CN112694532B - Antibody against Siglec-15 or antigen binding fragment thereof and application - Google Patents

Abstract

The present invention provides antibodies or antigen binding fragments thereof, particularly humanized monoclonal antibodies to Siglec-15, which antibodies comprise a heavy chain and a light chain and specifically bind to Siglec-15 with high affinity. The invention also provides nucleic acid sequences encoding the antibodies of the invention, cloning or expression vectors, host cells, and anti-tumor effects in vitro experiments. The invention further provides the use of anti-Siglec-15 antibodies for the treatment of various cancers.

Description

Antibody against Siglec-15 or antigen binding fragment thereof and application

Technical Field

The invention relates to the biomedical field, in particular to the antibody technical field and the immune field, more particularly to the preparation and application of an antibody against Siglec-15, in particular to the treatment of solid tumors by regulating the Siglec-15 mediated immunosuppression, in particular the inhibition of T cell proliferation, in the tumor microenvironment.

Background

The Siglec sialic acid binding Ig-like lectin family is a member of the Ig superfamily, expressed on a variety of leukocytes, and recognizes glycoprotein sialic acid structures expressed on the cell surface, mediating signal transduction. The Siglec family is the largest known group of vertebrate lectins (Varki angata 2006;Crocker et al.2007) that recognize sialylated glycans. Most mammalian Siglecs expressed in the immune system have an Immunoreceptor Tyrosine Inhibitory Motif (ITIM) in the cytoplasmic domain, which has been demonstrated to have negative regulation on cells expressing them (Otipoby et al 1996; sato et al 1996; nitschke et al 1997; mingari et al 2001; ulyanova et al 2001; nutku et al 2003; von Gunten et al 2005). Recently, siglecs have been found to be associated with the signal adapter molecule DNAX activation protein DAP12, which has an immunoreceptor tyrosine-based activation motif (ITAM) and is involved in immune cell activation (Takamiya et al, glycobiology, 23 (2): 178-87 (2013)), revealing that the subset of Siglecs has the potential to activate signal pathways (Angata et al 2006; blasius et al 2006).

Siglec-15, also known as CD33L3, is a newly discovered member of the Siglec family. Siglec-15 is a type I transmembrane protein having an extracellular domain of 244 amino acids, including Ig-like V-type domains and Ig-like C2-type domains. Siglec-15 is expressed on cells of the immune system, recognizes sialylated ligands, and can interact with the signal adapter molecules DNAX activation proteins DAP12 and DAP10 through lysine residues in the transmembrane domain. Siglec-15 is the immune system Siglec conserved during vertebrate evolution (TakashiAngata et al glycobiology, 2007). Several previous studies have shown that Siglec-15 plays an important role in osteoclast differentiation (Yoshiharu Hiruma et al. Biochemical and biophysical research Communications 2011;Norihiro Ishida-Kitagawa et al. J. Biochem., 2012). Siglec-15 on osteoclast precursors recognizes CD44 on neighboring osteoclast precursors and transduces signals through the DAP12-Syk pathway, cross-talk with the RANK-TRAF6 pathway and enhance downstream signals (e.g., ERK and PI 3K-AKT).

Siglec-15 is also involved in the regulation of immune cells and tumor microenvironment. Studies have shown that Siglec-15 preferentially binds to sialic acid-Tn (Neu 5 Ac. Alpha.2-6 GalNAc. Alpha.1) structure, which is found in several tumor associated antigens such as MUC-1, whose high expression is associated with poor prognosis (TakashiAngata et al, glycobiology, 2007;Jennifer Munkley, J.International molecular science, 2016). Siglec-15 is expressed on tumor-associated macrophages (TAMs) of various human tumor tissues, which significantly elevated expression in macrophage colony stimulating factor-induced M2-like macrophages, siglec-15 produces more transforming growth factor-beta (TGF-beta) on sTn positive cells than negative cells, indicating that Siglec-15 recognizes tumor sTn antigen and transduces signals of enhanced TGF-beta secretion in TAMs. Recent studies (Jun Wang et al, nature medicine, 2019) show that Siglec-15 may be an important immune checkpoint, expressed only on PD-L1 expressing tumor cells, and interacting with unknown receptors expressed on T cells, down regulating T cell proliferation. In a mouse tumor model, an antibody against Siglec-15 has been shown to have anti-tumor efficacy in vitro T cell proliferation rescue experiments.

Disclosure of Invention

The present invention provides antibodies or antigen binding fragments thereof, particularly humanized monoclonal antibodies to Siglec-15, which antibodies comprise a heavy chain and a light chain and specifically bind to Siglec-15 with high affinity. The invention also provides a nucleic acid sequence for encoding the antibody, a cloning or expression vector, a host cell and an anti-tumor effect in vitro experiments. The invention further provides the use of Siglec-15 antibodies for the treatment of various cancers.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

in some embodiments, an antibody or antigen binding fragment thereof provided herein that is directed against Siglec-15 comprises: a heavy chain variable region VH sequence having at least 95% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO:1, seq ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, seq ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, seq ID NO:23, SEQ ID NO:25, SEQ ID NO:27, seq ID NO:29, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47; a light chain variable region VL sequence having at least 95% identity to an amino acid sequence selected from the group consisting of: SEQ ID NO:2, seq ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, seq ID NO:22, seq ID NO:24, SEQ ID NO:26, seq ID NO:28, seq ID NO:30, SEQ ID NO:33, seq ID NO:34, SEQ ID NO:38, seq id NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48.

in some embodiments, an antibody or antigen binding fragment thereof provided herein that is directed against Siglec-15 comprises: a VH/VL sequence pair having at least 95% identity to a VH and VL amino acid sequence pair selected from the group consisting of: SEQ ID NO:1 and SEQ ID NO:2, seq ID NO:3 and SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:13, SEQ ID NO:16 and SEQ ID NO:13, SEQ ID NO:17 and SEQ ID NO:13, SEQ ID NO:18 and SEQ ID NO:13, SEQ ID NO:17 and SEQ ID NO:19, SEQ ID NO:18 and SEQ ID NO:19, SEQ ID NO:17 and SEQ ID NO:20, SEQ ID NO:18 and SEQ ID NO:20, SEQ ID NO:12 and SEQ ID NO:21, seq ID NO:14 and SEQ ID NO:22, seq ID NO:15 and SEQ ID NO:22, seq ID NO:16 and SEQ ID NO:22, seq ID NO:17 and SEQ ID NO:22, seq ID NO:18 and SEQ ID NO:22, seq ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, seq ID NO:27 and SEQ ID NO:28, seq ID NO:29 and SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:11, SEQ ID NO:32 and SEQ ID NO:33, seq ID NO:32 and SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:33, seq ID NO:32 and SEQ ID NO:11, SEQ ID NO:36 and SEQ ID NO:11, SEQ ID NO:37 and SEQ ID NO:38, seq ID NO:39 and SEQ ID NO:38, seq ID NO:40 and SEQ ID NO:38, seq ID NO:41 and SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:48.

the sequences are shown in the following table:

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in some embodiments, an antibody or antigen binding fragment thereof against Siglec-15 provided herein comprises or consists of a sequence of one or more heavy chain complementarity determining regions (HCDRs) selected from the group consisting of (1) HCDR1 comprising or consisting of an amino acid sequence having at least 95% identity to the amino acid sequence selected from the group consisting of SEQ ID NO. 49,SEQ ID NO:55,SEQ ID NO:61,SEQ ID NO:67,SEQ ID NO:69,SEQ ID NO:74,SEQ ID NO:77,SEQ ID NO:90; (2) HCDR2 comprising or consisting of an amino acid sequence having at least 95% identity to an amino acid sequence selected from SEQ ID No. 56,SEQ ID NO:62,SEQ ID NO:68,SEQ ID NO:70,SEQ ID NO:75,SEQ ID NO:78,SEQ ID NO:80,SEQ ID NO:83,SEQ ID NO:85,SEQ ID NO:87,SEQ ID NO:91,SEQ ID NO:92,SEQ ID NO:95,SEQ ID NO:97,SEQ ID NO:98; (3) HCDR3 comprising or consisting of an amino acid sequence having at least 95% identity to an amino acid sequence selected from SEQ ID No. 51,SEQ ID NO:57,SEQ ID NO:63,SEQ ID NO:71; (4) HCDR of (1), (2) and (3) above containing one or more amino acid substitutions, deletions or insertions of not more than 5 amino acids.

