an antigen-binding molecule in a unit dosage form used in standard pharmaceutical practice, in appropriate combination with pharmaceutically acceptable carriers or media, primarily sterile water, saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavor, excipient, carrier, preservative, binding agent, and the like. Specific examples of the carrier may include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinyl pyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, saccharide , carboxymethyl cellulose, corn starch and inorganic salts. The amount of active ingredient in such a preparation is determined so as to provide an appropriate dose within the prescribed range.

A sterile composition for injection can be prepared according to standard pharmaceutical practice using a carrier such as distilled water for injection. Examples of aqueous solutions for injection include saline, isotonic solutions containing glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride. These solutions can be used in combination with an appropriate stabilizer, such as an alcohol (primarily ethanol) or a polyhydric alcohol (such as propylene glycol and polyethylene glycol), or a non-ionic surfactant, such as polysorbate 80(TM) or HCO-50.

Examples of oil solutions include sesame oil and soybean oil. These solutions can be used in combination with benzyl benzoate or benzyl alcohol as a stabilizer. The solutions can be further mixed with a buffer (eg, phosphate buffer and sodium acetate buffer), a sedative (eg, procaine hydrochloride), a stabilizer (eg, benzyl alcohol and phenol), and an antioxidant. The injection solutions thus obtained are usually filled in appropriate ampoules. The pharmaceutical composition of the present invention is preferably administered parenterally. Specific examples of these unit dosage forms include injections, intranasal agents, transpulmonary agents, and transdermal agents. Examples of injections include intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection, which can be used to administer the pharmaceutical composition systemically or topically.

The route of administration can be appropriately selected depending on the age and symptoms of the patient. The dose of a pharmaceutical composition containing a polypeptide or a polynucleotide encoding a polypeptide can be selected in the range, for example, from 0.0001 mg/kg to 1000 mg/kg of body weight per dose. Alternatively, the dose may be selected in the range of, for example, 0.001 to 100,000 mg/kg of the patient's body weight, although the dose is not necessarily limited to these numerical values. Although the dose and route of administration vary depending on the body weight, age, symptoms of the patient, and the like, those skilled in the art can appropriately select the dose and method.

The present invention also provides a method for treating cancer, comprising the step of administering an antigen-binding molecule of the present invention, wherein the antigen-binding molecule of the present invention is for use in the treatment of cancer, and comprising the use of an antigen-binding molecule of the present invention in the preparation of a therapeutic agent for treating cancer. , and a method for producing a therapeutic agent for the treatment of cancer, comprising the step of using an antigen-binding molecule of the present invention.

The three-letter codes and the corresponding one-letter codes used to designate amino acids in this context are defined as follows: alanine: Ala and A, arginine: Arg and R, asparagine: Asn and N, aspartic acid: Asp and D, cysteine: Cys and C, glutamine: Gln and Q, glutamic acid: Glu and E, glycine: Gly and G, histidine: His and H, isoleucine: Ile and I, leucine: Leu and L, lysine: Lys and K, methionine: Met and M, phenylalanine : Phe and F, proline: Pro and P, series: Ser and S, threonine: Thr and T, tryptophan: Trp and W, tyrosine: Tyr and Y, and valine: Val and V.

Those skilled in the art will appreciate that the present invention also includes one or any combination of two or more of the aspects described herein, unless there is a technical conflict based on general technical principles known to those skilled in the art.

All documents cited in this context are incorporated herein by reference in their entirety.

Further, the present invention is illustrated by the following examples. However, the following examples do not limit the present invention.

Examples

Example 1

The concept of an altered variable domain (Fab fragment) of an immunoglobulin that binds to CD3 (first antigen) and another antigen (second antigen), but does not bind simultaneously to CD3 (first antigen) and another antigen (second antigen) on different cells.

Binding of an immunoglobulin to two or more activating FcyR receptors simultaneously, or to an activating FcyR receptor and another antigen simultaneously, causes an activating FcyR crosslinking reaction, which in turn can signal ITAM FcyR and lead to eventual activation of immunocytes. One IgG antibody molecule is only capable of binding to one FcyR molecule as described above. Therefore, it is only in the presence of an antigen that crosslinking of two or more activating FcyR molecules causes immunocyte activation.

When an IgG type antibody binds to an antigen via its variable domain (Fab), said antibody is also capable of simultaneously binding to one or more FcyR molecules via its Fc region. This causes the cell expressing the antigen to fuse with the cell expressing the FcyR. Depending on the cell expressing the antigen, such crosslinking of the antigen and FcyR may have an undesirable effect. First of all, if the antigen is, for example, CD3, fusion of a T cell with an FcyR expressing cell can cause immune activation such as cytokine release (J. Immunol., 163 (3), c. 1246-1252 (1999 Aug. 1)). In this case, the Fc region may lose its binding activity to FcyR due to the inclusion of a change that prevents the reaction of cross-linking of the antigen with FcyR (Advanced Drug Delivery Reviews, 58, pp. 640-656 (2006)). Similarly, when antigens bind to antibodies of the IgG type, such as molecules of the TNFR subfamily (eg, CD40, OX40, and CD27) or CD3 and TLR (eg, TLR2, TLR4, TLR8, and TLR9), FcyR-mediated crosslinking causes systemic immune activation . Thus, the simultaneous binding of these molecules expressed on different cells is undesirable.

Meanwhile, a standard multispecific antibody binds simultaneously to multiple antigens. Depending on the combination of antigens, simultaneous binding to multiple antigens may be undesirable. For example, the αΎβ3 integrin, known as a cell adhesion molecule, is expressed in many cancer cells and surrounding tumor blood vessels, and, therefore, it can be used as a target molecule for targeted drug delivery to the tumor (Haubner R., PLoS Med., 2, e70 (2005)), despite the fact that this molecule is also known to be expressed in various normal cells (Thromb. Haemost., 80 (5), c. 726-734

- 37 042507 (1998 Nov.)) - In this regard, the simultaneous binding of a multispecific antibody to CD3 and integrin ανβ3 can damage normal cells due to strong cytotoxic activity mediated by T cells.

Accordingly, a possible method of controlling such an undesirable cross-linking reaction is to use a Fab molecule with dual binding activity, one part of the variable domain (Fab) of which binds to the first antigen, and the other part, which does not take part in the specified binding to the first antigen, binds to the second antigen (Fig. 1). Provided that two fragments located as closely as possible in the same variable domain (Fab) play an important role in binding to their respective antigens, as shown in FIG. 1, binding to the first antigen inhibits binding to the second antigen, while binding to the second antigen also inhibits binding to the first antigen. Thus, a modified antibody having the properties of such a dual-binding Fab molecule is unable to bind to the first antigen and the second antigen at the same time and therefore may not elicit a crosslinking reaction of the first antigen and the second antigen (FIG. 2). It is also believed that a Fab molecule with dual binding activity is capable of simultaneously binding to both antigens - the first antigen and the second antigen, when the first antigen and the second antigen are not expressed on cell membranes, as in the case of soluble proteins, or both antigens are on the same the same cell, but does not bind simultaneously to these antigens when each antigen is expressed on different cells, and also does not cross-link these two cells (Fig. 3). On the other hand, an antigen (third antigen) binding to another variable domain (Fab) may be crosslinked with the first antigen (FIG. 4) or may be crosslinked with the second antigen (FIG. 5). For this antibody, an Fc region that binds to FcyR can be used as a conserved domain, or an Fc region having reduced FcyR binding activity can be used as a conserved domain.

In addition, by using the properties of said Fab molecule with dual binding activity, it is possible, for example, to propose a method for damaging cancer cells expressing a cancer antigen by antibody-mediated influx of T cells using an integrin as a target in cancer tissues, and thus provide a higher anticancer specificity.

Brief Description of the Method: If a variable domain (Fab) can be modified into a Fab with dual binding activity to provide the following properties, an antibody having the properties as shown in FIG. 1:

1) binding activity with the first antigen;

2) binding activity with the second antigen; And

3) lack of ability to simultaneously bind to the first antigen and the second antigen.

The expression not binding to the first antigen and the second antigen simultaneously also includes both the absence of crosslinking of the cell expressing the first antigen with the cell expressing the second antigen, and the absence of simultaneous binding to the first antigen and the second antigen when each antigen is expressed on different cells. This expression also includes the case when the variable domain is capable of simultaneously binding to both antigens - to the first antigen and the second antigen, when the first antigen and the second antigen are not expressed on cell membranes, as in the case of soluble proteins, or both antigens are on the same and the same cell, but is unable to bind simultaneously to the first antigen and the second antigen when each antigen is expressed on different cells.

Similarly, if a variable domain (Fab) can be modified into a Fab with dual binding activity to provide the following properties, an antibody having the properties as shown in FIG. 6:

1) binding activity with the first antigen on the T cell;

2) binding activity with the second antigen on the antigen-presenting cell; And

3) lack of ability to simultaneously bind to the first antigen and the second antigen. Example 2

Preparation of CE115 antibody against human CD3ε and cynomolgus macaque CD3ε.

(2-1) Preparation of a hybridoma using a rat immunized with cells expressing human CD3 and cynomolgus macaque CD3.

Each SD rat (female, 6 weeks old at start of immunization, Charles River Laboratories Japan, Inc.) was immunized with Ba/F3 cells expressing human CD3εy or cynomolgus macaque CD3εy as follows: on day 0 (original date was defined as day 0) , the rat was injected intraperitoneally with 5×10 ⁷ Ba/F3 cells expressing human CD3εy along with Freund's complete adjuvant (Difco Laboratories, Inc.). On day 14, 5 x 10 ⁷ Ba/F3 cells expressing cynomolgus monkey CD3εy were injected intraperitoneally along with incomplete Freund's adjuvant (Difco Laboratories, Inc.). The rat was then alternately intraperitoneally injected one week later (4 times in total) with 5×10 ⁷ Ba/F3 cells expressing human CD3εy and Ba/F3 cells expressing CD3εy of the cynomolgus macaque.

- 38 042507

One week later (Day 49), CD3εγ, Ba/F3 cells expressing human CD3εγ, were administered intravenously for the last time to enhance the immune response. After 3 days, rat spleen cells were mixed with SP2/0 mouse myeloma cells by a standard method using PEG1500 (Roche Diagnostics KK) to obtain hybridomas. Hybrid cells, i.e. hybridomas were cultured in RPMI1640 medium containing 10% fetal bovine serum (FBS, abbreviated as 10% FBS/RPMI1640 in this context).

One day after hybridization (1), the hybrid cells were suspended in a semi-liquid medium (Stemcell Technologies, Inc.). Hybridomas were selectively cultured and cell colonies were also obtained.

9 or 10 days after hybridization, hybridoma colonies were harvested and plated at 1 colony/well in a 96-well plate containing HAT selective medium (10% PBS/RPMI1640, 2 vol% HAT 50x concentrate (Sumitomo Dainippon Pharma Co., Ltd. .), and 5 vol.% BM-Condimed H1 (Roche Diagnostics K.K.)). After 3-4 days of cultivation, the culture supernatant was taken from each well and the concentration of rat IgG was measured in it. The culture supernatate, in which the presence of rat IgG was confirmed, was analyzed to detect a clone producing an antibody specifically binding to human CD3εγ by whole cell ELISA using immobilized Ba/F3 cells expressing human CD3εγ or immobilized Ba/F3 cells not expressing CD3εγ human (Fig. 7). The clone was also evaluated for cross-reactivity with cynomolgus macaque CD3εγ by whole cell ELISA using immobilized Ba/F3 cells expressing cynomolgus macaque CD3εγ (FIG. 7).

(2-2) Obtaining a chimeric antibody against human CD3ε and macaque CD3ε.

Full-length RNA was extracted from each hybridoma cell using RNeasy Mini Kits (Qiagen N.V.) and cDNA was synthesized using the SMART RACE cDNA Amplification Kit (BD Biosciences). The resulting cDNA was used in the PCR method to insert the antibody variable domain gene into a cloning vector. The nucleotide sequence of each DNA fragment was determined using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Inc.) and the ABI PRISM 3700 DNA Sequencer (Applied Biosystems, Inc.) according to the method described in the instruction manual supplied with the instrument. . The CDR and FR fragments of the CE115 H chain variable domain (SEQ ID NO: 13) and the CE115 L chain variable domain (SEQ ID NO: 14) were determined according to the Kabat numbering system.

A gene encoding the H chain of a chimeric antibody containing a rat antibody H chain variable domain fused to a conserved domain of a human IgG1 antibody and a gene encoding a chimeric antibody L chain containing a rat antibody L chain variable domain fused to a conserved kappa domain human antibody chains were inserted into animal cell expression vectors. The resulting expression vectors were used to express and purify the CE115 chimeric antibody (comparative example 1).

(2-3) Preparation of EGFR_ERY22_CE115 antibody.

Then, IgG against cancer antigen (EGFR) was used as the main chain to obtain a molecule in the form containing one Fab fragment, replaced by CD3ε-binding domains. In this operation, a silent Fc with reduced binding activity to FcgR (with the Fcy receptor) was used as the Fc of the IgG main chain, as in the case mentioned above. Cetuximab VH (SEQ ID NO: 15) and cetutximab VL (SEQ ID NO: 16), which are part of the variable domain of cetuximab, were used as EGFR binding domains. As conserved domains of the H chain of the antibody, Gld derived from IgG1 by deleting the C-terminal residues of Gly and Lys, A5 derived from Gld by incorporating the D356K and H435R mutations, and B3 derived from Gld by incorporating the K439E mutation were used, and each was combined with cetuximab VH, producing cetuximab VH-Gld (SEQ ID NO: 17), cetuximab VH-A5 (SEQ ID NO: 18), and cetuximab VH-B3 (SEQ ID NO: 19) as described in Comparative Example 1 When the conserved domain of the antibody H chain is designated as H1, the sequence corresponding to the antibody H chain containing cetuximab VH as the variable domain is designated as cetuximab VH-H1.

In this context, an amino acid change is represented, for example, as D356K. The first character (ie D) denotes the amino acid residue before the change in the one letter code. The number (i.e. 356) following the symbol denotes the position of the specified amended residue according to the Kabat numbering. The last character (ie K) denotes the character which is the amino acid residue after the change in the one letter code.

The EGFR_ERY22_CE115 antibody (FIG. 8) was generated by an exchange between the VH domain and the VL domain of an anti-EGFR Fab. In more detail, a series of expression vectors comprising the insertion of each polynucleotide encoding EGFR_ERY22_Hk (SEQ ID NO: 20), EGFR_ERY22_L (SEQ ID NO: 21), CE115_ERY22_Hh (SEQ ID NO: 22) or CE115_ERY22_L (SEQ ID NO: 23) was prepared by a known method, such as the PCR method, using primers containing the appropriate sequence added in the same manner as above.

- 39 042507

Expression vectors were transferred in the following combination to FreeStyle 293-F cells, where each test molecule was transiently expressed.

Molecule of interest: EGFR_ERY22_CE115.

Polypeptides encoded by polynucleotides inserted into expression vectors:

EGFR_ERY22_Hk, EGFR_ERY22_L, CE115_ERY22_Hh and CE115_ERY22_L.

(2-4) Purification of the EGFR_ERY22_CE115 antibody

The resulting culture supernatant was applied to an Anti FLAG M2 column (Sigma-Aldrich Corp.) and washed, then the test antibody was eluted with a FLAG peptide solution (0.1 mg/ml, Sigma-Aldrich Corp.). The fraction containing the desired antibody was applied to a HisTrap HP column (GE Healthcare Japan Corp.) and the column was washed, then eluted with an imidazole concentration gradient. The fraction containing the desired antibody was concentrated by ultrafiltration. This fraction was then applied to a Superdex 200 column (GE Healthcare Japan Corp.). Only the monomeric fraction was isolated from the eluate, and the purified molecule under study was obtained.

(2-5) Measurement of cytotoxic activity using human peripheral blood mononuclear cells.

(2-5-1) Obtaining a solution of human peripheral blood mononuclear cells (PBMC).

From each healthy volunteer (adult), 50 ml of peripheral blood was collected using a syringe prefilled with 100 μl of heparin solution (1000 units/ml, Novo-Heparin 5000 units for injection, Novo Nordisk A/S). Peripheral blood was diluted 2-fold with PBS(-) phosphate buffered saline and then divided into 4 equal parts, which were then added to Leucosep lymphocyte isolation tubes (catalog number 227290, Greiner Bio-One GmbH) pre-filled with 15 ml Ficoll- Paque PLUS and centrifuged beforehand. After centrifugation (2150 rpm, 10 min, room temperature), a layer of mononuclear cell fraction was separated in isolation tubes. Cells in the mononuclear cell fraction were washed once with Dulbecco's Modified Eagle's Medium (D-MEM) containing 10% PBS (Sigma-Aldrich Corp., in this context 10% PBS/D-MEM). The cells were then diluted with 10% FBS/D-MEM to a density of 4 x 10 ⁶ cells/ml. The cell solution thus obtained was used as human PBMC in the following analysis.

(2-5-2) Measurement of cytotoxic activity.

Cytotoxic activity was evaluated based on the rate of cell growth suppression using the xCELLigence real-time cell analyzer (Roche Diagnostics). As target cells, we used the SK-pca13a cell line created as a result of forced expression of human EGFR by SK-HEP-1 cells. SK-pca13a cells were separated from the plate, and seeded at 100 μl/well (1 x 10 ⁴ cells/well) in a 96-well E-Plate (Roche Diagnostics), and analysis of live cells was started using a real-time cell analyzer. xCELLigence real-time cell analyzer. The next day, the plate was removed from the xCELLigence real-time cell analyzer and 50 μl of each antibody was added to the plate at each concentration: 0.004 nM, 0.04 nM, 0.4 nM, and 4 nM. The reaction was carried out at room temperature for 15 minutes, then 50 μl (2x105 cells/well) of the human PBMC solution prepared as described in the previous section (2-5-1) was added to the plate. This plate was placed back into the xCELLigence real-time cell analyzer and live cell analysis was started. The reaction was carried out at 37°C in an atmosphere of 5% CO ₂ . 72 hours after the addition of human PBMC, the degree of cell growth inhibition (%) was determined from the cell index value according to the equation below. In the calculation, as the value of the cell index, the numerical value obtained after normalization to the value of the cell index obtained immediately before the addition of the antibody, which was determined as equal to 1, was used.

Degree of cell growth inhibition (%)=(A-B)x100/(A-1), where A is the average cell index value obtained for wells in which no antibody was added (containing only target cells and human PBMC), and B denotes the average value of the cell index obtained for the wells to which each antibody was added. The test was carried out in triplicate.

The cytotoxic activity of the CE115 antibody containing EGFR_ERY22_CE115 was determined using human blood-derived PBMCs as effector cells. As a result, extremely high activity was confirmed (FIG. 9).

Example 3

Obtaining an antibody that binds to CD3 and to human αΎβ3 integrin, but does not bind to these antigens simultaneously.

As shown in FIG. 1-6, a Fab with dual binding activity is a molecule that binds to CD3 (first antigen) and test antigen (second antigen) through its variable domain (Fab), but does not bind to CD3 (first antigen) and test antigen (second antigen). antigen) at the same time. When a change in amino acid residues is included for binding to a second antigen in the Fab region of an antibody that binds to CD3 (the first antigen), the change in amino acid residues is usually carried out in both H chains or L chains. By incorporating a change in either the H chain or the L chain, the two Fab regions of the antibody acquire the ability to bind to two antigens, respectively. Thus, these two Fab regions can bind to CD3 (first antigen) and test antigen (second antigen) simultaneously and crosslink them. In this regard, one Fab region of the antibody is obtained as a Fab region that binds to a third antigen or does not bind to any antigen, and the other Fab region is obtained as a Fab with dual binding activity to prevent cross-linking reaction between CD3 ( the first antigen) and the test antigen (the second antigen).

(3-1) Obtaining an antibody that binds to CD3 and to human αγβ3 integrin, but does not bind to these antigens simultaneously.

The αγβ3 integrin, known as a cell adhesion molecule, is expressed in many cancer cells and surrounding tumor blood vessels and, therefore, can be used as a target molecule for targeted drug delivery to the tumor, despite the fact that this molecule, is also known to be expressed in various normal cells (Thromb. Haemost., 80 (5), c. 726-734 (1998 Nov.)) - Thus, simultaneous binding to CD3 and integrin αγβ3 can damage normal cells due to strong cytotoxic activity mediated by T cells. Accordingly, it has been suggested that an anti-EGFR antibody molecule can target tumor cells expressing αγβ3 integrin without damaging normal cells if one can obtain a molecule that does not bind to CD3 and αγβ3 integrin simultaneously. In this regard, a study was conducted to obtain a Fab molecule with dual binding activity, which is able to bind to EGFR through one variable domain (Fab) and bind to the first CD3 antigen and the second antigen, αγβ3 integrin, through another variable domain, but is not able to bind to CD3 and integrin αγβ3 simultaneously.

Provided that a molecule that binds to CD3 through one Fab region in the absence of αγβ3 integrin and binds to αγβ3 integrin through another Fab region in the absence of CD3 can be represented as a molecule that does not bind to αγβ3 integrin in the CD3 binding state or not. binds to CD3 in a state of binding to the αγβ3 integrin, it can be concluded that a Fab molecule with dual binding activity has been successfully constructed, with the desired properties of the investigated Fab with dual binding activity (i.e., characterized by the ability to bind to CD3 and a second antigen and the lack of the ability to simultaneously bind with CD3 and a second antigen).

(3-2) Obtaining an antibody comprising a Fab region that binds to integrin αγβ3

Possible methods for obtaining a Fab molecule with dual binding activity include the use of libraries and the method of inserting a peptide with known protein binding activity. The RGD peptide (Arg-Gly-Asp) is known as a peptide exhibiting binding activity to the αγβ3 integrin. Therefore, the RGD peptide was inserted into the heavy chain loop of the CD3ε-binding antibody CE115 (heavy chain variable domain: SEQ ID NO: 13, light chain variable domain: SEQ ID NO: 14) to obtain a heterodimerized antibody that included EGFR binding domains in one Fab fragment, and a CD3-binding domain and an αγβ3 integrin-binding domain in another Fab fragment, as described in Comparative Example 1. In more detail, a series of expression vectors were generated such that each polynucleotide encoding

EGFR ERY22_Hk (SEQ ID NO: 20), EGFR ERY22 L (SEQ ID NO: 21) or

CE115 ERY22 L (SEQ ID NO: 23), as well as a polynucleotide encoding any of the following fragments: CE115 2 ERY22_Hh (SEQ ID NO. 24, positions 52b and 53 according to the Kabat numbering system replaced by K and N, respectively), CE115 4 ERY22_Hh (SEQ ID NO: 25, positions 52b and 54 according to the Kabat numbering system replaced by S and N, respectively), CE115_9 ERY22_Hh (SEQ ID NO. 26, insertion of RGD between positions 52a and 52b according to the Kabat numbering system), CE11510 ERY22_Hh ( SEQ ID NO. 27, RGD insertion between positions 52b and 52c according to Kabat numbering system), CE11512 ERY22_Hh (SEQ ID NO. 28, RGD insertion between positions 72 and 73 according to Kabat numbering system), CE115_17 ERY22_Hh (SEQ ID NO. 29, positions 52b and 52c according to the Kabat numbering system are replaced by K and S, respectively), CE115_47 ERY22_Hh (SEQ ID NO: 30, insert RGD between positions 98 and 99 according to Kabat numbering system), CE115_48 ERY22_Hh (SEQ ID NO. 31, insert RGD between positions 99 and 100 according to the K numbering system abata), and CE115 49 ERY22_Hh (SEQ ID NO. 32, insert RGD between positions 100 and 100a according to the Kabat numbering system).

An antibody (EH240-Knl25/EH240-H1076/L73, SEQ ID NO: 33/34/35) inserting an RGD peptide (ArgGly-Asp) in the CH3 region of the antibody described in J. Biotech., 155, c. 193-202 (2011), also received in

- 41 042507 as a control as described in Comparative Example 1. It can be assumed that this molecule, which binds to the αvβ3 integrin via its CH3 region, is able to simultaneously bind to CD3 and to the αγβ3 integrin.

(3-3) Confirmation of antibody binding to αvβ3 integrin.

The binding of each molecule, including an RGD (Arg-Gly-Asp) insert in the Fab region, to the αvβ3 integrin was assessed by electrochemiluminescence (ECL method). In more detail, a biotin-conjugated anti-human IgG Ab (Southern Biotech) diluted with a TBS solution containing 0.1% bovine serum albumin (BSA), 0.1 g/l calcium chloride and 0.1 g/l magnesium chloride (in in this context, a dilution solution (+)), while the concentration of each antibody solution was adjusted to 5 μg / ml or 1 μg / ml, as well as ογβ3 integrin (R&D Systems, Inc.) containing a sulfo-label, each of these solutions was added at 25 μl/well to a 96-well Nunc-Immuno(TM) MicroWell(TM) round bottom microplate (Nunc) and mixed, then the plate was incubated at 4° C. overnight to obtain an antibody-antigen complex. A TBS solution containing 0.5% BSA, 0.1 g/L calcium chloride, and 0.1 g/L magnesium chloride (in this context blocking(+) solution) was added at 150 µl/well to the streptavidin-immobilized plate ( firm MSD KK) and the tablet was incubated at 4°C during the night. The blocking solution was removed and then each well was washed three times with 250 μl of a TBS solution containing 0.1 g/l calcium chloride and 0.1 g/l magnesium chloride (in this context, TBS(+) solution). An antibody-antigen complex solution was added to the plate in an amount of 75 μl/well, and the plate was incubated at room temperature for 2 hours, while the biotin-conjugated anti-human IgG Ab bound to the streptavidin immobilized on the plate. The antibody-antigen complex solution was removed, then each well was washed three times with TBS(+) solution, and READ buffer solution (MSD K.K.) was added to each well in an amount of 150 μl/well, then the luminescent signal of the sulfo-label was recorded using a Sector Imager 2400 instrument ( from MSD K.K.).

The results shown in FIG. 11 indicate that the original antibody EGFRERY22 Hk/EGFR ERY22 L/CE115ERY22 Hh/CE115 ERY22 L „

- - - - - does not show binding activity with integrin αvβ3, while all antibodies

EGFRERY22 Hk/EGFR ERY22 L/CE115 2

ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_4

ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_9

ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_10

ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_12

ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_17

ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_47

ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_48

ERY22_Hh/CEl 15ERY22L and EGFR ERY22_Hk/EGFR ERY22_L/CE115_49

ERY22_Hh/CEl 15ERY22L bind to integrin αγβ3.

(3-4) Confirmation of antibody binding to CD3 (CD3ε).

The ability of each antibody comprising the ογβ3 integrin-binding Fab region prepared as described in the previous section to retain CD3 binding activity was then evaluated by ECL. In more detail, a biotin-conjugated anti-human IgG Ab (Southern Biotech) diluted with a TBS solution containing 0.1% BSA (herein the dilution solution(-)), with the concentration of each of these antibody solutions adjusted to 5 μg/mL or 1 μg/mL, as well as CD3ε homodimer protein containing a sulfo tag, were each added at 25 μL/well to a 96-well Nunc-Immuno(TM) MicroWell(TM) round bottom microplate (Nunc) and stirred, then the tablet was incubated at 4°C overnight, while receiving an antibody-antigen complex. A TBS solution containing 0.5% BSA (in this context blocking(-) solution) was added at 150 μl/well to a streptavidin immobilized plate (MSD K.K.) and the plate was incubated at 4° C. overnight. The blocking solution was removed and then each well was washed three times with 250 μl of TBS(-) solution. An antibody-antigen complex solution was added to the plate in an amount of 75 μl/well, and the plate was incubated at room temperature for 2 hours, while the conjugated anti-human IgG Ab bound to the streptavidin immobilized on the plate. The antibody-antigen complex solution was removed, then each well was washed three times with a TBS(-) solution, and READ buffer solution (MSD K.K.) was added to each well in an amount of 150 μl/well, then the luminescent signal of the sulfo-label was recorded using a Sector Imager 2400 (MSD K.K.).

- 42 042507

The results shown in FIG. 12 indicate that the original antibody

EGFR ERY22_Hk/EGFR ERY22L/CE115 ERY22_Hh/CEl 15ERY22L and antibodies

EGFR ERY22_Hk/EGFR ERY22_L/CE115_2

ERY22_Hh/CE115_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_4

ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_9

ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_10

ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_12

ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_17

ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_47

ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_48

ERY22 Hh/CEl 15 ERY22 L and EGFR ERY22_Hk/EGFR ERY22L/CE115 49

ERY22_Hh/CEl 15_ERY22_L bind to CD3.

(3-5) Confirmation of the absence of simultaneous binding of the Fab region to the αΎβ3 integrin and to CD3 by the ECL method.

As evident from the results presented in the sections above, the resulting molecules exhibit ave3 integrin binding activity and CD3 binding activity. Then, each Fab region, obtained as described in the sections above, was evaluated for simultaneous binding to CD3 (CD3ε) and ave3 integrin.

When the molecule with the RGD peptide (Arg-Gly-Asp) insert in the Fab region simultaneously binds to the αγβ3 integrin and to CD3, its binding to both antigens can be detected by the ECL method when the av33 integrin and biotinylated CD3 are added to the antibody solution. In more detail, biotinylated human CD3ε homodimeric protein diluted in diluent(+), each antibody solution adjusted to 10 µg/mL or 5 µg/mL, and αγβ3 integrin (R&D Systems, Inc.) containing sulfo α-tag, each was added at 25 μl/well to a 96-well Nunc-Immuno(TM) MicroWell(TM) round bottom microplate (Nunc) and mixed, then the plate was incubated at 4° C. overnight to obtain antibody-antigen complex.

The blocking(+) solution was added at 150 μl/well to a streptavidin immobilized plate (MSD K.K.) and the plate was incubated at 4° C. overnight. The blocking solution was removed and then each well was washed three times with 250 μl of a TBS(+) solution containing 0.1 g/l calcium chloride and 0.1 g/l magnesium chloride. A solution of the antibody-antigen complex was added to the plate at 75 μl/well, and the plate was incubated at room temperature for 2 hours, while the biotin-conjugated anti-human IgG Ab bound to the streptavidin immobilized on the plate. The antibody-antigen complex solution was removed, then each well was washed three times with TBS(+) solution, and READ buffer solution (MSD K.K.) was added to each well in an amount of 150 μl/well, then the luminescent signal of the sulfo-label was recorded using a Sector Imager 2400 (MSD K.K.).

The results shown in FIG. 13 and 14 indicate that antibodies

EGFR ERY22_Hk/EGFR ERY22 L/CE115 2

ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_12

ERY22_Hh/CEl 15ERY22L and EGFR ERY22_Hk/EGFR

ERY22 L/CE11517 ERY22_Hh/CEl 15ERY22L, including the RGD peptide (Arg-Gly-Asp) inserted into the Fab region, bind to the ave3 integrin and CD3 simultaneously, causing a strong signal recorded by the ECL analysis method. On the contrary, antibodies

EGFR ERY22_Hk/EGFR ERY22L/CE115 9

ERY22_Hh/CEl 15 ERY22 L and EGFR ERY22_Hk/EGFR ERY22 L/CE115 48

ERY22_Hh/CEl 15ERY22L produce only a weak signal in this assay (FIG. 13). All antibodies

- 43 042507

EGFR ERY22_Hk/EGFR

ERY22_L/CE115_4 ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR

ERY22_L/CE115_10 ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR

ERY22_L/CE115_47 ERY22_Hh/CEl 15ERY22L and also EGFR

ERY22_Hk/EGFR ERY22 L/CE115 49 ERY22_Hh/CEl 15 ERY22 L rarely produce an ECL signal (FIG. 14). These results indicate that these antibodies do not bind to the ave3 integrin in the CD3 binding state.

(3-6) Discussion of ECL analysis results showing that the Fab region does not bind to αγβ3 integrin and to CD3 at the same time.

As follows from the above results, the resulting antibody is characterized by the properties of a Fab molecule with dual binding activity, binding to each of the components - to CD3 (CD3ε) and to integrin, αγβ3 through one Fab region, but which does not bind to CD3 (CD3ε) and with ave3 integrin at the same time. In this example, the RGD peptide that binds to the second antigen, ave3 integrin, was inserted into the variable domain (Fab) of an antibody comprising the specified variable domain that binds to the first CD3 antigen, and a molecule was successfully obtained that had binding activity to the second antigen, but which did not bind to CD3 and the second antigen at the same time. By analogous methods, a peptide having protein binding activity as described in WO 2006/036834 can be inserted into a Fab loop, resulting in a Fab molecule with dual binding activity that binds to an arbitrary second antigen. A peptide exhibiting protein-binding activity can be obtained by creating a peptide library according to known methods and selecting from the library a peptide having the desired activity (Pasqualini R., Nature, 380 (6572) cc. 364-366 (1996)). In addition, a library of antigen-binding molecules obtained by altering the Fab loops as described in Example 5 can be used to generate a Fab molecule with dual binding activity that binds to an arbitrary second antigen. Variable domains that bind to the first antigen can be obtained by various known methods. Therefore, it can be concluded that such libraries can be used to generate Fab molecules with dual binding activity that bind to an arbitrary first antigen and an arbitrary second antigen, but which are unable to bind to the first antigen and the second antigen at the same time.

The above results indicate that antibodies

EGFR ERY22_Hk/EGFR ERY22_L/CE115_4 ERY22_Hh/CEl 15_ERY22_L, EGFR

ERY22_Hk/EGFR ERY22_L/CE115_10 ERY22_Hh/CEl 15_ERY22_L, EGFR

ERY22_Hk/EGFR ERY22_L/CE115_47 ERY22_Hh/CEl 15 ERY22 L and also

EGFR ERY22_Hk/EGFR ERY22_L/CE115_49 ERY22_Hh/CEl 15_ERY22_L bind to CD3 and αγβ3 integrin, but not to CD3 and ave3 integrin simultaneously. These results indicate that antibodies

EGFR ERY22_Hk/EGFR ERY22_L/CE115_4

ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_10

ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_47

ERY22 Hh/CEl 15 ERY22 L and also EGFR ERY22_Hk/EGFR

ERY22 L/CE115 49 ERY22_Hh/CEl 15 ERY22 L are Fab molecules with dual binding activity, and also confirm the possibility of obtaining such molecules.

Example 4

Obtaining antibodies that bind to human CD3 and Toll-like receptor 2 (TLR2) but do not bind to these antigens simultaneously.

(4-1) Obtaining antibodies that bind to human CD3 and TLR2, but do not bind to these antigens simultaneously.

TLR2 is known to be a pattern recognition receptor and is mainly expressed on immunocytes such as macrophages, dendritic cells or B cells and is a molecular target that activates immunocytes. It is known that TLR2 is also expressed on normal cells that are not immunocytes, such as epithelial cells or endothelial cells. Binding of the cancer antigen and CD3 simultaneously causes the migration (mobilization) of CD3-expressing T cells to the tumor site, whereby the T cells damage the cancer cells. In this case, the simultaneous binding of the cancer antigen and TLR2 can also cause the migration of TLR2-expressing immunocytes to the cancer site, apparently as a result of its activation.

- 44 042507 munocytes. Cancer cells damaged by T-cells are captured by TLR2-mobilized immunocytes. The antigen can be processed and present on HLA, activating T cells. In this case, stronger T-cell activation and induction can occur, as well as the development of acquired immunity. However, the binding of CD3 and TLR2 at the same time can lead to damage to immunocytes and normal cells due to the high cytotoxic activity mediated by T cells. Accordingly, it is expected that immunocytes and normal cells expressing TLR2 can be mobilized without damaging said cells if a molecule can be obtained that does not bind to CD3 and TLR2 simultaneously. Thus, studies have been carried out with the aim of developing a molecule containing a Fab fragment with dual binding activity that binds to EGFR (via one variable region (Fab)) and binds to the first antigen, CD3, and to the second antigen, TLR2, through another variable region, but which is unable to bind to CD3 and TLR2 at the same time.

Provided that a molecule that binds to CD3 through one Fab region in the absence of TLR2, and binds to TLR2 through another Fab region in the absence of CD3, can be represented as a molecule that does not bind to TLR2 in the CD3-binding state, or does not bind to CD3 in the TLR2 binding state, it can be concluded that a molecule containing a Fab region with dual binding activity has been successfully constructed (i.e., a molecule that has binding properties for CD3 and the second antigen, but which does not bind to CD3 and the second antigen at the same time ).

(4-2) Obtaining antibodies containing a Fab region that binds to TLR2

It is known that the peptide RWGYHLRDRKYKGVRSHKGVPR (SEQ ID NO: 36) is a peptide that has a binding activity with human TLR2. Thus, to obtain each heterodimerized antibody containing EGFR binding domains in one Fab region and a CD3 binding domain and a TRL2 binding domain in another Fab region, the TRL2 binding peptide was inserted into the heavy chain loop of the CD3ε binding antibody. CE115 (heavy chain variable domain: SEQ ID NO: 13, light chain variable domain: SEQ ID NO: 14) as described in Comparative Example 1. In more detail, a series of expression vectors were prepared in such a way as to ensure the insertion of each polynucleotide encoding

EGFR ERY22_Hk (SEQ ID no: 20), EGFR ERY22 L (SEQ ID no: 21) or

CE115ERY22L (SEQ ID no: 23), as well as a polynucleotide encoding any of the following fragments: CE115 DU21 ERY22_Hh (SEQ ID no. 37, where a TRL2-binding peptide is inserted between positions 52b and 52c according to Kabat numbering), CE115_DU22 ERY22_Hh (SEQ ID no: 38, where a TRL2 binding peptide is inserted between positions 52b and 52c according to Kabat numbering), CE115 DU26 ERY22_Hh (SEQ ID no. 39, where a TRL2 binding peptide is inserted between positions 72 and 73 according to Kabat numbering), and also CE115 DU27 ERY22_Hh (SEQ ID no. 40, where a TRL2 binding peptide is inserted between positions 72 and 73 according to Kabat numbering).

In addition, as controls, as described in Comparative Example 1, an antibody (CE115_ERY22_DU42_Hh, SEQ ID No. 41) wherein the TLR2 binding peptide is attached to the C-terminus of the CH3 region, and an antibody (CE115_ERY22_DU43_Hh, SEQ ID No. 42) were obtained , where the -N-binding peptide containing Cys residues at both ends is attached to the C-terminus of the CH3 region. These molecules, which bind to TLR2 through their CH3 regions, are expected to be able to bind to CD3 and TLR2 simultaneously. (4-3) Confirmation of antibody binding to TLR2 The binding activity of each molecule containing the TLR2 binding peptide inserted in the Fab region against TLR2 was evaluated by the ECL method. In more detail, biotin-conjugated antibodies against human IgG Ab (Southern Biotech) were diluted with TBS solution containing 0.1% BSA (dilution solution (-)), each antibody solution diluted to a concentration of 5 μg/ml or 1 μg/ml , and TLR2 (Abnova Corp.) with a sulfo-label included were added to the wells of a 96-well Nunc-Immuno MicroWell round bottom plate (Nunc), each at 25 μl/well, and mixed, then the plate was incubated for overnight at 4°C to form an antibody-antigen complex. To a streptavidin immobilized plate (MSD K.K.) was added a TBS solution containing 0.5% BSA (blocking solution (-)), in an amount of 150 μl/well, and the plate was incubated overnight at 4°C. After removal of the blocking solution, each well was washed three times with 250 μl of TBS(-) solution. A solution containing the antibody-antigen complex was added to the plate in an amount of 75 μl/well, and the plate was incubated at room temperature for 2 h to bind the biotin-conjugated anti-human IgG Ab to the immobilized streptavidin on the plate. After removing the solution containing the antibody-antigen complex, each well was washed three times with a TBS(-) solution, and READ buffer solution (MSD K.K.) was added to them in an amount of 150 μl/well, then the fluorescent signal of the sulfo-label was recorded using the Sector instrument Imager 2400 (MSD K.K.).

- 45 042507

The results are shown in FIG. 15.

Parent EGFR antibody

ERY22_Hk/EGFR ERY22L/CE115 ERY22_Hh/CEl 15ERY22L did not show binding activity with

TLR2, while all

EGFRERY22 Hk/EGFR

ERY22_L/CE115_DU21 ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR

ERY22_L/CE115_DU22 ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR

ERY22L/CE115 DU26 ERY22_Hh/CEl 15ERY22L and also EGFR

ERY22_Hk/EGFR ERY22_L/CE115_DU27 ERY22_Hh/CEl 15_ERY22_L bind to TLR2.

(4-4) Confirmation of antibody binding to CD3 (CD3ε).

Then, the persistence of the binding activity of each type of antibody containing TRL2 binding Fab regions obtained as described in the previous section against CD3 (CD3ε) was assessed by the ECL method. In more detail, biotinylated anti-human IgG Ab (Southern Biotech) was diluted with a TBS solution containing 0.1% BSA (dilution solution (-)), then each antibody solution diluted to a concentration of 5 μg/ml or 1 μg/ ml, as well as CD3ε protein homodimer with a sulfo-label included, were added to the wells of a 96-well Nunc-Immuno MicroWell round bottom plate (Nunc), each in an amount of 25 μl/well, and mixed, then the plate was incubated overnight at 4 °C to form an antibody-antigen complex. To a streptavidin immobilized plate (MSD K.K.) was added a TBS solution containing 0.5% BSA (blocking solution (-)), in an amount of 150 μl/well, and the plate was incubated overnight at 4°C. After removal of the blocking solution, each well was washed three times with 250 μl of TBS(-) solution. The solution containing the antibody-antigen complex was added to the plate in an amount of 75 μl/well, and the plate was incubated at room temperature for 2 hours to bind the biotin-conjugated anti-human IgG Ab to the immobilized streptavidin on the plate. After removing the solution containing the antibody-antigen complex, each well was washed three times with a TBS(-) solution, and READ buffer solution (MSD K.K.) was added to them in an amount of 150 μl/well, then the fluorescent signal of the sulfo-label was recorded using the Sector instrument. Imager 2400 (MSD K.K.).

The results are shown in FIG. 16. All antibodies: original EGFR

ERY22_Hk/EGFR ERY22L/CE115 ERY22_Hh/CEl 15_ERY22_L and also EGFR

ERY22_Hk/EGFR ERY22_L/CE115_DU21 ERY22_Hh/CEl 15_ERY22_L, EGFR

ERY22_Hk/EGFR ERY22_L/CE115_DU22 ERY22_Hh/CEl 15_ERY22_L, EGFR

ERY22_Hk/EGFR ERY22 L/CE115 DU26 ERY22_Hh/CEl 15 ERY22 L and EGFR

ERY22_Hk/EGFR ERY22_L/CE115_DU27 ERY22_Hh/CEl 15_ERY22_L associated with CD3.

(4-5) Confirmation of the absence of Fab region binding to TLR2 and CD3 at the same time by the ECL method.

According to the data presented in the previous section, the resulting molecules have TLR2 binding activity as well as CD3 binding activity. Then, each Fab region, obtained as described in the previous sections, was evaluated for binding to CD3 and TLR2 simultaneously.

If a molecule containing a TRL2 binding peptide inserted into the Fab region binds to TLR2 and CD3 simultaneously, then their binding to both antigens can be detected by ECL by adding TLR2 and biotin-conjugated CD3 to the antibody solution. In more detail, biotinylated human CD3ε protein homodimer diluted in dilution solution (-), each antibody solution diluted to 10 µl/ml or 5 µl/ml, and TLR2 (R&D Systems, Inc.) with sulfo label included was added to the wells of a 96-well round bottom Nunc-Immuno MicroWell plate (Nunc) each at 25 μl/well and mixed, then the plate was incubated overnight at 4°C to form an antibody-antigen complex. Blocking solution (-) was added to a streptavidin immobilized plate (MSD K.K.) at 150 μl/well and the plate was incubated overnight at 4°C.

After removal of the blocking solution, each well was washed three times with 250 μl of TBS(-) solution, which contained 0.1 g/l calcium chloride and 0.1 g/l magnesium chloride. A solution containing an antibody-antigen complex was added to the plate in an amount of 75 μl/well, and the plate was incubated at room temperature for 2 hours to bind the biotin-conjugated anti-human IgG Ab to the immobilized streptavidin on the plate. After removing the solution containing the antibody-antigen complex, each well was washed three times with a TBS(-) solution, and READ buffer solution (MSD K.K.) was added to them in an amount of 150 μl/well, then the fluorescent signal of the sulfo-label was recorded. using a Sector Imager 2400 (MSD K.K.).

The results are shown in FIG. 17. Antibodies

EGFR ERY22_Hk/EGFR

ERY22 L/CE115_DU42 ERY22_Hh/CEl 15ERY22L and EGFR ERY22_Hk/EGFR

ERY22L/CE115_DU43 ERY22_Hh/CEl 15ERY22L containing a TLR2 binding peptide inserted in the CH3 region bound TLR2 and CD3 simultaneously, as evidenced by a strong signal detected during the ECL assay. And vice versa for everyone

EGFR ERY22_Hk/EGFR

ERY22_L/CE115_DU21 ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR ERY22_L/CE115_DU22 ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR ERY22 L/CE115 DU26 ERY22_Hh/CEl 2 and EFREGRY2k L 2

ERY22 L/CE115 DU27 ERY22_Hh/CEl 15 ERY22 L rarely detected a signal during ECL analysis. These results indicate that these antibodies do not bind to TLR2 in the CD3 binding state.

(4-6) Discussion of the results obtained by the ECL method indicating that the Fab region does not bind to TLR2 and CD3 at the same time.

According to the results described above, the obtained antibodies have the properties of a molecule in which the Fab region has dual binding activity, and binds to each CD3 and TLR2 through one Fab region, but does not bind to CD3 and TLR2 at the same time. In this example, the peptide RWGYHLRDRKYKGVRSHKGVPR that binds to the second antigen, TLR2, was inserted into the variable region (Fab) of an antibody that contains the variable region that allows binding to the first antigen, CD3, in order to successfully construct a molecule that has binding activity to the second antigen, but which does not bind to CD3 and the second antigen at the same time. In a similar manner, a peptide with protein binding activity as described in WO2006036834 can be inserted into a Fab region loop to provide a Fab region with dual binding activity that binds to an arbitrary second antigen. A peptide exhibiting protein binding activity can be obtained by creating a peptide library according to known methods and selecting from the library a peptide having the desired activity (see Pasqualini R., Nature, 380 (6572), 364-366 (1996)). In addition, a library of antigen-binding molecules obtained by changing the length of the loops in the Fab region, as described in example 5, can be used to obtain a Fab with dual binding activity that binds to an arbitrary second antigen. Variable regions that bind to the first antigen can be obtained by various known methods. Thus, it can be concluded that these libraries can be used to generate a Fab region with dual binding activity that binds to an arbitrary first antigen and an arbitrary second antigen, but which does not bind to the first antigen and the second antigen at the same time.

The results described above indicate that EGFR antibodies

ERY22_Hk/EGFR ERY22_L/CE115_DU21 ERY22_Hh/CEl 15_ERY22_L, EGFR

ERY22_Hk/EGFR ERY22_L/CE115_DU22 ERY22_Hh/CEl 15_ERY22_L, EGFR

ERY22_Hk/EGFR ERY22_L/CE115_DU26 ERY22_Hh/CEl 15 ERY22 L and EGFR

ERY22_Hk/EGFR ERY22_L/CE115_DU27 ERY22_Hh/CEl 15_ERY22_L bind to CD3 and TLR2, but do not bind to CD3 and TLR2 at the same time. These results indicate that

EGFR ERY22_Hk/EGFR

ERY22_L/CE115_DU21 ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR

ERY22_L/CE115_DU22 ERY22_Hh/CEl 15_ERY22_L, EGFR ERY22_Hk/EGFR

ERY22 L/CE115_DU26 ERY22_Hh/CEl 15 ERY22 L and EGFR ERY22 Hk/EGFR

ERY22 L/CE115 DU27 ERY22_Hh/CEl 15 ERY22 L are molecules in which the Fab region has a dual binding activity, and also confirm the possibility of obtaining such molecules.

Example 5

Alteration of antibodies to obtain antibodies that bind to CD3 and the second antigen.

(5-1) Examination of the insertion site and length of the peptide capable of binding to the second antigen.

Studies have been conducted to obtain a molecule in which the Fab region is characterized by dual binding activity to the cancer antigen through one variable region (Fab), and also bind to the first antigen, CD3, and the second antigen through another variable region, but which is not able to bind to CD3 and the second antigen at the same time. The GGS peptide was inserted into the heavy chain loop of the CD3ε binding antibody CE115 to generate each heterodimerized antibody containing EGFR binding domains in one Fab region and CD3 binding domains in another Fab region, as described in Comparative Example 1.

_with . . EGFRERY22 Hk/EGFR

In more detail, antibodies ERY22.L/CE115.CE31 ERY22_Hh/CEl 15_ERY22_L ((SEQ ID no: 20/21/43/23) with a GGS insert between

K52B

S52c

CDR2, antibodies

EGFRERY22 Hk/EGFR

ERY22L/CE115CE32 ERY22 Hh/CEl 15 ERY22 L ((SEQ ID no: 20/21/44/23)

GGSGGS (SEQ ID NO: 90) as specified

EGFR ERY22 Hk/EGFR ERY22 L/CE115 CE3 3 ERY22 Hh/CEl 15 ERY22 L ((SEQ ID no: 20/21/45/23) position, with insert as well as antibody peptide with insert

GGSGGSGGS (SEQ ID No: 91) to the indicated position. Antibody peptides were obtained in a similar way

EGFR ERY22 Hk/EGFR ERY22L/CE115CE34 ERY22 Hh/CEl 15 ERY22L ((SEQ ID no: 20/21/46/23) with GGS insert between D72 and D73 (loop) in FR3, antibodies ERY22L/CE115CE35 ERY22_Hh/CE 115 ERY22 L ((SEQ ID NO: 20/21/47/23)

EGFRERY22 Hk/EGFR

GGSGGS (SEQ ID NO: 90) with peptide insert at position indicated, also antibodies

EGFR ERY22 Hk/EGFR ERY22 L/CE115 CE36

ERY22 Hh/CEl 15 ERY22L ((SEQ ID no: 20/21/48/23)

GGSGGSGGS (SEQ ID No. 91) to the indicated position.

EGFR ERY22_Hk/EGFR ERY22_L/CE115_CE37

ERY22 Hh/CEl 15_ERY22_L ((SEQ ID no: 20/21/49/23) with peptide insert

Additionally, antibodies were obtained by inserting GGS between A99 and Y100 in CDR3, antibodies

EGFRERY22 Hk/EGFR ERY22 L/CE115 CE38

ERY22_Hh/CEl 15 ERY22 L ((SEQ ID no. 20/21/50/23) with insertion of GGSGGS peptide at the indicated position, also antibodies

EGFRERY22 Hk/EGFR

ERY22 L/CE115 CE39 ERY22 Hh/CEl 15 ERY22 L ((SEQ ID no: 20/21/51/23) _stucco pep— — — — — j with insertion of the GGSGGSGGS peptide _at the indicated position.

(5-2) Confirmation of binding of CE115 antibodies to the inserted GGS peptide to CD3ε.

The binding activity of each obtained antibody to CD3s was confirmed using the Biacore T100 instrument. The biotin-conjugated peptide epitope of CD3s was immobilized onto a CM5 chip through streptavidin, treated with the resulting antibodies, which were used as an analyte, and the binding affinity was determined.

The results are presented in table. 2. The binding affinity of CE35, CE36, CE37, CE38 and CE39 to CD3ε is equivalent to the original CE115 antibody. These data indicate that the peptide that binds to the second antigen can be inserted into the loops of these antibodies. Binding affinity was not reduced for CE36 or CE39 containing the GGSGGSGGS insert. These data indicate that the insertion of a peptide containing at least 9 amino acid residues at these sites does not affect CD3ε binding activity.

table 2

Sample I ka kd KD J Position ι j of insert ι Linker CE115J! \ 1 5E+05 9.8E-03 6. 7E-08 CE31 2 WE+05 3. 5E-02 1.5E-07 K52b-S52c GS3 CE32 8 5E+04 _; 1.8E-02 2.1E-07 'K52b-S52c GS6 SEZZ I 1.1E-01 2.ZE-07 K52b-S52c GS9 CE34 1 1E+05 ^! 1.ZE-02 1.2E-07 1 D72-073 GS3 CE35 1.3EU5 j 1.1Е-02 8.7E-08 1 D72-D73 1 GS6 CE36 1.2EU5 j 1.2Е-02 9.9Е-08 : D72--D73 GS9 CE37 2.2EU5 '2.0E 02 9.4E 08 A99 Y100' GS3 CE38' 2.0EW5 | 1.7E-02 8. 7E 08 : A99ΥΙΟΟ GS6 .......CE39......... 1.6E+05 ?......1.4E-02 9. 1E 08 i A99-Y100 GS9'

These results indicate that antibodies capable of binding to CD3 and the second antigen, but which do not bind to said antigens simultaneously, can be obtained by generating an antibody that binds to the second antigen using a CE115 antibody that contains an insert of said peptides.

In this regard, the library can be obtained by making changes to the random amino acid sequence of the peptide used for insertion or replacement, according to a known method, for example,

- 48 042507 such as site-directed mutagenesis (see Kunkel et al., Proc. Natl. Acad. Sci. U.S.A., 82, 488-492 (1985)) or PCR with overlapping primers, and comparison of binding activity, etc. p., each modified form by a known method, to identify an insertion or substitution site that allows the desired activity to be shown even after a change in the amino acid sequence, as well as to determine the type and length of the amino acid sequence of this site.

Example 6

Obtaining a library to obtain an antibody that binds to CD3 and the second antigen.

(6-1) Antibody library for producing an antibody binding to CD3 and a second antigen (Fab library with dual binding activity).

In the case of choosing CD3 (CD3ε) as the first antigen, examples of a method for producing an antibody that binds to CD3 (CD3ε) and to an arbitrary second antigen include the following 6 methods:

1) a method that includes inserting a peptide or polypeptide that binds to a second antigen into a Fab domain that binds to the first antigen (this method involves inserting a peptide as described in example 3 or 4, as well as a G-CSF insertion method, as described in Angew Chem. Int. Ed. Engl., 52 (32), 8295-8298 (2013, 5 Aug.)), where the binding peptide or polypeptide can be obtained from a peptide or polypeptide display library, or a full-length natural protein or part thereof;

2) a method that includes obtaining an antibody library in such a way that various amino acids are in such positions that allow you to change the length (increase) of the loops in the composition of the Fab region, as described in example 5, and obtain Fab regions with binding activity with arbitrary a second antigen from an antibody library, using antigen binding activity as an index;

3) a method that includes identifying amino acids that maintain binding activity to CD3 using an antibody obtained by site-directed mutagenesis of antibodies for which their Fab domain is known to bind to CD3 in advance, and obtaining a Fab domain with binding activity with an arbitrary a second antigen from an antibody library that displays the identified amino acids using antigen-binding activity as an index;

4) method 3, which further includes obtaining an antibody library that displays the positions of various amino acids in positions that allow you to change the length (increase) of the loops of the Fab domains, and obtaining Fab domains with binding activity with an arbitrary second antigen from the antibody library using a binding activity with antigen as an index;

5) Methods 1, 2, 3, or 4, which further comprises modifying the antibodies such that glycosylated sequences (e.g., NxS and NxT, where x is an amino acid other than P) are displayed, into which carbohydrate chains that are recognized by receptors can be inserted. , which recognize carbohydrate chains (for example, insert high mannose carbohydrate chains that are recognized by receptors for high mannose compounds, and high mannose carbohydrate chains are known to be produced by the addition of kifunzin during antibody expression (see mAbs., 4 (4), 475-487 (2012 Jul.-Aug.)); and

6) method 1, 2, 3, or 4, which further comprises inserting domains (polypeptides, carbohydrate chains, and nucleic acids typed using TLR agonists) each binding to a second antigen via a covalent bond by inserting Cys, Lys, or a non-natural amino acid into loops or sites found to be subject to alteration of various amino acids or to be replaced by specified sites with Cys, Lys or non-natural amino acid association (see mAbs, 6:1, 34-45 (2014 Jan.-Fev.), WO 2009/134891 A2 and Bioconjug Chem., 25 (2), 351-361 (2014 19 Feb.)).

A Fab fragment having dual binding activity that binds to the first antigen and the second antigen, but does not bind to said antigens simultaneously, was obtained using any of the indicated methods, and these Fab domains can be combined with domains (another variable region, such as described in Example 1) binding to an arbitrary third antigen by known methods, eg common L chains, CrossMab or Fab region exchange.

(6-2) Obtaining an antibody with one amino acid residue changed in a CD3 (CD3ε) binding antibody using site-directed mutagenesis.

The VH domain of CE115HA000 (SEQ ID NO: 52) and the VL domain of GLS3000 (SEQ ID NO: 53) were chosen as template sequences for the CD3 (CD3ε) binding antibody. In each domain, a change was made to the amino acid residue at the site that is believed to be involved in antigen binding, as described in Comparative Example 1. In addition, pE22Hh (sequence derived from natural IgG1 CH1 and the following sequences by substitutions L234A, L235A, N297A, D356C, T366S , L368A and Y407V, deletion of the C-terminal GK sequence, and insertion of the sequence

- 49 042507 DYKDDDDK (SEQ ID NO: 89), SEQ ID NO: 54) was used as the H chain of the conserved domain, and a kappa chain (SEQ ID NO: 55) was used as the L chain of the conserved domain. Sites for changes are presented in Table. 3. To evaluate CD3 (CD3ε) binding activity, each antibody with a single amino acid residue change was generated as single arm antibodies (natural IgG antibodies containing only one of the Fab domains). In more detail, in the case of an H chain change, the changed H chain was attached to the conserved domain of pE22Hh, and Kn010G3 (amino acid sequence of natural IgG1 from position 216 to the C-terminus with substitutions C220S, Y349C, T366W and H435R, SEQ ID NO: 56) was used as H chains and GLS3000 attached to the 3' end of the kappa chain was used as the L chain. In the case of an L chain change, the changed L chain attached to the 3' end of the kappa chain was used as the L chain, and CE115HA000 attached to the 3' end of pE22Hh and also Kn010G3 were used as the H chains. These sequences were expressed and purified in FreeStyle 293 cells (which were used in the method described in Comparative Example 1).

Table 3

Substitution site in the H-chain

Domain FR1 CDR1 FR2 CDR2 Kabat numbering eleven 16 19 28 29 thirty 31 32 33 35 43 50 51 52 52a 52b 52s 53 54 55 56 57 58 59 60 61 62 64 65 Amino acid before replacement V R R T F S N A W H TO Q I TO A TO S N N Y A T Y Y A E S TO G

Domain FR3 CDR3 FR4 Kabat numbering 72 73 74 75 76 77 78 82a 95 96 97 98 99 100 100a 100b 100s 101 102 105 Amino acid before replacement D D S TO N S L N V H Y G A Y γ G V D A Q

Substitution site in the L chain

Domain CDR1 FR2 Kabat numbering 24 25 26 27 27a 27b 27s 27d 27th 28 29 thirty 31 32 33 34 45 Amino acid before replacement R S S Q S L V H S N R N T Y L H Q

Domain CDR2 FR3 CDR3 FR4 Kabat numbering 50 51 52 53 54 55 56 74 77 89 90 91 92 93 94 95 96 97 107 Amino acid before replacement TO V S N R F S TO R G Q G T Q V R Y τ TO

(6-3) Assessing the binding of antibodies with a single altered amino acid residue to CD3.

Each form of antibody with one amino acid residue changed, obtained, expressed and purified as described in section (6-2), was examined using a Biacore T200 instrument (manufactured by GE Healthcare Japan Corp.). An appropriate amount of CD3ε protein homodimer was immobilized on a CM4 sensor chip (GE Healthcare Japan Corp.) by amino condensation. The immobilized homodimers were then treated with antibodies used as an analyte at the required concentration to evaluate their interaction with the CD3ε protein homodimer on the surface of the sensor chip. The sensor chip was then regenerated by pumping a solution of glycine-HCl (10 mmol/l, pH 1.5) through it. The analysis was carried out at 25° C., HBS-EP+ buffer solution (manufactured by GE Healthcare Japan Corp.) was used as a working buffer solution. Sensorgrams were obtained during the analysis, the results of the analyzes were used to calculate the dissociation constant (K _D (M)), using a model of the kinetics of one interaction cycle for a certain amount of bound substance (binding 1:1, RI=0). Each parameter was calculated using Biacore T200 Evaluation Software (GE Healthcare Japan Corp.).

(6-3-1) Changing the H-chain.

In table. 4 shows the ratio of the amount of each bound mutated form of the H chain to the determined amount of bound corresponding unaltered CE115HA000 antibody. In more detail, if the amount of bound antibody containing CE115HA000 is X, and the amount of bound form in which one amino acid residue is changed in the H chain is Y, then the Z value (the ratio of the amounts of bound components) can be calculated by the equation Z=Y/ x. As shown in FIG. 8, the sensorgram shows very little analyte binding at Z less than 0.8, indicating that the dissociation constant K _D (M) cannot be calculated correctly. In table. 5 shows the ratio of the dissociation constant K _d (M) of each H-chain modified form and for CE115HA000 (=KD value of CE115HA000/KD value of the modified form).

If the Z value presented in Table. 4 is equal to 0.8 or more, then the modified form is considered to retain binding activity at the level of the corresponding unchanged CE115HA000 antibody. Thus, an antibody library with such a mapping of the indicated amino acids can be considered as a double Fab library.

- 50 042507

Table 4

Table 5

(6-3-2) L-chain change.

In table. 6 shows the ratio of the amount of each bound mutated form of the L chain to the determined amount of bound corresponding unaltered GLS3000 antibody. In more detail, if the amount of bound antibody containing GLS3000 is X, and the amount of bound form in which one amino acid residue is changed in the L chain is Y, then the Z value (the ratio of the amounts of bound components) can be calculated by the equation Z=Y/ x. As shown in FIG. 18, the sensorgram shows a very small amount of analyte binding at Z less than 0.8, indicating that the constant cannot be calculated correctly.

- 51 042507 dissociation KD(M). In table. 7 shows the ratio of the dissociation constant KD (M) of each L-chain modified form and GLS3000.

If the Z value presented in Table. 4 is 0.8 or more, then the modified form is considered to retain binding activity at the level of the corresponding unaltered GLS3000 antibody. Thus, an antibody library with such a mapping of the indicated amino acids can be considered as a double Fab library.

Table 6

- 52 042507

Table 7

(6-4) Assessing the binding of an antibody with one amino acid residue changed to ECM (extracellular matrix).

ECM (extracellular matrix) is an extracellular component and is present at various sites in vivo. Therefore, antibodies with high ECM binding activity are known to have poor blood kinetics (short half-life) (WO2012093704 A1).

Thus, amino acids that do not enhance ECM binding are preferably selected as amino acids that appear in the antibody library.

Each antibody was obtained in the form with a modified H-chain or L-chain according to the method described in section (6-2). Then, their ECM binding activity was evaluated by the method described in Comparative Example 2. The ECM binding activity value (ECL reaction) of each modified form was divided by the ECM binding activity value of the MRA antibody (H chain: SEQ ID NO: 57, L -chain: SEQ ID NO: 58), obtained in the same tablet or on the same day, the values obtained are presented in table. 8 (H-chain) and 9 (L-chain). According to the data presented in table. 8 and 9, some changes resulted in increased binding to ECM.

Based on the values presented in table. 8 (H-chain) and 9 (L-chain), up to a 10-fold effective value was included in the double Fab library, taking into account the effect of enhancing binding to ECM as a result of many changes.

- 53 042507

Table 8

Table 9

CDR1 FR2 domain

(6-5) Investigation of the role of insertion site and peptide length to enhance library diversity.

According to the data presented in example 5, the peptide can be inserted at each site using the GGS sequence without changing the binding activity against CD3 (CD3ε). If it is possible to extend the loop for a double Fab library, then the resulting library can include more types of molecules (or be more diverse) and provide Fab fragments that bind to a variety of second antigens. In this regard, given the expected decrease in binding activity as a result of the insertion of the peptide,

- 54 042507 V11L/D72A/L78I/D101Q inserts were introduced into the CE115HA000 to increase binding activity to CD3ε, which was then attached to pE22Hh. The molecule was obtained by inserting a GGS linker into the indicated sequence as described in Example 5, and the binding of the resulting molecules to CD3 was evaluated. The GGS sequence was inserted between positions 99 and 100 according to Kabat numbering. The antibodies were expressed as single arm antibodies. In more detail, the GGS linker-containing H chain described above and Kn010G3 (SEQ ID NO: 56) were used as H chains, and GLS3000 (SEQ ID NO: 53) fused to the kappa sequence (SEQ ID NO: : 55) was used as the L chain. These sequences were expressed and purified as described in Comparative Example 1.

(6-6) Confirmation of binding of the CE115 antibody containing the GGS peptide insert to CD3.

Binding of the modified antibody containing the GGS peptide insert to CD3ε was confirmed using the Biacore instrument, following the procedure described in Example 6. The data presented in Table 1. 10 indicate that the GGS linker can be inserted into loops. In more detail, the GGS linker can be inserted into the CDR3 region of the H chain, which is important for antigen binding, and binding to CD3ε was maintained when 3-, 6-, or 9-membered amino acid insertions were made. While this assay was performed using a GGS linker, an antibody library displaying different amino acids other than GGS is acceptable.

Table 10

Inserted amino acid sequence CD3_KD[M] GGS 6.31 E-08 GGSGGS (SEQIDNO: 90) 3.46 E-08 GGSGGS (SEQIDNO: 90) ZL05E-08 GGSGGGS (SEQIDNO: 9 2) 4.352E-08 GGSGGGS (SEQIDNO: 92) 3.429E-O8 GGGSGGGS( SEQIDNO: 9 3) 4.1 29E-O8 GGGSGGGS ( SEQIDNO: 9 3) 3.753E-O8 GGSGGSGGS ( SEQIDNO: 9 1) 4.39 E-08 GGSGGSGGS (SEQIDNO: 91) 3.537E-O8 Without insert 6.961E-O9 CE115HA000 I.O97E-O7

(6-7) Investigation of the influence of the library insert to the CDR3 H chain using the NNS nucleotide sequence.

Section (6-6) shows that 3-, 6-, or 9-mer amino acid sequences can be inserted using a GGS linker, and it can be concluded that a library containing 3-, 6-, or 9-mer amino acid sequences can be used to obtaining antibodies that bind to the second antigen using a standard method for obtaining antibodies, for example, the method of phage display. Thus, a study was made of the effect of a 6-mer amino acid insert in CDR3 on retaining binding to CD3, even if different amino acids are displayed at the 6-mer amino acid insert site, using the NNS nucleotide sequence (which allows each type of amino acid to be displayed). In view of the expected reduction in binding activity, primers were prepared using the NNS nucleotide sequence such that 6 amino acid residues were inserted between positions 99 and 100 (Kabat numbering) in the CDR3 sequence of CE115HA340 (SEQ ID NO: 59), which has a higher CD3ε- binding activity compared to CE115HA000. The antibody was expressed as single arm antibodies. In more detail, the modified H chain described above and Kn010G3 (SEQ ID NO: 56) were used as H chains, and GLS3000 (SEQ ID NO: 53) fused to the kappa sequence (SEQ ID NO: 55) was used in as an L-chain. These sequences were expressed and purified as described in Comparative Example 1. The binding activity of the obtained altered antibody was evaluated by the method described in section (6-3). The results are presented in table. 11. The results indicate that CD3 (CD3ε) binding activity is retained even when different amino acids are displayed at the site extended by amino acid residues. In table. 12 shows the results of a subsequent assessment of the presence or absence of non-specific binding enhancement as described in Comparative Example 2. The results indicate that ECM binding is enhanced when the extended CDR3 loop is enriched in amino acid residues that contain a positively charged side chain. Thus, it is desirable that three or more amino acid residues that contain a positively charged side chain are not present in the loop.

- 55 042507

Table 11

vh CD3_ KD[M] 9' 8 9 1 0 0 e ............................................... k 1 1 2 a b c d SE115HA340 2.0E-08 5 6 7 Hl tQ h i SE115HA340 2.7E-08 V H Y A A X XXX X X Y Y G V - - D A NNS6f17 7.4ED8 w G E G V V NNS6f27 3.8E-08 V W G S V w NNS6f29 9.0E-08 1 Y Y P T N NNS6f47 3.1 E-08 H F M W w G NNS6f50 7.1E-08 L T G G L G NNS6f51 3.1 E-08 G F L V L W NNS6f52 5.2E-08 Y M L G L G NNS6f54 2.9E-08 F E W V G W NNS6f55 3.1 E-08 A G R W L A —_ NNS6f56 2.1 E-08 R EAT R W NNS6f58 4.4E-08 S W Q V S R NNS6f59 2.0E-07 L L V Q E G NNS6f62 6.1 E-08 N G G T R H NNS6f63 6.9E-08 G G G G W V —— NNS6f64 7.8E-08 L V S L T V . NNS6f67 3.6E-08 G L L R A A —_ NNS6f68 4.5E-08 V E W G R w —— NNS6f71 5.1 E-08 G W V L G s NNS6f72 1.5E-07 E G 1 W W G —— NNS6f73 2.6E-08 W V V G V R

Looking for "

SEI5NL34O NVSH7 NMSSI27

NNSSP9~^ N4S5W

N4S3M NMSGfS' ΝΊ5»52 NVSWM N-ZHME

HNsass NWSO ' NNSSK9 tzhg N'ISWS iwsso ~ tWSSftV NVSS€8 ' NMS^7' NVS5f72 NMSS73

Table 12

(6-7) Designing and creating a library of double fab.

Based on the studies described in Example 6, the antibody library (double library)

Fab) designed to obtain an antibody that binds to CD3 and the second antigen was designed as follows:

step 1: selection of amino acids that provide retention of binding to CD3 (CD3ε) (providing 80% or more binding of CE115HA000 to CD3), step 2: selection of amino acids that provide binding to ECM at a level 10 times greater than that for MRA antibodies prior to substitutions, and step 3: insertion of 6 amino acid residues between positions 99 and 100 (Kabat numbering) in the CDR3 H chain.

The diversity of the antigen-binding site of the Fab fragments can be expanded by simply performing step 1. The resulting library can be used to identify an antigen-binding molecule that binds to a second antigen. The diversity of the antigen-binding site of Fab fragments can be expanded by simply performing steps 1 and 3. The resulting library can be used to identify an antigen-binding molecule that binds to a second antigen. Even a library created without performing step 2 makes it possible to obtain a molecule suitable for research.

- 56 042507 research and evaluation of binding activity with ECM.

Thus, the double Fab library, the sequences derived from CE115HA000 by adding the V11L/L78I mutation to the FR (framework) and then expanding the diversity of the CDR regions, as shown in Table. 13 were used as H chains, and the sequences obtained from GLS3000 during the expansion of the diversity of CDR regions, as presented in table. 14 were used as L chains. These antibody library components can be synthesized using known DNA synthesis techniques. The double fab library can be obtained as:

(1) library, which expanded the diversity of H-chains, as presented in table. 13 while the L chains are attached to the original GLS3000 sequence or the L chain enhances binding to CD3ε as described in Example 6, (2) the library in which the H chains are attached to the original sequence (CE115HA000) or the H chain enhances binding to CD3ε, as described in example 6, while the diversity of L-chains expanded, as presented in table. 14, and (3) a library in which the diversity of H-chains is expanded, as presented in table. 13, while the diversity of L-chains is expanded, as presented in table. 14.

A library of H chain sequences derived from CE115HA000 by adding the V11L/L78I mutation to the FR (framework region) followed by the expansion of CDR diversity, as shown in Table 1. 13 was obtained from DNA2.0, Inc., where DNA synthesis is carried out, and DNA fragments for the library were obtained. The resulting antibody library fragments were inserted into phage display phagemids, which were amplified by PCR. GLS3000 was chosen as the L-chains. The obtained phagemids for phage display were transferred to E. coli using the electroporation method, and E. coli cells containing antibody library fragments were obtained.

Table 13

3 5 5 6 9 1 0 Before replacement 1 2 3 4 5 0 1 2 A b from 3 4 5 6 7 8 5 0 1 2 3 4 5 5 5 7 ' 8 9 0 A b WITH d b f ίh i 1 2 Library N A W M H Q 1 TO A TO S N N Y A T Y Y A E S V TO G V H Y G A X X X X X X Y Y G V D 1 1 A W m n Q 1 TO D R A Q A V L A Y Y A R S V TO G V n Y ^! A A A A G A L R A Y G V D 1 N TO G S G N N E G L V V S V G G S F S L N L A T R S G' G T 1 S S Q G Q Q S Q S S Y G Y Y To V S T T T T F S F F Y 5 N Q Y Y D Y G H F F F D

Obtaining a Fab domain that binds to CD3 and a second antigen (IL6R) from a double Fab library.