In some embodiments, an antibody or antigen binding fragment thereof provided herein against Siglec-15 comprises a sequence of one or more light chain complementarity determining regions LCDR selected from (1) LCDR1 comprising a sequence selected from the group consisting of: 52, SEQ ID NO:58, SEQ ID NO:64, SEQ ID NO:72,SEQ ID NO:81,SEQ ID NO:100, seq id no:93, SEQ ID NO:99 or consists of an amino acid sequence having at least 95% identity to the amino acid sequence of seq id no; (2) LCDR2 comprising or consisting of an amino acid sequence having at least 95% identity to an amino acid sequence selected from SEQ ID No. 59,SEQ ID NO:65,SEQ ID NO:73,SEQ ID NO:76,SEQ ID NO:79,SEQ ID NO:82,SEQ ID NO:84,SEQ ID NO:88; (3) LCDR3 comprising or consisting of an amino acid sequence having at least 95% identity to an amino acid sequence selected from SEQ ID No. 60,SEQ ID NO:66,SEQ ID NO:89,SEQ ID NO:94,SEQ ID NO:96; (4) LCDR of the above (1), (2) and (3) containing one or more amino acid substitutions, deletions or insertions of not more than 5 amino acids.

In some embodiments, an antibody or antigen binding fragment thereof provided herein that is directed against Siglec-15 comprises an HCDR1/HCDR2/HCDR3 combination and/or an LCDR1/LCDR2/LCDR3 combination: (1) The HCDR1/HCDR2/HCDR3 combination comprises a sequence of one or more sets of heavy chain complementarity determining regions HCDR selected from the group consisting of: the HCDR1/HCDR2/HCDR3 is selected from the group consisting of SEQ ID NO 49/SEQ ID NO 50/SEQ ID NO 51, SEQ ID NO 55/SEQ ID NO 56/SEQ ID NO 57, SEQ ID NO 61/SEQ ID NO 62/SEQ ID NO 63, SEQ ID NO 67/SEQ ID NO 68/SEQ ID NO 63, SEQ ID NO 69/SEQ ID NO 70/SEQ ID NO 71, SEQ ID NO 74/SEQ ID NO 75/SEQ ID NO 63, SEQ ID NO 77/SEQ ID NO 78/SEQ ID NO 63, SEQ ID NO 74/SEQ ID NO 80/SEQ ID NO 63, SEQ ID NO 74/SEQ ID NO 83/SEQ ID NO 63, SEQ ID NO 74/SEQ ID NO 78/SEQ ID NO 63, SEQ ID NO 85/SEQ ID NO 63, SEQ ID NO 78/SEQ ID NO 87, SEQ ID NO 74/SEQ ID NO 87, SEQ ID NO 78/SEQ ID NO 63, SEQ ID NO 78/SEQ ID NO 63, 78/SEQ ID NO 63, 74/SEQ ID NO 63, and SEQ ID NO 63, and SEQ ID NO 74/SEQ ID NO 63, 74/98/63; (2) The LCDR1/LCDR2/LCDR3 combination comprises a sequence of one or more light chain complementarity determining regions LCDR selected from the group consisting of: LCDR1/LCDR2/LCDR3 is selected from the group consisting of SEQ ID NO 52/SEQ ID NO 53/SEQ ID NO 54, SEQ ID NO 58/SEQ ID NO 59/SEQ ID NO 60, SEQ ID NO 64/SEQ ID NO 65/SEQ ID NO 66, SEQ ID NO 72/SEQ ID NO 73/SEQ ID NO 66, SEQ ID NO 64/SEQ ID NO 76/SEQ ID NO 66, SEQ ID NO 64/SEQ ID NO 79/SEQ ID NO 66, SEQ ID NO 81/SEQ ID NO 82/SEQ ID NO 66, SEQ ID NO 64/SEQ ID NO 84/SEQ ID NO 66, SEQ ID NO 64/SEQ ID NO 82/SEQ ID NO 66, SEQ ID NO 88/SEQ ID NO 66, SEQ ID NO 100/SEQ ID NO 88/SEQ ID NO 89, SEQ ID NO 93/SEQ ID NO 88/SEQ ID NO 94, SEQ ID NO 82/SEQ ID NO 66, SEQ ID NO 84/SEQ ID NO 88/SEQ ID NO 66, SEQ ID NO 64/SEQ ID NO 88/SEQ ID NO 96; (3) Substitution, deletion or insertion of the amino acid sequence of the CDRs described above containing one or more than 5 amino acids.

The sequences are shown in the following table:

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in some embodiments, an antibody or antigen binding fragment thereof provided herein that is directed against Siglec-15 comprises: a human constant domain which is IgA, igD, igE, igG or IgM domain.

In some embodiments, the human IgG constant domain is an IgG1, igG2, igG3, or IgG4 domain.

In some embodiments, the antibody against Siglec-15 is a murine monoclonal antibody, a chimeric monoclonal antibody, a humanized antibody, a single chain antibody, a light chain constant region thereof, optionally a human antibody kappa chain or lambda chain constant region, a heavy chain constant region, optionally a human antibody IgG1 or IgG4 constant region, and a mutant of an IgG1 or IgG4 constant region.

In some embodiments, the antibody comprises a monospecific antibody, bispecific antibody or fusion protein, trispecific antibody or fusion protein, or multispecific antibody.

In some embodiments, the invention provides a bispecific antibody or bispecific fusion protein, the other end of which targets a tumor-associated antigen of a tumor cell or targets an immunoassay target of an immune cell, comprising EGFR, HER2, CD47, PD-1, PD-L1, CTLA4, tgfβ, VEGF, HER3, TIGIT, CLAUDIN18.2, CD3, TIM3, LAG3, and the like.

In some embodiments, the invention provides a trispecific antibody or trispecific fusion protein, the other two ends of which target a tumor-associated antigen of a tumor cell or target an immunoassay target of an immune cell, comprising EGFR, HER2, CD47, PD-1, PD-L1, CTLA4, tgfβ, VEGF, HER3, TIGIT, CLAUDIN18.2, CD3, TIM3, LAG3, and the like.

In some embodiments, the invention provides a nucleic acid sequence encoding an antibody or antigen binding fragment thereof against Siglec-15 as described above.

Preferably, the nucleic acid sequence provided by the invention comprises SEQ ID NO:101, SEQ ID NO: 102. SEQ ID NO: 103. SEQ ID NO. 104 sequence. The following table shows:

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in some embodiments, the invention provides an expression vector comprising the nucleic acid sequence described above.

In some embodiments, the present invention provides a host cell comprising the above expression vector, preferably the host cell is selected from mammalian cells.

In some embodiments, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof (also including bispecific antibodies or fusion proteins, trispecific antibodies or fusion proteins) as described above against Siglec-15, and a pharmaceutically acceptable carrier.

In some embodiments, an antibody or antigen binding fragment thereof provided herein that is directed against Siglec-15 is therapeutically active in expressing or over-expressing Siglec-15 cancer cells, macrophages, or cancer.

In some embodiments, the antibodies provided herein against Siglec-15, or antigen binding fragments thereof, inhibit osteoclastogenesis and are useful in the treatment of osteoporosis.

In some embodiments, the invention provides a method of inhibiting osteoclastogenesis comprising administering to a subject in need thereof an effective amount of an antibody to Siglec-15, or an antigen binding fragment thereof, described above.

In some embodiments, the invention provides the use of an antibody, antigen binding fragment thereof, bispecific antibody, bispecific fusion protein, trispecific antibody, trispecific fusion protein, and a pharmaceutically optional pharmaceutical carrier against Siglec-15 in the manufacture of a medicament for the treatment of a tumor selected from the group consisting of melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, and rectal cancer.

Furthermore, in some embodiments, the antibodies provided herein against Siglec-15, or antigen binding fragments thereof, are capable of enhancing an immune response, blocking or preventing tumor growth, inhibiting tumor-mediated immunosuppression, eliminating tumors, depleting or blocking the activity of tumor-associated macrophages (TAMs) so as to alter their activity, reduce TAM-mediated immunosuppression, reduce or reverse T cell inhibition; can enhance the activation of cytokines such as IFNgamma, TNF-alpha secretion and CD4+ T cells or CDT cells; siglec-15 binding to its ligand can be increased and/or Siglec-15 mediated signal transduction can be increased or enhanced. Antibodies will also exhibit impaired, enhanced or Fc receptor (FcR) binding activity as well as enhanced antibody dependent cell-mediated cytotoxicity (ADCC) or Complement Dependent Cytotoxicity (CDC) activity upon modification.

The beneficial effects of the invention are as follows:

the anti-Siglec 15 antibody provided by the invention is screened from a series of technical routes such as antigen design and synthesis, mouse immunity, cell fusion, hybridoma screening, subcloning identification, in-vitro protein binding experiments, affinity identification, epitope identification, cell binding experiments with human/mouse/cynomolgus monkey, T cell related function experiments, antibody humanization, antibody affinity maturation, osteoclast differentiation experiments and the like to obtain a murine antibody (11H 5E6, 48B5B1 and 69C4D 8) with good biological activity and a series of humanized and affinity matured antibody molecules, wherein the L2H1-58-PTM, 14-58-2 and other antibody molecules show better affinity than the control antibody 5G12 in the cell binding experiments, and show good activity in-vitro T cell proliferation experiments and the like. There were significant differences in binding epitopes and sequences from the control antibodies. Has a great application prospect in the fields of cancer and loosening.