(7-1) Obtaining a Fab domain that binds to human IL6R.

Fab domains (antibody fragments) binding to human IL6R were selected from a library of double Fabs, designed and generated as described in Example 6. Biotinylated human IL6R was used as antigen and antibody fragments capable of binding to human IL6R were accumulated.

Phages were obtained from E. coli containing engineered phagemids for phage display. 2.5 M NaCl/10% PEG was added to the E. coli culture medium where phages were produced and the precipitated pool of phages was diluted with TBS to give a phage library solution. BSA was then added to the phage library solution (final concentration: 4%). Panning was carried out according to the standard method, with reference to the general method of panning, using antigens immobilized on magnetic particles (see articles J. Immunol. Methods., 332 (1-2), 2-9 (2008), J. Immunol. Methods ., 247 (1-2), 191-203 (2001), Biotechnol Prog., 18 (2), 212-220 (2002) and Mol Cell Proteomics 2 (2), 61-69 (2003)). NeutrAvidin particles with immobilized avidin (Sera-Mag SpeedBeads NeutrAvidin) or particles with immobilized streptavidin (Dynabeads M-280) were used as magnetic particles.

In more detail, 250 pmoles of the biotinylated antigen was added to the resulting phage library solution, while contacting the antigen with the phage library solution was carried out at room temperature for 60 minutes. After the addition of BSA-blocked magnetic particles, the antigen-phage complexes were immobilized on magnetic particles at room temperature for 15 min. The particles were washed three times with TBST (TBS (Takara Bio Inc.) containing 0.1% Tween 20), then washed twice more with 1 ml TBS. After adding 0.5 ml trypsin solution (1 mg/ml), the particles were suspended for 15 min at room temperature, after which the particles were immediately separated using a magnetic stand to regenerate the phage solution. The regenerated phage solution was added to E. coli (strain ER2738, 10 ml) in logarithmic growth phase (OD 600: 0.4-0.5).

The E. coli strain was infected with phages, while the cells were incubated at 37°C for 1 h with constant slow stirring. E. coli infected were transferred to a 225 mm x 225 mm plate. Phages were then isolated from the culture medium of the infected E. coli to obtain a phage library solution. This cycle (another name for panning) was repeated several times. In the second and subsequent rounds of panning, 40 pmoles of biotinylated antigen were used. In the fourth round of panning, phages were enriched in the ability to bind to CD3 as an index. In more detail, 250 pmoles of the biotinylated CD3ε peptide antigen (amino acid sequence: SEQ ID NO: 60) was added to the resulting phage library solution, and the antigen and phage library solution were contacted for 60 minutes at room temperature. After the addition of BSA-blocked magnetic particles, the antigen-phage complexes were immobilized on magnetic particles for 15 min at room temperature. The particles were washed with 1 ml TBS containing 0.1% Tween 20 and TBS. Particles treated with 0.5 ml trypsin (1 mg/ml) were suspended at room temperature for 15 min, then the particles were immediately separated using a magnetic stand, while regenerating the phage solution. Phages isolated from trypsin-treated phage solution were added to E. coli (strain ER2738, 10 ml) in logarithmic growth phase (OD 600: 0.4-0.7). The E. coli strain was infected with phages, while the cells were incubated at 37°C for 1 h with constant slow stirring. E. coli infected were transferred to a 225 mm x 225 mm plate. Phages were then isolated from the culture medium of the infected E. coli to obtain a phage library solution.

To prevent the production of multiple phages from a single E. coli cell, the phage library solution obtained from E. coli infected using phages isolated in the fifth panning cycle was again diluted 100,000-fold, and E. coli was infected using the resulting phage solution at this resulted in single colonies.

(7-2) Binding of Fab domain derived from phage display to CD3 or IL6R (phage ELISA).

Culture supernatant containing phages was obtained according to the standard method (see the article Methods Mol. Biol., 178, 133-145 (2002)) from each single colony of E. coli obtained as described above. After addition of BSA (final concentration: 4%), the culture supernatant containing phages was analyzed by solid phase ELISA according to the following procedure: A 96-well microtiter plate (StreptaWell 96, from F. Hoffmann-La Roche Ltd.) was treated overnight at 4°C. or for 1 hour at room temperature, 100 µl of PBS containing a biotinylated antigen (biotinylated CD3ε peptide or biotinylated human IL6R). Each well of the plate was washed with PBST to remove unbound antigens. The wells were then blocked with 250 μl of 4% BCATBS for 1 hour or more. After removal of 4% BSA-TBS, the resulting culture supernatant was added to each well and the plate was incubated at room temperature for 1 hour to bind the phage display antibody to the antigen present in each well. Each well was washed with TBST, then horseradish peroxidase-anti-M13 conjugate (Amersham Pharmacia Biotech Inc.) diluted with TBS containing BSA (final concentration: 4%) was added to each well. The plate was incubated for 1 hour. After washing with TBST, TMB solution (ZYMED Laboratories, Inc.) was added to the wells. The chromogenic reaction in the solution in each well was stopped by adding sulfuric acid. The staining was then assessed by optical density at 450 nm. The results are shown in FIG. 19. Clones #50 and #62 exhibit binding activity to human CD3ε and IL6R. In other words, clones exhibiting binding activity for the second antigen (in example 7, human IL6R) can be successfully selected using a library of double Fabs. Clones exhibiting binding activity can be selected from the library by further increasing the number of components included in it and their evaluation, followed by conversion to IgG (VH and VL sequences of each clone are attached to the H-chain and L-chain of conserved human antibody domains, respectively) , and evaluate their binding activity to CD3ε and a second antigen (human IL6R). In addition, the binding of the obtained molecule to CD3ε and the second antigen (human IL6R) simultaneously or its absence can be determined by the method described in examples 3 or 4 or by the method of competitive binding. The competitive binding method indicates that the antibodies do not bind to CD3ε and the second antigen at the same time, for example, if the level of their binding to

CD3ε is reduced in the presence of the second antigen compared to the level when using only one antibody.

Example 8

Obtaining a Fab domain binding to CD3 and a second antigen (human IgA) from a double Fab library.

(8-1) Obtaining a Fab domain that binds to human IgA.

Immunoglobulin IgA refers to an antibody isotype that is present in excess in vivo and is known to be involved in non-specific immune reactions of the intestines and mucous membranes of various organs, and also binds to FcaR (Fc alpha receptor) (see the article by J. Pathol ., 208, 270-282 (2006)).

Fab domains (antibody fragments) binding to human IgA were selected from a library of double Fabs designed and constructed as described in Example 6. Biotinylated human IgA (as described in Comparative Example 3) was used as antigen, and antibody fragments were accumulated, capable of binding to human IgA.

Phages were obtained from E. coli containing engineered phagemids for phage display. 2.5 M NaCl/10% PEG was added to the E. coli culture medium where phages were produced, the precipitated pool of phages was diluted with TBS to give a phage library solution. BSA was then added to the phage library solution (final concentration: 4%). Panning was performed according to the standard method with reference to the general method using antigens immobilized on magnetic particles (see articles J. Immunol. Methods., 332 (1-2), 2-9 (2008), J. Immunol. Methods., 247 (1-2), 191-203 (2001), Biotechnol Prog., 18 (2), 212-220 (2002) and Mol Cell Proteomics 2 (2), 61-69 (2003)). NeutrAvidin particles with immobilized avidin (Sera-Mag SpeedBeads NeutrAvidin) or particles with immobilized streptavidin (Dynabeads M-280) were used as magnetic particles.

In more detail, 250 pmoles of the biotinylated antigen was added to the resulting phage library solution, while contacting the antigen with the phage library solution was carried out at room temperature for 60 minutes. After the addition of BSA-blocked magnetic particles, the antigen-phage complexes were immobilized on magnetic particles at room temperature for 15 min. The particles were washed three times with TBST (TBS (Takara Bio Inc.)) containing 0.1% Tween 20), then washed twice with 1 ml TBS. After adding 0.5 ml trypsin solution (1 mg/ml), the particles were suspended for 15 min at room temperature, after which the particles were immediately separated using a magnetic stand to regenerate the phage solution. The regenerated phage solution was added to E. coli (strain ER2738, 10 ml) in logarithmic growth phase (OD 600: 0.4-0.5). The E. coli strain was infected with phages, while the cells were incubated at 37°C for 1 h with constant slow stirring. E. coli infected were transferred to a 225 mm x 225 mm plate. Phages were then isolated from the culture medium of the infected E. coli to obtain a phage library solution. This cycle (another name for panning) was repeated 4 times. In the second and subsequent rounds of panning, 40 pmoles of human IgA were used.

(8-2) Binding of Fab domain derived from phage display to human CD3 or IgA.

Culture supernatant containing phages was obtained according to the standard method (see the article Methods Mol. Biol., 178, 133-145 (2002)) from each single colony of E. coli obtained as described above. After the addition of BSA (final concentration: 4%), the culture supernatant containing phages was analyzed using a solid-phase ELISA according to the following procedure: A 96-well microtiter plate (StreptaWell 96, F. Hoffmann-La Roche Ltd.) was treated overnight at 4° C. or for 1 hour at room temperature 100 μl of PBS containing a biotinylated antigen (biotinylated CD3ε peptide or biotinylated human IgA as described in Comparative Example 3). Each well of the plate was washed with PBST to remove unbound antigens. The wells were then blocked with 250 µl 0.1xTBS/150 mM NaCl/0.02% skim milk for 1 hour or more. After removal of the 0.1xTBS/150 mM NaCl/0.02% skim milk solution, the obtained culture supernatant was added to each well and the plate was incubated at room temperature for 1 h to bind the antibody displayed on the phage displays to the antigen present in each hole. Each well was washed with 0.1xTBS/150 mM NaCl/0.01% Tween 20, then horseradish peroxidase anti-M13 conjugate (Amersham Pharmacia Biotech Inc.) diluted with 0.1xTBS/150 mM NaCl/0 was added to each well. 01% Tween 20. The plate was incubated for 1 hour. After washing with TBST, TMB solution (ZYMED Laboratories, Inc.) was added to the wells. The chromogenic reaction in the solution in each well was stopped by adding sulfuric acid. The staining was then assessed by optical density at 450 nm. The results are shown in FIG. 20. As shown in FIG. 20, some clones bind to human CD3 and IgA. Thus, clones exhibiting binding activity for the second antigen (Example 8, human IgA) can be successfully selected using a library of double Fabs.

(8-3) Binding of IgGs containing the derived Fab domain to human CD3 or IgA.

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The VH fragment of each clone that binds to human CD3 and IgA as described in section (8-2) was amplified from E. soi containing the indicated sequence by PCR using primers specifically binding to the H chain in the double Fab library. The amplified VH fragment was attached to plasmids containing pE22Hh for expression in animal cells by the method described in Comparative Example 1, expression and purification were performed as described in Example 6 (section 6-2) for single arm antibodies. The name of each clone and SEQ ID number of the sequence of their Nchain are presented in table. 15. In more detail, each H-chain presented in table. 15 and Kn010G3 (SEQ ID NO: 56) were used as H chains, and GLS3000 (SEQ ID NO: 53) fused to a kappa sequence (SEQ ID NO: 55) was used as L chain. These sequences were expressed and purified as described in Comparative Example 1.

Table 15

Sample name SEQID No. IGAR4C09#l 61 IGAR4C12#4 62 IGAR6(2)B02#l 63

The binding activity of antibodies containing the resulting Fab fragment against human CD3s and IgA was assessed by the electrochemiluminescent method (ECL method). In more detail, biotinylated CD3s peptide (as described in Example 7) or biotinylated human IgA (Comparative Example 3) were diluted with TBST solution (TBS (Takara Bio Inc.) containing 0.1% Tween 20), then each antibody solution diluted to a concentration of 2 μg / ml, as well as antibodies to human IgG (Invitrogen Corp., #628400) with the included sulfo-label were added to the wells of a 96-well Nunc-Immuno MicroWell round bottom plate (Nunc), each in an amount of 25 µl/well, and mixed, then the plates were incubated in the dark for 1 hour or more at room temperature to form the antibody-antigen complex. TBST solution containing 0.5% BSA (another name for blocking solution) was added to a streptavidin immobilized plate (MSD K.K.) at 150 μl/well, and the plate was incubated for 1 hour or more at room temperature. After removal of the blocking solution, each well was washed three times with 250 μl of TBS(-) solution. A solution containing the antibody-antigen complex was added to the plate in an amount of 50 μl/well, and the plate was incubated at room temperature for 1 h to bind the biotinylated antigen-detection antibody complex containing the sulfo-label through the biotinylated antigen with immobilized streptavidin on tablet. After removing the solution containing the antibody-antigen complex, each well was washed three times with TBST solution, and 4x READ buffer solution (MSD K.K.), diluted with water twice, was added to them in an amount of 150 μl/well, then fluorescent sulfo-label signal using Sector Imager 2400 (MSD K.K.).

The results are shown in FIG. 21. Clones with binding activity as determined by phage ELISA were each converted to IgG and also included clones that contained some amino acid mutations. Therefore, these sequences, which have binding activity according to phage-based ELISA, bind to human CD3s and IgA even in IgG form.

The results obtained indicate that antibodies binding to the second antigen can be obtained from the library of double Fab, and clones with confirmed binding can be enriched even in the form of IgG containing the Fc region, despite the fact that standard panning is enriched using a phage library only Fab domains. Thus, it can be concluded that the double Fab library can be used as a library that allows one to obtain a Fab domain capable of binding to a second antigen while retaining the ability to bind to CD3.

(8-4) Assessing the binding of IgG containing the derived Fab domain to CD3 (CD3s) and human IgA simultaneously.

According to section (8-3), clones derived from the double Fab library have binding activity even in IgG form. Subsequently, the ability of each obtained IgG to bind to POPs (CD3s) and human IgA was evaluated simultaneously by the method of competitive binding (electrochemiluminescent method (ECL method)). It can be expected that if IgG binds to CD3 (CD3g) and human IgA simultaneously, then the addition of POPs (CD3e) to antibodies with bound IgA will not lead to a change in the ECL signal, and if IgG does not bind to these antigens simultaneously, then the addition of POPs ( CD3s) will decrease the ECL signal due to some antibody binding to CD3 (CD3e).

In more detail, 25 µl of biotinylated human IgA was diluted with TBST solution, 12.5 µl of each antibody solution diluted to 1 µl/ml, 12.5 µl of TBST or CD3e protein homodimer (9.4 pmol/µl) for competitive binding, and 25 µl of anti-human IgG antibody (Invitrogen Corp., #628400) with a sulfo label included was added to each well of a 96-well Nunc plate.

-60042507

Immuno™ Micro Well™ round bottom (Nunc) and mixed, then the plates were incubated in the dark for 1 hour or more at room temperature to form an antibody-antigen complex. TBST solution containing 0.5% BSA (another name for blocking solution, TBS (Takara Bio Inc.) containing 0.1% Tween 20) was added to a plate with immobilized streptavidin (manufactured by MSD K.K.) in an amount of 150 μl / the well and plate were incubated for 1 hour or more at room temperature. After removal of the blocking solution, each well was washed three times with 250 µl of TBST solution. A solution containing the antibody-antigen complex was added to the plate in an amount of 50 μl/well, and the plate was incubated at room temperature for 1 h to bind the biotinylated antigen-detection antibody complex containing the sulfo-label through the biotinylated antigen with immobilized streptavidin on tablet. After removing the solution containing the antibody-antigen complex, each well was washed three times with TBST solution, and 4x READ buffer solution (MSD K.K.) diluted with water twice, in the amount of 150 μl/well, was added to them, then the fluorescent signal of sulfo- marks using the Sector Imager 2400 (MSD K.K.).

The results are shown in FIG. 22. The addition of the CD3ε protein homodimer for competitive binding was found to decrease the ECL signal compared to the addition of TBST. The results indicate that the CD3 and human IgA binding molecule is a dual-Fab molecule that does not bind to human IgA in the CD3-associated state. Thus, the results indicate that an antibody capable of binding to the second antigen can be obtained from a library of double Fab and, among others, can be obtained in the form of a double Fab molecule that does not bind to the second antigen in the CD3-associated state (or does not bind to CD3 in the state if it is associated with the second antigen), i.e. cannot bind to multiple types of antigens at the same time.

One of skill in the art will appreciate that the diversity of binding molecule sequences found by assessing binding activity using phages as described in section (8-2) can be expanded by increasing the number of library components to be assessed. Thus, it was concluded that the double Fab library could be used as a library to generate a Fab domain with second antigen binding activity while maintaining CD3 binding activity. In this example, the double Fab library was expanded with H chains only. Increasing the size of the library (also known as diversity, meaning that the library includes many different sequences) usually allows more antigen-binding molecules to be obtained. Thus, this example shows that a library of double Fabs can also be used to generate double Fabs, with extensions to share H and L chains.

Provided that molecules with two Fabs can be produced as described in Example 8, the Fab fragment or antigen-binding domain that binds to the third antigen can be identified by a method known to one of skill in the art, such as the hybridoma method or the binding selection method. antibodies (or binding domains) from an antibody library. An antibody containing an identified antigen-binding domain (e.g., a Fab fragment) that binds to a third antigen and the Fab domain of a double-Fab molecule can be prepared as a multispecific antibody using a known method for making multispecific antibodies, for example, a method for making antibodies with common L- chains and two different H chains (method of controlling the interaction of each domain in the Fc region), CrossMab, or a method of exchanging Fab arms. In other words, provided that a molecule with two Fabs can be identified, the desired multispecific antibody can be obtained in a known manner by combining Fabs that bind to a third antigen with two Fabs that bind to the first and second antigens, as described in Example 8.

(8-5) A molecule with two Fab binding to CD3/IgA.

According to the data presented in Example 8, the resulting dual-Fab molecule binds to human CD3ε and IgA but does not bind to human CD3ε and IgA simultaneously. The antigen-binding domain that binds to the third antigen can be further included in its composition in a known manner.

In recent years, it has been found that IgA modified to bind to EGFR, i.e. with a cancer antigen cause the death of cancer cells that express EGFR (J Immunol., 179: 2936-2943 (2007)). This death mechanism is that the IgA receptor FcaR is expressed on polymorphonuclear cells and is reported to induce autophagy in cancer cells (see J. Immunol., 187, 726-732 (2011)). In this example, it was shown that it is possible to construct molecules with two Fab binding to CD3 and IgA. When searching for molecules that bind to FcaR via IgA, in a manner known to those skilled in the art (eg, solid phase ELISA or ECL method), one would expect the identification of molecules that exhibit antitumor effects mediated by FcaR. In other words, these double Fabs can elicit both a T cell-mediated cytotoxic response by binding to CD3ε and a cytotoxic response mediated by binding of FcaR-expressing cells to IgA compared to cells expressing an arbitrary third antigen, resulting in expect the development of a strong cytotoxic reaction.

Example 9

Obtaining a Fab domain binding to CD3 and a second antigen (human CD154) from a double Fab library.

(9-1) Obtaining a Fab domain that binds to human CD154.

Fab domains (antibody fragments) binding to human CD154 were selected from a library of double Fabs designed and constructed as described in Example 6. Antibody fragments capable of binding to human CD154 were enriched using biotinylated human CD154 as antigen.

Phages were obtained from E. coli containing engineered phagemids for phage display. 2.5 M NaCl/10% PEG was added to the E. coli culture medium where phages were produced, the precipitated pool of phages was diluted with TBS to give a phage library solution. BSA was then added to the phage library solution (final concentration: 4%). Panning was carried out according to the standard method, with reference to the general method, using antigens immobilized on magnetic particles (see articles J. Immunol. Methods., 332 (1-2), 2-9 (2008), J. Immunol. Methods. , 247 (1-2), 191-203 (2001), Biotechnol Prog., 18 (2), 212-220 (2002) and Mol Cell Proteomics 2 (2), 61-69 (2003)). NeutrAvidin particles with immobilized avidin (Sera-Mag SpeedBeads NeutrAvidin) or particles with immobilized streptavidin (Dynabeads M-280) were used as magnetic particles.

In more detail, 250 pmoles of the biotinylated antigen was added to the resulting phage library solution, while contacting the antigen with the phage library solution was carried out at room temperature for 60 minutes. After the addition of BSA-blocked magnetic particles, the antigen-phage complexes were immobilized on magnetic particles at room temperature for 15 min. The particles were washed three times with TBST (TBS (Takara Bio Inc.)) containing 0.1% Tween 20), then washed twice with 1 ml TBS. After adding 0.5 ml trypsin solution (1 mg/ml), the particles were suspended for 15 min at room temperature, after which the particles were immediately separated using a magnetic stand to regenerate the phage solution. The regenerated phage solution was added to E. coli (strain ER2738, 10 ml) in logarithmic growth phase (OD 600: 0.4-0.5). The E. coli strain was infected with phages, while the cells were incubated at 37°C for 1 h with constant slow stirring. E. coli infected were transferred to a 225 mm x 225 mm plate. Phages were then isolated from the culture medium of the infected E. coli to obtain a phage library solution. This cycle (another name for panning) was repeated 5 times. In the second and subsequent rounds of panning, 40 pmoles of human CD154 were used.

(9-2) Binding of the Fab domain derived from phage display to human CD3 or CD154.

Culture supernatant containing phages was obtained according to the standard method (see the article Methods Mol. Biol., 178, 133-145 (2002)) from each single colony of E. coli obtained as described above. After the addition of BSA (final concentration: 4%), the culture supernatant containing phages was analyzed using a solid-phase ELISA according to the following procedure: A 96-well microtiter plate (StreptaWell 96, F. Hoffmann-La Roche Ltd.) was treated overnight at 4°C or for 1 hour at room temperature 100 µl of PBS containing biotinylated antigen (biotinylated CD3ε peptide or biotinylated human CD154). Each well of the plate was washed with PBST to remove unbound antigens. The wells were then blocked with 250 µl 0.1xTBS/150 mM NaCl/0.02% skim milk for 1 hour or more. After removal of the 0.1xTBS/150 mM NaCl/0.02% skim milk solution, the resulting culture supernatant was added to each well and the plate was incubated at room temperature for 1 hour to bind the antibody displayed on the phage display to the antigen present in each hole. Each well was washed with 0.1xTBS/150 mM NaCl/0.01% Tween 20, then horseradish peroxidase anti-M13 conjugate (Amersham Pharmacia Biotech Inc.) diluted with 0.1xTBS/150 mM NaCl/0 was added to each well. 01% Tween 20. The plate was incubated for 1 hour. After washing with TBST, TMB solution (ZYMED Laboratories, Inc.) was added to the wells. The chromogenic reaction in the solution in each well was stopped by adding sulfuric acid. The staining was then assessed by optical density at 450 nm. The results are shown in FIG. 23. As shown in FIG. 23, some clones bind to human CD3 and CD154. Thus, clones exhibiting binding activity to the second antigen can be successfully selected using a library of double Fabs (Example 9, CD154IgA).

As shown in examples 7, 8 and 9, Fab domains can be obtained that bind to 3 different antigens, indicating that a library of double Fabs can be used to obtain a molecule that binds to a second antigen.

(9-3) Binding of IgGs containing the derived Fab domain to human CD3 or CD154.

VH fragment of each clone that binds to human CD3 and CD154 as described in section (8-62 042507

2) were amplified from E. coli containing the indicated sequence by PCR using primers that specifically bind to the H chain in the double Fab library. The amplified VH fragment was attached to plasmids containing pE22Hh for expression in animal cells by the method described in Comparative Example 1, expression and purification were performed as described in Example 6 (section 6-2) for single arm antibodies. The name of each resulting sequence and SEQ ID NO their H-chain are presented in table. 16. In more detail, each H-chain presented in table. 16 and Kn010G3 (SEQ ID NO: 56) were used as H chains, and GLS3000 (SEQ ID NO: 53) fused to a kappa sequence (SEQ ID NO: 55) was used as L chain. These sequences were expressed and purified as described in Comparative Example 1.

Table 16

Sample name SEQID No. Sample name SEQID No. 154R3A01#1 64 154R4B07#1 72 154R3A01#2 65 154R4F08#1 73 154R3A01#3 66 154R5A02#1 74 154R3A01#4 67 154R5A02#2 75 154R3B01#1 68 154R5F08#1 76 154R3F02#2 69 154R5F08#3 77 154R4B03#1 70 154R5E11#3 78 154R4B03#4 71

The binding activity of antibodies containing a Fab region that binds to human CD3ε and CD154, as determined by phage-based ELISA as described in section (9-2), against human CD3 and CD154 was assessed by an electrochemiluminescent method (ECL method). In more detail, 25 µl of biotinylated human CD3 or biotinylated human CD154 was diluted with TBST solution, followed by 25 µl of each antibody solution diluted to 2 µg/ml, and 25 µl of anti-human IgG antibody (Invitrogen Corp., #628400) with sulfo included. The α-label was added to each well of a 96-well Nunc-Immuno™ MicroWell™ round bottom plate (Nunc) and mixed, then the plates were incubated in the dark for 1 hour or more at room temperature to form an antibody-antigen complex. TBST solution containing 0.5% BSA (blocking solution, TBST solution is TBS (Takara Bio Inc.) containing 0.1% Tween 20) was added to the streptavidin immobilized plate (MSD K.K.) in an amount of 150 µl /well, and the tablet was incubated for 1 hour or more at room temperature. After removal of the blocking solution, each well was washed three times with 250 µl of TBST solution. A solution containing the antibody-antigen complex was added to the plate in an amount of 50 μl/well, and the plate was incubated at room temperature for 1 h to bind the biotinylated antigen-detection antibody complex containing the sulfo-label through the biotinylated antigen with immobilized streptavidin on tablet. After removing the solution containing the antibody-antigen complex, each well was washed three times with a TBST solution, and 4x READ buffer solution (MSD K.K.), diluted with water twice, was added to them in an amount of 150 μl/well, then the fluorescent signal of the sulfo-label was recorded with using a Sector Imager 2400 (MSD K.K.).

The results are shown in FIG. 24. Clones with binding activity as determined by phage-solid-phase ELISA were each converted to IgG, and also included clones that contained some amino acid mutations. Therefore, these sequences, which have binding activity according to phage-based ELISA, bind to human CD3s and CD154 even in IgG form.

The results indicate that the antibody that binds to the second antigen can be obtained from the library of double Fab, and clones with confirmed binding can be enriched not only for human IgA, but also for human CD154, even as IgG containing the Fc region, despite the fact that only Fab domains are enriched with standard panning using a phage library. Thus, it can be concluded that the library of double Fab can be used as a library that allows you to get a Fab domain capable of binding to the second antigen, while maintaining the ability to bind to CD3.

(9-4) Assessing the co-binding of IgGs containing the derived Fab domain to human CD3ε and CD154.

According to section (9-3), clones derived from the double Fab library have binding activity even in IgG form. Then, the ability of each obtained IgG to simultaneously bind to human CD3 and CD154 was evaluated by competitive binding method (electrochemiluminescent method (ECL method)). It can be expected that if IgG simultaneously binds to human CD3 and CD154, then the addition of CD3 to antibodies that bind to CD154 will not lead to a change in the ECL signal, and if IgG does not simultaneously bind to these antigens, then the addition of CD3 will reduce the ECL signal. due to the binding of some antibodies to CD3.

- 63 042507

In more detail, 25 µl of biotinylated human CD154 was diluted with TBST solution, 12.5 µl of each antibody solution diluted to 1 µl/ml, 12.5 µl of TBST or CD3ε protein homodimer (9.4 pmol/µl) for competitive binding, and 25 µl of anti-human IgG antibody (Invitrogen Corp., #628400) with a sulfo-label included was added to each well of a 96-well NuncImmuno™ MicroWell™ round bottom plate (Nunc) and mixed, then the plates were incubated in the dark for 1 h or more at room temperature to form an antibody-antigen complex. TBST solution containing 0.5% BSA (blocking solution, TBS (Takara Bio Inc.) containing 0.1% Tween 20)) was added to a streptavidin-immobilized plate (MSD K.K.) at a rate of 150 μl/well and the plate was incubated for 1 hour or more at room temperature. After removal of the blocking solution, each well was washed three times with 250 µl of TBST solution. A solution containing the antibody-antigen complex was added to the plate in an amount of 50 μl/well, and the plate was incubated at room temperature for 1 h to bind the biotinylated antigen-detection antibody complex containing the sulfo-label through the biotinylated antigen with immobilized streptavidin on tablet. After removing the solution containing the antibody-antigen complex, each well was washed three times with TBST solution, and 4x READ buffer solution (MSD K.K.) diluted with water twice, in the amount of 150 μl/well, was added to them, then the fluorescent signal of sulfo- marks using the Sector Imager 2400 (MSD K.K.).

The results are shown in FIG. 25. The addition of the CD3ε protein homodimer for competitive binding was found to reduce the ECL signal compared to the addition of TBST. These results indicate that the human CD3 and CD154 binding molecule is a dual-Fab molecule that does not bind to human CD154 in the CD3 binding state. Thus, the results indicate that antibodies capable of binding to the second antigen can be obtained from a library of double Fab and, among others, can be obtained in the form of a double Fab molecule that does not bind to the second antigen in the state when it is associated with CD3 (or does not bind to CD3 in the state when it is associated with the second antigen), i.e. cannot simultaneously bind to multiple types of antigens.

One of skill in the art will appreciate that the diversity of binding molecule sequences found by assessing binding activity using phages as described in section (9-2) can be expanded by increasing the number of library components to be assessed. Thus, it was concluded that the double Fab library could be used as a library to generate a Fab domain with second antigen binding activity while maintaining CD3 binding activity. In this example, a double Fab library was used, extended only with H chains. Increasing the size of the library (also known as diversity, meaning that the library includes many different sequences) usually allows for a greater number of antigen-binding molecules. Thus, this example shows that a library of double Fabs can also be used to generate double Fabs, with extensions to share H and L chains.

Provided that two-Fab molecules can be prepared as described in Example 9, the Fab fragment or antigen-binding domain that binds to the third antigen can be identified by a known method, e.g., a hybridoma method or a method of selecting binding antibodies or binding domains from a library. antibodies. Antibodies containing an identified antigen-binding domain (e.g., a Fab fragment) that binds to a third antigen and the Fab domain of a double-Fab molecule can be produced as a multispecific antibody using a known method for producing multispecific antibodies, for example, the method for producing antibodies with common L chains and two different H-chains (a way to control the interaction of each domain in the Fc region), CrossMab or a way to exchange Fab-arms. In other words, provided that a molecule with two Fabs can be identified, the desired multispecific antibody can be obtained in a known manner by combining the Fabs that bind to the third antigen with two Fabs that bind to the first and second antigens, as described in example 9.

These examples show that a molecule that binds to CD3ε and a second antigen can be obtained by tailoring a library of double Fabs to a variety of antigen types, and also shows that a molecule obtained as described in examples 8 or 9 binds to the first antigen ( CD3ε) and the second antigen, but does not simultaneously bind to the first antigen and the second antigen. As described above, the Fab that binds to the third antigen can be identified in a known manner. Thus, the desired antibodies described in example 1 can be obtained using a library of double Fabs.

(9-5) Molecule with two Fabs to human CD3/CD154.

According to the data presented in Example 9, the resulting dual-Fab molecule binds to human CD3ε and CD154 but does not simultaneously bind to human CD3ε and CD154. The antigen-binding domain that binds to the third antigen can be further included in its composition known

- 64 042507 way.

In recent years, CD154 receptor agonist antibodies have been found to enhance antitumor activity in a method for transfection of cancer antigen sensitive T cells (see J. Immunother., 35 (3), 276-282 (2012 Apr.). In this example, it has been shown that it is possible to design a molecule with two Fab binding to CD3 and CD154.Antitumor effects mediated by CD40 can be expected to be observed during the selection of antibodies that exhibit CD40 agonist activity via CD154.In other words, one can expect that these dual Fabs can induce a T-cell mediated cytotoxic response by binding to CD3ε compared to cells expressing an arbitrary third antigen, as well as enhancing antitumor effects mediated by CD40 agonist signals via binding to CD154.

Comparison examples

Comparison example 1.

Obtaining an expression vector of the antibody, expression and purification of the antibody.

The amino acid substitution was carried out in a known manner using QuikChange site-directed mutagenesis kit (Stratagene Corp.), PCR or Infusion Advantage PCR cloning kit (Takara Bio Inc.) and the like to obtain expression vectors. The resulting expression vectors were sequenced according to known methods. The resulting plasmids were immediately used to transfect HEK293H cell line (Invitrogen Corp.) derived from human kidney cancer embryonic cells or FreeStyle 293 cell line (Invitrogen Corp.) for antibody expression. Each antibody was purified from the components of the resulting culture supernatant by a known method using rProtein A Sepharose(TM) Fast Flow (GE Healthcare Japan Corp.). To determine the concentration of the purified antibody, the optical density of its solution was measured at 280 nm using a spectrophotometer, and the antibody concentration was calculated using the extinction coefficient from the values obtained by the PACE method (see Protein Science, 4, 2411-2423 (1995)).

Comparison example 2.

Evaluation of antibody binding to ECM (extracellular matrix).

The binding of each antibody to the ECM was evaluated according to the following methods described in the application WO 2012093704 A1. Phenol red free ECM (BD Matrigel, #356237) was diluted with TBS to 2 mg/ml and added dropwise to the center of each well (5 µl/well) of an ECL assay plate (L15XB-3, MSD K.K., high level of binding), when cooled with ice. The plate was then capped and incubated at 4°C overnight. The plate with immobilized ECM was heated to room temperature. Then ECL blocking buffer (PBS containing 0.5% BSA and 0.05% Tween 20) was added to the wells at 150 µl/well, and the plate was incubated at room temperature for 2 hours or more or overnight at 4 °C. Each antibody sample was then diluted with PBS-T (PBS containing 0.05% Tween 20) to 9 μg/ml. The second antibody was diluted to a concentration of 2 μg/ml with ECLDB solution (PBS containing 0.1% BSA and 0.01% Tween 20). The antibody solution (20 µl) and the second antibody solution (30 µl) were added to each well of a round bottom plate containing ECLDB solution (10 µl/well) in the wells and mixed in the dark at room temperature for 1 hour. for ECL was removed by inverting the ECM plate containing ECL blocking buffer. To this plate was added a mixture of the above antibodies and the second antibody (50 μl/well). The plate was then incubated in the dark at room temperature for 1 hour. The sample was removed by inverting the plate, and then READ buffer solution (MSD K.K., 150 μl/well) was added to the wells, and the fluorescent signal of the sulfo-label was detected using a Sector Imager 2400 (MSD K.K.).

Comparison example 3.

Obtaining human IgA.

The Fc region of the natural human IgA sequence was used as human IgA (human IgA Fc). For C-terminal biotinylation of human IgA Fc, a gene fragment encoding a specific sequence (AviTag sequence, SEQ ID NO: 79) for biotin ligase-mediated biotinylation was attached via a linker to the gene fragment encoding human Fc IgA. A gene fragment encoding a protein containing a human IgA Fc and an affixed AviTag sequence (SEQ ID NO: 80) was inserted into a vector for expression in animal cells, and the constructed plasmid vectors were transfected into FreeStyle 293 cells (Invitrogen Corp.) using 293Fectin (Company Invitrogen Corp.). During this operation, cells were co-transfected with the expression vector and the gene encoding EBNA1 (SEQ ID NO: 81) and the gene encoding biotin ligase (BirA, SEQ ID NO: 82), and in addition biotin was added to biotinylate human Fc IgA . Cells transfected according to the methods described above were cultured at 37°C for 6 days in an atmosphere of 8% CO2, while the desired protein was secreted into the culture supernatant.

The cell culture solution containing the desired human IgA Fc was filtered through a 0.22 µm filter placed on the neck of the vial to obtain a culture supernatant. The culture supernatant was diluted with 20 mM Tris-HCl (pH 7.4), applied to

- 65 042507 HiTrap Q HP column (GE Healthcare Japan Corp.) equilibrated with 20 mM Tris-HCl (pH 7.4), then the desired human IgA Fc was eluted with a NaCl gradient. The eluate from the HiTrap Q HP column was then diluted with 50 mM Tris-HCl (pH 8.0) and applied to a SoftLink Avidin column (Promega Corp.) equilibrated with 50 mM Tris-HCl (pH 8.0) followed by the desired Fc IgA the human was eluted with 5 mM biotin, 150 mM NaCl and 50 mM Tris-HCl (pH 8.0). Then associates (undesirable impurities) were removed by gel filtration using Superdex 200 (GE Healthcare Japan Corp.) and after changing the buffer solution to a solution of 20 mM histidine-HCl and 150 mM NaCl (pH 6.0), purified human IgA Fcs were obtained.