Drawings

FIG. 1 shows the results of SDS-PAGE experimental identification of BPSL15-C protein.

FIG. 2a shows the results of ELISA experiments for detecting antibody titers by binding of mouse serum to human Siglec-15 protein; FIG. 2b shows the results of FACS experiments to detect antibody titers from mouse serum binding to human Siglec-15 protein.

FIG. 3 shows the results of FACS experiments identifying the binding of selected clones to CHOS cells expressing human siglec-15 after screening by antigen binding ELISA.

FIGS. 4a and 4b show the results of FACS comparison screening of blank CHOS cells and human siglc-15 expressing CHOS cells on subcloned hybridoma cell lines.

FIGS. 5a, 5b, 5c and 5d show experimental results of cell binding assays of the 26 subcloned cell lines identified by antibody expression, respectively with blank CHOS cells, CHOS cells expressing human Siglec-15, CHOS cells expressing cynomolgus monkey Siglec-15 and CHOS cells expressing mouse Siglec-15, respectively.

FIG. 6 shows the results of a recovery CD4/CD 8T cell proliferation assay of antibody supernatants from 26 hybridoma cell lines with 4 blood cell donors.

FIGS. 7a and 7b show osteoclast differentiation experiments with antibodies purified from 26 hybridoma cell supernatants.

FIG. 8 shows the affinity results of three antibodies 11H5E6, 48B5B1 and 69C4D8 as determined by Fortebio.

Fig. 9a, 9B, and 9C show the experimental results of FACS cell binding of the 11H5E6, 48B5B1, and 69C4D8 chimeric antibodies to CHOS cells expressing human Siglec-15, CHOS cells expressing cynomolgus monkey Siglec-15, and CHOS cells expressing mouse Siglec-15, respectively.

FIGS. 10a and 10B show the results of T cell proliferation experiments with the 30C2B12 chimeric antibody and the negative control antibody.

FIG. 11 shows the results of an experiment for detecting the relative binding epitope of a candidate antibody to 5G12 by BLI technique (forteBio).

Fig. 12a, 12b, and 12c show the results of thermal stability experiments performed by melting curves.

Figure 13 shows the affinity results of humanized antibodies as determined by Fortebio.

FIG. 14 shows the results of FACS affinity experiments of humanized antibodies with CHOS cells expressing human Siglec-15.

FIG. 15 shows the results of an experiment for the proliferation of humanized antibodies in recovering T cells.

FIG. 16 shows the results of an experiment in which affinity matured antibodies were FACS cell-bound to CHOS cells expressing human Siglec-15.

FIG. 17 shows the results of an experiment in which affinity matured antibodies were FACS cell-bound to murine Siglec-15-expressing CHOS cells.

FIG. 18 shows the results of an experiment in which affinity matured antibodies were FACS cell-bound to cynomolgus monkey Siglec-15-expressing CHOS cells.

FIG. 19 shows the affinity results of affinity matured antibody antibodies by Fortebio assay

FIG. 20 shows the results of experiments designed to optimize FACS cell binding of antibodies to CHOS cells expressing human Siglec-15.

FIG. 21 shows the results of experiments designed for FACS cell binding of optimized antibodies to cynomolgus monkey Siglec-15 expressing CHOS cells.

FIG. 22 shows the results of experiments designed for FACS cell binding of optimized antibodies to mouse Siglec-15 expressing CHOS cells.

FIGS. 23, 24 and 25 show the results of experiments for recovering T cell proliferation from L2H1-58-PTM and 14-58-2 antibody molecules.

Detailed Description

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Definition of terms:

the term "antibody" is used herein in its broadest sense and encompasses a variety of antibody constructs, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. An "antibody" (or "Abs") and an "immunoglobulin" (or "Igs") are glycoproteins having the same structural characteristics, an antibody will typically comprise at least two full length heavy chains and two full length light chains, but may in some cases comprise fewer chains, e.g. an antibody naturally occurring in a camel may comprise only heavy chains. Each light chain is linked to a heavy chain (also referred to as a "VH/VL pair") by a covalent disulfide bond, each heavy and light chain also having regularly arranged intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by multiple constant domains. One variable domain (VL) at one end of each light chain and one constant domain at the other; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain.

The term "monoclonal antibody" refers to a preparation of antibody molecules having a single amino acid composition, and does not relate to a method of preparation thereof. Monoclonal antibodies or immunologically active fragments thereof may be produced by hybridoma techniques, recombinant techniques, phage display techniques, synthetic techniques, and the like, or other production techniques known in the art, and methods of the invention involving monoclonal antibody production include in vitro culture of hybridoma cells or by DNA recombination techniques. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Each monoclonal antibody is directed against a single determinant on the antigen.

The term "variable" refers to the fact that certain portions of the sequence of the variable domains of an antibody vary to a relatively high degree and are used for the binding and specificity of each particular antibody for its particular antigen. However, variability is unevenly distributed throughout the variable region of the antibody. It concentrates in three segments in the light and heavy chain variable regions, known as Complementarity Determining Regions (CDRs) or hypervariable regions. The more highly conserved parts in the variable domain are called Frameworks (FR). The variable domains of the natural heavy and light chains each comprise four FR regions, and the two light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the N-terminus to the C-terminus. Most FR regions adopt a β -sheet configuration, are connected by three CDRs, form loop junctions, and in some cases form part of a β -sheet structure. The CDRs in each chain are in close proximity by the FR region and together with the CDRs from the other chain contribute to the formation of the antigen binding site of the antibody. Please refer to, for example, kabat et al, immunological protein sequences, fifth edition, national institutes of health, bescens, maryland (1991). The constant region is not directly involved in binding of an antibody to an antigen, but exhibits various biological effector functions such as antibody-dependent cellular cytotoxicity (ADCC), complement killing pathway (CDC), and the like.

Antibodies can be classified into 5 classes according to the amino acid sequence of the heavy chain constant region of the antibody: igA, igD, igE, igG and IgM, and several isotypes in these classes can be further divided into subclasses, e.g., igG1, igG2, igG3 and IgG4, igA1, and IgA2. The constant domains of the heavy chain corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. Kappa and lambda can be distinguished by the difference in antibody light chain constant regions (CL). Within full length light and heavy chains, typically the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids.

An "antigen binding fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that is capable of binding to an antigen to which the intact antibody binds. Wherein the portion does not contain constant heavy chain domains (i.e., CH2, CH3, and CH4, depending on the antibody isotype) in the Fc region of the intact antibody. Examples of immunologically active fragments of antibodies include Fab, fab ', fab ' -SH, F (ab ') 2, scFv and Fv fragments. The Fab fragment comprises the heavy/light chain variable region and the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments in that several residues are added at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. F (ab ') 2 antibody fragments were originally produced as pairs of Fab ' fragments with hinge cysteines between the Fab ' fragments. Fv fragments are the smallest fragments of the products of antibodies of the IgG and IgM types after enzymatic analysis. Fv fragment antigen binding regions consisting of VH and VC regions, but they lack CH1 and CL regions. VH and VL are joined together in Fv fragments by a non-covalent bond. ScFv is an Fv-type fragment comprising VH and VL regions linked together by a flexible polypeptide.

The term "chimeric antibody" as used herein refers to a portion of the heavy and/or light chain (generally referred to as the variable region) that is derived from a particular species or from an antibody belonging to a particular antibody class or subclass, and the remainder of the chain (generally referred to as the constant region) that is derived from an antibody of another species or from an antibody belonging to another antibody class or subclass, and fragments of such antibodies, that are identical or homologous so long as they exhibit the desired biological activity. Chimeric antibodies referred to in the present invention, such as the heavy/light chain variable region from murine Siglec-15 antibody, are grafted to the constant region of the heavy/light chain of a human antibody by antibody engineering techniques, which exhibit similar biological activity.

The term "humanized antibody" refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to sequences of a non-human antibody and all or substantially all of the FRs correspond to sequences of a human antibody. The humanized antibody is mainly a murine monoclonal antibody which is reformed and re-expressed by gene cloning and DNA recombination technology, most of amino acid sequences (mainly comprising a constant region and an FR region) of the humanized antibody are replaced by human sequences, and the CDR region is subjected to no change or a small amount of mutation, so that the affinity and the specificity of the parent murine monoclonal antibody are basically reserved, the heterology of the humanized antibody is reduced, and the immunogenicity caused in a human body is reduced.

The term "bispecific antibody" is an artificial antibody containing 2 specific antigen binding sites, which can bridge the bridge between target cells and functional molecules (cells), and excite immune response with guidance, and is one of genetically engineered antibodies. Diabodies may be bivalent or bispecific. Diabodies are more fully described in, for example, EP 404,097; WO 1993/01161; hudson et al, nat. Med.9:129-134 (2003); and Hollinger et al, proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

The term "trispecific antibody" is an artificial antibody containing 3 specific antigen binding sites, which can bridge the bridge between target cells and functional molecules (cells), and excite immune response with guidance, which is one of genetically engineered antibodies. The tri-antibodies may be trivalent or tri-specific.

The term "epitope" refers to any antigenic determinant on an antigen that binds to the paratope of an antibody. Epitope determinants generally consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and generally have specific three dimensional structural characteristics as well as specific charge characteristics.

The term "vector" as used herein refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked by transformation. The term includes vectors that are self-replicating nucleic acid structures and that bind to the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors".

The term "host cell" refers to a cell that is foreign to nucleic acid, including the progeny of such a cell. And is capable of expressing the exogenous nucleic acid in a cell or cell membrane or released outside the cell.

The term "cancer" refers to a neoplasm or tumor resulting from abnormal uncontrolled growth of cells. As used herein, cancer expressly includes leukemia and lymphoma. The term "cancer" refers to a disease involving cells that have the potential to metastasize to distant sites and that exhibit a different phenotypic trait than non-cancerous cells, such as colony formation in a three-dimensional matrix such as soft agar or tubular or reticulum formation in a three-dimensional basement membrane or extracellular matrix formulation. Non-cancerous cells do not form colonies in soft agar, but rather form distinct globular structures in a three-dimensional basement membrane or extracellular matrix preparation.

The terms "immunity", "immunological" or "immune" response are humoral (antibody-mediated) and/or cellular (mediated by antigen-specific T cells or secretion products thereof) responses that are beneficial for peptide development in a recipient patient. Such a response may be an active response induced by administration of an immunogen or a passive response induced by administration of an antibody or priming T cells. Cellular immune responses are elicited by presentation of polypeptide epitopes associated with class I or class II MHC molecules to activate antigen-specific cd4+ T helper cells and/or cd8+ cytotoxic T cells. The response may also involve activation or recruitment of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, neutrophils or other components of innate immunity. The presence of a cell-mediated immune response can be determined by proliferation assays (cd4+ T cells) or CTL (cytotoxic T lymphocytes) assays. The relative contributions of the humoral and cellular responses to the protective or therapeutic effects of the immunogens can be distinguished by isolating antibodies and T cells from the immunized syngeneic animals alone and measuring the protective or therapeutic effects in the second subject.

The term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains substantial properties. Typical variants of a polypeptide differ in amino acid sequence from another reference polypeptide. Typically, the differences are limited such that the sequences of the reference polypeptide and the variant are very similar overall and are identical in many regions. Variants and reference polypeptides may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). The substituted or inserted amino acid residue may or may not be an amino acid residue encoded by the genetic code. Variants of the polypeptide may be naturally occurring, such as allelic variants, or may be variants that are not known to occur naturally.

In making such changes, the hydropathic index of amino acids may be considered. The importance of amino acid hydropathic index in conferring interactive biological function on a polypeptide is generally understood in the art. It is known that certain amino acids may be substituted for other amino acids having similar hydropathic indices or scores and still produce polypeptides having similar biological activities. The hydropathic index has been assigned to each amino acid based on its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The relative hydrophilic nature of amino acids is believed to determine the secondary structure of the resulting polypeptide, which in turn defines the interaction of the polypeptide with other molecules such as enzymes, substrates, receptors, antibodies, antigens and cofactors. It is known in the art that an amino acid may be substituted with another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such a modification, amino acids whose hydropathic index is within.+ -. 2, particularly those within.+ -. 1, and even more particularly those within.+ -. 0.5 are preferably substituted.

Substitution of similar amino acids may also be made based on hydrophilicity, particularly where the resulting biologically functionally equivalent polypeptide or peptide is intended for use in immunological embodiments. Amino acid residues have been assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5±1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It will be appreciated that an amino acid may be substituted for another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent, in particular immunologically equivalent, polypeptide. In such a modification, amino acids having hydrophilicity values within.+ -. 2 are preferably substituted, those within.+ -. 1 are particularly preferred, and those within.+ -. 0.5 are even more particularly preferred.

As noted above, amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into account various of the foregoing features are well known to those skilled in the art and include (original residues: exemplary substitutions): (Ala: gly, ser), (Arg: lys), (Asn: gln, his), (Asp: glu, cys, ser), (Gln: asn), (Glu: asp), (Gly: ala), (His: asn, gln), (Ile: leu, val), (Leu: ile, val), (Lys: arg), (Met: leu, tyr), (Ser: thr), (Thr: ser), (Tip: tyr), (Tyr: trp, phe) and (Val: ile, leu). Thus, embodiments of the present disclosure contemplate functional or biological equivalents of the antibody sequences described above. In particular, large embodiments may include variants having about 95% or more sequence identity to an antibody sequence.

The experimental methods in the following examples are conventional methods unless otherwise specified.

Examples

Example 1 production of lead anti-Siglec-15 antibodies

1.1 antigen design and Synthesis: the extracellular domain of the human Siglec-15 protein (amino acids 20-263, https:// www.uniprot.org/blast/: "FVRTKIDTTENLLNTEVHSSPAQRWSMQVPPEVSAEAGDAAVLPCTFTHPHRHYDGPLTAIWRAGEPYAGPQVFRCAAARGSELCQTALSLHGRFRLLGNPRRNDLSLRVERLALADDRRYFCRVEFAGDVHDRYESRHGVRLHVTAAPRIVNISVLPSPAHAFRALCTAEGEPPPALAWSGPALGNSLAAVRSPREGHGHLVTAELPALTHDGRYTCTAANSLGRSEASVYLFRFHGASGAST". The DNA encoding the above amino acid sequence was called together, and ligated with the DNA encoding camel FC, and the synthesized gene was constructed into a PCDNA3.4 vector. The plasmid transformed E.coli TOP10 was amplified overnight in LB medium containing 1% ampicillin after picking up the monoclonal. After centrifugation at 10000G for 10 minutes, the plasmid was extracted by alkaline lysis (Biomiga, PD 1511).

When the animal immunization is carried out by adopting the antigen of human Fc and the extracellular domain of Siglec15, the antigen expression yield is not high, and the animal serum is not easy to transfer to the sun. In order to overcome the limitations of protein immunization in the immunization process and the problem of positive transfer efficiency, camel Fc is adopted to connect with the extracellular domain of Siglec15, and three linear immune peptides of NH2-CTFTHPHRHYDGPLT-CONH2, NH2-CRVEFAGDVHDRYESR-CONH2NH2-CWRAGEPYAGPQV-CONH2 are creatively designed aiming at the Siglec15 protein to couple with KLH protein, and one cyclic peptide NH2-CTFTHPHRHYDGPLTC-CONH2 is adopted to carry out auxiliary boost immunization so as to break through the immune barrier.

Using thermo Expi293 ^TM Expression System Kit (A14635) produces antigen. The Expi293 cells were resuscitated, plasmid transfection was performed using the kit recommended method, and cell culture was performed. The culture medium was collected and centrifuged at 5000G for 10 minutes to remove cells and cell debris. Protein A/G (50% each, 1:1 mix; protein A:18-0010-02, protein G: 16-0010-025) microspheres were used to purify antigen. 1ml of the microspheres were packed in a column after being mixed upside down, and washed with 10 column volumes of equilibration buffer (0.15M sodium chloride, 0.02M PB, pH 7.0). Adding the centrifuged culture supernatant, slowly adding an adsorption column, allowing the mixture to flow through by gravity, and repeating the steps twice. After washing with 10 column volumes of equilibration buffer, elution buffer (0.15M sodium chloride, 0.1M glycine, 0.02M PB, pH 3.0) was added. Coomassie Brilliant blue (10. Mu.l per well, 96 well plate) was used to continuously check for protein washout, i.e.10. Mu.l per well was added. When coomassie brilliant blue changed significantly to blue, the eluted fraction was collected until no blue. The chromatographic column was passed through 10 column volumes of eluent and 10 column volumes of equilibration liquid and stored in 20% ethanol. The eluted protein was dialyzed against preparation buffer (25 mM sodium citrate, 50 mM sodium chloride, 100 mM arginine, 5% sucrose) and the protein concentration was checked by BCA method (4.55 mg/ml), and the protein purity was checked by SDS-PAGE (FIG. 1) and Coomassie brilliant blue staining and stored at-20℃for use, designated BPSL15-C. The SDS-PAGE bands in FIG. 1 show that the molecular weight of the antigen protein is 70Kd, which is in line with the molecular weight expectations of the protein.