Industrial Applicability

The present invention can increase the activity of antigen-binding molecules and provide a polypeptide that avoids cross-linking of different cells by binding to antigens expressed by different cells, which is known to cause negative reactions, and these antigen-binding molecules can be used as a drug.

List of sequences <110> Chugai Seiyaku Kabushiki Kaisha <120> Antigenuming molecule containing a variable area of antibodies <130> С1-a1309r <140> ЗС/jp2014/079785 <141> 2014-11-11 <150> jp 2013-232803 <51 > 2013-11-11 <160> 93 < 170> Patentin version 3.5 <210>1 <211>330 < 212> PROTEIN <213> Homo sapiens <400>1

Ala Ser Thr Lys Gly Pro Ser Vai Phe Pro Leu Ala Pro Ser Ser Lys 15 1015

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Vai Lys Asp Tyr 20 2530

Phe Pro Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 4045

Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser 50 5560

Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr 65 70 7580

Tyr lie Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp Lys 85 9095

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

100 105110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Vai Phe Leu Phe Pro Pro

115 120125

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys

130 135140

Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn Trp

145 150 155160

Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu

165 170175

Glu Gin Tyr Asn Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu

180 185190

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn

195 200205

- 66 042507

Lys Ala Leu Pro Ala Pro lie Glu

210 215

Lys Thr lie Ser Lys Ala Lys Gly

220

Gin Pro Arg Glu Pro Gin Vai Tyr 225 230

Thr Leu Pro Pro Ser Arg Asp Glu

235 240

Leu Thr Lys Asn Gin Vai Ser Leu

245

Thr Cys Leu Vai Lys Gly Phe Tyr

250 255

Pro Ser Asp lie Ala Vai Glu Trp

260

Glu Ser Asn Gly Gin Pro Glu Asn 265 270

Asn Tyr Lys Thr Thr Pro Pro Vai

275 280

Leu Asp Ser Asp Gly Ser Phe Phe 285

Leu Tyr Ser Lys Leu Thr Vai Asp

290 295

Lys Ser Arg Trp Gin Gin Gly Asn

300

Vai Phe Ser Cys Ser Vai Met His 305 310

Glu Ala Leu His Asn His Tyr Thr

315 320

Gin Lys Ser Leu Ser Leu Ser Pro 325 <210>2 <211>326 <212> PROTEIN <213> Homo sapiens <400>2

Ala Ser Thr Lys Gly Pro Ser Vai

Gly Lys

330

Phe Pro Leu Ala Pro Cys Ser Arg

15

Ser Thr Ser Glu Ser Thr Ala Ala

Leu Gly Cys Leu Vai Lys Asp Tyr 25 30

Phe Pro Glu Pro Vai Thr Vai Ser

40

Trp Asn Ser Gly Ala Leu Thr Ser 45

Gly Vai His Thr Phe Pro Ala Vai

55

Leu Gin Ser Ser Gly Leu Tyr Ser 60

Leu Ser Ser Vai Vai Thr Vai Pro

70

Ser Ser Asn Phe Gly Thr Gin Thr

80

Tyr Thr Cys Asn Vai Asp His Lys 85

Pro Ser Asn Thr Lys Vai Asp Lys

95

Thr Vai Glu Arg Lys Cys Cys Vai

100

Glu Cys Pro Pro Cys Pro Ala Pro 105 110

Pro Vai Ala Gly Pro Ser Vai Phe

115 120

Leu Phe Pro Pro Lys Pro Lys Asp

125

Thr Leu Met lie Ser Arg Thr Pro

130 135

Glu Vai Thr Cys Vai Vai Vai Asp 140

Vai Ser His Glu Asp Pro Glu Vai

Gin Phe Asn Trp Tyr Vai Asp Gly

- 67 042507

145

150

155

160

Vai

Vai

His

Asn

165

Lys

Thr

Lys

Pro

170

Arg

gin

Phe

175

Asn

Ser

Thr

Phe

Arg

180

Vai

Vai

Ser

Vai

Leu

185

Thr

Vai

Vai

His

gin

190

asp

trp

Leu

Asn

Gly 195

Lys

Tyr

Lys

Cys 200

Lys

Vai

Ser

Asn

Lys

205

gly

Leu

Pro

Pro

225

Pro

210

gin

Vai

Tyr

Lys

Thr

Thr

Leu

230

Ser

215

Pro

Pro

Lys

Ser

Thr

Arg

Lys

Gly 220

gin

Met

Pro

Thr

235

Arg

Lys

Asn

240

gin

Vai

Leu

Thr

245

Cys

Leu

Vai

Lys

Gly 250

Phe

Tyr

Pro

Ser

Asp 255

Vai

TRP 260

Ser

Asn

gly

gin

265

Pro

Asn

Asn

Tyr

270

Lys

Thr

Thr

Pro

Pro

275

Met

Leu

asp

Ser

Asp 280

gly

Ser

Phe

Phe

Leu

285

Tyr

Ser

Lys

Leu

Thr

290

Vai

asp

Lys

Ser

Arg 295

trp

gin

gin

gly

Asn

300

Vai

Phe

Ser

Cys

Ser

305

Vai

Met

His

Leu

His

Asn

His

310

Tyr 315

Thr

gin

Lys

Ser

Leu

320

Ser

Leu

Pro

Gly 325

Lys <210> 3 <211> 377 <212> PROTEIN <213> Homo sapiens <400> 3

Ala Ser Thr Lys Gly Pro Ser Vai 1 5

Phe Pro Leu Ala Pro Cys Ser Arg

15

Ser Thr Ser Gly Gly Thr Ala Ala 20

Leu Gly Cys Leu Vai Lys Asp Tyr 25 30

Phe Pro Glu Pro Vai Thr Vai Ser

40

Trp Asn Ser Gly Ala Leu Thr Ser 45

Gly Vai His Thr Phe Pro Ala Vai

55

Leu Gin Ser Ser Gly Leu Tyr Ser 60

Leu Ser Ser Vai Vai Thr Vai Pro

70

Ser Ser Ser Leu Gly Thr Gin Thr

80

Tyr Thr Cys Asn Vai Asn His Lys 85

Pro Ser Asn Thr Lys Vai Asp Lys

95

- 68 042507

Arg Vai Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro

100 105110

Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120125

Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys

130 135140

Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

145 150 155160

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

165 170175

Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys Vai

180 185190

Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Gin Phe Lys Trp Tyr

195 200205

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

210 215220

Gin Tyr Asn Ser Thr Phe Arg Vai Vai Ser Vai Leu Thr Vai Leu His

225 230 235240

Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn Lys

245 250255

Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Thr Lys Gly Gin

260 265270

Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Ser Arg Glu Glu Met

275 280285

Thr Lys Asn Gin Vai Ser Leu Thr Cys Leu Vai Lys Gly Phe Tyr Pro

290 295300

Ser Asp lie Ala Vai Glu Trp Glu Ser Ser Gly Gin Pro Glu Asn Asn

305 310 315320

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

325 330335

Tyr Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly Asn lie

340 345350

Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn Arg Phe Thr Gin

355 360365

Lys Ser Leu Ser Leu Ser Pro Gly Lys

370375 <210>4 <211>327 <212> PROTEIN <213> Homo sapiens

- 69 042507 <4OO> 4

Ser

Th g

Lys

Gly 5

Pro

Ser

Vai

Phe

Pro

Leu

Pro

Cys

Ser 15

Arg

Ser

Thr

Ser

Ser

Thr

Phe

Pro

Pro

Vai

Thr

Vai

Ser 40

gly

Vai 50

His

Thr

Phe

Pro

Vai

Leu

trp

Leu

gly

Asn

gin

Cys

Ser

Ser

Leu

Vai

Lys 30

asp

Tyr

gly

Leu

Thr

Ser

Ser

gly

Leu

Tyr

Ser

Leu 65

Ser

Ser

Vai

Vai

Thr

Vai

Pro

Ser

Ser

Ser

Leu

gly

Thr

Lys

Thr

Tyr

Thr

Cys

Asn

Vai 85

asp

His

Lys

Pro

Ser 90

Asn

Thr

Lys

Vai

Asp 95

Lys

Arg

Vai

Ser

100

Lys

Tyr

gly

Pro

Pro

105

Cys

Pro

Ser

Cys

Pro

110

Pro

Phe

Leu

115

gly

gly

Pro

Ser

Vai

120

Phe

Leu

Phe

Pro

Pro

125

Lys

Pro

Lys

asp

Thr

130

Leu

Met

Ser

Arg 135

Thr

Pro

Vai

Thr

140

Cys

Vai

Vai

Vai

Asp 145

Vai

Ser

gin

Asp 150

Pro

Vai

gin

Phe

155

Asn

trp

Tyr

Vai

Asp 160

gly

Vai

Vai

His

165

Asn

Lys

Thr

Lys 170

Pro

Arg

gin

175

Phe

Asn

Ser

Thr

Tyr 180

Arg

Vai

Vai

Ser

Vai

185

Leu

Thr

Vai

Leu

His

190

gin

asp

trp

Leu

Asn

195

gly

Lys

Tyr

Lys

200

Cys

Lys

Vai

Ser

Asn

205

Lys

gly

Leu

Pro

Ser

210

Ser

Lys

Thr

215

Ser

Lys

Lys

220

gly

gin

Pro

Arg

Pro

gin

Vai

225

Tyr

Thr

230

Leu

Pro

Pro

Ser

gin

235

Met

Thr

Lys

240

Asn

gin

Vai

Ser

Leu

245

Thr

Cys

Leu

Vai

Lys

250

gly

Phe

Tyr

Pro

Ser

255

asp

Vai

260

trp

Ser

Asn

Gly 265

gin

Pro

Asn

Asn

270

Tyr

Lys

Thr

Thr

Pro

275

Pro

Vai

Leu

asp

Ser

280

asp

gly

Ser

Phe

Phe

285

Leu

Tyr

Ser

Arg

Leu

290

Thr

Vai

asp

Lys

Ser

295

Arg

trp

gin

Gly 300

Asn

Vai

Phe

Ser

- 70 042507

Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser

305 310 315320

Leu Ser Leu Ser Leu Gly Lys 325 <210>5 <211>4 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>5

Gly Gly Gly Ser 1 <210>6 <211>4 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>6

Ser Gly Gly Gly 1 <210>7 <211>5 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>7

Gly Gly Gly Gly Ser 15 <210>8 <211>5 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>8

Ser Gly Gly Gly Gly <210>9 <211>6 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 9

- 71 042507

Gly Gly Gly Gly Gly Ser 15 <210>10 <211>6 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>10

Ser Gly Gly Gly Gly Gly <210>11 <211>7 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>11

Gly Gly Gly Gly Gly Gly Ser <210>12 <211>7 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>12

Ser Gly Gly Gly Gly Gly Gly <210>13 <211>122 <212> PROTEIN <213> Rattus norvegicus <400>13

Glu 1 Val gin Leu Val 5 Glu Thr gly gly Ser 10 Leu Val gin Pro Gly 15 Lys Ser Leu Lys Leu 20 Thr Cys Ala Thr Ser 25 gly Phe Thr Phe Ser thirty Asn Ala trp Met His 35 trp Val Arg gin Ser 40 Pro Glu Lys gin Leu 45 Glu trp Val Ala Gin 50 lie Lys Ala Lys Ser 55 Asn Asn Tyr Ala Thr 60 Tyr Tyr Ala Glu Ser 65 Val Lys gly Arg Phe 70 Thr lie Ser Arg Asp 75 asp Ser Lys Ser Asn 80 lie Tyr Leu gin Met Asn Ser Leu Lys Glu Glu asp Thr Ala Val Tyr

- 72 042507

95

Tyr Cys Arg Tyr Val His Tyr Gly

100

Ala Tyr Tyr Gly Val Asp Ala Trp 105 110

Gly Gin Gly Thr Ser Val Thr Val

115120

Ser Ser <210>14 <211>112 <212> PROTEIN <213> Rattus norvegicus <400>14

Asp Val Val Met Thr Gin Thr Pro

Val Ser Met Ser Val Ser Leu Gly

15

Gly Gin Val Ser lie Ser Cys Arg 20

Ser Ser Gin Ser Leu Val His Asn

thirty

Asn Gly Asn Thr Tyr Val Ser Trp

40

Tyr lie Gin Lys Pro Ser Gin Ser 45

Pro Gin Leu Leu lie Tyr Lys Val

55

Ser Asn Arg Phe Ser Gly lie Ser 60

Asp Arg Phe Ser Gly Ser Gly Ser 65 70

Gly Thr Asp Phe Thr Leu Lys lie

80

Ser Arg Val Glu Pro Asp Asp Leu 85

Gly Val Tyr Tyr Cys Gly Gin Gly

95

Thr Gin Asp Pro Tyr Thr Phe Gly

100

Ala Gly Thr Lys Leu Glu Leu Lys 105 110 <210> 15 <211>119 <212> PROTEIN <213> Mus musculus <400>15

Gin Val Gin Leu Lys Gin Ser Gly

Pro Gly Leu Val Gin Pro Ser Gin

15

Ser Leu Ser lie Thr Cys Thr Val 20

Ser Gly Phe Ser Leu Thr Asn Tyr 25 30

Gly Val His Trp Val Arg Gin Ser

40

Pro Gly Lys Gly Leu Glu Trp Leu 45

Gly Val lie Trp Ser Gly Gly Asn 50 55

Thr Asp Tyr Asn Thr Pro Phe Thr 60

Ser Arg Leu Ser lie Asn Lys Asp 65 70

Asn Ser Lys Ser Gin Val Phe Phe

80

Lys Met Asn Ser Leu Gin Ser Asn 85

Asp Thr Ala lie Tyr Tyr Cys Ala

95

- 73 042507

Arg Ala Leu Thr Tyr Tyr Asp

100

Tyr Glu Phe Ala Tyr Trp Gly Gin Gly

105110

Thr Leu Vai Thr Vai Ser Ala

115 <210>16 <211>107 <212> PROTEIN <213> Mus musculus <400>16

Asp lie Leu Leu Thr Gin Ser Pro

Vai lie Leu Ser Vai Ser Pro Gly

15

Glu Arg Vai Ser Phe Ser Cys Arg 20

Ala Ser Gin Ser lie Gly Thr Asn 25 30 lie His Trp Tyr Gin Gin Arg Thr 35 40

Asn Gly Ser Pro Arg Leu Leu lie 45

Lys Tyr Ala Ser Glu Ser lie Ser

55

Gly lie Pro Ser Arg Phe Ser Gly 60

Ser Gly Ser Gly Thr Asp Phe Thr 65 70

Leu Ser lie Asn Ser Vai Glu Ser

80

Glu Asp lie Ala Asp Tyr Tyr Cys 85

Gin Gin Asn Asn Asn Trp Pro Thr

95

Thr Phe Gly Ala Gly Thr Lys Leu

100

Glu Leu Lys 105 <210> 17 <211> 447 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 17

Gin Vai Gin Leu Lys Gin Ser Gly 1 5

Pro Gly Leu Vai Gin Pro Ser Gin

15

Ser Leu Ser lie Thr Cys Thr Vai 20

Ser Gly Phe Ser Leu Thr Asn Tyr 25 30

Gly Vai His Trp Vai Arg Gin Ser

40

Pro Gly Lys Gly Leu Glu Trp Leu 45

Gly Vai lie Trp Ser Gly Gly Asn

55

Thr Asp Tyr Asn Thr Pro Phe Thr 60

Ser Arg Leu Ser lie Asn Lys Asp

70

Asn Ser Lys Ser Gin Vai Phe Phe

80

Lys Met Asn Ser Leu Gin Ser Asn 85

Asp Thr Ala lie Tyr Tyr Cys Ala

95

- 74 042507

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105110

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

115 120125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185190

Ser Ser Leu Gly Thr Gln Thr Tyr lie Cys Asn Val Asn His Lys Pro

195 200205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235240

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

245 250255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265270

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

275 280285

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

290 295300

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

305 310 315320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys

325 330335

Thr lie Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345350

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

355 360365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu

370 375380

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

385 390 395400

- 75 042507

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Vai Asp Lys

405 410415

Ser Arg Trp Gin Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu

420 425430

Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro

435 440445 <210>18 <211>447 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 18

Gin Vai Gin Leu Lys Gin Ser Gly 1 5

Pro Gly Leu Vai Gin Pro Ser Gin

15

Ser Leu Ser lie Thr Cys Thr Vai 20

Ser Gly Phe Ser Leu Thr Asn Tyr 25 30

Gly Vai His Trp Vai Arg Gin Ser

40

Pro Gly Lys Gly Leu Glu Trp Leu 45

Gly Vai lie Trp Ser Gly Gly Asn

55

Thr Asp Tyr Asn Thr Pro Phe Thr 60

Ser Arg Leu Ser lie Asn Lys Asp 65 70

Asn Ser Lys Ser Gin Vai Phe Phe

80

Lys Met Asn Ser Leu Gin Ser Asn 85

Asp Thr Ala lie Tyr Tyr Cys Ala

95

Arg Ala Leu Thr Tyr Tyr Asp Tyr

100

Glu Phe Ala Tyr Trp Gly Gin Gly 105 110

Thr Leu Vai Thr Vai Ser Ala Ala

115 120

Ser Thr Lys Gly Pro Ser Vai Phe

125

Pro Leu Ala Pro Ser Ser Lys Ser

130 135

Thr Ser Gly Gly Thr Ala Ala Leu

140

Gly Cys Leu Vai Lys Asp Tyr Phe 145 150

Pro Glu Pro Vai Thr Vai Ser Trp

155 160

Asn Ser Gly Ala Leu Thr Ser Gly

165

Vai His Thr Phe Pro Ala Vai Leu

170 175

Gin Ser Ser Gly Leu Tyr Ser Leu

180

Ser Ser Vai Vai Thr Vai Pro Ser

185 190

Ser Ser Leu Gly Thr Gin Thr Tyr

195 200 lie Cys Asn Vai Asn His Lys Pro

205

Ser Asn Thr Lys Vai Asp Lys Lys

210 215

Vai Glu Pro Lys Ser Cys Asp Lys

220

- 76 042507

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235240

Ser Vai Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser

245 250255

Arg Thr Pro Glu Vai Thr Cys Vai Vai Vai Asp Vai Ser His Glu Asp

260 265270

Pro Glu Vai Lys Phe Asn Trp Tyr Vai Asp Gly Vai Glu Vai His Asn

275 280285

Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Vai

290 295300

Vai Ser Vai Leu Thr Vai Leu His Gin Asp Trp Leu Asn Gly Lys Glu

305 310 315320

Tyr Lys Cys Lys Vai Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys

325 330335

Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr

340 345350

Leu Pro Pro Ser Arg Lys Glu Leu Thr Lys Asn Gin Vai Ser Leu Thr

355 360365

Cys Leu Vai Lys Gly Phe Tyr Pro Ser Asp He Ala Vai Glu Trp Glu

370 375380

Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Vai Leu

385 390 395400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Vai Asp Lys

405 410415

Ser Arg Trp Gin Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu

420 425430

Ala Leu His Asn Arg Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro

435 440445 <210>19 <211>447 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>19

Gin Vai Gin Leu Lys Gin Ser Gly Pro Gly Leu Vai Gin Pro Ser Gin 15 1015

Ser Leu Ser He Thr Cys Thr Vai Ser

2025

Gly Phe Ser Leu Thr

Asn Tyr

- 77 042507

Gly Vai His Trp Vai Arg Gin Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 4045

Gly Vai lie Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 5560

Ser Arg Leu Ser lie Asn Lys Asp Asn Ser Lys Ser Gin Vai Phe Phe

70 7580

Lys Met Asn Ser Leu Gin Ser Asn Asp Thr Ala lie Tyr Tyr Cys Ala 85 9095

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gin Gly

100 105110

Thr Leu Vai Thr Vai Ser Ala Ala Ser Thr Lys Gly Pro Ser Vai Phe

115 120125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135140

Gly Cys Leu Vai Lys Asp Tyr Phe Pro Glu Pro Vai Thr Vai Ser Trp

145 150 155160

Asn Ser Gly Ala Leu Thr Ser Gly Vai His Thr Phe Pro Ala Vai Leu

165 170175

Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Vai Vai Thr Vai Pro Ser

180 185190

Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Vai Asn His Lys Pro

195 200205

Ser Asn Thr Lys Vai Asp Lys Lys Vai Glu Pro Lys Ser Cys Asp Lys

210 215220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235240

Ser Vai Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser

245 250255

Arg Thr Pro Glu Vai Thr Cys Vai Vai Vai Asp Vai Ser His Glu Asp

260 265270

Pro Glu Vai Lys Phe Asn Trp Tyr Vai Asp Gly Vai Glu Vai His Asn

275 280285

Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Vai

290 295300

Vai Ser Vai Leu Thr Vai Leu His Gin Asp Trp Leu Asn Gly Lys Glu

305 310 315320

Tyr Lys Cys Lys Vai Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys

325 330335

Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr

- 78 042507

340

345 350

Leu Pro Pro Ser Arg Asp Glu Leu

355 360

Thr Lys Asn Gin Vai Ser Leu Thr 365

Cys Leu Vai Lys Gly Phe Tyr Pro

370 375

Ser Asp lie Ala Vai Glu Trp Glu 380

Ser Asn Gly Gin Pro Glu Asn Asn 385 390

Tyr Lys Thr Thr Pro Pro Vai Leu

395 400

Asp Ser Asp Gly Ser Phe Phe Leu

405

Tyr Ser Lys Leu Thr Vai Asp Lys

410 415

Ser Arg Trp Gin Gin Gly Asn Vai 420

Phe Ser Cys Ser Vai Met His Glu 425 430

Ala Leu His Asn His Tyr Thr Gin

435 440

Glu Ser Leu Ser Leu Ser Pro

445 <210> 20 <211> 445 <212> PROTEIN <213>

CTb<220>

<223> Artificially synthesized peptide sequence <400>20

Asp lie Leu Leu Thr Gin Ser Pro Vai lie Leu Ser Vai Ser Pro Gly 15 1015

Glu Arg Vai Ser Phe Ser Cys Arg Ala Ser Gin Ser lie Gly Thr Asn 20 2530 lie His Trp Tyr Gin Gin Arg Thr Asn Gly Ser Pro Arg Leu Leu lie 35 4045

Lys Tyr Ala Ser Glu Ser lie Ser Gly lie Pro Ser Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser lie Asn Ser Vai Glu Ser

70 7580

Glu Asp lie Ala Asp Tyr Tyr Cys Gin Gin Asn Asn Asn Trp Pro Thr 85 9095

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

100 105110

Lys Gly Pro Ser Vai Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Vai Lys Asp Tyr Phe Pro Glu

130 135140

Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Vai His

145 150 155160

- 79 042507

Thr Phe Pro Ala Vai Leu Gin Ser

165

Ser Gly Leu Tyr Ser Leu Ser Ser

170 175

Vai Vai Thr Vai Pro Ser Ser Ser

180

Leu Gly Thr Gin Thr Tyr lie Cys 185 190

Asn Vai Asn His Lys Pro Ser Asn

195 200

Thr Lys Vai Asp Lys Lys Vai Glu 205

Pro Lys Ser Cys Asp Lys Thr His

210 215

Thr Cys Pro Pro Cys Pro Ala Pro

220

Glu Ala Ala Gly Gly Pro Ser Vai 225 230

Phe Leu Phe Pro Pro Lys Pro Lys

235 240

Asp Thr Leu Met lie Ser Arg Thr

245

Pro Glu Vai Thr Cys Vai Vai Vai

250 255

Asp Vai Ser His Glu Asp Pro Glu

260

Vai Lys Phe Asn Trp Tyr Vai Asp 265 270

Gly Vai Glu Vai His Asn Ala Lys

275 280

Thr Lys Pro Arg Glu Glu Gin Tyr

285

Ala Ser Thr Tyr Arg Vai Vai Ser

290 295

Vai Leu Thr Vai Leu His Gin Asp

300

Trp Leu Asn Gly Lys Glu Tyr Lys 305 310

Cys Lys Vai Ser Asn Lys Ala Leu

315 320

Pro Ala Pro lie Glu Lys Thr lie

325

Ser Lys Ala Lys Gly Gin Pro Arg

330 335

Glu Pro Gin Vai Cys Thr Leu Pro

340

Pro Ser Arg Asp Glu Leu Thr Lys

345 350

Asn Gin Vai Ser Leu Trp Cys Leu

355 360

Vai Lys Gly Phe Tyr Pro Ser Asp

365 lie Ala Vai Glu Trp Glu Ser Asn

370 375

Gly Gin Pro Glu Asn Asn Tyr Lys 380

Thr Thr Pro Pro Vai Leu Asp Ser 385 390

Asp Gly Ser Phe Phe Leu Tyr Ser

395 400

Lys Leu Thr Vai Asp Lys Ser Arg 405

Trp Gin Gin Gly Asn Vai Phe Ser

410 415

Cys Ser Vai Met His Glu Ala Leu

420

His Asn Arg Tyr Thr Gin Lys Ser 425 430

Leu Ser Leu Ser Pro His His His

435440

His His His His His His

445 <210>21 <211>226 <212> PROTEIN <213> Artificial sequence

- 80 042507 <220>

<223>

Artificially synthesized peptide sequence <400>21

Gin Vai Gin Leu Lys Gin Ser Gly Pro Gly Leu Vai Gin Pro Ser Gin 15 1015

Ser Leu Ser lie Thr Cys Thr Vai Ser Gly Phe Ser Leu Thr Asn Tyr 20 2530

Gly Vai His Trp Vai Arg Gin Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 4045

Gly Vai lie Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 5560

Ser Arg Leu Ser lie Asn Lys Asp Asn Ser Lys Ser Gin Vai Phe Phe

70 7580

Lys Met Asn Ser Leu Gin Ser Asn Asp Thr Ala lie Tyr Tyr Cys Ala 85 9095

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gin Gly

100 105110

Thr Leu Vai Thr Vai Ser Ala Arg Thr Vai Ala Ala Pro Ser Vai Phe

115 120125

He Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr Ala Ser Vai

130 135140

Vai Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Vai Gin Trp

145 150 155160

Lys Vai Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu Ser Vai Thr

165 170175

Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr

180 185190

Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Vai Tyr Ala Cys Glu Vai

195 200205

Thr His Gin Gly Leu Ser Ser Pro Vai Thr Lys Ser Phe Asn Arg Gly

210 215220

Glu Cys 225 <210> 22 <211>458 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 22

- 81 042507

Glu Vai Gin Leu Vai Glu Thr Gly Gly Ser Leu Vai Gin Pro Gly Lys 15 1015

Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 2530

Trp Met His Trp Vai Arg Gin Ser Pro Glu Lys Gin Leu Glu Trp Vai 35 4045

Ala Gin lie Lys Ala Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Glu 50 5560

Ser Vai Lys Gly Arg Phe Thr lie Ser Arg Asp Asp Ser Lys Ser Asn

70 7580 lie Tyr Leu Gin Met Asn Ser Leu Lys Glu Glu Asp Thr Ala Vai Tyr 85 9095

Tyr Cys Arg Tyr Vai His Tyr Gly Ala Tyr Tyr Gly Vai Asp Ala Trp

100 105110

Gly Gin Gly Thr Ser Vai Thr Vai Ser Ser Ala Ser Thr Lys Gly Pro

115 120125

Ser Vai Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135140

Ala Ala Leu Gly Cys Leu Vai Lys Asp Tyr Phe Pro Glu Pro Vai Thr

145 150 155160

Vai Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Vai His Thr Phe Pro

165 170175

Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Vai Vai Thr

180 185190

Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Vai Asn

195 200205

His Lys Pro Ser Asn Thr Lys Vai Asp Lys Lys Vai Glu Pro Lys Ser

210 215220

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

225 230 235240

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

245 250255

Met lie Ser Arg Thr Pro Glu Vai Thr Cys Vai Vai Vai Asp Vai Ser

260 265270

His Glu Asp Pro Glu Vai Lys Phe Asn Trp Tyr Vai Asp Gly Vai Glu

275 280285

Vai His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Ala Ser Thr

290 295300

- 82 042507

Tyr Arg Val Val Ser Val Leu Thr 305 310

Val Leu His Gln Asp Trp Leu Asn

315 320

Gly Lys Glu Tyr Lys Cys Lys Val 325

Ser Asn Lys Ala Leu Pro Ala Pro

330 335 lie Glu Lys Thr lie Ser Lys Ala 340

Lys Gly Gln Pro Arg Glu Pro Gln

345 350

Val Tyr Thr Leu Pro Pro Ser Arg

355 360

Cys Glu Leu Thr Lys Asn Gln Val 365

Ser Leu Ser Cys Ala Val Lys Gly

370 375

Phe Tyr Pro Ser Asp lie Ala Val 380

Glu Trp Glu Ser Asn Gly Gln Pro 385 390

Glu Asn Asn Tyr Lys Thr Thr Pro

395 400

Pro Val Leu Asp Ser Asp Gly Ser 405

Phe Phe Leu Val Ser Lys Leu Thr

410 415

Val Asp Lys Ser Arg Trp Gln Gln 420

Gly Asn Val Phe Ser Cys Ser Val 425 430

Met His Glu Ala Leu His Asn His

435 440

Tyr Thr Gln Lys Ser Leu Ser Leu 445

Ser Pro Asp Tyr Lys Asp Asp Asp

450455

Asp Lys <210>23 <211>219 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>23

Asp Val Val Met Thr Gln Thr Pro Val Ser Met Ser Val Ser Leu Gly 15 1015

Gly Gln Val Ser lie Ser Cys Arg Ser Ser Gln Ser Leu Val His Asn 20 2530

Asn Gly Asn Thr Tyr Val Ser Trp Tyr lie Gln Lys Pro Ser Gln Ser 35 4045

Pro Gln Leu Leu lie Tyr Lys Val Ser Asn Arg Phe Ser Gly lie Ser 50 5560

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 7580

Ser Arg Val Glu Pro Asp Asp Leu Gly Val Tyr Tyr Cys Gly Gln Gly 85 9095

Thr Gln Asp Pro Tyr Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105110

- 83 042507

Arg Thr Val Ala Ala Pro Ser Val Phe lie Phe Pro Pro Ser Asp Glu

115 120125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170175

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

180 185190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210215 <210>24 <211>462 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>24

Glu Val Gln Leu Val Glu Thr Gly Gly Ser Leu Val Gln Pro Gly Lys 15 1015

Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 2530

Trp Met His Trp Val Arg Gln Ser Pro Glu Lys Gln Leu Glu Trp Val 35 4045

Ala Gln lie Lys Ala Gly Cys Arg Gly Asp Cys Leu Asn Tyr Ala Thr 50 5560

Tyr Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr lie Ser Arg Asp Asp

70 7580

Ser Lys Ser Asn lie Tyr Leu Gln Met Asn Ser Leu Lys Glu Glu Asp 85 9095

Thr Ala Val Tyr Tyr Cys Arg Tyr Val His Tyr Gly Ala Tyr Tyr Gly

100 105110

Val Asp Ala Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser

115 120125

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

130 135140

- 84 042507

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Vai Lys Asp Tyr Phe Pro

145 150 155160

Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Vai

165 170175

His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser

180 185190

Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr lie

195 200205

Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp Lys Lys Vai

210 215220

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

225 230 235240

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

245 250255

Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys Vai Vai

260 265270

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

275 280285

Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin

290 295300

Tyr Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu His Gin

305 310 315320

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

325 330335

Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro

340 345350

Arg Glu Pro Gin Vai Tyr Thr Leu Pro Ser Arg Cys Glu Leu Thr

355 360365

Lys Asn Gin Vai Ser Leu Ser Cys Ala Vai Lys Gly Phe Tyr Pro Ser

370 375380

Asp lie Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr

385 390 395400

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

405 410415

Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly Asn Vai Phe

420 425430

Ser Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys

435 440445

Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

- 85 042507

450 455460 <210>25 <211>462 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 25

Glu Val Gin Leu Val Glu Thr Gly 1 5

Gly Ser Leu Val Gin Pro Gly Lys

15

Ser Leu Lys Leu Thr Cys Ala Thr 20

Ser Gly Phe Thr Phe Ser Asn Ala 25 30

Trp Met His Trp Val Arg Gin Ser

40

Pro Glu Lys Gin Leu Glu Trp Val 45

Ala Gin lie Lys Ala Lys Gly Cys

55

Arg Gly Asp Cys Leu Tyr Ala Thr 60

Tyr Tyr Ala Glu Ser Val Lys Gly

70

Arg Phe Thr lie Ser Arg Asp Asp

80

Ser Lys Ser Asn lie Tyr Leu Gin 85

Met Asn Ser Leu Lys Glu Glu Asp

95

Thr Ala Val Tyr Tyr Cys Arg Tyr

100

Val His Tyr Gly Ala Tyr Tyr Gly

105 110

Val Asp Ala Trp Gly Gin Gly Thr

115 120

Ser Val Thr Val Ser Ser Ala Ser

125

Thr Lys Gly Pro Ser Val Phe Pro

130 135

Leu Ala Pro Ser Ser Lys Ser Thr 140

Ser Gly Gly Thr Ala Ala Leu Gly 145 150

Cys Leu Val Lys Asp Tyr Phe Pro

155 160

Glu Pro Val Thr Val Ser Trp Asn

165

Ser Gly Ala Leu Thr Ser Gly Val

170 175

His Thr Phe Pro Ala Val Leu Gin

180

Ser Ser Gly Leu Tyr Ser Leu Ser 185 190

Ser Val Val Thr Val Pro Ser Ser

195 200

Ser Leu Gly Thr Gin Thr Tyr lie 205

Cys Asn Val Asn His Lys Pro Ser

210 215

Asn Thr Lys Val Asp Lys Lys Val

220

Glu Pro Lys Ser Cys Asp Lys Thr 225 230

His Thr Cys Pro Pro Cys Pro Ala

235 240

Pro Glu Ala Ala Gly Gly Pro Ser

245

Val Phe Leu Phe Pro Pro Lys Pro

250 255

- 86 042507

Lys Asp Thr Leu Met lie Ser Arg

260

Thr Pro Glu Vai Thr Cys Vai Vai 265 270

Vai Asp Vai Ser His Glu Asp Pro

275 280

Glu Vai Lys Phe Asn Trp Tyr Vai 285

Asp Gly Vai Glu Vai His Asn Ala

290 295

Lys Thr Lys Pro Arg Glu Glu Gin

300

Tyr Ala Ser Thr Tyr Arg Vai Vai 305 310

Ser Vai Leu Thr Vai Leu His Gin

315 320

Asp Trp Leu Asn Gly Lys Glu Tyr

325

Lys Cys Lys Vai Ser Asn Lys Ala

330 335

Leu Pro Ala Pro lie Glu Lys Thr 340 lie Ser Lys Ala Lys Gly Gin Pro

345 350

Arg Glu Pro Gin Vai Tyr Thr Leu

355 360

Pro Pro Ser Arg Cys Glu Leu Thr 365

Lys Asn Gin Vai Ser Leu Ser Cys

370 375

Ala Vai Lys Gly Phe Tyr Pro Ser 380

Asp lie Ala Vai Glu Trp Glu Ser 385 390

Asn Gly Gin Pro Glu Asn Asn Tyr

395 400

Lys Thr Thr Pro Pro Vai Leu Asp 405

Ser Asp Gly Ser Phe Phe Leu Vai

410 415

Ser Lys Leu Thr Vai Asp Lys Ser 420

Arg Trp Gin Gin Gly Asn Vai Phe 425 430

Ser Cys Ser Vai Met His Glu Ala

435 440

Leu His Asn His Tyr Thr Gin Lys 445

Ser Leu Ser Leu Ser Pro Asp Tyr

450 455

Lys Asp Asp Asp Asp Lys

460 <210> 26 <211> 465 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 26

Glu 1 Vai gin Leu Wai 5 Glu Thr gly gly Ser 10 Leu Vai gin Pro Gly 15 Lys Ser Leu Lys Leu 20 Thr Cys Ala Thr Ser 25 gly Phe Thr Phe Ser 30 Asn Ala trp Met Hi s 35 trp Vai Arg gin Ser 40 Pro Glu Lys gin Leu 45 Glu trp Vai Ala gin He Lys Ala gly Cys Arg gly asp Cys Leu Lys Ser Asn Asn