1.2 immunization and fusion of mice: 6 female Balb/c mice of 6-8 weeks of age were divided into 2 groups: group a and group B, 3 each. Group A: 15C-FA-1/15C-FA-2/15C-FA-3, group B: 15C-SGZ-1/15C-SGZ-2/15C-SGZ-3. Mice were injected subcutaneously on their backs with 100 μg of antigen (BPSL 15-C). Group a mice were immunized for the first time with freund's complete adjuvant. Freund's incomplete adjuvant was used in the second and third immunizations. Group B mice were first immunized with freund's complete adjuvant. At the second and third timesThe solubility enhancing adjuvant PAP-1 (from SGE Biotech, 20181129) was used for immunization. On day 7 after 3 rd immunization, serum was extracted from the tail vein and antibody titers were detected by human siglec-15 binding ELISA and FACS. 15C-SGZ-2 and 15C-SGZ-3 were selected and further boosted (50. Mu.g per mouse) and hybridoma fusion was performed three days after boosting. Electrofusion method spleen cells of 15C-SGZ-2 and 15C-SGZ-3 were fused with SP2/0 cells (spleen cells: SP 2/0=1:1), and the fused cells were fused at 5% CO ₂ Cultures were performed in incubators (100 96-well plates, 20% FBS HAT medium) and screened by HAT (hypoxanthine aminopterin thymidine) medium.

Mouse serum and human Siglec-15 binding ELISA assay to detect antibody titers: mu.g/ml human Siglec-15his protein (ACRO SG5-H52H 3) was taken and ELISA plates were overcoated overnight in PBS. The antigen solution was aspirated the next day, and after three washes with PBST (0.5%o), 200. Mu.l of blocking solution (1% BSA in PBS, BSA: biorfox, 4240GR 100) was blocked for 2 hours at room temperature. After removing the blocking solution by pipetting, 100. Mu.l of serum from each mouse was added after three washes with PBST (0.5% by weight) and diluted with a gradient of blocking solution. The serum of the mice was diluted 4-fold at 8 spots with 1%o of the initial concentration. After incubation of the samples for 1 hour at room temperature, after three washes with PBST (0.5% by weight), 100. Mu.l of conjugated horseradish peroxidase anti-murine FC secondary antibody (Abcam, ab 97040) in blocking solution was added and incubated for 1 hour at room temperature. After three washes with PBST (0.5%o), 100. Mu.l TMB developing solution (Solabaio, PR 1200) was added, the reaction was stopped by developing at room temperature for 10 minutes, and 50. Mu.l stop solution (Solabaio, C1058) was added, and the OD450 absorbance was measured by a multifunctional microplate reader (FIG. 2 a). FIG. 2a shows the results of ELISA experiments for detecting antibody titers in mouse serum binding to human Siglec-15 protein, with the titers of 15C-SGZ-2 and 15C-SGZ-3 being the highest.

1.3 mouse serum binding to human Siglec-15 FACS experiments to detect antibody titres: human Siglec-15 full-length CHOS overexpressing cells were constructed. The synthesized encoded human Siglec-15 full length (MEKSIWLLACLAWVLPTGSFVRTKIDTTENLLNTEVHSSPAQRWSMQVPPEVSAEAGDAAVLPCTFTHPHRHYDGPLTAIWRAGEPYAGPQVFRCAAARGSELCQTALSLHGRFRLLGNPRRNDLSLRVERLALADDRRYFCRVEFAGDVHDRYESRHGVRLHVTAAPRIVNISVLPSPAHAFRALCTAEGEPPPALAWSGPALGNSLAAVRSPREGHGHLVTAELPALTHDGRYTCTAANSLGRSEASVYLFRFHGASGASTVALLLGALGFKALLLLGVLAARAARRRPEHLDTPDTPPRSQAQESNYENLSQMNPRSPPATMCSP) was constructed to ORF1 of the PCHO1.0 plasmid, and the plasmid was linearized overnight (total 100. Mu.g) by plasmid extraction (method with antigen expression) with NRUI restriction enzymes (NEB, R0192S) as recommended. CHOS cells were thawed using CD-forti CHO (Gibco, A1148301) +8 millimoles of glutamine (Gibco, 25030081) +anti-agglutinant (Gibco, 0010057 AE) 120rpm/37 ℃/5% CO ₂ And (5) shake cultivation. And selecting stable cells by transfecting the logarithmic phase CHOS cells (50. Mu.g plasmid/50. Mu.l Freestyle MAX, 1X 10. Mu.6/ml 30ml CHOS cells, 100 nanomole MTX (SIGMA, A6770-100 MG)/10. Mu.g/ml puromycin (Gibco, A1113803)) using the linearized plasmid and Freestyle MAX reagent (thermo, 16447100) in the prescribed manner, replacing the cells after 48 hours of transfection with fresh medium containing the above selection pressure, adjusting the cell density to 1X 10. Mu.6/ml, 120rpm/37 ℃/5% CO ₂ And (5) shake cultivation. After one week, cell viability/density was measured every other day. Maintaining the density of living cells to be less than 2.0 x 10-6/ml, passaging the cells at 0.5 x 10-6/ml, and freezing and storing partial cells (growth medium+10% DMSO) when the cell viability reaches more than 95%. The remaining cells were then subjected to pressure screening until the viability was greater than 99%, and the cells were re-frozen. Mouse serum combined with human Siglec-15 FACS: PBS 1 was used: 500 dilutions of mouse serum, 100 μl of diluted serum was incubated with 200000 human Siglec-15 overexpressing CHOS cells for 30 min at 4 ℃. After three washes with PBS, cells were washed with 1:200 anti-murine FC fluorescence secondary antibody (eBioscience, 17-4010-82) diluted in PBS was incubated at 4℃for 30 min in the absence of light, and after three PBS washes, the APC fluorescence channel mean-FI values were FACS detected (FIG. 2 b). FIG. 2b shows that 15C-SGZ-1, 15C-SGZ-2 and 15C-SGZ-3 bind to CHOS cells more effectively.

Example 2 subclone screening and identification

After screening by antigen binding ELISA, 138 clones were selected for FACS binding experimental identification of CHOS cells expressing human siglec-15 (FIG. 3). Subcloning was performed on 46 FACS positive clones, and subclones were successfully obtained for 30 clones, and screening was performed by human siglc-15 expressing CHOS cells and blank CHOS cells (see FIGS. 4a and 4b for screening results). 26 subclones were selected for antibody production. The supernatant of the hybridoma was purified by protein G. The 3 lead candidate murine anti-Siglec-15 antibodies (11H 5E6, 30C2B12 and 48B5B 1) were screened for sequence identification and further characterization by performing FACS binding experiments on blank CHOS cells (FIG. 5 a), CHOS cells expressing human Siglec-15 (FIG. 5B), CHOS cells expressing Cyno Siglec-15 (FIG. 5C) and CHOS cells expressing mouse Siglec-15 (FIG. 5 d), respectively, followed by T cell proliferation experiments (FIG. 6), osteoclast differentiation experiments (FIGS. 7a and 7B), fortebio affinity experiments (FIG. 8).

2.1 ELISA and FACS identification of hybridomas: positive clones were identified using antigen binding ELISA 10 days after hybridoma cell fusion plating. The sample was the supernatant of the hybridoma supernatant after centrifugation at 5000g for 10 minutes. Method reference mouse serum human Siglec-15 binding ELISA, antigen plating concentration was changed to 0.5 μg/ml. 138 clones with ELISA signals three times greater than the negative control signals were used as FACS binding experiments for human Siglec-15 from supernatants of these 138 clones, and the method was referred to the FACS binding experiments for mouse serum and human Siglec-15. The FACS binding experimental sample was 100. Mu.l supernatant. FIG. 3 shows that a total of 46 higher signal clones were picked as FACS positive and subcloned for the next step. Clone numbers were respectively: 8H6, 11E3, 11H5, 14H4, 15D1, 16A4, 17A3, 17F5, 23E8, 27G3, 30C2, 32A2, 37E11, 40A5, 46A2, 47C9, 48A12, 48B5, 48D1, 52G11, 54E10, 55E8, 56H8, 57F6, 58H9, 69C4, 69F2, 70E6, 74D4, 75G4, 76A10, 84F2, 87A9, 89F4, 89G3, 89G6, 90H7, 91B3, 92H7, 93B5, 96B2, 96C6, 97B6, 97H1, 98H9, 99F4.

2.2 subcloning of positive cell lines: the 46 clone wells selected above were subcloned once. The cells in the well were blown and evenly mixed, and the counting plate counted the number of living cells. At a density of 0.5 per well, 1 96-well plate was plated per clone subclone with 1640+10% fbs+ht (Hypoxanthine, thymidine) medium. Subcloning was performed 14 days later, and subclone supernatant ELISA was performed. A total of 30 subclones were successful for 46 master clones.