- 87 042507

Tyr 65

Thr

Tyr

Tyr

Ser

Vai

Lys

Gly 75

Arg

Phe

Thr

Ser

Arg

asp

asp

Ser

Lys 85

Ser

Asn

Tyr

Leu 90

gin

Met

Asn

Ser

Leu 95

Lys

asp

Thr

100

Vai

Tyr

Tyr

Cys 105

Arg

Tyr

Vai

His

Tyr 110

gly

Tyr

Tyr

Gly 115

Vai

asp

trp

Gly 120

gin

gly

Ser

Vai

125

Vai

Ser

Ser

Ser

Thr

130

Lys

gly

Pro

135

Ser

Vai

Phe

Pro

Leu

140

Pro

Ser

Ser

Lys 145

Ser

Thr

Ser

gly

Gly 150

Thr

Leu

Gly 155

Cys

Leu

Vai

Lys

Asp 160

Tyr

Phe

Pro

Pro

165

Vai

Thr

Vai

Ser

Trp 170

Asn

Ser

gly

Leu

175

Thr

Ser

gly

Vai

His

180

Thr

Phe

Pro

Vai

185

Leu

gin

Ser

Ser

Gly 190

Leu

Tyr

Ser

Leu

Ser

195

Ser

Vai

Vai

Thr

Vai

200

Pro

Ser

Ser

Ser

Leu

205

gly

Thr

gin

Thr

Tyr 210

Cys

Asn

Vai

Asn

215

His

Lys

Pro

Ser

Asn

220

Thr

Lys

Vai

asp

Lys

225

Lys

Vai

Pro

Lys

230

Ser

Cys

asp

Lys

Thr

235

His

Thr

Cys

Pro

Pro

240

Cys

Pro

Pro

245

gly

gly

Pro

250

Ser

Vai

Phe

Leu

Phe

255

Pro

Pro

Lys

Pro

Lys

260

asp

Thr

Leu

Met

Ser

265

Arg

Thr

Pro

270

Vai

Thr

Cys

Vai

Vai

275

Vai

asp

Vai

Ser

His

280

asp

Pro

Vai

285

Lys

Phe

Asn

trp

Tyr 290

Vai

asp

gly

Vai

Vai

His

Asn

gin

305

Tyr

Ser

310

Leu

His

gin

asp

Trp 325

Leu

Asn

Lys

Leu

340

Pro

295

Thr

Tyr

Arg

Vai

Vai

315

Asn

gly

Lys

330

Tyr

Pro

345

Lys

Thr

Lys

300

Ser

Lys

Thr

Vai

Cys

Ser

Lys

Leu

Lys

Lys 350

Pro

Thr

Vai

335

Arg

Vai

320

Ser

Lys

gly

gin

Pro

355

Arg

Pro

gin

Vai

360

Tyr

Thr

Leu

Pro

Pro

365

Ser

Arg

Cys

- 88 042507

Glu Leu Thr Lys Asn Gin Vai Ser

370 375

Leu Ser Cys Ala Vai Lys Gly Phe 380

Tyr Pro Ser Asp lie Ala Vai Glu 385 390

Trp Glu Ser Asn Gly Gin Pro Glu

395 400

Asn Asn Tyr Lys Thr Thr Pro Pro

405

Vai Leu Asp Ser Asp Gly Ser Phe

410 415

Phe Leu Vai Ser Lys Leu Thr Vai

420

Asp Lys Ser Arg Trp Gin Gin Gly 425 430

Asn Vai Phe Ser Cys Ser Vai Met

435 440

His Glu Ala Leu His Asn His Tyr 445

Thr Gin Lys Ser Leu Ser Leu Ser

450 455

Pro Asp Tyr Lys Asp Asp Asp Asp

460

Lys 465 <210> 27 <211> 465 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>27

Glu Vai Gin Leu Vai Glu Thr Gly Gly Ser Leu Vai Gin Pro Gly Lys 15 1015

Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 2530

Trp Met His Trp Vai Arg Gin Ser Pro Glu Lys Gin Leu Glu Trp Vai 35 4045

Ala Gin lie Lys Ala Lys Gly Cys Arg Gly Asp Cys Leu Ser Asn Asn 50 5560

Tyr Ala Thr Tyr Tyr Ala Glu Ser Vai Lys Gly Arg Phe Thr lie Ser

70 7580

Arg Asp Asp Ser Lys Ser Asn lie Tyr Leu Gin Met Asn Ser Leu Lys 85 9095

Glu Glu Asp Thr Ala Vai Tyr Tyr Cys Arg Tyr Vai His Tyr Gly Ala

100 105110

Tyr Tyr Gly Vai Asp Ala Trp Gly Gin Gly Thr Ser Vai Thr Vai Ser

115 120125

Ser Ala Ser Thr Lys Gly Pro Ser Vai Phe Pro Leu Ala Pro Ser Ser

130 135140

- 89 042507

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Vai Lys Asp

145 150 155160

Tyr Phe Pro Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu Thr

165 170175

Ser Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly Leu Tyr

180 185190

Ser Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr Gin

195 200205

Thr Tyr lie Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp

210 215220

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

225 230 235240

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

245 250255

Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr

260 265270

Cys Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn

275 280285

Trp Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg

290 295300

Glu Glu Gin Tyr Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai

305 310 315320

Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser

325 330335

Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys

340 345350

Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Pro Ser Arg Cys

355 360365

Glu Leu Thr Lys Asn Gin Vai Ser Leu Ser Cys Ala Vai Lys Gly Phe

370 375380

Tyr Pro Ser Asp lie Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu

385 390 395400

Asn Asn Tyr Lys Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly Ser Phe

405 410415

Phe Leu Vai Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly

420 425430

Asn Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr

435 440445

Thr Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp

- 90 042507

450

455

460

Lys 465 < 210> 28 < 211> 465 < 212> PROTEIN < 213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 28

Glu Vai Gin Leu Vai Glu Thr Gly 1 5

Gly Ser Leu Vai Gin Pro Gly Lys

15

Ser Leu Lys Leu Thr Cys Ala Thr 20

Ser Gly Phe Thr Phe Ser Asn Ala 25 30

Trp Met His Trp Vai Arg Gin Ser

40

Pro Glu Lys Gin Leu Glu Trp Vai 45

Ala Gin lie Lys Ala Lys Ser Asn

55

Asn Tyr Ala Thr Tyr Tyr Ala Glu 60

Ser Vai Lys Gly Arg Phe Thr lie

70

Ser Arg Asp Gly Cys Arg Gly Asp

80

Cys Leu Asp Ser Lys Ser Asn lie 85

Tyr Leu Gin Met Asn Ser Leu Lys

95

Glu Glu Asp Thr Ala Vai Tyr Tyr

100

Cys Arg Tyr Vai His Tyr Gly Ala

105 110

Tyr Tyr Gly Vai Asp Ala Trp Gly

115 120

Gin Gly Thr Ser Vai Thr Vai Ser 125

Ser Ala Ser Thr Lys Gly Pro Ser

130 135

Vai Phe Pro Leu Ala Pro Ser Ser

140

Lys Ser Thr Ser Gly Gly Thr Ala

145 150

Ala Leu Gly Cys Leu Vai Lys Asp

155 160

Tyr Phe Pro Glu Pro Vai Thr Vai

165

Ser Trp Asn Ser Gly Ala Leu Thr

170 175

Ser Gly Vai His Thr Phe Pro Ala

180

Vai Leu Gin Ser Ser Gly Leu Tyr

185 190

Ser Leu Ser Ser Vai Vai Thr Vai

195 200

Pro Ser Ser Ser Leu Gly Thr Gin

205

Thr Tyr lie Cys Asn Vai Asn His

210 215

Lys Pro Ser Asn Thr Lys Vai Asp

220

Lys Lys Vai Glu Pro Lys Ser Cys

225 230

Asp Lys Thr His Thr Cys Pro Pro

235 240

- 91 042507

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

245 250255

Pro Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro Glu Vai Thr

260 265270

Cys Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn

275 280285

Trp Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg

290 295300

Glu Glu Gin Tyr Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai

305 310 315320

Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser

325 330335

Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys

340 345350

Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Pro Ser Arg Cys

355 360365

Glu Leu Thr Lys Asn Gin Vai Ser Leu Ser Cys Ala Vai Lys Gly Phe

370 375380

Tyr Pro Ser Asp lie Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu

385 390 395400

Asn Asn Tyr Lys Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly Ser Phe

405 410415

Phe Leu Vai Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly 420 425430

Asn Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr

435 440445

Thr Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp

450 455460

Lys 465 <210>29 <2H>463 <212> PROTEIN <213> Artificial sequence <220>

< 223> Artificially synthesized peptide sequence < 400> 29

Glu Vai Gin Leu Vai Glu Thr Gly Gly Ser Leu Vai Gin Pro Gly Lys 15 10 15

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

- 92 042507

trp

Met

His

trp

Vai

Arg

gin

Ser 40

Pro

Lys

gin

Leu 45

trp

Vai

Gin 50 lie

Lys

gly

Cys 55

Arg

gly

asp

Cys

Leu 60

Asn

Asn

Tyr

Thr

Tyr

Tyr

Ser

Vai

Lys

gly

Arg

Phe

Thr

Ser

Arg

Asp 80

asp

Ser

Lys

Ser

Asn 85

Tyr

Leu

gin

Met 90

Asn

Ser

Leu

Lys

asp

Thr

Vai

100

Tyr

Tyr

Cys

Arg

Tyr 105

Vai

His

Tyr

gly

110

Tyr

Tyr

gly

Vai

Asp 115

trp

gly

gin

Gly 120

Thr

Ser

Vai

Thr

Vai

125

Ser

Ser

Ser

Thr

130

Lys

gly

Pro

Ser

Vai

135

Phe

Pro

Leu

Pro

140

Ser

Ser

Lys

Ser

Thr

145

Ser

gly

gly

Thr

Leu

150

gly

Cys

Leu

155

Vai

Lys

asp

Tyr

Phe

160

Pro

Pro

Vai

Thr

165

Vai

Ser

trp

Asn

Ser

170

gly

Leu

Thr

Ser

175

gly

Vai

His

Thr

Phe

180

Pro

Vai

Leu

gin

185

Ser

Ser

gly

Leu

Tyr 190

Ser

Leu

Ser

Ser

Vai

195

Vai

Thr

Vai

Pro

Ser

200

Ser

Ser

Leu

gly

Thr

205

gin

Thr

Tyr

Cys 210

Asn

Vai

Asn

His

Lys 215

Pro

Ser

Asn

Thr

Lys

220

Vai

asp

Lys

Lys

Vai

225

Pro

Lys

Ser

Cys 230

asp

Lys

Thr

His

Thr

235

Cys

Pro

Pro

Cys

Pro

240

Pro

245

gly

gly

Pro

Ser

Vai

250

Phe

Leu

Phe

Pro

Pro

255

Lys

Pro

Lys

asp

Thr

260

Leu

Met

Ser

Arg 265

Thr

Pro

Vai

Thr

270

Cys

Vai

Vai

Vai

Asp 275

Vai

Ser

His

Glu

Asp 280

Pro

Glu

Vai

Lys

Phe

285

Asn

trp

Tyr

Vai

Asp 290

gly

Vai

Glu

Vai

His

295

Asn

Lys

Thr

Lys

300

Pro

Arg

Glu

Glu

gin

305

Tyr

Ser

Thr

Tyr 310

Arg

Vai

Vai

Ser

Vai

315

Leu

Thr

Vai

Leu

His

320

gin

asp

trp

Leu

Asn

325

gly

Lys

Tyr

Lys 330

Cys

Lys

Vai

Ser

Asn

335

Lys

- 93 042507

Ala Leu Pro Ala Pro lie Glu Lys

340

Thr lie Ser Lys Ala Lys Gly Gin 345 350

Pro Arg Glu Pro Gin Vai Tyr Thr

355 360

Leu Pro Pro Ser Arg Cys Glu Leu 365

Thr Lys Asn Gin Vai Ser Leu Ser

370 375

Cys Ala Vai Lys Gly Phe Tyr Pro

380

Ser Asp lie Ala Vai Glu Trp Glu 385 390

Ser Asn Gly Gin Pro Glu Asn Asn

395 400

Tyr Lys Thr Thr Pro Pro Vai Leu 405

Asp Ser Asp Gly Ser Phe Phe Leu

410 415

Vai Ser Lys Leu Thr Vai Asp Lys 420

Ser Arg Trp Gin Gin Gly Asn Vai 425 430

Phe Ser Cys Ser Vai Met His Glu

435 440

Ala Leu His Asn His Tyr Thr Gin 445

Lys Ser Leu Ser Leu Ser Pro Asp

450 455

Tyr Lys Asp Asp Asp Asp Lys

460 <210> 30 <211> 465 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>30

Glu Vai Gin Leu Vai Glu Thr Gly Gly Ser Leu Vai Gin Pro Gly Lys 15 1015

Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 2530

Trp Met His Trp Vai Arg Gin Ser Pro Glu Lys Gin Leu Glu Trp Vai 35 4045

Ala Gin lie Lys Ala Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Glu 50 5560

Ser Vai Lys Gly Arg Phe Thr lie Ser Arg Asp Asp Ser Lys Ser Asn

70 7580 lie Tyr Leu Gin Met Asn Ser Leu Lys Glu Glu Asp Thr Ala Vai Tyr 85 9095

Tyr Cys Arg Tyr Vai His Tyr Gly Gly Cys Arg Gly Asp Cys Leu Ala

100 105110

Tyr Tyr Gly Vai Asp Ala Trp Gly Gin Gly Thr Ser Vai Thr Vai Ser

115 120125

- 94 042507

Ser Ala Ser Thr Lys Gly Pro Ser Vai Phe Pro Leu Ala Pro Ser Ser

130 135140

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Vai Lys Asp

145 150 155160

Tyr Phe Pro Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu Thr

165 170175

Ser Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly Leu Tyr

180 185190

Ser Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr Gin

195 200205

Thr Tyr lie Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp

210 215220

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

225 230 235240

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

245 250255

Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr

260 265270

Cys Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn

275 280285

Trp Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg

290 295300

Glu Glu Gin Tyr Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai

305 310 315320

Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser

325 330335

Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys

340 345350

Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Pro Ser Arg Cys

355 360365

Glu Leu Thr Lys Asn Gin Vai Ser Leu Ser Cys Ala Vai Lys Gly Phe

370 375380

Tyr Pro Ser Asp lie Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu

385 390 395400

Asn Asn Tyr Lys Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly Ser Phe

405 410415

Phe Leu Vai Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly

420 425430

Asn Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr

- 95 042507

435 440445

Thr Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp

450 455460

Lys 465 <210>31 <211>465 <212> PROTEIN <213> Artificial sequence <220>

< 223> Artificially synthesized peptide sequence < 400> 31

Glu Vai Gin Leu Vai Glu Thr Gly 1 5

Gly Ser Leu Vai Gin Pro Gly Lys

15

Ser Leu Lys Leu Thr Cys Ala Thr 20

Ser Gly Phe Thr Phe Ser Asn Ala 25 30

Trp Met His Trp Vai Arg Gin Ser

40

Pro Glu Lys Gin Leu Glu Trp Vai 45

Ala Gin lie Lys Ala Lys Ser Asn

55

Asn Tyr Ala Thr Tyr Tyr Ala Glu 60

Ser Vai Lys Gly Arg Phe Thr lie

70

Ser Arg Asp Asp Ser Lys Ser Asn

80 lie Tyr Leu Gin Met Asn Ser Leu 85

Lys Glu Glu Asp Thr Ala Vai Tyr

95

Tyr Cys Arg Tyr Vai His Tyr Gly

100

Ala Gly Cys Arg Gly Asp Cys Leu 105 110

Tyr Tyr Gly Vai Asp Ala Trp Gly

115 120

Gin Gly Thr Ser Vai Thr Vai Ser

125

Ser Ala Ser Thr Lys Gly Pro Ser

130 135

Vai Phe Pro Leu Ala Pro Ser Ser

140

Lys Ser Thr Ser Gly Gly Thr Ala

145 150

Ala Leu Gly Cys Leu Vai Lys Asp

155 160

Tyr Phe Pro Glu Pro Vai Thr Vai

165

Ser Trp Asn Ser Gly Ala Leu Thr

170 175

Ser Gly Vai His Thr Phe Pro Ala

180

Vai Leu Gin Ser Ser Gly Leu Tyr 185 190

Ser Leu Ser Ser Vai Vai Thr Vai

195 200

Pro Ser Ser Ser Leu Gly Thr Gin

205

Thr Tyr lie Cys Asn Vai Asn His

210 215

Lys Pro Ser Asn Thr Lys Vai Asp

220

- 96 042507

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

225 230 235240

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

245 250255

Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr

260 265270

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

275 280285

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

290 295300

Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

305 310 315320

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

325 330335

Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys

340 345350

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys

355 360365

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

370 375380

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

385 390 395400

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

405 410415

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

420 425430

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

435 440445

Thr Gln Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp

450 455460

Lys 465 <210>32 <211>465 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 32

Glu Val Gln Leu Val Glu Thr Gly Gly Ser Leu Val Gln Pro Gly Lys

- 97 042507

10 15

Ser

Leu

Lys

Leu

Thr

Cys

Thr

Ser

gly

Phe

Thr

Phe

Ser

Asn

trp

Met

His 35

trp

Val

Arg

Gln

Ser 40

Pro

Lys

Gln

Leu 45

trp

Val

Glin 50

Lys

Lys

Ser 55

Asn

Asn

Tyr

Thr

Tyr

Tyr

Ser 65

Val

Lys

gly

Arg

Phe

Thr

Ser

Arg

Asp 75

asp

Ser

Lys

Ser

Asn 80

Tyr

Leu

Gln

Met 85

Asn

Ser

Leu

Lys

asp

Thr

Val 95

Tyr

Tyr

Cys

Arg

Tyr

100

Val

His

Tyr

gly

105

Tyr

gly

Cys

Arg

Gly 110

asp

Cys

Leu

Tyr

Gly 115

Val

asp

trp

Gly 120

Gln

gly

Thr

Ser

Val

125

Thr

Val

Ser

Ser

Ser

Thr

130

Lys

gly

Pro

135

Ser

Val

Phe

Pro

Leu

140

Pro

Ser

Ser

Lys 145

Ser

Thr

Ser

gly

Gly 150

Thr

Leu

Gly 155

Cys

Leu

Val

Lys

Asp 160

Tyr

Phe

Pro

Pro

165

Val

Thr

Val

Ser

Trp 170

Asn

Ser

gly

Leu

175

Thr

Ser

gly

Val

His

180

Thr

Phe

Pro

Val

185

Leu

Gln

Ser

Ser

Gly 190

Leu

Tyr

Ser

Leu

Ser

195

Ser

Val

Val

Thr

Val

200

Pro

Ser

Ser

Ser

Leu

205

gly

Thr

Gln

Thr

Tyr 210

Cys

Asn

Val

Asn

215

His

Lys

Pro

Ser

Asn

220

Thr

Lys

Val

asp

Lys

225

Lys

Val

Pro

Lys

230

Ser

Cys

asp

Lys

Thr

235

His

Thr

Cys

Pro

Pro

240

Cys

Pro

Pro

245

gly

gly

Pro

250

Ser

Val

Phe

Leu

Phe

255

Pro

Pro

Lys

Pro

Lys

260

asp

Thr

Leu

Met lie

265

Ser

Arg

Thr

Pro

Glu

270

Val

Thr

Cys

Val

Val

275

Val

asp

Val

Ser

His

280

Glu

asp

Pro

Glu

Val

285

Lys

Phe

Asn

trp

Tyr 290

Val

asp

gly

Val

Glu

295

Val

His

Asn

Lys

300

Thr

Lys

Pro

Arg

Gln

305

Tyr

Ser

310

Thr

Tyr

Arg

Val

Val

315

Ser

Val

Leu

Thr

Val

320

- 98 042507

Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser

325 330335

Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys

340 345350

Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Pro Ser Arg Cys

355 360365

Glu Leu Thr Lys Asn Gin Vai Ser Leu Ser Cys Ala Vai Lys Gly Phe

370 375380

Tyr Pro Ser Asp lie Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu

385 390 395400

Asn Asn Tyr Lys Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly Ser Phe

405 410415

Phe Leu Vai Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly

420 425430

Asn Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr

435 440445

Thr Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp

450 455460

Lys 465 <210>33 <2H>444 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 33

Gin Asp Gin Leu Vai Gin Ser Gly 1 5

Ala Glu Vai Lys Lys Pro Gly Ala

15

Ser Vai Lys Vai Ser Cys Lys Ala 20

Ser Gly Phe Asn He Lys Asp Thr 25 30

Tyr lie Gin Trp Vai Arg Gin Ala

40

Pro Gly Gin Gly Leu Glu Trp Met 45

Gly Arg lie Asp Pro Leu Arg Lys 50 55

Gin Thr Lys Tyr Arg Glu Lys Phe 60

Glu Gly Arg Vai Thr He Thr Ala 65 70

Asp Thr Ser Thr Asn Thr Ala Tyr

80

Met Glu Leu Ser Ser Leu Arg Ser 85

Glu Asp Thr Ala Vai Tyr Tyr Cys

95

- 99 042507

Val Arg Ser Gly Arg Glu Phe Asp Tyr Trp Gly Gin Gly Thr Leu Vai

100 105110

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

115 120125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser

165 170175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185190

Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200205

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

210 215220

Cys Pro Pro Cys Pro Ala Pro Glu Tyr Gin Trp Gly Pro Met Val Phe

225 230 235240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro

245 250255

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

260 265270

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

275 280285

Lys Pro Arg Glu Glu Gin Tyr Asn Ala Thr Tyr Arg Val Val Ser Val

290 295300

Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser

325 330335

Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Cys Thr Leu Pro Pro

340 345350

Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Trp Cys Leu Val

355 360365

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

370 375380

Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Val Leu Asp Ser Asp

385 390 395400

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

- 100 042507

405 410415

Gin Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His

420 425430

Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro

435440 <210>34 <211>444 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 34

Gin Asp Gin Leu Vai Gin Ser Gly 1 5

Ala Glu Vai Lys Lys Pro Gly Ala

15

Ser Vai Lys Vai Ser Cys Lys Ala 20

Ser Gly Phe Asn lie Lys Asp Thr 25 30

Tyr He Gin Trp Vai Arg Gin Ala

40

Pro Gly Gin Gly Leu Glu Trp Met 45

Gly Arg He Asp Pro Leu Arg Lys

55

Gin Thr Lys Tyr Arg Glu Lys Phe 60

Glu Gly Arg Vai Thr He Thr Ala 65 70

Asp Thr Ser Thr Asn Thr Ala Tyr

80

Met Glu Leu Ser Ser Leu Arg Ser 85

Glu Asp Thr Ala Vai Tyr Tyr Cys

95

Vai Arg Ser Gly Arg Glu Phe Asp

100

Tyr Trp Gly Gin Gly Thr Leu Vai 105 110

Thr Vai Ser Ser Ala Ser Thr Lys

115 120

Gly Pro Ser Vai Phe Pro Leu Ala

125

Pro Ser Ser Lys Ser Thr Ser Gly

130 135

Gly Thr Ala Ala Leu Gly Cys Leu 140

Vai Lys Asp Tyr Phe Pro Glu Pro

145 150

Vai Thr Vai Ser Trp Asn Ser Gly

155 160

Ala Leu Thr Ser Gly Vai His Thr

165

Phe Pro Ala Vai Leu Gin Ser Ser

170 175

Gly Leu Tyr Ser Leu Ser Ser Vai 180

Vai Thr Vai Pro Ser Ser Ser Leu

185 190

Gly Thr Gin Thr Tyr He Cys Asn

195 200

Vai Asn His Lys Pro Ser Asn Thr

205

Lys Vai Asp Lys Lys Vai Glu Pro

210 215

Lys Ser Cys Asp Lys Thr His Thr

220

- 101 042507

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

225 230 235240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro

245 250255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Glu Pro Glu Val

260 265270

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

275 280285

Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295300

Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315320

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

325 330335

Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro

340 345350

Ser Arg Cys Glu Leu Thr Lys Asn Gin Val Ser Leu Ser Cys Ala Val

355 360365

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

370 375380

Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Val Leu Asp Ser Asp

385 390 395400

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

405 410415

Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425430

Asn His

Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 435440 <210>35 <211>213 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 35

Asp lie Gin Met Thr Gin Ser Pro 1 5

Ser Ser Leu Ser Ala Ser Val Gly

15

Asp Arg Val Thr lie Thr Cys Arg 20

Ala Ser Gin Asp lie His Lys Tyr 25 30 lie Ala Trp Tyr Gin Gin Lys Pro

Gly Gin Ala Pro Arg Leu Leu lie

- 102 042507

4045

Gin Tyr Thr Ser Thr Leu Gin Pro Gly Val Pro Ser Arg Phe Ser Gly 50 5560

Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr lie Ser Ser Leu Gin Pro

70 7580

Glu Asp lie Ala Thr Tyr Tyr Cys Leu Gin Tyr Glu Gin Leu Arg Thr 85 9095

Phe Gly Gly Gly Thr Lys Val Glu lie Lys Arg Thr Val Ala Ala Pro

100 105110

Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr

115 120125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135140

Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu

145 150 155160

Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170175

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

180 185190

Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200205

Asn Arg Gly Glu Cys 210 <210> 36 <211>22 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>36

Arg Trp Gly Tyr His Leu Arg Asp Arg Lys Tyr Lys Gly Val Arg Ser 15 1015

His Lys Gly Val Pro Arg < 210> 37 <211>480 < 212> PROTEIN < 213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 37

- 103 042507

Glu Vai Gin Leu Vai Glu Thr Gly Gly Ser Leu Vai Gin Pro Gly Lys 15 1015

Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 2530

Trp Met His Trp Vai Arg Gin Ser Pro Glu Lys Gin Leu Glu Trp Vai 35 4045

Ala Gin He Lys Ala Lys Arg Trp Gly Tyr His Leu Arg Asp Arg Lys 50 5560

Tyr Lys Gly Vai Arg Ser His Lys Gly Vai Pro Arg Ser Asn Asn Tyr

70 7580

Ala Thr Tyr Tyr Ala Glu Ser Vai Lys Gly Arg Phe Thr He Ser Arg 85 9095

Asp Asp Ser Lys Ser Asn lie Tyr Leu Gin Met Asn Ser Leu Lys Glu

100 105110

Glu Asp Thr Ala Vai Tyr Tyr Cys Arg Tyr Vai His Tyr Gly Ala Tyr

115 120125

Tyr Gly Vai Asp Ala Trp Gly Gin Gly Thr Ser Vai Thr Vai Ser Ser

130 135140

Ala Ser Thr Lys Gly Pro Ser Vai Phe Pro Leu Ala Pro Ser Ser Lys

145 150 155160

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Vai Lys Asp Tyr

165 170175

Phe Pro Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu Thr Ser

180 185190

Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser

195 200205

Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr

210 215220

Tyr lie Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp Lys

225 230 235240

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

245 250255

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Vai Phe Leu Phe Pro Pro

260 265270

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys

275 280285

Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn Trp

290 295300

Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu

- 104 042507

305

310

315

320

Glu Gin Tug Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu

325 330335

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn

340 345350

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly

355 360365

Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Ser Arg Cys Glu

370 375380

Leu Thr Lys Asn Gin Vai Ser Leu Ser Cys Ala Vai Lys Gly Phe Tyr

385 390 395400

Pro Ser Asp lie Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn

405 410415

Asn Tyr Lys Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly Ser Phe Phe

420 425430

Leu Vai Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly Asn

435 440445

Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr Thr

450 455460

Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

465 470 475480 <210>38 <211>482 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 38

Glu Vai Gin Leu Vai Glu Thr Gly 1 5

Gly Ser Leu Vai Gin Pro Gly Lys

15

Ser Leu Lys Leu Thr Cys Ala Thr 20

Ser Gly Phe Thr Phe Ser Asn Ala 25 30

Trp Met His Trp Vai Arg Gin Ser

40

Pro Glu Lys Gin Leu Glu Trp Vai 45

Ala Gin lie Lys Ala Lys Cys Arg

55

Trp Gly Tyr His Leu Arg Asp Arg 60

Lys Tyr Lys Gly Vai Arg Ser His 65 70

Lys Gly Vai Pro Arg Cys Ser Asn

80

Asn Tyr Ala Thr Tyr Tyr Ala Glu 85

Ser Vai Lys Gly Arg Phe Thr lie

95

- 105 042507

Ser Arg Asp Asp Ser Lys Ser Asn lie Tyr Leu Gin Met Asn Ser Leu

100 105110

Lys Glu Glu Asp Thr Ala Vai Tyr Tyr Cys Arg Tyr Vai His Tyr Gly

115 120125

Ala Tyr Tyr Gly Vai Asp Ala Trp Gly Gin Gly Thr Ser Vai Thr Vai

130 135140

Ser Ser Ala Ser Thr Lys Gly Pro Ser Vai Phe Pro Leu Ala Pro Ser

145 150 155160

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Vai Lys

165 170175

Asp Tyr Phe Pro Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu

180 185190

Thr Ser Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly Leu

195 200205

Tyr Ser Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr

210 215220

Gin Thr Tyr lie Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai

225 230 235240

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

245 250255

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

260 265270

Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai

275 280285

Thr Cys Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe

290 295300

Asn Trp Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro

305 310 315320

Arg Glu Glu Gin Tyr Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr

325 330335

Vai Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai

340 345350

Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala

355 360365

Lys Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Pro Ser Arg

370 375380

Cys Glu Leu Thr Lys Asn Gin Vai Ser Leu Ser Cys Ala Vai Lys Gly

385 390 395400

- 106 042507

Phe Tyr Pro Ser Asp lie Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro

405 410415

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly Ser

420 425430

Phe Phe Leu Vai Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin

435 440445

Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn His

450 455460

Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp

465 470 475480

Asp Lys <210>39 <211>480 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 39

Glu Vai Gin Leu Vai Glu Thr Gly 1 5

Gly Ser Leu Vai Gin Pro Gly Lys

15

Ser Leu Lys Leu Thr Cys Ala Thr 20

Ser Gly Phe Thr Phe Ser Asn Ala 25 30

Trp Met His Trp Vai Arg Gin Ser

40

Pro Glu Lys Gin Leu Glu Trp Vai 45

Ala Gin lie Lys Ala Lys Ser Asn

55

Asn Tyr Ala Thr Tyr Tyr Ala Glu 60

Ser Vai Lys Gly Arg Phe Thr lie 65 70

Ser Arg Asp Arg Trp Gly Tyr His

80

Leu Arg Asp Arg Lys Tyr Lys Gly 85

Vai Arg Ser His Lys Gly Vai Pro

95

Arg Asp Ser Lys Ser Asn lie Tyr

100

Leu Gin Met Asn Ser Leu Lys Glu 105 110

Glu Asp Thr Ala Vai Tyr Tyr Cys

115 120

Arg Tyr Vai His Tyr Gly Ala Tyr

125

Tyr Gly Vai Asp Ala Trp Gly Gin

130 135

Gly Thr Ser Vai Thr Vai Ser Ser 140

Ala Ser Thr Lys Gly Pro Ser Vai 145 150

Phe Pro Leu Ala Pro Ser Ser Lys

155 160

Ser Thr Ser Gly Gly Thr Ala Ala

165

Leu Gly Cys Leu Vai Lys Asp Tyr

170 175

- 107 042507

Phe Pro Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu Thr Ser

180 185190

Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser

195 200205

Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr

210 215220

Tyr lie Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp Lys

225 230 235240

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

245 250255

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Vai Phe Leu Phe Pro Pro

260 265270

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys

275 280285

Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn Trp

290 295300

Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu

305 310 315320

Glu Gin Tyr Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu

325 330335

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn

340 345350

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly

355 360365

Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Ser Arg Cys Glu

370 375380

Leu Thr Lys Asn Gin Vai Ser Leu Ser Cys Ala Vai Lys Gly Phe Tyr

385 390 395400

Pro Ser Asp lie Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn

405 410415

Asn Tyr Lys Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly Ser Phe Phe

420 425430

Leu Vai Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly Asn

435 440445

Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr Thr

450 455460

Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

465 470 475480

- 108 042507 <210> 40 <211> 482 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 40

Glu Val Gln Leu Val Glu Thr Gly 1 5

Gly Ser Leu Val Gln Pro Gly Lys

15

Ser Leu Lys Leu Thr Cys Ala Thr 20

Ser Gly Phe Thr Phe Ser Asn Ala 25 30

Trp Met His Trp Val Arg Gln Ser

40

Pro Glu Lys Gln Leu Glu Trp Val 45

Ala Gln lie Lys Ala Lys Ser Asn

55

Asn Tyr Ala Thr Tyr Tyr Ala Glu 60

Ser Val Lys Gly Arg Phe Thr lie

70

Ser Arg Asp Cys Arg Trp Gly Tyr

80

His Leu Arg Asp Arg Lys Tyr Lys 85

Gly Val Arg Ser His Lys Gly Val

95

Pro Arg Cys Asp Ser Lys Ser Asn

100 lie Tyr Leu Gln Met Asn Ser Leu

105 110

Lys Glu Glu Asp Thr Ala Val Tyr

115 120

Tyr Cys Arg Tyr Val His Tyr Gly

125

Ala Tyr Tyr Gly Val Asp Ala Trp

130 135

Gly Gln Gly Thr Ser Val Thr Val 140

Ser Ser Ala Ser Thr Lys Gly Pro 145 150

Ser Val Phe Pro Leu Ala Pro Ser

155 160

Ser Lys Ser Thr Ser Gly Gly Thr

165

Ala Ala Leu Gly Cys Leu Val Lys

170 175

Asp Tyr Phe Pro Glu Pro Val Thr

180

Val Ser Trp Asn Ser Gly Ala Leu

185 190

Thr Ser Gly Val His Thr Phe Pro

195 200

Ala Val Leu Gln Ser Ser Gly Leu 205

Tyr Ser Leu Ser Ser Val Val Thr

210 215

Val Pro Ser Ser Ser Leu Gly Thr

220

Gln Thr Tyr lie Cys Asn Val Asn 225 230

His Lys Pro Ser Asn Thr Lys Val

235 240

Asp Lys Lys Val Glu Pro Lys Ser 245

Cys Asp Lys Thr His Thr Cys Pro

250 255

Pro Cys Pro Ala Pro Glu Ala Ala

Gly Gly Pro Ser Val Phe Leu Phe

- 109 042507

260 265270

Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val

275 280285

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

290 295300

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

305 310 315320

Arg Glu Glu Gin Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr

325 330335

Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

340 345350

Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala

355 360365

Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg

370 375380

Cys Glu Leu Thr Lys Asn Gin Val Ser Leu Ser Cys Ala Val Lys Gly

385 390 395400

Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro

405 410415

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

420 425430

Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin

435 440445

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

450 455460

Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp

465 470 475480

Asp Lys <210>41 <211>477 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 41

Glu Val Gin Leu Val Glu Thr Gly Gly Ser Leu Val Gin Pro Gly Lys 15 10 15

Ser Leu Lys Leu Thr Cys Ala Thr Ser

25

Gly Phe Thr Phe Ser Asn Ala 30

- 110 042507

Trp Met His Trp Vai Arg Gin Ser Pro Glu Lys Gin Leu Glu Trp Vai 35 4045

Ala Gin He Lys Ala Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Glu 50 5560

Ser Vai Lys Gly Arg Phe Thr lie Ser Arg Asp Asp Ser Lys Ser Asn

70 7580 lie Tyr Leu Gin Met Asn Ser Leu Lys Glu Glu Asp Thr Ala Vai Tyr 85 9095

Tyr Cys Arg Tyr Vai His Tyr Gly Ala Tyr Tyr Gly Vai Asp Ala Trp

100 105110

Gly Gin Gly Thr Ser Vai Thr Vai Ser Ser Ala Ser Thr Lys Gly Pro

115 120125

Ser Vai Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

130 135140

Ala Ala Leu Gly Cys Leu Vai Lys Asp Tyr Phe Pro Glu Pro Vai Thr

145 150 155160

Vai Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Vai His Thr Phe Pro

165 170175

Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Vai Vai Thr

180 185190

Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Vai Asn

195 200205

His Lys Pro Ser Asn Thr Lys Vai Asp Lys Lys Vai Glu Pro Lys Ser

210 215220

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

225 230 235240

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

245 250255

Met lie Ser Arg Thr Pro Glu Vai Thr Cys Vai Vai Vai Asp Vai Ser

260 265270

His Glu Glu Pro Glu Vai Lys Phe Asn Trp Tyr Vai Asp Gly Vai Glu

275 280285

Vai His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr

290 295300

Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu His Gin Asp Trp Leu Asn

305 310 315320

Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn Asp Ala Leu Pro Met Pro

325 330335

- 111 042507

He Glu Glu Thr lie Ser Lys Ala

340

Lys Gly Gin Pro Arg Glu Pro Gin

345 350

Vai Tyr Thr Leu Pro Pro Ser Arg

355 360

Cys Glu Leu Thr Lys Asn Gin Vai 365

Ser Leu Ser Cys Ala Vai Lys Gly

370 375

Phe Tyr Pro Ser Asp lie Ala Vai

380

Glu Trp Glu Ser Asn Gly Gin Pro 385 390

Glu Asn Asn Tyr Lys Thr Thr Pro

395 400

Pro Vai Leu Asp Ser Asp Gly Ser

405

Phe Phe Leu Vai Ser Lys Leu Thr

410 415

Vai Asp Lys Ser Arg Trp Gin Gin 420

Gly Asn Vai Phe Ser Cys Ser Vai 425 430

Met His Glu Ala Leu His Asn His

435 440

Tyr Thr Gin Lys Ser Leu Ser Leu 445

Ser Pro Gly Gly Gly Gly Ser Arg

450 455

Trp Gly Tyr His Leu Arg Asp Arg

460

Lys Tyr Lys Gly Vai Arg Ser His

465470

Lys Gly Vai Pro Arg 475 <210>42 <2H>479 <212> PROTEIN <213> Artificial sequence <220>