2.3 flow cytometry identification of subclones: 2-3 subclones with stronger signals were selected for FACS detection per master clone, including empty cells and CHOS cells overexpressing human SIGEC-15. Methods reference mouse serum combined with human Siglec-15 for FACS experiments. . The comparative screening results of FIGS. 4a, 4b show that 26 subclones in total have better binding activity to CHOS cells overexpressing human SIGEC-15.

2.4 production purification of subclone culture supernatant, flow cytometry combined functional verification: the 26 subcloned cell lines selected by the expansion culture are cultured until a considerable number of cells begin to die, and culture supernatants are collected by centrifugation. The antibody in the supernatant was purified using protein G, method reference antigen synthesis. CHOS cells overexpressing mouse and cynomolgus monkey Siglec-15 were constructed by referring to the method of construction of cells overexpressing human Siglec-15 CHOS. The purification of murine antibodies was used to perform functional verification of the binding of empty CHOS cells, CHOS overexpressing human Siglec-15, CHOS overexpressing murine Siglec-15, CHOS overexpressing cynomolgus monkey Siglec-15 by flow cytometry, the method referenced mouse serum to bind FACS to human Siglec-15, and the samples were changed to gradient diluted purified antibodies. FIGS. 5a, 5b, 5c, and 5d show that the antibody supernatants generated by some of the 26 subclone cell lines bind well to CHOS cells overexpressing murine, cynomolgus Siglec-15. Based on this, comprehensive screening will be performed in conjunction with subsequent T cell proliferation experiments.

2.5 hybridoma supernatant purified antibody T cell proliferation assay: the antibodies purified from the supernatants of 26 hybridoma cells were tested for T cell proliferation. Clone 30C2B12 was found to stably restore Siglec-15 inhibited T cell proliferation in experiments with multiple different blood cell donors (fig. 6). The experimental method is as follows:

(1) Frozen PBMCs (ALLCELLS, PB 004F) were thawed from liquid nitrogen by rapidly shaking the tube in a 37 ℃ water bath.

(2) Cells were added to 10mL of pre-warmed T cell medium (AIM-V+5% human AB serum; AIM-V: gibco, 12055091). Human AB serum: GEMINI, 100-512), 400G/10min. Cells were resuspended in 15mL T cell medium (Invitrogen, 18047019) containing 0.01% DNase I, placed in T75 flasks and incubated for 2 hours at 37 ℃.

(3) Non-adherent cells were collected and counted. Cells were centrifuged (400 g/10 min) and resuspended in PBS (10M/ml) and labeled with 5. Mu. MCFSE (Invitrogen, C34554) for 10min at 37 ℃. Then washed 2 times with PBS+10% BSA. Cells were centrifuged (400 g/10 min), resuspended in T cell medium (1.5M/mL) and anti-CD 3 coating (50 ng/mL) was added. Overnight at 4℃and 2 washes with PBS. 96-well plates (200. Mu.L/well).

(4) 5 μg/mL Siglec-15 (ACRO, SG 5-H5253) was added to the wells. anti-Siglec-15 antibodies were then added to the wells at 45 μg/mL. Cells were cultured at 37℃for 72h.

(5) After 72 hours, cells were transferred to round bottom plates for FACS analysis.

(6) Fc receptor was blocked by TruStain-Fcx (Biolegend, 422302,2. Mu.L/well) and then by APC-Cy ^TM 7 mice anti-human CD3 (BD, 557832)/BV 421 mice anti-human CD4 (BD, 562842)/APC mice anti-human CD8 (BD, 555369) staining.

(7) Proliferation rates of CD4 and CD 8T cells were analyzed by CFSE labeling and CD3/CD4/CD8 gating.

2.6 osteoclast differentiation experiments: the antibodies purified from the supernatants of 26 hybridomas were used for osteoclast differentiation experiments. FIGS. 7a and 7B show that clone 11H5E6/48B5B1 and 69C4D8 were found to reduce TRAP enzyme activity in the supernatant and reduce osteoclast differentiation, clone 48B5B1 being more pronounced but weaker than the positive control. The experimental method refers to the published patent of Next cure company: WO2018057735. The experimental method is as follows: PBMCs were resuscitated from liquid nitrogen in fresh medium and monocytes were purified according to the kit (meitian and gentle, 130-096-537) recommendations. Three parallel wells were plated at a density of 10 ten thousand cells per well on 96-well flat bottom cell culture plates. The cell culture medium was alpha-MEM medium (Gibco, 32571-036), 10% FBS,1 mM sodium pyruvate, 25ng/ml human M-CSF (Meitian, 103-096-491), 30ng/ml human RANKL (Meitian, 130-093-988). After incubation at 37℃for 3 hours for adherence, an antibody (containing positive control antibody 25E9, from Alethia company patent US 8741289) was added to the antibody against human Siglec-15 at a final concentration of 50. Mu.g/ml. After three days of culture, fresh medium containing the stimulating factors was changed. Seven days after incubation, the supernatant was transferred and centrifuged to remove cells. The adherent mononuclear/osteoclasts were stained for TRAP (B-Bridge, PMC-AK04F-COS, FIG. 7B) and the supernatant assayed for TRAP enzyme activity (FIG. 7 a) according to the kit recommended. The microscope is used for photographing a set of cameras for photographing and dyeing, and a multifunctional enzyme-labeling instrument is used for detecting the OD540 absorbance.

2.7 antibody affinity assay: the affinity of the candidate antibodies in the different screening states was detected by the BLI technique (Fortebio or Gator). 100nM candidate antibody was loaded onto an AHC or AMC biosensor capable of capturing its FC (mouse or human) and Siglec-15 was then interacted with a 100nM his tag (Kactus, SIG-HM 415) in solution. Association and dissociation rates were measured and K-on/K-off and KD were calculated by fitting signal curves. FIG. 8 shows that the 11H5E6/48B5B1 and 69C4D8 antibodies have higher affinity for antigen.

2.8 molecular epitope identification of lead chimeric antibodies: the relative binding of the candidate antibody to 5G12 was detected by BLI technology (Fortebio). For example, 100nM 5G12-IgG4 antibody was loaded onto the AHC sensor; siglec-15his protein (Kactus, SIG-HM 415) was associated with the 5G12 antibody. The sensor is then blocked by the human Fc containing protein, another antibody being associated with his antigen. If the secondary antibody is capable of binding to the antigen, the secondary antibody is on another box. If not, the two antibodies are placed on the same box. FIG. 11 shows that the three antibody molecules 11H5E6, 30C2B12, 48B5B1 and 5G12 are not in the same epitope, 11H5E6 and 30C2B12 have the same epitope

2.9 thermostability experiments of lead chimeric antibodies: materials and methods: melting curves were detected using a Luo Shiguang cycler 480 real-time PCR instrument. Briefly, 45 μl of 5 μΜ protein was added to PCR tubes (or plates) in triplicate. Fresh 200 XSPRO orange solution (from 5000 Xstock solution, 5000 Xconcentration in DMSO; life Technologies S-6650) was prepared in the selected buffer. To each sample was added 5. Mu.L of 200 XSYPRO orange solution and mixed well. The sample was kept at 25 ℃ for 30s and the reading was taken; raised by 0.5 ℃, raised from 25 ℃ to 99 ℃ and then returned to 25 ℃. Tm was measured using melting curves (fig. 12a, 12b, 12 c), 3 chimeric antibodies Tm1 were: 11H5E6 is 60.5 ℃,30C2B12 is 62.5 ℃,48B5B1 is 62 ℃, and the thermal stability is good.

Example 3 Gene cloning of anti-Siglec-15 antibody, chimeric antibody construction expression purification, humanized engineering, affinity maturation, engineering

3.1 Gene cloning of lead anti-Siglec-15 antibody, chimeric antibody construction, expression, purification

3.1.1 Gene cloning: the sequences of the antibody heavy/light chains were obtained using Rapid Amplification of CDNA Ends (RACE). The method comprises the following steps: (1) The hybridoma cells were expanded to 80% by blowing them off from the flask, centrifuging at 1500rpm for 5 minutes, and resuspending the cells in 200 μl PBS. (2) 800. Mu.l Trizol lysate was added, vigorously shaken, and allowed to stand at 4℃for 10 minutes. (3) After 250. Mu.l of chloroform was added thereto, the mixture was vigorously shaken for 5 minutes and allowed to stand at 4℃for 5 minutes. (4) The sample was centrifuged at 12000rpm at 4℃for 15 minutes, and the supernatant was added to 600. Mu.l of isopropyl alcohol, and the mixture was gently mixed up and down and allowed to stand at 4℃for 10 minutes. (5) After centrifugation at 12000rpm/4℃for 10 minutes, the supernatant was carefully decanted. 800 μl of pre-cooled 75% ethanol was added for washing, and after pouring off the ethanol, the mixture was dried at 72 ℃. (6) cDNA was obtained using a reverse transcription kit. (7) cDNA was PCR amplified with degenerate primers that angle either the heavy or light chain variable regions. (8) The PCR amplified products were used to construct T vectors and to transform TOP10 competent cells, and monoclonal sequencing was performed. Three single clones were sequenced together: 11H5E6/30C2B12/48B5B1, the corresponding VH and VL sequences were obtained.

3.1.2 chimeric antibody construction: the DNA sequence of the heavy chain variable region was optimized and synthesized, and the sequence was ligated into a PCDNA3.4 vector containing the IgG4 heavy chain constant region sequence by PCR, cleavage, and construction of the complete heavy chain. The DNA sequence of the light chain variable region was optimized and synthesized, and the sequence was ligated into a PCDNA3.4 vector containing the KAPPA light chain constant region by PCR, cleavage, and construction of a complete light chain. The two plasmids were transformed into TOP10 competent cells, and plasmids were extracted in large quantities (methods reference antigen synthesis).

3.1.3 expression: the above mass extracted seed light chain plasmid was electrotransfected into CHOS cells and cultured with feed for 7 days.

3.1.4 purification: the above cultured cells were collected, and the corresponding antibodies were purified using protein A, and the purity was detected by SEC/SDS-PAGE (method reference antigen synthesis).

3.1.5 in vitro binding Activity identification by chimeric antibody flow cytometry: the chimeric antibody and the negative control antibody are taken as a binding experiment with CHOS cells which over-express human/cynomolgus monkey/mouse Siglec-15, the method refers to a FACS experiment of the binding of mouse serum with human Siglec-15, and the results are shown in FIG. 9a, FIG. 9B and FIG. 9C, and the results show that three chimeric antibodies 11H5E6, 48B5B1 and 69C4D8 respectively have higher binding activity with CHOS cells which express human Siglec-15, CHOS cells which express cynomolgus monkey Siglec-15 and CHOS cells which express mouse Siglec-15. 3.1.6T cell proliferation assay of chimeric antibodies: the chimeric antibody and the negative control antibody are taken as T cell proliferation experiments, the method refers to hybridoma supernatant purified antibody T cell proliferation experiments, and the results are shown in figures 10a and 10B, and show that the titer of the 30C2B12 chimeric antibody in different donors in the recovery T cell proliferation experiments is better than that of the control 5G12.

3.2 humanized engineering of lead anti-Siglec-15 antibodies

3.2.1 molecular design:

humanization of lead candidate 30C2B12 used CDR grafting and back mutation. The FRs and CDRs regions are defined using CDRs numbering rules. Based on the antibody sequence database, IGBLAST searches were performed on VL and VH of the 30C2B12 sequence, respectively. According to the search result, IGHV1-46 x 01 is selected as a template of VH, and IGKV7-3 x 01, IGKV4-B3 or IGKV4-39 x 01 is selected as a template of VL. CDRs of the mouse-derived 30C2B12 sequence were transplanted into VL and VH templates, respectively, and homology modeling was performed by the Modeller open source program. Comparing the mouse source sequences of the 3D model 30C2B123D model, and selecting RMSD<

As a Grafted 3D model.

The transplanted 3D model was energy minimized by NAMD open source procedure and few residues were mutated back to mouse-derived residues according to rules of three-dimensional structure, physicochemical properties, and energy minimization. The sequences of the above models are optimized according to isoelectric point, PTMs, aggregation, expansibility, immunogenicity, etc.

3.2.2 antibody expression purification: the humanized engineered molecule designed above was constructed into the IgG 4-S228P/kappa constant region and purified by expression of the humanized molecule (30 ml) according to the method recommended by the expi-293 transfection system (Gibco, A14635), protein A affinity adsorption purified antibody (reference antigen synthesis).

3.2.3 characterization of antibodies: affinity of humanized molecules was measured using fortebio, and figure 13 shows that both antibodies remained consistent with the binding activity of murine molecules after humanization.

3.2.4 in vitro binding Activity identification by flow cytometry of humanized antibodies: the above purified antibodies and negative control antibodies were used for binding experiments with CHOS cells overexpressing human Siglec-15, and the results of the method were shown in FIG. 14 by referring to FACS binding experiments of mouse serum with human Siglec-15. The humanized antibodies can keep consistent binding activity with murine antibody molecules in the CHOS cell binding experiments over-expressing human Siglec-15.

T cell proliferation assay of 3.2.5 humanized antibodies: the purified antibodies and the negative control antibodies were taken as T cell proliferation experiments, and the method was used for referring to the hybridoma supernatant purified antibody T cell proliferation experiments, and the results are shown in FIG. 15, wherein most of the daughter molecules show the equivalent functional activities to the mother molecule 30C2B 12.

3.3 affinity maturation of humanized anti-Siglec-15 antibodies

3.3.1 affinity maturation of antibodies using phage display technology with L2H1 as parent molecule: (1) preparation of materials: i.e., antigen-antibody detection (ELISA/SDS-PAGE). (2) Gene Synthesis: based on hamster codon optimized sequences, primers were designed for insertion into expression vectors. (3) female parent verification: the sequence of the constructed female parent and the activity ELISA of the Fab antibody were verified. (4) construction of antibody engineering library: by means of single-point saturation mutation, 8 mutation libraries were constructed by four mutation schemes of LCDR1+LCDR3+HCDR3/LCDR2+HCDR1+HCDR2/LCDR3/HCDR3 and two frameworks, respectively, and quality and antibody diversity analysis of the libraries were verified. (5) Antibody selection, i.e., screening phage that bind to antibodies bound to the immune tubes by a multiplex panning method based on the immune tubes, and detecting their affinity properties by phage ELISA and prokaryotic expression supernatant ELISA. The clones with better affinity activity obtained by sequencing are totally 38. (6) Based on the primary ELISA signal and the prokaryotic expression supernatant ELSIA EC50 and whether the sequence has a PTM site, more preferably 8 clones were constructed into the IgG 4S 228P constant region as full field antibodies. The 8 cloning plasmids were transfected into CHOS cells and purified by protein a to obtain antibodies.

3.3.2 in vitro binding Activity identification of antibodies after affinity maturation by flow cytometry: the above purified antibodies and negative control antibodies were used as binding experiments with CHOS cells overexpressing human/cynomolgus monkey/mouse Siglec-15, and the results of the method were shown in fig. 16, 17 and 18 by referring to FACS experiments in which mouse serum was bound to human Siglec-15. The results show that affinity matured antibodies show better binding activity in CHOS cell binding experiments with overexpressing human/cynomolgus monkey/mouse Siglec-15.

3.3.3 antibody characterization: affinity of affinity maturation molecules was detected using fortebio and binding of affinity maturation molecules was detected using CHOS cells overexpressing human Siglec-15, and the results in fig. 19 show that the binding level of the affinity-matured antibody molecules to antigen was greatly improved.

Example 4 Gene cloning construction and expression purification of candidate antibodies

4.1 cloning construction: respectively will be numbered

The DNA of the VH region of the antibody, 5G12/30C2B12/L2H1/L2H1-58/L2H1-58-PTM/14-58-1/14-58-2/16-58-1, was PCR/restriction digested/ligated to the PCDNA3.4 plasmid containing the CH region of IgG1 to form a complete heavy chain, and the VL region of these clones was ligated to the PCDNA3.4 plasmid containing the IgG1-Kappa CL region to form a complete light chain.

4.2 expression purification: the plasmid is subjected to mass extraction by transforming TOP10 competent cells, and the expi-293 cells are transfected and cultured for fermentation according to the recommended method of the kit. The antibodies were purified using a protein a adsorption column.

Example 5 Activity identification and functional experiments of optimized antibodies

5.1 in vitro binding Activity identification of optimized antibodies by flow cytometry: the purified antibodies and negative control antibodies were used for binding experiments with CHOS cells overexpressing human/cynomolgus monkey/mouse Siglec-15. The antibodies were diluted 3-fold at 12 spots at an initial concentration of 100 nM. (methods reference mouse serum combined with human Siglec-15 FACS experiments). The results of figures 20, 21, 22 show that the affinity-matured antibody molecules have higher affinity in binding experiments with CHOS cells overexpressing human/cynomolgus monkey/mouse Siglec-15 than before the non-affinity maturation, even beyond the positive control antibody 5G12 molecule.