<223>

Artificially synthesized peptide sequence <400>

Vai

gin

Leu

Wai 5

Thr

gly

gly

Ser 10

Leu

Vai

gin

Pro

Gly 15

Lys

Ser

Leu

Lys

Leu

Cys

Thr

Ser

gly

Phe

Thr

Phe

Ser

Asn

trp

Met

His

trp

Vai

Arg

gin

Ser 40

Pro

Lys

gin

Leu 45

trp

Vai

Gin 50

Lys

Lys

Ser 55

Asn

Asn

Tyr

Thr

Tyr

Tyr

Ser 65

Vai

Lys

gly

Arg

Phe

Thr

Ser

Arg

Asp 75

asp

Ser

Lys

Asn 80

Tyr

Tyr

Cys

Leu

Arg

gin

Met 85

Asn

Ser

Leu

Lys

asp

Thr

Wai 95

Tyr

Tyr

100

Vai

His

Tyr

gly

105

Tyr

Tyr

gly

Vai

AspHO

trp

gly

gin

Gly 115

Thr

Ser

Vai

Thr

Vai

120

Ser

Ser

Ser

Thr

125

Lys

gly

Pro

- 112 042507

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

130 135140

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

145 150 155160

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

165 170175

Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

180 185190

Val Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys Asn Val Asn

195 200205

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

210 215220

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

225 230 235240

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

245 250255

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

260 265270

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

275 280285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr

290 295300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn

305 310 315320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Asp Ala Leu Pro Met Pro

325 330335 lie Glu Glu Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin

340 345350

Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gin Val

355 360365

Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val

370 375380

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

385 390 395400

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

405 410415

Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val

420 425430

- 113 042507

Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu

435 440445

Ser Pro Gly Gly Gly Gly Ser Cys Arg Trp Gly Tyr His Leu Arg Asp

450 455460

Arg Lys Tyr Lys Gly Vai Arg Ser His Lys Gly Vai Pro Arg Cys 465 470475 <210>43 <211>461 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 43

Glu Vai Gin Leu Vai Glu Thr Gly 1 5

Gly Ser Leu Vai Gin Pro Gly Lys

15

Ser Leu Lys Leu Thr Cys Ala Thr 20

Ser Gly Phe Thr Phe Ser Asn Ala 25 30

Trp Met His Trp Vai Arg Gin Ser

40

Pro Glu Lys Gin Leu Glu Trp Vai 45

Ala Gin lie Lys Ala Lys Gly Gly

55

Ser Ser Asn Asn Tyr Ala Thr Tyr 60

Tyr Ala Glu Ser Vai Lys Gly Arg

70

Phe Thr lie Ser Arg Asp Asp Ser

80

Lys Ser Asn lie Tyr Leu Gin Met 85

Asn Ser Leu Lys Glu Glu Asp Thr

95

Ala Vai Tyr Tyr Cys Arg Tyr Vai

100

His Tyr Gly Ala Tyr Tyr Gly Vai

105 110

Asp Ala Trp Gly Gin Gly Thr Ser

115 120

Vai Thr Vai Ser Ser Ala Ser Thr

125

Lys Gly Pro Ser Vai Phe Pro Leu

130 135

Ala Pro Ser Ser Lys Ser Thr Ser 140

Gly Gly Thr Ala Ala Leu Gly Cys 145 150

Leu Vai Lys Asp Tyr Phe Pro Glu

155 160

Pro Vai Thr Vai Ser Trp Asn Ser

165

Gly Ala Leu Thr Ser Gly Vai His

170 175

Thr Phe Pro Ala Vai Leu Gin Ser

180

Ser Gly Leu Tyr Ser Leu Ser Ser 185 190

Vai Vai Thr Vai Pro Ser Ser Ser

195 200

Leu Gly Thr Gin Thr Tyr lie Cys 205

Asn Vai Asn His Lys Pro Ser Asn

Thr Lys Vai Asp Lys Lys Vai Glu

- 114 042507

210

215

220

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

225 230 235240

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

245 250255

Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys Vai Vai Vai

260 265270

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

275 280285

Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr

290 295300

Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu His Gin Asp

305 310 315320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn Lys Ala Leu

325 330335

Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg

340 345350

Glu Pro Gin Vai Tyr Thr Leu Pro Ser Arg Cys Glu Leu Thr Lys

355 360365

Asn Gin Vai Ser Leu Ser Cys Ala Vai Lys Gly Phe Tyr Pro Ser Asp

370 375380 lie Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys

385 390 395400

Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly Ser Phe Phe Leu Vai Ser

405 410415

Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly Asn Vai Phe Ser

420 425430

Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser

435 440445

Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

450 455460 <210>44 <211>464 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 44

Glu Vai Gin Leu Vai Glu Thr Gly Gly Ser Leu Vai Gin Pro Gly Lys 15 10 15

- 115 042507

Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 2530

Trp Met His Trp Vai Arg Gin Ser Pro Glu Lys Gin Leu Glu Trp Vai 35 4045

Ala Gin He Lys Ala Lys Gly Gly Ser Gly Gly Ser Ser Asn Asn Tyr 50 5560

Ala Thr Tyr Tyr Ala Glu Ser Vai Lys Gly Arg Phe Thr He Ser Arg 65 70 7580

Asp Asp Ser Lys Ser Asn lie Tyr Leu Gin Met Asn Ser Leu Lys Glu 85 9095

Glu Asp Thr Ala Vai Tyr Tyr Cys Arg Tyr Vai His Tyr Gly Ala Tyr

100 105110

Tyr Gly Vai Asp Ala Trp Gly Gin Gly Thr Ser Vai Thr Vai Ser Ser

115 120125

Ala Ser Thr Lys Gly Pro Ser Vai Phe Pro Leu Ala Pro Ser Ser Lys

130 135140

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Vai Lys Asp Tyr

145 150 155160

Phe Pro Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170175

Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser

180 185190

Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr

195 200205

Tyr lie Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp Lys

210 215220

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

225 230 235240

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Vai Phe Leu Phe Pro Pro

245 250255

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys

260 265270

Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn Trp

275 280285

Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu

290 295300

Glu Gin Tyr Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu

305 310 315320

- 116 042507

His Gin Asp Trp Leu Asn Gly Lys 325

Glu Tyr Lys Cys Lys Vai Ser Asn

330 335

Lys Ala Leu Pro Ala Pro lie Glu

340

Lys Thr lie Ser Lys Ala Lys Gly

345 350

Gin Pro Arg Glu Pro Gin Vai Tyr

355 360

Thr Leu Pro Pro Ser Arg Cys Glu 365

Leu Thr Lys Asn Gin Vai Ser Leu

370 375

Ser Cys Ala Vai Lys Gly Phe Tyr 380

Pro Ser Asp lie Ala Vai Glu Trp

385 390

Glu Ser Asn Gly Gin Pro Glu Asn

395 400

Asn Tyr Lys Thr Thr Pro Pro Vai 405

Leu Asp Ser Asp Gly Ser Phe Phe

410 415

Leu Vai Ser Lys Leu Thr Vai Asp 420

Lys Ser Arg Trp Gin Gin Gly Asn

425 430

Vai Phe Ser Cys Ser Vai Met His

435 440

Glu Ala Leu His Asn His Tyr Thr 445

Gin Lys Ser Leu Ser Leu Ser Pro

450 455

Asp Tyr Lys Asp Asp Asp Asp Lys 460 <210> 45 <211> 467 <212> PROTEIN <213> Artificial sequence <220>

<223>

Artificially synthesized peptide sequence <400>

Vai

gin

Leu

Wai 5

Thr

gly

gly

Ser 10

Leu

Vai

gin

Pro

Gly 15

Lys

Ser

Leu

Lys

Leu

Thr

Cys

Thr

Ser

gly

Phe

Thr

Phe

Ser

Asn

trp

Met

His

trp

Vai

Arg

gin

Ser 40

Pro

Lys

gin

Leu 45

trp

Vai

Gin 50

Lys

Lys

Gly 55

gly

Ser

gly

gly

Ser

gly

gly

Ser

Ser

Asn 65

Asn

Tyr

Thr

Tyr 70

Tyr

Ser

Vai 75

Lys

gly

Arg

Phe

Thr

Ser

Arg

asp

Asp 85

Ser

Lys

Ser

Asn

Tyr

Leu

gin

Met

Asn 95

Ser

Leu

Lys

100

asp

Thr

Vai

Tyr 105

Tyr

Cys

Arg

Tyr

Vai

110

His

Tyr

gly

Tyr 115

Tyr

gly

Vai

asp

120

trp

gly

gin

gly

Thr

125

Ser

Vai

Thr

- 117 042507

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

130 135140

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

145 150 155160

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

165 170175

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly

180 185190

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

195 200205

Thr Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

210 215220

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

225 230 235240

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

245 250255

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu

260 265270

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

275 280285

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

290 295300

Pro Arg Glu Glu Gin Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu

305 310 315320

Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

325 330335

Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys

340 345350

Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser

355 360365

Arg Cys Glu Leu Thr Lys Asn Gin Val Ser Leu Ser Cys Ala Val Lys

370 375380

Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin

385 390 395400

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

405 410415

Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin

420 425430

- 118 042507

Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn

435 440445

His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp

450 455460

Asp Asp Lys 465 <210>46 <211>461 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 46

Glu Vai Gin Leu Vai Glu Thr Gly 1 5

Gly Ser Leu Vai Gin Pro Gly Lys

15

Ser Leu Lys Leu Thr Cys Ala Thr 20

Ser Gly Phe Thr Phe Ser Asn Ala 25 30

Trp Met His Trp Vai Arg Gin Ser

40

Pro Glu Lys Gin Leu Glu Trp Vai 45

Ala Gin He Lys Ala Lys Ser Asn

55

Asn Tyr Ala Thr Tyr Tyr Ala Glu 60

Ser Vai Lys Gly Arg Phe Thr lie

70

Ser Arg Asp Gly Gly Ser Asp Ser

80

Lys Ser Asn lie Tyr Leu Gin Met 85

Asn Ser Leu Lys Glu Glu Asp Thr

95

Ala Vai Tyr Tyr Cys Arg Tyr Vai

100

His Tyr Gly Ala Tyr Tyr Gly Vai

105 110

Asp Ala Trp Gly Gin Gly Thr Ser

115 120

Vai Thr Vai Ser Ser Ala Ser Thr

125

Lys Gly Pro Ser Vai Phe Pro Leu

130 135

Ala Pro Ser Ser Lys Ser Thr Ser 140

Gly Gly Thr Ala Ala Leu Gly Cys 145 150

Leu Vai Lys Asp Tyr Phe Pro Glu

155 160

Pro Vai Thr Vai Ser Trp Asn Ser

165

Gly Ala Leu Thr Ser Gly Vai His

170 175

Thr Phe Pro Ala Vai Leu Gin Ser

180

Ser Gly Leu Tyr Ser Leu Ser Ser 185 190

Vai Vai Thr Vai Pro Ser Ser Ser

195 200

Leu Gly Thr Gin Thr Tyr He Cys 205

Asn Vai Asn His Lys Pro Ser Asn

Thr Lys Vai Asp Lys Lys Vai Glu

- 119 042507

210

215

220

Pro Lys Ser Cys Asp Lys Thr His 225 230

Thr Cys Pro Pro Cys Pro Ala Pro

235 240

Glu Ala Ala Gly Gly Pro Ser Vai

245

Phe Leu Phe Pro Pro Lys Pro Lys

250 255

Asp Thr Leu Met lie Ser Arg Thr

260

Pro Glu Vai Thr Cys Vai Vai Vai 265 270

Asp Vai Ser His Glu Asp Pro Glu

275 280

Vai Lys Phe Asn Trp Tyr Vai Asp

285

Gly Vai Glu Vai His Asn Ala Lys

290 295

Thr Lys Pro Arg Glu Glu Gin Tyr

300

Ala Ser Thr Tyr Arg Vai Vai Ser 305 310

Vai Leu Thr Vai Leu His Gin Asp

315 320

Trp Leu Asn Gly Lys Glu Tyr Lys 325

Cys Lys Vai Ser Asn Lys Ala Leu

330 335

Pro Ala Pro lie Glu Lys Thr lie 340

Ser Lys Ala Lys Gly Gin Pro Arg 345 350

Glu Pro Gin Vai Tyr Thr Leu Pro

355 360

Pro Ser Arg Cys Glu Leu Thr Lys 365

Asn Gin Vai Ser Leu Ser Cys Ala

370 375

Vai Lys Gly Phe Tyr Pro Ser Asp 380 lie Ala Vai Glu Trp Glu Ser Asn

385 390

Gly Gin Pro Glu Asn Asn Tyr Lys

395 400

Thr Thr Pro Pro Vai Leu Asp Ser 405

Asp Gly Ser Phe Phe Leu Vai Ser

410 415

Lys Leu Thr Vai Asp Lys Ser Arg

420

Trp Gin Gin Gly Asn Vai Phe Ser 425 430

Cys Ser Vai Met His Glu Ala Leu

435 440

His Asn His Tyr Thr Gin Lys Ser 445

Leu Ser Leu Ser Pro Asp Tyr Lys

450 455

Asp Asp Asp Asp Lys

460 <210> 47 <211> 464 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 47

Glu Vai Gin Leu Vai Glu Thr Gly Gly Ser Leu Vai Gin Pro Gly Lys 15 10 15

- 120 042507

Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 2530

Trp Met His Trp Vai Arg Gin Ser Pro Glu Lys Gin Leu Glu Trp Vai 35 4045

Ala Gin lie Lys Ala Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Glu 50 5560

Ser Vai Lys Gly Arg Phe Thr lie Ser Arg Asp Gly Gly Ser Gly Gly

70 7580

Ser Asp Ser Lys Ser Asn lie Tyr Leu Gin Met Asn Ser Leu Lys Glu 85 9095

Glu Asp Thr Ala Vai Tyr Tyr Cys Arg Tyr Vai His Tyr Gly Ala Tyr

100 105110

Tyr Gly Vai Asp Ala Trp Gly Gin Gly Thr Ser Vai Thr Vai Ser Ser

115 120125

Ala Ser Thr Lys Gly Pro Ser Vai Phe Pro Leu Ala Pro Ser Ser Lys

130 135140

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Vai Lys Asp Tyr

145 150 155160

Phe Pro Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170175

Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser

180 185190

Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr

195 200205

Tyr lie Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp Lys

210 215220

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

225 230 235240

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Vai Phe Leu Phe Pro Pro

245 250255

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys

260 265270

Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn Trp

275 280285

Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu

290 295300

Glu Gin Tyr Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu

305 310 315320

- 121 042507

His Gin Asp Trp Leu Asn Gly Lys 325

Glu Tyr Lys Cys Lys Vai Ser Asn

330 335

Lys Ala Leu Pro Ala Pro He Glu

340

Lys Thr lie Ser Lys Ala Lys Gly

345 350

Gin Pro Arg Glu Pro Gin Vai Tyr

355 360

Thr Leu Pro Pro Ser Arg Cys Glu 365

Leu Thr Lys Asn Gin Vai Ser Leu

370 375

Ser Cys Ala Vai Lys Gly Phe Tyr 380

Pro Ser Asp lie Ala Vai Glu Trp

385 390

Glu Ser Asn Gly Gin Pro Glu Asn

395 400

Asn Tyr Lys Thr Thr Pro Pro Vai 405

Leu Asp Ser Asp Gly Ser Phe Phe

410 415

Leu Vai Ser Lys Leu Thr Vai Asp 420

Lys Ser Arg Trp Gin Gin Gly Asn

425 430

Vai Phe Ser Cys Ser Vai Met His

435 440

Glu Ala Leu His Asn His Tyr Thr 445

Gin Lys Ser Leu Ser Leu Ser Pro

450 455

Asp Tyr Lys Asp Asp Asp Asp Lys 460 <210> 48 <2H> 467 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 48

Glu Vai Gin Leu Vai Glu Thr Gly 1 5

Gly Ser Leu Vai Gin Pro Gly Lys

15

Ser Leu Lys Leu Thr Cys Ala Thr 20

Ser Gly Phe Thr Phe Ser Asn Ala 25 30

Trp Met His Trp Vai Arg Gin Ser

40

Pro Glu Lys Gin Leu Glu Trp Vai 45

Ala Gin lie Lys Ala Lys Ser Asn

55

Asn Tyr Ala Thr Tyr Tyr Ala Glu 60

Ser Vai Lys Gly Arg Phe Thr lie 65 70

Ser Arg Asp Gly Gly Ser Gly Gly

80

Ser Gly Gly Ser Asp Ser Lys Ser 85

Asn lie Tyr Leu Gin Met Asn Ser

95

Leu Lys Glu Glu Asp Thr Ala Vai

100

Tyr Tyr Cys Arg Tyr Vai His Tyr 105 110

Gly Ala Tyr Tyr Gly Vai Asp Ala

115 120

Trp Gly Gin Gly Thr Ser Vai Thr

125

- 122 042507

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

130 135140

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

145 150 155160

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

165 170175

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly

180 185190

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

195 200205

Thr Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

210 215220

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

225 230 235240

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

245 250255

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu

260 265270

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

275 280285

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

290 295300

Pro Arg Glu Glu Gin Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu

305 310 315320

Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

325 330335

Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys

340 345350

Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser

355 360365

Arg Cys Glu Leu Thr Lys Asn Gin Val Ser Leu Ser Cys Ala Val Lys

370 375380

Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin

385 390 395400

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

405 410415

Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin

420 425430

- 123 042507

Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn

435 440445

His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp

450 455460

Asp Asp Lys 465 <210>49 <211>461 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>49

Glu Vai Gin Leu Vai Glu Thr Gly Gly Ser Leu Vai Gin Pro Gly Lys 15 1015

Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 2530

Trp Met His Trp Vai Arg Gin Ser Pro Glu Lys Gin Leu Glu Trp Vai 35 4045

Ala Gin lie Lys Ala Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Glu 50 5560

Ser Vai Lys Gly Arg Phe Thr lie Ser Arg Asp Asp Ser Lys Ser Asn

70 7580 lie Tyr Leu Gin Met Asn Ser Leu Lys Glu Glu Asp Thr Ala Vai Tyr 85 9095

Tyr Cys Arg Tyr Vai His Tyr Gly Ala Gly Gly Ser Tyr Tyr Gly Vai

100 105110

Asp Ala Trp Gly Gin Gly Thr Ser Vai Thr Vai Ser Ser Ala Ser Thr

115 120125

Lys Gly Pro Ser Vai Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Vai Lys Asp Tyr Phe Pro Glu

145 150 155160

Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Vai His

165 170175

Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185190

Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr lie Cys

195 200205

Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp Lys Lys Vai Glu

- 124 042507

210

215

220

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

225 230 235240

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

245 250255

Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys Vai Vai Vai

260 265270

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

275 280285

Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr

290 295300

Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu His Gin Asp

305 310 315320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn Lys Ala Leu

325 330335

Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg

340 345350

Glu Pro Gin Vai Tyr Thr Leu Pro Ser Arg Cys Glu Leu Thr Lys

355 360365

Asn Gin Vai Ser Leu Ser Cys Ala Vai Lys Gly Phe Tyr Pro Ser Asp

370 375380 lie Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys

385 390 395400

Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly Ser Phe Phe Leu Vai Ser

405 410415

Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly Asn Vai Phe Ser

420 425430

Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser

435 440445

Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

450 455460 <210>50 <211>464 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 50

Glu Vai Gin Leu Vai Glu Thr Gly Gly Ser Leu Vai Gin Pro Gly Lys 15 10 15

- 125 042507

Ser Leu Lys Leu Thr Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Ala 20 2530

Trp Met His Trp Val Arg Gin Ser Pro Glu Lys Gin Leu Glu Trp Val 35 4045

Ala Gin lie Lys Ala Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Glu 50 5560

Ser Val Lys Gly Arg Phe Thr lie Ser Arg Asp Asp Ser Lys Ser Asn

70 7580 lie Tyr Leu Gin Met Asn Ser Leu Lys Glu Glu Asp Thr Ala Val Tyr 85 9095

Tyr Cys Arg Tyr Val His Tyr Gly Ala Gly Gly Ser Gly Gly Ser Tyr

100 105110

Tyr Gly Val Asp Ala Trp Gly Gin Gly Thr Ser Val Thr Val Ser Ser

115 120125

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

130 135140

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

145 150 155160

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170175

Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser

180 185190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr

195 200205

Tyr lie Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215220

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

225 230 235240

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

245 250255

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys

260 265270

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

275 280285

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

290 295300

Glu Gin Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

305 310 315320

- 126 042507

His Gin Asp Trp Leu Asn Gly Lys 325

Glu Tyr Lys Cys Lys Vai Ser Asn

330 335

Lys Ala Leu Pro Ala Pro He Glu

340

Lys Thr lie Ser Lys Ala Lys Gly

345 350

Gin Pro Arg Glu Pro Gin Vai Tyr

355 360

Thr Leu Pro Pro Ser Arg Cys Glu 365

Leu Thr Lys Asn Gin Vai Ser Leu

370 375

Ser Cys Ala Vai Lys Gly Phe Tyr

380

Pro Ser Asp lie Ala Vai Glu Trp

385 390

Glu Ser Asn Gly Gin Pro Glu Asn

395 400

Asn Tyr Lys Thr Thr Pro Pro Vai 405

Leu Asp Ser Asp Gly Ser Phe Phe

410 415

Leu Vai Ser Lys Leu Thr Vai Asp 420

Lys Ser Arg Trp Gin Gin Gly Asn

425 430

Vai Phe Ser Cys Ser Vai Met His

435 440

Glu Ala Leu His Asn His Tyr Thr 445

Gin Lys Ser Leu Ser Leu Ser Pro

450 455

Asp Tyr Lys Asp Asp Asp Asp Lys 460 <210> 51 <2H> 467 <212> PROTEIN <213> Artificial sequence <220>

<223>

Artificially synthesized peptide sequence <400>

Vai

gin

Leu

Wai 5

Thr

gly

gly

Ser 10

Leu

Vai

gin

Pro

Gly 15

Lys

Ser

Leu

Lys

Leu

Cys

Thr

Ser

gly

Phe

Thr

Phe

Ser

Asn

trp

Met

His

trp

Vai

Arg

gin

Ser 40

Pro

Lys

gin

Leu 45

trp

Vai

Gin 50

Lys

Lys

Ser 55

Asn

Asn

Tyr

Thr

Tyr

Tyr

Ser 65

Vai

Lys

gly

Arg

Phe

Thr

Ser

Arg

Asp 75

asp

Ser

Lys

Asn 80

Tyr

gly

Tyr

Cys

Ser

Leu

Arg

Tyr 115

gin

Tyr

100

Tyr

Met 85

Vai

gly

Asn

His

Vai

Ser

Tyr

asp

Leu

gly

120

Lys

105

trp

gly

gly

gly

gin

asp

Ser

gly

Thr

gly

Thr

125

Wai 95

Tyr

Gly 110

Ser

Vai

gly

Thr

- 127 042507

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Vai Phe Pro Leu Ala Pro

130 135140

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

145 150 155160

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

165 170175

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly

180 185190

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

195 200205

Thr Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

210 215220

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

225 230 235240

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

245 250255

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu

260 265270

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

275 280285

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

290 295300

Pro Arg Glu Glu Gin Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu

305 310 315320

Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

325 330335

Val Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys

340 345350

Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser

355 360365

Arg Cys Glu Leu Thr Lys Asn Gin Val Ser Leu Ser Cys Ala Val Lys

370 375380

Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin

385 390 395400

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

405 410415

Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin

420 425430

- 128 042507

Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn

435 440445

His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp

450 455460

Asp Asp Lys 465 <210>52 <211>122 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 52

Gin Vai Gin Leu Vai Glu Ser Gly 1 5

Gly Gly Vai Vai Gin Pro Gly Arg

15

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ser Gly Phe Thr Phe Ser Asn Ala 25 30

Trp Met His Trp Vai Arg Gin Ala

40

Pro Gly Lys Gly Leu Glu Trp Vai 45

Ala Gin lie Lys Ala Lys Ser Asn

55

Asn Tyr Ala Thr Tyr Tyr Ala Glu 60

Ser Vai Lys Gly Arg Phe Thr lie

70

Ser Arg Asp Asp Ser Lys Asn Ser

80

Leu Tyr Leu Gin Met Asn Ser Leu 85

Lys Thr Glu Asp Thr Ala Vai Tyr

95

Tyr Cys Arg Tyr Vai His Tyr Gly

100

Ala Tyr Tyr Gly Vai Asp Ala Trp 105 110

Gly Gin Gly Thr Thr Vai Thr Vai Ser Ser

115120 <210>53 <211>112 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 53

Asp lie Vai Met Thr Gin Ser Pro Leu Ser Leu Pro Vai Thr Pro Gly 15 1015

Glu Pro Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Vai His Ser 20 2530

Asn Arg Asn Thr Tyr Leu His Trp Tyr Gin Gin Lys Pro Gly Gin Ala 35 4045

- 129 042507

Pro Arg Leu Leu lie Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 5560

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 7580

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gly Gin Gly 85 9095

Thr Gin Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys

100 105110 <210>54 <2H>336 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 54

Ala Ser Thr Lys Gly Pro Ser Val 1 5

Phe Pro Leu Ala Pro Ser Ser Lys

15

Ser Thr Ser Gly Gly Thr Ala Ala 20

Leu Gly Cys Leu Val Lys Asp Tyr 25 30

Phe Pro Glu Pro Val Thr Val Ser

40

Trp Asn Ser Gly Ala Leu Thr Ser 45

Gly Val His Thr Phe Pro Ala Val

55

Leu Gin Ser Ser Gly Leu Tyr Ser 60

Leu Ser Ser Val Val Thr Val Pro

70

Ser Ser Ser Leu Gly Thr Gin Thr

80

Tyr He Cys Asn Val Asn His Lys 85

Pro Ser Asn Thr Lys Val Asp Lys

95

Lys Val Glu Pro Lys Ser Cys Asp

100

Lys Thr His Thr Cys Pro Pro Cys 105 HO

Pro Ala Pro Glu Ala Ala Gly Gly

115 120

Pro Ser Val Phe Leu Phe Pro Pro

125

Lys Pro Lys Asp Thr Leu Met lie

130 135

Ser Arg Thr Pro Glu Val Thr Cys

140

Val Val Val Asp Val Ser His Glu 145 150

Asp Pro Glu Val Lys Phe Asn Trp

155 160

Tyr Val Asp Gly Val Glu Val His 165

Asn Ala Lys Thr Lys Pro Arg Glu

170 175

Glu Gin Tyr Ala Ser Thr Tyr Arg 180

Val Val Ser Val Leu Thr Val Leu

185 190

- 130 042507

His Gin Asp Trp Leu Asn Gly Lys

195 200

Glu Tyr Lys Cys Lys Val Ser Asn 205

Lys Ala Leu Pro Ala Pro lie Glu

210 215

Lys Thr lie Ser Lys Ala Lys Gly

220

Gin Pro Arg Glu Pro Gin Val Tyr 225 230

Thr Leu Pro Pro Ser Arg Cys Glu

235 240

Leu Thr Lys Asn Gin Val Ser Leu

245

Ser Cys Ala Val Lys Gly Phe Tyr

250 255

Pro Ser Asp lie Ala Val Glu Trp

260

Glu Ser Asn Gly Gin Pro Glu Asn

265 270

Asn Tyr Lys Thr Thr Pro Pro Val

275 280

Leu Asp Ser Asp Gly Ser Phe Phe 285

Leu Val Ser Lys Leu Thr Val Asp

290 295

Lys Ser Arg Trp Gin Gin Gly Asn

300

Val Phe Ser Cys Ser Val Met His 305 310

Glu Ala Leu His Asn His Tyr Thr

315 320

Gin Lys Ser Leu Ser Leu Ser Pro 325

Asp Tyr Lys Asp Asp Asp Asp Lys

330 335 <210>55 <211>107 <212> PROTEIN <213> Homo sapiens <400>55

Arg Thr Val Ala Ala Pro Ser Val

Phe lie Phe Pro Pro Ser Asp Glu

15

Gin Leu Lys Ser Gly Thr Ala Ser 20

Val Val Cys Leu Leu Asn Asn Phe 25 30

Tyr Pro Arg Glu Ala Lys Val Gin

40

Trp Lys Val Asp Asn Ala Leu Gin 45

Ser Gly Asn Ser Gin Glu Ser Val 50 55

Thr Glu Gin Asp Ser Lys Asp Ser 60

Thr Tyr Ser Leu Ser Ser Thr Leu 65 70

Thr Leu Ser Lys Ala Asp Tyr Glu

80

Lys His Lys Val Tyr Ala Cys Glu 85

Val Thr His Gin Gly Leu Ser Ser

95

Pro Val Thr Lys Ser Phe Asn Arg

100

Gly Glu Cys 105 <210> 56 <211> 230 <212> PROTEIN <213> Artificial sequence

- 131 042507 <220>

<223>

Artificially synthesized peptide sequence <400> 56

Glu Pro Lys Ser Ser Asp Lys Thr 1 5

Pro Glu Leu Leu Gly Gly Pro Ser 20

His Thr Cys Pro Pro Cys Pro Ala 10 15

Val Phe Leu Phe Pro Pro Lys Pro 25 30

Lys Asp Thr Leu Met lie Ser Arg

40

Thr Pro Glu Val Thr Cys Val Val 45

Val Asp Val Ser His Glu Asp Pro

55

Glu Val Lys Phe Asn Trp Tyr Val 60

Asp Gly Val Glu Val His Asn Ala 65 70

Lys Thr Lys Pro Arg Glu Glu Gln

80

Tyr Asn Ser Thr Tyr Arg Val Val 85

Ser Val Leu Thr Val Leu His Gln

95

Asp Trp Leu Asn Gly Lys Glu Tyr

100

Lys Cys Lys Val Ser Asn Lys Ala 105 110

Leu Pro Ala Pro lie Glu Lys Thr

115 120 lie Ser Lys Ala Lys Gly Gln Pro 125

Arg Glu Pro Gln Val Cys Thr Leu

130 135

Pro Pro Ser Arg Asp Glu Leu Thr

140

Lys Asn Gln Val Ser Leu Trp Cys

145 150

Leu Val Lys Gly Phe Tyr Pro Ser

155 160

Asp lie Ala Val Glu Trp Glu Ser 165

Asn Gly Gln Pro Glu Asn Asn Tyr

170 175

Lys Thr Thr Pro Pro Val Leu Asp

180

Ser Asp Gly Ser Phe Phe Leu Tyr 185 190

Ser Lys Leu Thr Val Asp Lys Ser

195 200

Arg Trp Gln Gln Gly Asn Val Phe

205

Ser Cys Ser Val Met His Glu Ala

210 215

Leu His Asn Arg Tyr Thr Gln Lys

220

Ser Leu Ser Leu Ser Pro

225230 <210>57 <211>449 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 57

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

- 132 042507

Thr

Leu

Ser

Leu

Thr

Cys

Thr

Val

Ser

gly

Tyr

Ser

Thr

Ser

asp

His

Trp 35

Ser

trp

Val

Arg

Glin 40

Pro

Pro

gly

Arg

Gly 45

Leu

trp

Gly 50

Tyr

Ser

Tyr

Ser 55

gly

Thr

Thr

Tyr 60

Asn

Pro

Ser

Leu

Lys 65

Ser

Arg

Val

Thr

Met

Leu

Arg

asp

Thr

Ser

Lys

Asn

Gln

Phe

Ser

Leu

Arg

Leu

Ser

Ser 85

Val

Thr

Asp 90

Thr

Val

Tyr

Tyr 95

Cys

Ser

Pro

Arg

Leu

Leu

130

Ser

Leu

100

Arg

Thr

Thr

Met

105

asp

Tyr

trp

Gly 110

Gln

gly

Val

115

Thr

Val

Ser

Ser

Ser

Thr

Pro

Ser

Ser

Lys 135

120

Lys

gly

Pro

125

Ser

Val

Phe

Ser

Thr

Ser

gly

Gly 140

Thr

Leu

Gly 145

Cys

Leu

Val

Lys

Asp 150

Tyr

Phe

Pro

Pro

155

Val

Thr

Val

Ser

TRP 160

Asn

Ser

gly

Leu

165

Thr

Ser

gly

Val

His

170

Thr

Phe

Pro

Val

175

Leu

Gln

Ser

Ser

Gly 180

Leu

Tyr

Ser

Leu

Ser

185

Ser

Val

Val

Thr

Val

190

Pro

Ser

Ser

Ser

Leu

195

gly

Thr

Gln

Thr

Tyr 200

Cys

Asn

Val

Asn

205

His

Lys

Pro

Ser

Asn

210

Thr

Lys

Val

asp

Lys 215

Lys

Val

Pro

Lys

220

Ser

Cys

asp

Lys

Thr

225

His

Thr

Cys

Pro

Pro

230

Cys

Pro

Pro

Leu

Leu

Ser

Val

Phe

Leu

Phe

245

Pro

Pro

Lys

Pro

Lys

250

235

asp

Thr

Leu

gly

Met

gly

Pro

240

Ser

255

Arg

Thr

Pro

Val

Thr

Pro

Val

275

260

Cys

Val

Val

265

Val

asp

Val

Ser

His

270

asp

Lys

Phe

Asn

trp

Tyr 280

Val

asp

gly

Val

Val

His

Asn

285

Lys

290

Thr

Lys

Pro

Arg

Gln

295

Tyr

Asn

Ser

300

Thr

Tyr

Arg

Val

Val

305

Ser

Val

Leu

Thr

Val

310

Leu

His

Gln

asp

Trp 315

Leu

Asn

gly

Lys

320

- 133 042507

Tyr Lys Cys Lys Vai Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys

325 330335

Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Vai Tyr Thr

340 345350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Vai Ser Leu Thr

355 360365

Cys Leu Vai Lys Gly Phe Tyr Pro Ser Asp lie Ala Vai Glu Trp Glu

370 375380

Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Vai Leu

385 390 395400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Vai Asp Lys

405 410415

Ser Arg Trp Gin Gin Gly Asn Vai Phe Ser Cys Ser Vai Met His Glu

420 425430

Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly

435 440445

Lys <210>58 <2H>214 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 58

Asp 1 He gin Met Thr 5 gin Ser Pro Ser Ser 10 Leu Ser Ala Ser Wai 15 gly asp Arg Vai Thr 20 He Thr Cys Arg Ala 25 Ser gin asp He Ser thirty Ser Tyr Leu Asn Trp 35 Tyr gin gin Lys Pro 40 gly Lys Ala Pro Lys 45 Leu Leu He Tugh Tyr 50 Thr Ser Arg Leu His 55 Ser gly Vai Pro Ser 60 Arg Phe Ser gly Ser 65 gly Ser gly Thr Asp 70 Phe Thr Phe Thr He 75 Ser Ser Leu gin Pro 80 Glu asp He Ala Thr 85 Tyr Tyr Cys gin Gin 90 gly Asn Thr Leu Pro 95 Tyr Thr Phe gly gin 100 gly Thr Lys Vai Glu 105 He Lys Arg Thr Vai HO Ala Ala

- 134 042507

Pro Ser Vai Phe He Phe Pro Ser Asp Glu Gin Leu Lys Ser Gly

115 120125

Thr Ala Ser Vai Vai Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135140

Lys Vai Gin Trp Lys Vai Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin

145 150 155160

Glu Ser Vai Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Vai Tyr

180 185190

Ala Cys Glu Vai Thr His Gin Gly Leu Ser Ser Pro Vai Thr Lys Ser

195 200205

Phe Asn Arg Gly Glu Cys

210 <210>59 <2H>458 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 59

Gin Vai Gin Leu Vai Glu Ser Gly 1 5

Gly Gly Leu Vai Gin Pro Gly Arg

15

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ser Gly Phe Thr Phe Ser Asn Ala 25 30

Trp Met His Trp Vai Arg Gin Ala

40

Pro Gly Lys Gly Leu Glu Trp Vai 45

Ala Gin lie Lys Asp Lys Ser Asn 50 55

Asn Tyr Ala Thr Tyr Tyr Ala Glu 60

Ser Vai Lys Gly Arg Phe Thr lie 65 70

Ser Arg Asp Asp Ser Lys Asn Ser

80 lie Tyr Leu Gin Met Asn Ser Leu 85

Lys Thr Glu Asp Thr Ala Vai Tyr

95

Tyr Cys Arg Tyr Vai His Tyr Ala

100

Ala Tyr Tyr Gly Vai Asp Ala Trp 105 110

Gly Gin Gly Thr Thr Vai Thr Vai

115 120

Ser Ser Ala Ser Thr Lys Gly Pro

125

Ser Vai Phe Pro Leu Ala Pro Ser

130 135

Ser Lys Ser Thr Ser Gly Gly Thr

140

Ala Ala Leu Gly Cys Leu Vai Lys 145 150

Asp Tyr Phe Pro Glu Pro Vai Thr

155 160

- 135 042507

Val

Ser

trp

Asn

Ser

165

gly

Leu

Thr

Ser

170

gly

Val

His

Thr

Phe

175

Pro

Val

His

Cys 225

gly

Met

His

Val

Tyr 305

gly

Val

Ser

385

Pro

Val

Met

Val

Leu

gin

180

Ser

Ser

gly

Leu

Tyr 185

Ser

Leu

Ser

Ser

Val

190

Val

Thr

Pro

Lys 210

asp

gly

His

290

Arg

Lys

Tyr

Leu

370

trp

Val

asp

His

Ser 195

Ser

Ser

Leu

gly

Thr

200

gin

Thr

Tyr

Cys 205

Asn

Val

Asn

Pro

Lys

Pro

Ser

Asp 275

Asn

Val

Lys

Thr

355

Ser

Leu

Lys

435

Ser

Thr

Ser

Arg

260

Pro

Val

Tyr

Thr

340

Leu

Cys

Ser

asp

Ser

420

Asn

His

Val

245

Thr

Lys

Ser

Lys 325

Pro

Asn

Ser

405

Arg

Leu

Thr

Thr

230

Phe

Pro

Val

Thr

Val

310

Cys

Ser

Pro

Val

Gly 390

asp

trp

His

Lys 215

Cys

Leu

Lys

Lys 295

Leu

Lys

Lys

Ser

Lys 375

gin

gly

gin

Asn

Val

Pro

Phe

Val

Phe

280

Pro

Thr

Val

Arg

360

gly

Pro

Ser

gin

His

440

asp

Lys

Lys

Val

220

Pro

Lys

Ser

Pro

Pro

Thr

265

Asn

Arg

Val

Ser

Lys 345

Cys

Phe

Phe

Gly 425

Tyr

Cys

Pro

235

Pro

240

Pro

250

Cys

trp

Leu

Asn

330

gly

Tyr

Asn

Phe

410

Asn

Thr

Lys

Pro

Lys

asp

Thr

255

Leu

Val

Val

Val

asp

270

Val

Ser

Tyr

His

315

Lys

gin

Leu

Pro

Asn

395

Leu

Val

gin

Val

gin

300

gin

Pro

Thr

Ser

380

Tyr

Val

Phe

Lys

Asp 285

gly

Val

Tyr

Ser

Thr

asp

Leu

Arg

Lys 365

asp

Lys

Ser

Ser

Ser

445

trp

Leu

Asn

320

Pro

Pro

335

Pro

gin

350

Asn

gin

Val

Val

Thr

Thr

Pro

400

Lys

Cys 430

Leu

Leu

415

Ser

Ser

Thr

Val

Leu

Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

450 455

- 136 042507 <210> 60 <211> 15 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>60

Gin Asp Gly Asn Glu Glu Met Gly Gly lie Thr Gin Thr Pro Tyr

1015 <210>61 <211>467 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 61

Gin Val Gin Leu Val Glu Ser Gly 1 5

Gly Gly Leu Val Gin Pro Gly Arg 10 15

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ser Gly Phe Thr Phe Ser Ser Ala 25 30

Trp Met His Trp Val Arg Gin Ala

40

Pro Gly Lys Gly Leu Glu Trp Val 45

Ala Gin lie Lys Asp Lys Gly Asn 50 55

Gly Tyr Asn Ala Tyr Tyr Ala Pro 60

Ser Val Lys Gly Arg Phe Thr lie 65 70

Ser Arg Asp Asp Ser Lys Asn Ser

80 lie Tyr Leu Gin Met Asn Ser Leu 85

Lys Thr Glu Asp Thr Ala Val Tyr

95

Tyr Cys Arg Tyr Val His Tyr Thr

100

Thr Ser Tyr Phe Gly Leu Phe Gly 105 110

Gly Gly Gly Gly Gly Val Asp Ala

115 120

Trp Gly Gin Gly Thr Thr Val Thr

125

Val Ser Ser Ala Ser Thr Lys Gly

130 135

Pro Ser Val Phe Pro Leu Ala Pro

140

Ser Ser Lys Ser Thr Ser Gly Gly 145 150

Thr Ala Ala Leu Gly Cys Leu Val

155 160

Lys Asp Tyr Phe Pro Glu Pro Val

165

Thr Val Ser Trp Asn Ser Gly Ala

170 175

Leu Thr Ser Gly Val His Thr Phe 180

Pro Ala Val Leu Gin Ser Ser Gly 185 190

Leu Tyr Ser Leu Ser Ser Val Val

195 200

Thr Val Pro Ser Ser Ser Leu Gly 205

- 137 042507

Thr Gin Thr Tyr lie Cys Asn Val

210 215

Asn His Lys Pro Ser Asn Thr Lys

220

Val Asp Lys Lys Val Glu Pro Lys 225 230

Ser Cys Asp Lys Thr His Thr Cys

235 240

Pro Pro Cys Pro Ala Pro Glu Ala

245

Ala Gly Gly Pro Ser Val Phe Leu

250 255

Phe Pro Pro Lys Pro Lys Asp Thr

260

Leu Met lie Ser Arg Thr Pro Glu

265 270

Val Thr Cys Val Val Val Asp Val

275 280

Ser His Glu Asp Pro Glu Val Lys 285

Phe Asn Trp Tyr Val Asp Gly Val

290 295

Glu Val His Asn Ala Lys Thr Lys 300

Pro Arg Glu Glu Gin Tyr Ala Ser

305 310

Thr Tyr Arg Val Val Ser Val Leu

315 320

Thr Val Leu His Gin Asp Trp Leu 325

Asn Gly Lys Glu Tyr Lys Cys Lys

330 335

Val Ser Asn Lys Ala Leu Pro Ala

340

Pro lie Glu Lys Thr lie Ser Lys

345 350

Ala Lys Gly Gin Pro Arg Glu Pro

355 360

Gin Val Tyr Thr Leu Pro Ser 365

Arg Cys Glu Leu Thr Lys Asn Gin

370 375

Val Ser Leu Ser Cys Ala Val Lys 380

Gly Phe Tyr Pro Ser Asp lie Ala 385 390

Val Glu Trp Glu Ser Asn Gly Gin

395 400

Pro Glu Asn Asn Tyr Lys Thr Thr 405

Pro Pro Val Leu Asp Ser Asp Gly

410 415

Ser Phe Phe Leu Val Ser Lys Leu 420

Thr Val Asp Lys Ser Arg Trp Gin 425 430

Gin Gly Asn Val Phe Ser Cys Ser

435 440

Val Met His Glu Ala Leu His Asn

445

His Tyr Thr Gin Lys Ser Leu Ser

450 455

Leu Ser Pro Asp Tyr Lys Asp Asp 460

Asp Asp Lys 465 <210> 62 <211> 467 <212> PROTEIN <213> Artificial sequence

CTb<220>

- 138 042507 <223>

Artificially synthesized peptide sequence <400> 62

Gin 1

Vai

gin

Leu

Wai 5

Ser

gly

gly

Gly 10

Leu

Vai

gin

Pro

Gly 15

Arg

Ser

Leu

Arg

Leu

Ser

Cys

Ser

gly

Phe

Thr

Phe

Ser

Ser

trp

Met

Hi s 35

trp

Vai

Arg

gin

Pro

gly

Lys

gly

Leu 45

trp

Vai

Gin 50

Lys

asp

Lys

Gly 55

Asn

gly

Tyr

Asn

Tyr

Tyr

Pro

Ser 65

Vai

Lys

gly

Arg

Phe

Thr

Ser

Arg

Asp 75

asp

Ser

Lys

Asn

Ser

Tyr

Leu

gin

Met 85

Asn

Ser

Leu

Lys

Thr 90

asp

Thr

Wai 95

Tyr

Tyr

Cys

Arg

Tyr 100

Vai

His

Tyr

Thr

Thr

105

Ser

Tyr

Phe

gly

Leu

110

Phe

gly

gly

gly

Gly 115

gly

gly

Vai

asp

120

trp

gly

gin

gly

Thr

125

Thr

Vai

Thr

Vai

Ser

130

Ser

Ser

Thr

Lys 135

gly

Pro

Ser

Vai

Phe

140

Pro

Leu

Pro

Ser

145

Ser

Lys

Ser

Thr

Ser

150

gly

gly

Thr

Leu

155

gly

Cys

Leu

Vai

160

Lys

asp

Tyr

Phe

Pro

165

Pro

Vai

Thr

Vai

170

Ser

trp

Asn

Ser

Gly 175

Leu

Thr

Ser

Gly 180

Vai

His

Thr

Phe

Pro

185

Vai

Leu

gin

Ser

190

Ser

gly

Leu

Tyr

Ser

195

Leu

Ser

Ser

Vai

Vai

200

Thr

Vai

Pro

Ser

Ser

205

Ser

Leu

gly

Thr

gin

210

Thr

Tyr

Cys

Asn

215

Vai

Asn

His

Lys

Pro

220

Ser

Asn

Thr

Lys

Vai

225

asp

Lys

Lys

Vai

Pro

Pro

Pro

Cys

Pro

245

230

Pro

Lys

Ser

Cys

Gly 250

Asp 235

gly

Lys

Pro

Thr

Ser

His

Vai

Thr

Phe

255

Cys 240

Leu

Phe

Pro

Pro

Lys 260

Pro

Lys

asp

Thr

Leu

265

Met

Ser

Arg

Thr

270

Pro

Vai

Thr

Cys 275

Vai

Vai

Vai

asp

Vai

280

Ser

His

asp

Pro

285

Vai

Lys

Phe

Asn

trp

Tyr

Vai

asp

gly

Vai

Vai

His

Asn

Lys

Thr

Lys

- 139 042507

290 295

300

Pro Arg Glu Glu Gin Tyr Ala Ser 305 310

Thr Tyr Arg Vai Vai Ser Vai Leu

315 320

Thr Vai Leu His Gin Asp Trp Leu 325

Asn Gly Lys Glu Tyr Lys Cys Lys

330 335

Vai Ser Asn Lys Ala Leu Pro Ala

340

Pro lie Glu Lys Thr lie Ser Lys 345 350

Ala Lys Gly Gin Pro Arg Glu Pro

355 360

Gin Vai Tyr Thr Leu Pro Ser 365

Arg Cys Glu Leu Thr Lys Asn Gin

370 375

Vai Ser Leu Ser Cys Ala Vai Lys 380

Gly Phe Tyr Pro Ser Asp lie Ala 385 390

Vai Glu Trp Glu Ser Asn Gly Gin

395 400

Pro Glu Asn Asn Tyr Lys Thr Thr 405

Pro Pro Vai Leu Asp Ser Asp Gly

410 415

Ser Phe Phe Leu Vai Ser Lys Leu 420

Thr Vai Asp Lys Ser Arg Trp Gin 425 430

Gin Gly Asn Vai Phe Ser Cys Ser

435 440

Vai Met His Glu Ala Leu His Asn

445

His Tyr Thr Gin Lys Ser Leu Ser

450 455

Leu Ser Pro Asp Tyr Lys Asp Asp 460

Asp Asp Lys 465 <210> 63 <211> 464 <212> PROTEIN <213> Artificial sequence

CTb<220>

<223> Artificially synthesized peptide sequence <400> 63

Gin Vai Gin Leu Vai Glu Ser Gly 1 5

Gly Gly Leu Vai Gin Pro Gly Arg

15

Ser Leu Arg Leu Ser Cys Ala Ala

Ser Gly Phe Thr Phe Ser Asn Ala 25 30

Trp Met His Trp Vai Arg Gin Ala

40

Pro Gly Lys Gly Leu Glu Trp Vai 45

Ala Gin lie Lys Asp Lys Gly Gin

55

Gin Tyr Ala Ala Tyr Tyr Ala Pro 60

Ser Vai Lys Gly Arg Phe Thr lie

70

Ser Arg Asp Asp Ser Lys Asn Ser

80

- 140 042507

Ne Tug Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Vai Tyr 85 9095

Tyr Cys Arg Tyr Vai His Tyr Gin Thr Tyr Ser Asp Gly Ser Phe Tyr

100 105110

Phe Gly Vai Asp Ala Trp Gly Gin Gly Thr Thr Vai Thr Vai Ser Ser

115 120125

Ala Ser Thr Lys Gly Pro Ser Vai Phe Pro Leu Ala Pro Ser Ser Lys

130 135140

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Vai Lys Asp Tyr

145 150 155160

Phe Pro Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170175

Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser

180 185190

Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr

195 200205

Tyr He Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp Lys

210 215220

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

225 230 235240

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Vai Phe Leu Phe Pro Pro

245 250255

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys

260 265270

Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn Trp

275 280285

Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu

290 295300

Glu Gin Tyr Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu

305 310 315320

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn

325 330335

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly

340 345350

Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Ser Arg Cys Glu

355 360365

Leu Thr Lys Asn Gin Vai Ser Leu Ser Cys Ala Vai Lys Gly Phe Tyr

370 375380

- 141 042507

Pro Ser Asp He Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn

385 390 395400

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

405 410415

Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn

420 425430

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

435 440445

Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

450 455460 <210>64 <2H>464 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 64

Gin Val Gin Leu Val Glu Ser Gly 1 5

Gly Gly Leu Val Gin Pro Gly Arg

15

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ser Gly Phe Thr Phe Ser Asn Ala 25 30

Trp Met His Trp Val Arg Gin Ala

40

Pro Gly Lys Gly Leu Glu Trp Val 45

Ala Gin lie Lys Asp Arg Val Asn

55

Gin Tyr Ala Asn Tyr Tyr Ala Glu 60

Ser Val Lys Gly Arg Phe Thr lie 65 70

Ser Arg Asp Asp Ser Lys Asn Ser

80 lie Tyr Leu Gin Met Asn Ser Leu 85

Lys Thr Glu Asp Thr Ala Val Tyr

95

Tyr Cys Arg Tyr Val His Tyr Gly

100

Leu Thr Gly Tyr Phe Ser Gly Gin 105HO

Gly Gly Val Asp Ala Trp Gly Gin

115 120

Gly Thr Thr Val Thr Val Ser Ser

125

Ala Ser Thr Lys Gly Pro Ser Val

130 135

Phe Pro Leu Ala Pro Ser Ser Lys

140

Ser Thr Ser Gly Gly Thr Ala Ala 145 150

Leu Gly Cys Leu Val Lys Asp Tyr

155 160

Phe Pro Glu Pro Val Thr Val Ser

165

Trp Asn Ser Gly Ala Leu Thr Ser

170 175

Gly Val His Thr Phe Pro Ala Val 180

Leu Gin Ser Ser Gly Leu Tyr Ser 185 190

- 142 042507

Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr

195 200205

Tyr lie Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp Lys

210 215220

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

225 230 235240

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Vai Phe Leu Phe Pro Pro

245 250255

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys

260 265270

Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn Trp

275 280285

Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu

290 295300

Glu Gin Tyr Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu

305 310 315320

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn

325 330335

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly

340 345350

Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Ser Arg Cys Glu

355 360365

Leu Thr Lys Asn Gin Vai Ser Leu Ser Cys Ala Vai Lys Gly Phe Tyr

370 375380

Pro Ser Asp lie Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn

385 390 395400

Asn Tyr Lys Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly Ser Phe Phe

405 410415

Leu Vai Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly Asn

420 425430

Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr Thr

435 440445

Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

450 455460 <210>65 <211>464 <212> PROTEIN <213> Artificial sequence <220>

- 143 042507 <223> Artificially synthesized peptide sequence <400> 65

Gin 1 Vai gin Leu Wai 5 Glu Ser gly gly Gly 10 Leu Vai gin Pro Gly 15 Arg Ser Leu Arg Leu 20 Ser Cys Ala Ala Ser 25 gly Phe Thr Phe Ser thirty Asn Ala trp Met His 35 trp Vai Arg gin Ala 40 Pro gly Lys gly Leu 45 Glu trp Vai Ala Gin 50 He Lys asp Arg Wai 55 Asn gin Tyr Ala Asn 60 Tyr Tyr Ala Glu Ser 65 Vai Lys gly Arg Leu 70 Thr He Ser Arg Asp 75 asp Ser Lys Asn Ser 80 He Tyr Leu gin Met 85 Asn Ser Leu Lys Thr 90 Glu asp Thr Ala Wai 95 Tyr Tyr Cys Arg Tyr 100 Vai His Tyr gly Leu 105 Thr gly Tyr Phe Ser 110 gly gin gly gly Vai 115 asp Ala trp gly gin 120 gly Thr Thr Vai Thr 125 Vai Ser Ser Ala Ser 130 Thr Lys gly Pro Ser 135 Vai Phe Pro Leu Ala 140 Pro Ser Ser Lys Ser 145 Thr Ser gly gly Thr 150 Ala Ala Leu gly Cys 155 Leu Vai Lys asp Tyr 160 Phe Pro Glu Pro Vai 165 Thr Vai Ser trp Asn 170 Ser gly Ala Leu Thr 175 Ser gly Vai His Thr 180 Phe Pro Ala Vai Leu 185 gin Ser Ser gly Leu 190 Tyr Ser Leu Ser Ser 195 Vai Vai Thr Vai Pro 200 Ser Ser Ser Leu Gly 205 Thr gin Thr Tyr He 210 Cys Asn Vai Asn His 215 Lys Pro Ser Asn Thr 220 Lys Vai asp Lys Lys 225 Vai Glu Pro Lys Ser 230 Cys asp Lys Thr His 235 Thr Cys Pro Pro Cys 240 Pro Ala Pro Glu Ala 245 Ala gly gly Pro Ser 250 Vai Phe Leu Phe Pro 255 Pro Lys Pro Lys Asp 260 Thr Leu Met He Ser 265 Arg Thr Pro Glu Vai 270 Thr Cys Vai Vai Vai 275 asp Vai Ser His Glu 280 asp Pro Glu Vai Lys 285 Phe Asn trp Tyr Vai asp gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu

- 144 042507

290

295

300

Glu Gin Tyr Ala Ser Thr Tyr Arg 305 310

Vai Vai Ser Vai Leu Thr Vai Leu

315 320

His Gin Asp Trp Leu Asn Gly Lys 325

Glu Tyr Lys Cys Lys Vai Ser Asn

330 335

Lys Ala Leu Pro Ala Pro He Glu

340

Lys Thr lie Ser Lys Ala Lys Gly 345 350

Gin Pro Arg Glu Pro Gin Vai Tyr

355 360

Thr Leu Pro Pro Ser Arg Cys Glu 365

Leu Thr Lys Asn Gin Vai Ser Leu

370 375

Ser Cys Ala Vai Lys Gly Phe Tyr 380

Pro Ser Asp lie Ala Vai Glu Trp 385 390

Glu Ser Asn Gly Gin Pro Glu Asn

395 400

Asn Tyr Lys Thr Thr Pro Pro Vai 405

Leu Asp Ser Asp Gly Ser Phe Phe

410 415

Leu Vai Ser Lys Leu Thr Vai Asp 420

Lys Ser Arg Trp Gin Gin Gly Asn 425 430

Vai Phe Ser Cys Ser Vai Met His

435 440

Glu Ala Leu His Asn His Tyr Thr 445

Gin Lys Ser Leu Ser Leu Ser Pro

450 455

Asp Tyr Lys Asp Asp Asp Asp Lys 460 <210> 66 <2H> 464 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 66

Gin Vai Gin Leu Vai Glu Ser Gly 1 5

Gly Gly Leu Vai Gin Pro Gly Arg

15

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ser Gly Phe Ala Phe Ser Asn Ala 25 30

Trp Met His Trp Vai Arg Gin Ala

40

Pro Gly Lys Gly Leu Glu Trp Vai 45

Ala Gin lie Lys Asp Arg Vai Asn

55

Gin Tyr Ala Asn Tyr Tyr Ala Glu 60

Ser Vai Lys Gly Arg Phe Thr lie

70

Ser Arg Asp Asp Ser Lys Asn Ser

80 lie Tyr Leu Gin Met Asn Ser Leu 85

Lys Thr Glu Asp Thr Ala Vai Tyr

95

- 145 042507

Tyr Cys Arg Tyr Vai His Tyr Gly Leu Thr Gly Tyr Phe Ser Gly Gin

100 105110

Gly Gly Vai Asp Ala Trp Gly Gin Gly Thr Thr Vai Thr Vai Ser Ser

115 120125

Ala Ser Thr Lys Gly Pro Ser Vai Phe Pro Leu Ala Pro Ser Ser Lys

130 135140

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Vai Lys Asp Tyr

145 150 155160

Phe Pro Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170175

Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser

180 185190

Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr

195 200205

Tyr He Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp Lys

210 215220

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

225 230 235240

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Vai Phe Leu Phe Pro Pro

245 250255

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys

260 265270

Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn Trp

275 280285

Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu

290 295300

Glu Gin Tyr Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu

305 310 315320

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn

325 330335

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly

340 345350

Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Ser Arg Cys Glu

355 360365

Leu Thr Lys Asn Gin Vai Ser Leu Ser Cys Ala Vai Lys Gly Phe Tyr

370 375380

Pro Ser Asp lie Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn

385 390 395400

- 146 042507

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

405 410415

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

420 425430

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

435 440445

Gln Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

450 455460 <210>67 <211>464 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 67

Gln Val Gln Leu Val Glu Ser Gly 1 5

Gly Gly Leu Val Gln Pro Gly Arg

15

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ser Gly Phe Thr Phe Ser Asn Ala 25 30

Trp Met His Trp Val Arg Gln Ala

40

Pro Gly Lys Gly Leu Glu Trp Val 45

Ala Gln lie Lys Asp Arg Val Asn

55

Gln Tyr Ala Asn Tyr Tyr Ala Glu 60

Cys Val Lys Gly Arg Phe Thr lie 65 70

Ser Arg Asp Asp Ser Lys Asn Ser

80 lie Tyr Leu Gln Met Asn Ser Leu 85

Lys Thr Glu Asp Thr Ala Val Tyr

95

Tyr Cys Arg Tyr Val His Tyr Gly

100

Leu Thr Gly Tyr Phe Ser Gly Gln 105 110

Gly Gly Val Asp Ala Trp Gly Gln

115 120

Gly Thr Thr Val Thr Val Ser Ser

125

Ala Ser Thr Lys Gly Pro Ser Val

130 135

Phe Pro Leu Ala Pro Ser Ser Lys

140

Ser Thr Ser Gly Gly Thr Ala Ala 145 150

Leu Gly Cys Leu Val Lys Asp Tyr

155 160

Phe Pro Glu Pro Val Thr Val Ser

165

Trp Asn Ser Gly Ala Leu Thr Ser

170 175

Gly Val His Thr Phe Pro Ala Val 180

Leu Gln Ser Ser Gly Leu Tyr Ser 185 190

Leu Ser Ser Val Val Thr Val Pro

195 200

Ser Ser Ser Leu Gly Thr Gln Thr 205

- 147 042507

Tyg Ne Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp Lys

210 215220

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

225 230 235240

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Vai Phe Leu Phe Pro Pro

245 250255

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys

260 265270

Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn Trp

275 280285

Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu

290 295300

Glu Gin Tyr Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu

305 310 315320

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn

325 330335

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly

340 345350

Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Ser Arg Cys Glu

355 360365

Leu Thr Lys Asn Gin Vai Ser Leu Ser Cys Ala Vai Lys Gly Phe Tyr

370 375380

Pro Ser Asp lie Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn

385 390 395400

Asn Tyr Lys Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly Ser Phe Phe

405 410415

Leu Vai Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly Asn

420 425430

Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr Thr

435 440445

Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

450 455460 <210> 68 <2H>464 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 68

- 148 042507

Gin Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Arg 15 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ala 20 2530

Trp Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045

Ala Gin lie Lys Asp Lys Gin Asn Gin Tyr Asn Ala Tyr Tyr Ala Glu 50 5560

Ser Val Lys Gly Arg Phe Thr lie Ser Arg Asp Asp Ser Lys Asn Ser

70 7580 lie Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 9095

Tyr Cys Arg Tyr Val His Tyr Ala Thr Ser Asp Gly Phe Leu Pro Tyr

100 105110

Phe Gly Val Asp Ala Trp Gly Gin Gly Thr Thr Thr Val Thr Val Ser Ser

115 120125

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

130 135140

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

145 150 155160

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170175

Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser

180 185190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr

195 200205

Tyr lie Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215220

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

225 230 235240

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

245 250255

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys

260 265270

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

275 280285

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

290 295300

Glu Gin Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

- 149 042507

305 310

315 320

His Gin Asp Trp Leu Asn Gly Lys 325

Glu Tyr Lys Cys Lys Vai Ser Asn

330 335

Lys Ala Leu Pro Ala Pro He Glu

340

Lys Thr lie Ser Lys Ala Lys Gly 345 350

Gin Pro Arg Glu Pro Gin Vai Tyr

355 360

Thr Leu Pro Pro Ser Arg Cys Glu 365

Leu Thr Lys Asn Gin Vai Ser Leu

370 375

Ser Cys Ala Vai Lys Gly Phe Tyr 380

Pro Ser Asp lie Ala Vai Glu Trp 385 390

Glu Ser Asn Gly Gin Pro Glu Asn

395 400

Asn Tyr Lys Thr Thr Pro Pro Vai 405

Leu Asp Ser Asp Gly Ser Phe Phe

410 415

Leu Vai Ser Lys Leu Thr Vai Asp 420

Lys Ser Arg Trp Gin Gin Gly Asn 425 430

Vai Phe Ser Cys Ser Vai Met His

435 440

Glu Ala Leu His Asn His Tyr Thr 445

Gin Lys Ser Leu Ser Leu Ser Pro

450 455

Asp Tyr Lys Asp Asp Asp Asp Lys 460 <210> 69 <211> 464 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 69

Gin Vai Gin Leu Vai Glu Ser Gly 1 5

Gly Gly Leu Vai Gin Pro Gly Arg

15

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ser Gly Phe Thr Phe Ser Asn Ala 25 30

Trp Met His Trp Vai Arg Gin Ala

40

Pro Gly Lys Gly Leu Glu Trp Vai 45

Ala Gin He Lys Asp Arg Ala Ser

55

Gly Tyr Asn Asn Tyr Tyr Ala Glu 60

Ser Vai Lys Gly Arg Phe Thr He

70

Ser Arg Asp Asp Ser Lys Asn Ser

80 lie Tyr Leu Gin Met Asn Ser Leu 85

Lys Thr Glu Asp Thr Ala Vai Tyr

95

Tyr Cys Arg Tyr Vai His Tyr Ala

100

Gin Ser Vai Ser Ala Phe Phe Gin

105 110

- 150 042507

Tyr Gly Val Asp Ala Trp Gly Gin Gly Thr Thr Thr Val Thr Val Ser Ser

115 120125

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

130 135140

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

145 150 155160

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170175

Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser

180 185190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr

195 200205

Tyr lie Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215220

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

225 230 235240

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

245 250255

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys

260 265270

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

275 280285

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

290 295300

Glu Gin Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

305 310 315320

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

325 330335

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly

340 345350

Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Ser Arg Cys Glu

355 360365

Leu Thr Lys Asn Gin Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr

370 375380

Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn

385 390 395400

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

405 410415

- 151 042507

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

420 425430

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

435 440445

Gln Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

450 455460 <210>70 <211>464 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 70

Gln Val Gln Leu Val Glu Ser Gly 1 5

Gly Gly Leu Val Gln Pro Gly Arg

15

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ser Gly Phe Thr Phe Ser Asn Ala 25 30

Trp Met His Trp Val Arg Gln Ala

40

Pro Gly Lys Gly Leu Glu Trp Val 45

Ala Gln lie Lys Asp Lys Gln Ser

55

Gln Tyr Ala Asn Tyr Tyr Ala Pro 60

Ser Val Lys Gly Arg Phe Thr lie 65 70

Ser Arg Asp Asp Ser Lys Asn Ser

80 lie Tyr Leu Gln Met Asn Ser Leu 85

Lys Thr Glu Asp Thr Ala Val Tyr

95

Tyr Cys Arg Tyr Val His Tyr Gln

100

Ser Thr Ser Tyr Ser Phe Tyr Gln 105 110

Gly Gly Val Asp Ala Trp Gly Gln

115 120

Gly Thr Thr Val Thr Val Ser Ser

125

Ala Ser Thr Lys Gly Pro Ser Val

130 135

Phe Pro Leu Ala Pro Ser Ser Lys

140

Ser Thr Ser Gly Gly Thr Ala Ala 145 150

Leu Gly Cys Leu Val Lys Asp Tyr

155 160

Phe Pro Glu Pro Val Thr Val Ser

165

Trp Asn Ser Gly Ala Leu Thr Ser

170 175

Gly Val His Thr Phe Pro Ala Val 180

Leu Gln Ser Ser Gly Leu Tyr Ser 185 190

Leu Ser Ser Val Val Thr Val Pro

195 200

Ser Ser Ser Leu Gly Thr Gln Thr 205

Tyr lie Cys Asn Val Asn His Lys

210 215

Pro Ser Asn Thr Lys Val Asp Lys

220

- 152 042507

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

225 230 235240

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Vai Phe Leu Phe Pro Pro

245 250255

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys

260 265270

Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn Trp

275 280285

Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu

290 295300

Glu Gin Tyr Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu

305 310 315320

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn

325 330335

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly

340 345350

Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Ser Arg Cys Glu

355 360365

Leu Thr Lys Asn Gin Vai Ser Leu Ser Cys Ala Vai Lys Gly Phe Tyr

370 375380

Pro Ser Asp lie Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn

385 390 395400

Asn Tyr Lys Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly Ser Phe Phe

405 410415

Leu Vai Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly Asn

420 425430

Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr Thr

435 440445

Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

450 455460 <210>71 <211>464 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 71

Gin Vai Gin Leu Vai Glu Ser Gly Gly Gly Leu Vai Gin Pro Gly Arg 15 10 15

- 153 042507

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 2530

Trp Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045

Ala Gin He Lys Asp Lys Gin Ser Gin Tyr Ala Asn Tyr Tyr Ala Pro 50 5560

Ser Val Lys Gly Arg Phe Thr lie Ser Arg Asp Asp Ser Lys Ser Ser

70 7580 lie Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 9095

Tyr Cys Arg Tyr Val His Tyr Gin Ser Thr Ser Tyr Ser Phe Tyr Gin 100 105HO

Gly Gly Val Asp Ala Trp Gly Gin Gly Thr Thr Thr Val Thr Val Ser Ser

115 120125

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

130 135140

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

145 150 155160

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170175

Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser

180 185190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr

195 200205

Tyr lie Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215220

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

225 230 235240

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

245 250255

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys

260 265270

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

275 280285

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

290 295300

Glu Gin Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

305 310 315320

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

- 154 042507

325

330 335

Lys Ala Leu Pro Ala Pro lie Glu

340

Lys Thr lie Ser Lys Ala Lys Gly 345 350

Gin Pro Arg Glu Pro Gin Val Tyr

355 360

Thr Leu Pro Pro Ser Arg Cys Glu 365

Leu Thr Lys Asn Gin Val Ser Leu

370 375

Ser Cys Ala Val Lys Gly Phe Tyr 380

Pro Ser Asp lie Ala Val Glu Trp 385 390

Glu Ser Asn Gly Gin Pro Glu Asn

395 400

Asn Tyr Lys Thr Thr Pro Pro Val 405

Leu Asp Ser Asp Gly Ser Phe Phe

410 415

Leu Val Ser Lys Leu Thr Val Asp 420

Lys Ser Arg Trp Gin Gin Gly Asn 425 430

Val Phe Ser Cys Ser Val Met His

435 440

Glu Ala Leu His Asn His Tyr Thr 445

Gin Lys Ser Leu Ser Leu Ser Pro

450 455

Asp Tyr Lys Asp Asp Asp Asp Lys 460 <210> 72 <211> 464 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>72

Gin Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Arg 15 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 2530

Trp Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045

Ala Gin lie Lys Asp Arg Gly Asn Asn Tyr Ala Ala Tyr Tyr Ala Glu 50 5560

Ser Val Lys Gly Arg Phe Thr lie Ser Arg Asp Asp Ser Lys Asn Ser

70 7580 lie Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 9095

Tyr Cys Arg Tyr Val His Tyr Gly Ser Tyr Phe Phe Ala Ser Phe Tyr

100 105110

Phe Gly Val Asp He Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser

115 120125

- 155 042507

Ala Ser Thr Lys Gly Pro Ser Vai Phe Pro Leu Ala Pro Ser Ser Lys

130 135140

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Vai Lys Asp Tyr

145 150 155160

Phe Pro Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170175

Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser

180 185190

Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr

195 200205

Tyr lie Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp Lys

210 215220

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

225 230 235240

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Vai Phe Leu Phe Pro Pro

245 250255

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys

260 265270

Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn Trp

275 280285

Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu

290 295300

Glu Gin Tyr Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu

305 310 315320

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn

325 330335

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly

340 345350

Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Ser Arg Cys Glu

355 360365

Leu Thr Lys Asn Gin Vai Ser Leu Ser Cys Ala Vai Lys Gly Phe Tyr

370 375380

Pro Ser Asp lie Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn

385 390 395400

Asn Tyr Lys Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly Ser Phe Phe

405 410415

Leu Vai Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly Asn

420 425430

- 156 042507

Val Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr Thr

435 440445

Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

450 455460 <210>73 <211> 464 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 73

Gin Val Gin Leu Val Glu Ser Gly 1 5

Gly Gly Leu Val Gin Pro Gly His

15

Ser Leu Arg Leu Ser Cys Ala Ala

Ser Gly Phe Thr Phe Ser Asn Ala 25 30

Trp Met His Trp Val Arg Gin Ala

40

Pro Gly Lys Gly Leu Glu Trp Val 45

Ala Gin lie Lys Asp Arg Gly Asn

55

Asn Tyr Ala Ala Tyr Tyr Ala Glu 60

Ser Val Lys Gly Arg Phe Thr lie 65 70

Ser Arg Asp Asp Ser Lys Asn Ser

80 lie Tyr Leu Gin Met Asn Ser Leu 85

Lys Thr Glu Asp Thr Ala Val Tyr

95

Tyr Cys Arg Tyr Val His Tyr Gly

100

Ser Tyr Phe Phe Ala Ser Phe Tyr 105 110

Phe Gly Val Asp lie Trp Gly Gin

115 120

Gly Thr Thr Val Thr Val Ser Ser

125

Ala Ser Thr Lys Gly Pro Ser Val

130 135

Phe Pro Leu Ala Pro Ser Ser Lys

140

Ser Thr Ser Gly Gly Thr Ala Ala 145 150

Leu Gly Cys Leu Val Lys Asp Tyr

155 160

Phe Pro Glu Pro Val Thr Val Ser

165

Trp Asn Ser Gly Ala Leu Thr Ser

170 175

Gly Val His Thr Phe Pro Ala Val 180

Leu Gin Ser Ser Gly Leu Tyr Ser 185 190

Leu Ser Ser Val Val Thr Val Pro

195 200

Ser Ser Ser Leu Gly Thr Gin Thr

205

Tyr lie Cys Asn Val Asn His Lys

210 215

Pro Ser Asn Thr Lys Val Asp Lys

220

Lys Val Glu Pro Lys Ser Cys Asp 225 230

Lys Thr His Thr Cys Pro Pro Cys

235 240

- 157 042507

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Vai Phe Leu Phe Pro Pro

245 250255

Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro Glu Vai Thr Cys

260 265270

Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn Trp

275 280285

Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu

290 295300

Glu Gin Tyr Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu

305 310 315320

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn

325 330335

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly

340 345350

Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Ser Arg Cys Glu

355 360365

Leu Thr Lys Asn Gin Vai Ser Leu Ser Cys Ala Vai Lys Gly Phe Tyr

370 375380

Pro Ser Asp He Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn

385 390 395400

Asn Tyr Lys Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly Ser Phe Phe

405 410415

Leu Vai Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly Asn

420 425430

Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr Thr

435 440445

Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

450 455460 <210>74 <211>464 <212> PROTEIN <213> Artificial sequence <220>

< 223> Artificially synthesized peptide sequence < 400> 74

Gin Vai Gin Leu Vai Glu Ser Gly Gly Gly Leu Vai Gin Pro Gly Arg 15 10 15

Ser Leu Arg

Leu Ser Cys Ala Ala Ser

25

Gly Phe Thr Phe Ser

Asn Ala

- 158 042507

Trp Met His Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Vai 35 4045

Ala Gin He Lys Asp Arg Gly Asn Asn Tyr Asn Thr Tyr Tyr Ala Pro 50 5560

Ser Vai Lys Gly Arg Phe Thr lie Ser Arg Asp Asp Ser Lys Asn Ser

70 7580 lie Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Vai Tyr 85 9095

Tyr Cys Arg Tyr Vai His Tyr Gly Ser Tyr Phe Phe Ser Gly Ser Ser

100 105110

Phe Gly Vai Asp He Trp Gly Gin Gly Thr Thr Vai Thr Vai Ser Ser

115 120125

Ala Ser Thr Lys Gly Pro Ser Vai Phe Pro Leu Ala Pro Ser Ser Lys

130 135140

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Vai Lys Asp Tyr

145 150 155160

Phe Pro Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170175

Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser

180 185190

Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr

195 200205

Tyr lie Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp Lys

210 215220

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

225 230 235240

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Vai Phe Leu Phe Pro Pro

245 250255

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys

260 265270

Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn Trp

275 280285

Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu

290 295300

Glu Gin Tyr Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu

305 310 315320

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn

325 330335

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly

- 159 042507

340

345 350

Gin Pro Arg Glu Pro Gin Vai Tyr

355 360

Thr Leu Pro Pro Ser Arg Cys Glu 365

Leu Thr Lys Asn Gin Vai Ser Leu

370 375

Ser Cys Ala Vai Lys Gly Phe Tyr 380

Pro Ser Asp lie Ala Vai Glu Trp 385 390

Glu Ser Asn Gly Gin Pro Glu Asn

395 400

Asn Tyr Lys Thr Thr Pro Pro Vai 405

Leu Asp Ser Asp Gly Ser Phe Phe

410 415

Leu Vai Ser Lys Leu Thr Vai Asp 420

Lys Ser Arg Trp Gin Gin Gly Asn 425 430

Vai Phe Ser Cys Ser Vai Met His

435 440

Glu Ala Leu His Asn His Tyr Thr 445

Gin Lys Ser Leu Ser Leu Ser Pro

450 455

Asp Tyr Lys Asp Asp Asp Asp Lys 460 <210> 75 <211> 464 <212> PROTEIN <213> Artificial sequence

CTb<220>

<223> Artificially synthesized peptide sequence <400>75

Gin Vai Gin Leu Vai Glu Ser Gly Gly Gly Leu Vai Gin Pro Gly Arg 15 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 2530

Trp Met His Trp Vai Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Vai 35 4045

Ala Gin lie Lys Asp Arg Gly Asn Asn Tyr Asn Thr Tyr Tyr Ala Pro 50 5560

Ser Vai Lys Gly Arg Phe Thr lie Ser Arg Asp Asp Ser Lys Asn Ser

70 7580 lie Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Vai Tyr 85 9095

Tyr Cys Arg Tyr Ala His Tyr Gly Ser Tyr Phe Phe Ser Gly Ser Ser

100 105110

Phe Gly Vai Asp He Trp Gly Gin Gly Thr Thr Vai Thr Vai Ser Ser

115 120125

Ala Ser Thr Lys Gly Pro Ser Vai Phe Pro Leu Ala Pro Ser Ser Lys

130 135140

- 160 042507

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Vai Lys Asp Tyr

145 150 155160

Phe Pro Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170175

Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser

180 185190

Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr

195 200205

Tyr He Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp Lys

210 215220

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

225 230 235240

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Vai Phe Leu Phe Pro Pro

245 250255

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys

260 265270

Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn Trp

275 280285

Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu

290 295300

Glu Gin Tyr Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu

305 310 315320

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn

325 330335

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly

340 345350

Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Ser Arg Cys Glu

355 360365

Leu Thr Lys Asn Gin Vai Ser Leu Ser Cys Ala Vai Lys Gly Phe Tyr

370 375380

Pro Ser Asp lie Ala Vai Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn

385 390 395400

Asn Tyr Lys Thr Thr Pro Pro Vai Leu Asp Ser Asp Gly Ser Phe Phe

405 410415

Leu Vai Ser Lys Leu Thr Vai Asp Lys Ser Arg Trp Gin Gin Gly Asn

420 425430

Vai Phe Ser Cys Ser Vai Met His Glu Ala Leu His Asn His Tyr Thr

435 440445

- 161 042507

Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

450 455460 <210>76 <211>464 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 76

Gin Val Gin Leu Val Glu Ser Gly 1 5

Gly Gly Leu Val Gin Pro Gly Arg

15

Ser Leu Arg Leu Ser Cys Ala Ala 20

Ser Gly Phe Thr Phe Ser Asn Ala 25 30

Trp Met His Trp Val Arg Gin Ala

40

Pro Gly Lys Gly Leu Glu Trp Val 45

Ala Gin lie Lys Asp Lys Gly Asn 50 55

Gin Tyr Ala Thr Tyr Tyr Ala Glu 60

Ser Val Lys Gly Arg Phe Thr lie 65 70

Ser Arg Asp Asp Ser Lys Asn Ser

80 lie Tyr Leu Gin Met Asn Ser Leu 85

Lys Thr Glu Asp Thr Ala Val Tyr

95

Tyr Cys Arg Tyr Val His Tyr Gly

100

Thr Tyr Phe Phe Ala Leu Pro Ala 105 110

Phe Gly Val Asp lie Trp Gly Gin

115 120

Gly Thr Thr Val Thr Val Ser Ser

125

Ala Ser Thr Lys Gly Pro Ser Val

130 135

Phe Pro Leu Ala Pro Ser Ser Lys

140

Ser Thr Ser Gly Gly Thr Ala Ala 145 150

Leu Gly Cys Leu Val Lys Asp Tyr

155 160

Phe Pro Glu Pro Val Thr Val Ser

165

Trp Asn Ser Gly Ala Leu Thr Ser

170 175

Gly Val His Thr Phe Pro Ala Val 180

Leu Gin Ser Ser Gly Leu Tyr Ser 185 190

Leu Ser Ser Val Val Thr Val Pro

195 200

Ser Ser Ser Leu Gly Thr Gin Thr 205

Tyr lie Cys Asn Val Asn His Lys

210 215

Pro Ser Asn Thr Lys Val Asp Lys

220

Lys Val Glu Pro Lys Ser Cys Asp 225 230

Lys Thr His Thr Cys Pro Pro Cys

235 240

Pro Ala Pro Glu Ala Ala Gly Gly

245

Pro Ser Val Phe Leu Phe Pro Pro

250 255

- 162 042507

Lys Pro Lys Asp Thr Leu Met lie

260

Ser Arg Thr Pro Glu Val Thr Cys

265 270

Val Val Val Asp Val Ser His Glu

275 280

Asp Pro Glu Val Lys Phe Asn Trp

285

Tyr Val Asp Gly Val Glu Val His

290 295

Asn Ala Lys Thr Lys Pro Arg Glu

300

Glu Gin Tyr Ala Ser Thr Tyr Arg 305 310

Val Val Ser Val Leu Thr Val Leu

315 320

His Gin Asp Trp Leu Asn Gly Lys 325

Glu Tyr Lys Cys Lys Val Ser Asn

330 335

Lys Ala Leu Pro Ala Pro lie Glu

340

Lys Thr lie Ser Lys Ala Lys Gly

345 350

Gin Pro Arg Glu Pro Gin Val Tyr

355 360

Thr Leu Pro Pro Ser Arg Cys Glu 365

Leu Thr Lys Asn Gin Val Ser Leu

370 375

Ser Cys Ala Val Lys Gly Phe Tyr 380

Pro Ser Asp lie Ala Val Glu Trp

385 390

Glu Ser Asn Gly Gin Pro Glu Asn

395 400

Asn Tyr Lys Thr Thr Pro Pro Val 405

Leu Asp Ser Asp Gly Ser Phe Phe

410 415

Leu Val Ser Lys Leu Thr Val Asp 420

Lys Ser Arg Trp Gin Gin Gly Asn

425 430

Val Phe Ser Cys Ser Val Met His

435 440

Glu Ala Leu His Asn His Tyr Thr 445

Gin Lys Ser Leu Ser Leu Ser Pro

450 455

Asp Tyr Lys Asp Asp Asp Asp Lys 460 <210> 77 <211> 464 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>77

Gin Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Arg 15 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 2530

Trp Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045

- 163 042507

Ala Gin He Lys Asp Lys Gly Asn Gin Tyr Ala Thr Tyr Tyr Ala Glu 50 5560

Ser Vai Lys Gly Arg Tyr Thr lie Ser Arg Asp Asp Ser Lys Asn Ser

70 7580 lie Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Vai Tyr 85 9095

Tyr Cys Arg Tyr Vai His Tyr Gly Thr Tyr Phe Phe Ala Leu Pro Ala

100 105110

Phe Gly Vai Asp He Trp Gly Gin Gly Thr Thr Vai Thr Vai Ser Ser

115 120125

Ala Ser Thr Lys Gly Pro Ser Vai Phe Pro Leu Ala Pro Ser Ser Lys

130 135140

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Vai Lys Asp Tyr

145 150 155160

Phe Pro Glu Pro Vai Thr Vai Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170175

Gly Vai His Thr Phe Pro Ala Vai Leu Gin Ser Ser Gly Leu Tyr Ser

180 185190

Leu Ser Ser Vai Vai Thr Vai Pro Ser Ser Ser Leu Gly Thr Gin Thr

195 200205

Tyr He Cys Asn Vai Asn His Lys Pro Ser Asn Thr Lys Vai Asp Lys

210 215220

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

225 230 235240

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Vai Phe Leu Phe Pro Pro

245 250255

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Vai Thr Cys

260 265270

Vai Vai Vai Asp Vai Ser His Glu Asp Pro Glu Vai Lys Phe Asn Trp

275 280285

Tyr Vai Asp Gly Vai Glu Vai His Asn Ala Lys Thr Lys Pro Arg Glu

290 295300

Glu Gin Tyr Ala Ser Thr Tyr Arg Vai Vai Ser Vai Leu Thr Vai Leu

305 310 315320

His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Vai Ser Asn

325 330335

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly

340 345350

Gin Pro Arg Glu Pro Gin Vai Tyr Thr Leu Pro Ser Arg Cys Glu

- 164 042507

355

360

365

Leu Thr Lys Asn Gin Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr

370 375380

Pro Ser Asp He Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn

385 390 395400

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

405 410415

Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn

420 425430

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

435 440445

Gin Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

450 455460 <210>78 <2H> 464 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>78

Gin Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Arg 15 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 2530

Trp Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045

Ala Gin lie Lys Asp Arg Gly Asn Asn Tyr Ala Ala Tyr Tyr Ala Glu 50 5560

Ser Val Lys Gly Arg Phe Thr lie Ser Arg Asp Asp Ser Lys Asn Ser

70 7580 lie Tyr Leu Gin Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 9095

Tyr Cys Arg Tyr Val His Tyr Gly Ser Tyr Phe Phe Ala Ser Phe Tyr 100 105HO

Phe Gly Val Asp He Trp Gly Gin Gly Thr Ala Val Thr Val Ser Ser

115 120125

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

130 135140

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

145 150 155160

- 165 042507

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170175

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 180 185190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

195 200205

Tyr lie Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215220

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

225 230 235240

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

245 250255

Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys

260 265270

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

275 280285

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

290 295300

Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

305 310 315320

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

325 330335

Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly

340 345350

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

355 360365

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

370 375380

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

385 390 395400

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

405 410415

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

420 425430

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

435 440445

Gln Lys Ser Leu Ser Leu Ser Pro Asp Tyr Lys Asp Asp Asp Asp Lys

450 455460

- 166 042507 <210> 79 <211> 15 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 79

Gly Leu Asn Asp lie Phe Glu Ala Gin Lys lie Glu Trp His Glu 15 1015 <210>80 <211>233 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400> 80

Cys His Pro Arg Leu Ser Leu His 1 5

Arg Pro Ala Leu Glu Asp Leu Leu

15

Leu Gly Ser Glu Ala Asn Leu Thr 20

Cys Thr Leu Thr Gly Leu Arg Asp 25 30

Ala Ser Gly Vai Thr Phe Thr Trp

40

Thr Pro Ser Ser Gly Lys Ser Ala 45

Vai Gin Gly Pro Glu Arg Asp

55

Leu Cys Gly Cys Tyr Ser Vai Ser 60

Ser Vai Leu Pro Gly Cys Ala Glu

70

Pro Trp Asn His Gly Lys Thr Phe

80

Thr Cys Thr Ala Ala Tyr Pro Glu 85

Ser Lys Thr Pro Leu Thr Ala Thr

95

Leu Ser Lys Ser Gly Asn Thr Phe

100

Arg Pro Glu Vai His Leu Leu Pro 105 110

Pro Pro Ser Glu Glu Leu Ala Leu

115 120

Asn Glu Leu Vai Thr Leu Thr Cys

125

Leu Ala Arg Gly Phe Ser Pro Lys

130 135

Asp Vai Leu Vai Arg Trp Leu Gin 140

Gly Ser Gin Glu Leu Pro Arg Glu 145 150

Lys Tyr Leu Thr Trp Ala Ser Arg

155 160

Gin Glu Pro Ser Gin Gly Thr Thr 165

Thr Phe Ala Vai Thr Ser lie Leu

170 175

Arg Vai Ala Ala Glu Asp Trp Lys

180

Lys Gly Asp Thr Phe Ser Cys Met

185 190

Vai Gly His Glu Ala Leu Pro Leu

195 200

Ala Phe Thr Gin Lys Thr lie Asp 205

- 167 042507

Arg Leu Ala Gly Lys Gly Gly Gly Gly

210215

Ser Gly Leu Asn Asp He Phe

220

Glu Ala Gin Lys lie Glu Trp His Glu 225230 <210>81 <2H>641 <212> PROTEIN <213> Human herpesvirus 4 <400>81

Met Ser Asp Glu Gly Pro Gly Thr 15

Gly Pro Gly Asn Gly Leu Gly Glu

15

Lys Gly Asp Thr Ser Gly Pro Glu 20

Gly Ser Gly Gly Ser Gly Pro Gin 25 30

Arg Arg Gly Gly Asp Asn His Gly

40

Arg Gly Arg Gly Arg Gly Arg Gly 45

Arg Gly Gly Gly Arg Pro Gly Ala

55

Pro Gly Gly Ser Gly Ser Gly Pro 60

Arg His Arg Asp Gly Vai Arg Arg 65 70

Pro Gin Lys Arg Pro Ser Cys lie

80

Gly Cys Lys Gly Thr His Gly Gly 85

Thr Gly Ala Gly Ala Gly Ala Gly

95

Gly Ala Gly Ala Gly Gly Ala Gly 100

Ala Gly Gly Gly Ala Gly Ala Gly

105 110

Gly Gly Ala Gly Gly Ala Gly Gly

115 120

Ala Gly Gly Ala Gly Ala Gly Gly

125

Gly Ala Gly Ala Gly Gly Gly Ala

130 135

Gly Gly Ala Gly Gly Ala Gly Ala 140

Gly Gly Gly Ala Gly Ala Gly Gly 145 150

Gly Ala Gly Gly Ala Gly Ala Gly

155 160

Gly Gly Ala Gly Gly Ala Gly Gly

165

Ala Gly Ala Gly Gly Gly Ala Gly

170 175

Ala Gly Gly Gly Ala Gly Gly Ala 180

Gly Ala Gly Gly Gly Ala Gly Gly 185 190

Ala Gly Gly Ala Gly Ala Gly Gly

195 200

Gly Ala Gly Ala Gly Gly Ala Gly 205

Gly Ala Gly Gly Ala Gly Ala Gly

210 215

Gly Ala Gly Ala Gly Gly Gly Ala

220

Gly Gly Ala Gly Gly Ala Gly Ala 225 230

Gly Gly Ala Gly Ala Gly Gly Ala

235 240

Gly Ala Gly Gly Ala Gly Ala Gly

Gly Ala Gly Gly Ala Gly Ala Gly

- 168 042507

245 250 255

Gly Ala gly Gly 260 Ala gly Ala gly Gly 265 Ala gly gly Ala Gly 270 Ala gly Gly Gly Ala 275 gly gly Ala gly Ala 280 gly gly gly Ala Gly 285 gly Ala gly Ala Gly 290 gly Ala gly gly Ala 295 gly Ala gly gly Ala 300 gly gly Ala gly Ala Gly 305 gly Ala gly Gly 310 Ala gly Ala gly Gly 315 gly Ala gly Ala Gly 320 Gly Ala gly Ala Gly 325 gly gly gly Arg Gly 330 Arg gly gly Ser Gly 335 gly Arg Gly Arg Gly 340 gly Ser gly gly Arg 345 gly Arg gly gly Ser 350 gly gly Arg Arg Gly 355 Arg gly Arg Glu Arg 360 Ala Arg gly gly Ser 365 Arg Glu Arg Ala Arg 370 gly Arg gly Arg Gly 375 Arg gly Glu Lys Arg 380 Pro Arg Ser Pro Ser Ser 385 gin Ser Ser Ser 390 Ser gly Ser Pro Pro 395 Arg Arg Pro Pro Pro 400 Gly Arg Arg Pro Phe 405 Phe His Pro Vai Gly 410 Glu Ala asp Tyr Phe 415 Glu Tyr His gin Glu 420 gly gly Pro asp Gly 425 Glu Pro asp Vai Pro 430 Pro gly Ala lie Glu 435 gin gly Pro Ala Asp 440 asp Pro gly Glu Gly 445 Pro Ser Thr GlyPro 450 Arg gly gin gly Asp 455 gly gly Arg Arg Lys 460 Lys gly gly trp Phe Gly 465 Lys His Arg Gly 470 gin gly gly Ser Asn 475 Pro Lys Phe Glu Asn 480 lie Ala Glu gly Leu 485 Arg Ala Leu Leu Ala 490 Arg Ser His Vai Glu 495 Arg Thr Thr asp Glu 500 gly Thr trp Vai Ala 505 gly Vai Phe Vai Tyr 510 gly gly Ser Lys Thr 515 Ser Leu Tyr Asn Leu 520 Arg Arg gly Thr Ala 525 Leu Ala lie Pro Gin 530 Cys Arg Leu Thr Pro 535 Leu Ser Arg Leu Pro 540 Phe gly Met Ala Pro Gly 545 Pro gly Pro gin 550 Pro gly Pro Leu Arg 555 Glu Ser lie Vai Cys 560

- 169 042507

Tyr Phe Met Val Phe Leu Gln Thr 565

His lie Phe Ala Glu Val Leu Lys

570 575

Asp Ala lie Lys Asp Leu Val Met 580

Thr Lys Pro Ala Pro Thr Cys Asn 585 590 lie Arg Val Thr Val Cys Ser Phe

595 600

Asp Asp Gly Val Asp Leu Pro Pro 605

Trp Phe Pro Pro Met Val Glu Gly

610 615

Ala Ala Ala Glu Gly Asp Asp Gly

620

Asp Asp Gly Asp Glu Gly Gly Asp 625 630

Gly Asp Glu Gly Glu Glu Gly Gln

635 640

Glu <210>82 <211>321 <212> PROTEIN <213> Escherichia coli <400>82

Met Lys Asp Asn Thr Val Pro Leu 15

Lys Leu lie Ala Leu Leu Ala Asn

15

Gly Glu Phe His Ser Gly Glu Gln 20

Leu Gly Glu Thr Leu Gly Met Ser 25 30

Arg Ala Ala lie Asn Lys His lie

40

Gln Thr Leu Arg Asp Trp Gly Val 45

Asp Val Phe Thr Val Pro Gly Lys

55

Gly Tyr Ser Leu Pro Glu Pro lie 60

Gln Leu Leu Asn Ala Lys Gln lie 65 70

Leu Gly Gln Leu Asp Gly Gly Ser

80

Val Ala Val Leu Pro Val lie Asp 85

Ser Thr Asn Gln Tyr Leu Leu Asp 90 95

Arg lie Gly Glu Leu Lys Ser Gly

100

Asp Ala Cys lie Ala Glu Tyr Gln

105 110

Gln Ala Gly Arg Gly Arg Arg Gly

115 120

Arg Lys Trp Phe Ser Pro Phe Gly 125

Ala Asn Leu Tyr Leu Ser Met Phe

130 135

Trp Arg Leu Glu Gln Gly Pro Ala 140

Ala Ala lie Gly Leu Ser Leu Val 145 150

He Gly He Val Met Ala Glu Val

155 160

Leu Arg Lys Leu Gly Ala Asp Lys

165

Val Arg Val Lys Trp Pro Asn Asp

170 175

- 170 042507

Leu Tyr Leu Gin Asp Arg Lys Leu Ala Gly He Leu Vai Glu Leu Thr

180 185190

Gly Lys Thr Gly Asp Ala Ala Gin lie Vai lie Gly Ala Gly lie Asn

195 200205

Met Ala Met Arg Arg Vai Glu Glu Ser Vai Vai Asn Gin Gly Trp lie

210 215220

Thr Leu Gin Glu Ala Gly lie Asn Leu Asp Arg Asn Thr Leu Ala Ala

225 230 235240

Met Leu lie Arg Glu Leu Arg Ala Ala Leu Glu Leu Phe Glu Gin Glu

245 250255

Gly Leu Ala Pro Tyr Leu Ser Arg Trp Glu Lys Leu Asp Asn Phe lie

260 265270

Asn Arg Pro Vai Lys Leu lie lie Gly Asp Lys Glu lie Phe Gly lie

275 280285

Ser Arg Gly lie Asp Lys Gin Gly Ala Leu Leu Leu Glu Gin Asp Gly

290 295300 lie lie Lys Pro Trp Met Gly Gly Glu lie Ser Leu Arg Ser Ala Glu

305 310 315320

Lys <210>83 <2H>549 <212> DNA <213> Homo sapiens

400>83

atggaacagg ggaagggcct ggctgtcctc atcctggcta tcattcttct tcaaggtact 60 ttggcccagt caatcaaagg aaaccacttg gttaaggtgt atgactatca agaagatggt 120 tcggtacttc tgacttgtga tgcagaagcc aaaaatatca catggtttaa agatgggaag 180 atgatcggct tcctaactga agataaaaaa aaatggaatc tgggaagtaa tgccaaggac 240 cctcgaggga tgtatcagtg taaaggatca cagaacaagt caaaaccact ccaagtgtat 300 tacagaatgt gtcagaactg cattgaacta aatgcagcca ccatatctgg ctttctcttt 360 gctgaaatcg tcagcatttt cgtccttgct gttggggtct acttcattgc tggacaggat 420 ggagttcgcc agtcgagagc ttcagacaag cagactctgt tgcccaatga ccagctctac 480 cagcccctca aggatcgaga agatgaccag tacagccacc ttcaaggaaa ccagttgagg 540 aggaattga 549

<210>84 <2H> 182 <212> PROTEIN <213> Homo sapiens <400>84

Met Glu Gin Gly Lys Gly Leu Ala Vai Leu lie Leu Ala lie lie Leu

1015

- 171 042507

Leu Gin Gly Thr Leu Ala Gin Ser He Lys Gly Asn His Leu Vai Lys 20 2530

Vai Tyr Asp Tyr Gin Glu Asp Gly Ser Vai Leu Leu Thr Cys Asp Ala 35 4045

Glu Ala Lys Asn lie Thr Trp Phe Lys Asp Gly Lys Met lie Gly Phe 50 5560

Leu Thr Glu Asp Lys Lys Lys Trp Asn Leu Gly Ser Asn Ala Lys Asp 65 70 7580

Pro Arg Gly Met Tyr Gin Cys Lys Gly Ser Gin Asn Lys Ser Lys Pro 85 9095

Leu Gin Vai Tyr Tyr Arg Met Cys Gin Asn Cys He Glu Leu Asn Ala 100 105110

Ala Thr He Ser Gly Phe Leu Phe Ala Glu lie Vai Ser lie Phe Vai 115 120125

Leu Ala Vai Gly Vai Tyr Phe lie Ala Gly Gin Asp Gly Vai Arg Gin

130 135140

Ser Arg Ala Ser Asp Lys Gin Thr Leu Leu Pro Asn Asp Gin Leu Tyr

145 150 155160

Gin Pro Leu Lys Asp Arg Glu Asp Asp Gin Tyr Ser His Leu Gin Gly

165 170175

Asn Gin Leu Arg Arg Asn

180 <210>85 <2H>516 < 212> ДНК <213> Homo sapiens <400>85 atggaacata gcacgtttct ctctggcctg gtactggcta cccttctctc gcaagtgagc60 cccttcaaga tacctataga ggaacttgag gacagagtgt ttgtgaattg caataccagc120 atcacatggg tagagggaac ggtgggaaca ctgctctcag acattacaag actggacctg180 ggaaaacgca tcctggaccc acgaggaata tataggtgta atgggacaga tatatacaag240 gacaaagaat ctaccgtgca agttcattat cgaatgtgcc agagctgtgt ggagctggat300 ccagccaccg tggctggcat cattgtcact gatgtcattg ccactctgct ccttgctttg360 ggagtcttct gctttgctgg acatgagact ggaaggctgt ctggggctgc cgacacacaa420 gctctgttga ggaatgacca ggtctatcag cccctccgag atcgagatga tgctcagtac480 agccaccttg gaggaaactg ggctcggaac aagtga516 <210>86 <2H>171 <212> БЕЛОК <213> Homo sapiens <400>86

- 172 042507

Met Glu His Ser Thr Phe Leu Ser Gly Leu Val Leu Ala Thr Leu Leu 15 1015

Ser Gin Val Ser Pro Phe Lys He Pro lie Glu Glu Leu Glu Asp Arg 20 2530

Val Phe Val Asn Cys Asn Thr Ser He Thr Trp Val Glu Gly Thr Val 35 4045

Gly Thr Leu Leu Ser Asp lie Thr Arg Leu Asp Leu Gly Lys Arg lie 50 5560

Leu Asp Pro Arg Gly He Tyr Arg Cys Asn Gly Thr Asp He Tyr Lys 65 70 7580

Asp Lys Glu Ser Thr Val Gin Val His Tyr Arg Met Cys Gin Ser Cys 85 9095

Val Glu Leu Asp Pro Ala Thr Val Ala Gly lie lie Val Thr Asp Val

100 105110 lie Ala Thr Leu Leu Leu Ala Leu Gly Val Phe Cys Phe Ala Gly His

115 120125

Glu Thr Gly Arg Leu Ser Gly Ala Ala Asp Thr Gin Ala Leu Leu Arg

130 135140

Asn Asp Gin Val Tyr Gin Pro Leu Arg Asp Arg Asp Asp Ala Gin Tyr

145 150 155160

Ser His Leu Gly Gly Asn Trp Ala Arg Asn Lys

165170 <210>87 <2H>624 <212> DNA <213> Homo sapiens <400>87 atgcagtcgg gcactcactg gagagttctg ggcctctgcc tcttatcagt tggcgtttgg60 gggcaagatg gtaatgaaga aatgggtggt attacacaga caccatataa agtctccatc120 tctggaacca cagtaatatt gacatgccct cagtatcctg gatctgaaat actatggcaa180 cacaatgata aaaacatagg cggtgatgag gatgataaaa acataggcag tgatgaggat240 cacctgtcac tgaaggaatt ttcagaattg gagcaaagtg gttattatgt ctgctacccc300 agaggaagca aaccagaaga tgcgaacttt tatctctacc tgagggcaag agtgtgtgag360 aactgcatgg agatggatgt gatgtcggtg gccacaattg tcatagtgga catctgcatc420 actgggggct tgctgctgct ggtttactac tggagcaaga atagaaaggc caaggccaag480 cctgtgacac gaggagcggg tgctggcggc aggcaaaggg gacaaaacaa ggagaggcca540 ccacctgttc ccaacccaga ctatgagccc atccggaaag gccagcggga cctgtattct 600 ggcctgaatc agagacgcat ctga624 <210>88 <2H>207 <212> БЕЛОК <213 > Homo sapiens

- 173 042507 <4OO>88

Met Gin Ser Gly Thr His Trp Arg Vai Leu Gly Leu Cys Leu Leu Ser 15 1015

Vai Gly Vai Trp Gly Gin Asp Gly Asn Glu Glu Met Gly Gly He Thr 20 2530

Gin Thr Pro Tyr Lys Vai Ser He Ser Gly Thr Thr Vai He Leu Thr 35 4045

Cys Pro Gin Tyr Pro Gly Ser Glu lie Leu Trp Gin His Asn Asp Lys 50 5560

Asn lie Gly Gly Asp Glu Asp Asp Lys Asn lie Gly Ser Asp Glu Asp 65 70 7580

His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gin Ser Gly Tyr Tyr 85 9095

Vai Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu

100 105110

Tyr Leu Arg Ala Arg Vai Cys Glu Asn Cys Met Glu Met Asp Vai Met

115 120125

Ser Vai Ala Thr lie Vai lie Vai Asp lie Cys lie Thr Gly Gly Leu

130 135140

Leu Leu Leu Vai Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys

145 150 155160

Pro Vai Thr Arg Gly Ala Gly Ala Gly Gly Arg Gin Arg Gly Gin Asn

165 170175

Lys Glu Arg Pro Pro Pro Vai Pro Asn Pro Asp Tyr Glu Pro He Arg

180 185190

Lys Gly Gin Arg Asp Leu Tyr Ser Gly Leu Asn Gin Arg Arg lie 195 200205 <210>89 <2H>8 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence <400>89

Asp Tyr Lys Asp Asp Asp Asp Lys <210>90 <2H>6 <212> PROTEIN <213> Artificial sequence <220>

<223> Artificially synthesized peptide sequence

- 174 -

Claims (15)

<4OO>90 Gly Gly Ser Gly Gly Ser 15 <210>91 <211>9 <212> PROTEIN <213> Artificial sequence <220> <223> Artificially synthesized peptide sequence <400>91 Gly Gly Ser Gly Gly Ser Gly Gly Ser 15 <210>92 <211>7 <212> PROTEIN <213> Artificial sequence <220> <223> Artificially synthesized peptide sequence <400>92 Gly Gly Ser Gly Gly Gly Ser 15 <210> 93 <211>8 <212> PROTEIN <213> Artificial sequence <220> <223> Artificially synthesized peptide sequence <400> 93 Gly Gly Gly Ser Gly Gly Gly Ser 1 5 CLAIM 1. An antigen-binding molecule for cancer treatment, comprising an antibody variable region having binding activity to a first antigen and a second antigen that is different from the first antigen but does not bind to the first antigen and the second antigen at the same time, wherein either of the first and second antigens is a molecule , specifically expressed on the surface of T cells, and the other antigen is a molecule expressed on the surface of T cells or any other immunocyte; and a variable region that binds to a third antigen, which is a cancer antigen, wherein the first antigen is CD3, and the variable region of an antibody that is capable of binding the first and second antigens is obtained by replacing a portion of the amino acid sequence of the variable region that binds to the first antigen, to the amino acid sequence that binds to the second antigen, or inserts of such a portion and vice versa, and is a variable region comprising from 1 to 25 amino acid substitutions or insertions, and the amino acid that is replaced or inserted is an amino acid in the CDR1, CDR2, CD3 or FR3 region , or an amino acid selected from amino acids located at positions 31-35, 50-65, 71-74 and 95-102 in the variable domain of the antibody H chain according to Kabat numbering and at positions 24-34, 50-56 and 8997 in variable domain of the L chain according to Kabat numbering, and the antigen-binding molecule does not contain an Fc region or contains Fc region with reduced FcyR binding activity. 2. The antigen-binding molecule of claim 1, wherein the variable region that does not bind to the first antigen and the second antigen simultaneously is a variable region that does not bind to the first antigen and the second antigen simultaneously, each of which is expressed on different cells. 3. An antigen-binding molecule according to claim 1 or 2, comprising an Fc region of an antibody, which is an Fc region with reduced binding activity to the FcyR receptor. compared to the Fc region of a natural human IgG1 antibody. 4. An antigen-binding molecule according to any one of claims 1 to 3, wherein the antigen-binding molecule is - 175 042507 multispecific antibody. 5. An antigen-binding molecule according to any one of claims 1 to 4, wherein the variable region of the antibody capable of binding to the first antigen and the second antigen is a variable region with at least one amino acid residue altered. 6. An antigen binding molecule according to any one of claims 1 to 5, wherein either the first antigen and the second antigen is CD3 and the other antigen is FcyR, TLR, lectin, IgA, immune checkpoint molecule, TNF subfamily molecule, TNFR subfamily molecule, or receptor molecule killer cells. 7. An antigen binding molecule according to any one of claims 1 to 6, wherein the third antigen is a molecule specifically expressed in cancer tissue. 8. A pharmaceutical composition for the treatment of cancer, comprising an antigen-binding molecule according to any one of claims 1 to 7, and a pharmaceutically acceptable carrier. 9. A method for producing an antigen-binding molecule according to any one of claims 1 to 7, wherein the method comprises steps (i) to (iv): (i) obtaining a library of antigen-binding molecules with at least one altered amino acid residue in their antibody variable regions, each of which binds to the first antigen or to the second antigen, wherein the portion of the amino acid sequence of the variable region binding to the first antigen is replaced with an amino acid a sequence that binds to the second antigen, or an insertion of such a part and vice versa, and the altered variable regions differ from each other by at least one amino acid residue, where at least one altered amino acid residue in the variable regions is a substituted or inserted amino acid residue, and the change is a change in at least one amino acid residue selected from amino acid positions 31-35, 50-65, 71-74, and 95102 according to Kabat numbering in the H heavy chain variable domain, and 24-34, 50-56, and 89-97 according to numbering according to Kabat in the variable domain l a light chain L, (ii) selecting from the resulting library an antigen-binding molecule comprising a variable region having binding activity to the first antigen and to the second antigen, but which does not bind to the first antigen and to the second antigen simultaneously, (iii) culturing the host cells, comprising a nucleic acid encoding the variable region of the antigen-binding molecule selected in step (ii) and a nucleic acid encoding the variable region of the antigen-binding molecule that binds to a third antigen for expressing an antigen-binding molecule comprising an antibody variable region that binds to the first antigen and to the second antigen, but does not bind to the first antigen and the second antigen at the same time, and/or a variable region that binds to the third antigen, and the host cells cultured at this stage may additionally include a nucleic acid encoding the Fc region of the antibody, which is Fc- area with lower significant binding activity for the FcyR receptor compared to the Fc region of a native human IgG1 antibody, and (iv) isolating the antigen-binding molecule from host cell cultures. 10. The method according to claim 9, wherein the variable region that does not bind both the first antigen and the second antigen at the same time contained in the antigen-binding molecule selected in step (ii) is the variable region that does not bind both the first antigen and the second antigen , each of which is expressed on different cells. 11. The method of any one of claims 9-10, wherein the antigen-binding molecule produced is a multispecific antibody. 12. The method of claim 9 wherein the number of amino acid residues inserted is between 1 and 25. 13. The method of any one of claims 9-12, wherein the change is a change in an amino acid residue in the CDR1, CDR2, CDR3, or FR3 regions of the variable region of the antibody. 14. The method of any one of claims 9-13, wherein the first antigen is CD3 and the second antigen is any other antigen selected from FcyR, TLR, lectin, IgA, 274 immune checkpoint molecule, TNF superfamily molecule, TNFR superfamily molecule, or killer cell receptor molecule. 15. The method according to any one of claims 9 to 14, wherein the third antigen is a molecule specifically expressed in cancer tissue. - 176 -