5.2T cell proliferation assay of the optimized antibodies: the purified antibodies and negative control antibodies were used as T cell proliferation experiments (methods refer to hybridoma supernatant purified antibody T cell proliferation experiments). Experiments were performed using PBMCs from three different donors in total. Wherein, blank is a blank, i.e. no CD3 stimulated group; CD3 group i.e. add CD3 panel as positive control; CD3+Siglec-15 groups were stimulated with CD3 plates while adding Siglec-15 protein to inhibit T cell proliferation. The 5G12-IgG1 is a positive control, irrelevant IgG1 is a negative control, and the functions of candidate antibodies L2H1-58-PTM and 14-58-2 are detected, and the results are shown in FIG. 23, FIG. 24 and FIG. 25, and show that the L2H1-58-PTM and 14-58-2 molecules have higher in-vitro functional activities.

Sequence listing

<110> times to the pharmaceutical industry (Suzhou) Co., ltd

<120> Siglec-15 monoclonal antibody or antigen binding fragment thereof and use thereof

<160> 104

<170> SIPOSequenceListing 1.0

<210> 1

<211> 117

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Lys Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Asp Ile Asn Pro Asn Asp Gly Asp Ser Ser Tyr Asn Gln Lys Phe

50 55 60

Thr Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Gly Thr Trp Gly Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ala

115

<210> 2

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly

1 5 10 15

Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Arg

20 25 30

Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Phe Tyr His Met Ser Lys Phe Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

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

100 105 110

<210> 3

<211> 119

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Tyr Thr Ser Thr Asp Tyr Tyr Ile

20 25 30

His Trp Val Met Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Trp

35 40 45

Ile Ser Pro Glu Asn Phe Asn Thr Tyr Tyr Asn Glu Lys Phe Lys Gly

50 55 60

Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln

65 70 75 80

Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

85 90 95

Asp His Gly Ser Ser Phe Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 4

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Glu Ile Gln Met Thr Gln Ser Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Asn Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Arg Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 5

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

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

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Ala Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Gly Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 6

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

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

1 5 10 15

Gln Arg Ala Thr Ile Phe Cys Arg Ala Ser Gln Ser Val Asp Tyr Asn

20 25 30

Gly Ile Ser Tyr Met Asp Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Ile

85 90 95

Gly Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 7

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 8

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asn

20 25 30

Gly Ile Ser Tyr Met Asp Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile His

65 70 75 80

Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Ile

85 90 95

Gly Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 9

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 9

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 10

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Val Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 11

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Gln Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asn

20 25 30

Gly Ile Ser Tyr Met Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

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

85 90 95

Gly Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 12

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 12

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Ala Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 13

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 13

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Asp Tyr Asn

20 25 30

Gly Ile Ser Tyr Met Asp Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

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

85 90 95

Gly Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 14

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 14

Gln Val Gln Leu Val Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Ala Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 15

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 15

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Ala Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 16

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 16

Gln Val Gln Leu Val Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Ala Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 17

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 17

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Ala Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Ser Ser Ser Glu Thr Arg Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 18

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 18

Gln Val Gln Leu Val Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Ala Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Ser Ser Ser Glu Thr Arg Leu Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 19

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 19

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Asp Tyr Ser

20 25 30

Gly Ile Ser Tyr Met Asp Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

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

85 90 95

Gly Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 20

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 20

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Asp Tyr Asn

20 25 30

Gly Ile Ser Tyr Met Asp Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

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

85 90 95

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

100 105 110

<210> 21

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 21

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Asp Tyr Ser

20 25 30

Gly Ile Ser Tyr Met Asp Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

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

85 90 95

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

100 105 110

<210> 22

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 22

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Asp Tyr Ser

20 25 30

Gly Ile Ser Tyr Met Asp Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

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

85 90 95

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

100 105 110

<210> 23

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 23

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ser Ile His Pro Ser Asp Ser Tyr Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 24

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Gln Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asn

20 25 30

Gly Ile Ser Tyr Met Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu His Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

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

85 90 95

Gly Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 25

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 25

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Tyr Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 26

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 26

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Gln Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asn

20 25 30

Gly Ile Ser Tyr Met Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Leu Glu Pro Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

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

85 90 95

Gly Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 27

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 27

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ser Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 28

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 28

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Gln Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Tyr Tyr Asn

20 25 30

Gly Ile Ser Tyr Met Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

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

85 90 95

Gly Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 29

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 29

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile His Pro Ser Asp Ser Phe Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 30

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 30

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Gln Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asn

20 25 30

Gly Ile Ser Tyr Met Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

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

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

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

85 90 95

Gly Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 31

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 31

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Tyr Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 32

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 32

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Phe Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 33

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 33

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Gln Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asn

20 25 30

Gly Ile Ser Tyr Met Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

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

85 90 95

Gly Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 34

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 34

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Gln Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asn

20 25 30

Gly Ile Ser Tyr Met Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr His Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

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

85 90 95

Gly Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 35

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 35

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met Val Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Tyr Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 36

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 36

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Ser Ser Phe Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 37

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 37

Gln Val Gln Leu Val Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Ser Asp Ser Phe Thr Arg Leu Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 38

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 38

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Asp Tyr Ser

20 25 30

Gly Ile Ser Tyr Met Asp Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr His Ala Ser Asn Leu Glu Ser Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

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

85 90 95

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

100 105 110

<210> 39

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 39

Gln Val Gln Leu Val Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Ala Tyr Ser Phe Ile Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Ser Asp Ser Phe Thr Arg Leu Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 40

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 40

Gln Val Gln Leu Val Gln Pro Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Ser Ser Ser Glu Thr Arg Leu Ala Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 41

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 41

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Glu Ser Phe Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 42

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 42

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

Gly Ile Ser Tyr Met Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr His Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

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

85 90 95

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

100 105 110

<210> 43

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 43

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asn Ser Phe Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 44

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 44

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

Gly Ile Ser Tyr Met Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr His Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

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

85 90 95

Gly Asn Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 45

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 45

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Thr Ser Phe Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 46

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 46

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

Gly Ile Ser Tyr Met Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr His Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

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

85 90 95

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

100 105 110

<210> 47

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 47

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Gly Ser Phe Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 48

<211> 111

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 48

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

Gly Ile Ser Tyr Met Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr His Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

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

85 90 95

Gly Asn Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 49

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 49

Gly Tyr Thr Phe Thr Asp Tyr Tyr

1 5

<210> 50

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 50

Ile Asn Pro Asn Asp Gly Asp Ser

1 5

<210> 51

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 51

Ser Arg Gly Thr Trp Gly Gly Phe Ala Tyr

1 5 10

<210> 52

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 52

Lys Ser Leu Leu His Arg Asn Gly Ile Thr Tyr

1 5 10

<210> 53

<211> 3

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 53

His Met Ser

1

<210> 54

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 54

Ala Gln Asn Leu Glu Leu Pro Thr

1 5

<210> 55

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 55

Ala Tyr Thr Ser Thr Asp Tyr Tyr

1 5

<210> 56

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 56

Ile Ser Pro Glu Asn Phe Asn Thr

1 5

<210> 57

<211> 14

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 57

Ala Arg Asp His Gly Ser Ser Phe Tyr Trp Tyr Phe Asp Val

1 5 10

<210> 58

<211> 6

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 58

Gln Asp Ile Asn Asn Tyr

1 5

<210> 59

<211> 3

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 59

Tyr Thr Ser

1

<210> 60

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 60

Gln Gln Gly Asn Ser Leu Pro Arg Thr

1 5

<210> 61

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 61

Ser Tyr Trp Met Asn

1 5

<210> 62

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 62

Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 63

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 63

Asn Tyr Pro Tyr Phe Pro Met Asp Phe

1 5

<210> 64

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 64

Arg Ala Ser Gln Ser Val Asp Tyr Asn Gly Ile Ser Tyr Met Asp

1 5 10 15

<210> 65

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 65

Ala Ala Ser Asn Leu Glu Ser

1 5

<210> 66

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 66

Gln Gln Ser Ile Gly Asp Pro Trp Thr

1 5

<210> 67

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 67

Ala Tyr Ser Phe Thr Ser Tyr

1 5

<210> 68

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 68

His Pro Ser Asp Ser Glu Thr Arg

1 5

<210> 69

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 69

Ala Tyr Ser Phe Thr Ser Tyr Trp

1 5

<210> 70

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 70

Ile His Pro Ser Asp Ser Glu Thr

1 5

<210> 71

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 71

Ala Arg Asn Tyr Pro Tyr Phe Pro Met Asp Phe

1 5 10

<210> 72

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 72

Gln Ser Val Asp Tyr Asn Gly Ile Ser Tyr

1 5 10

<210> 73

<211> 3

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 73

Ala Ala Ser

1

<210> 74

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 74

Gly Tyr Thr Phe Ile Ser Tyr Trp Met Asn

1 5 10

<210> 75

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 75

Ser Ile His Pro Ser Asp Ser Tyr Thr Arg

1 5 10

<210> 76

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 76

Ala Ala Ser Asn Leu Glu His

1 5

<210> 77

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 77

Gly Tyr Thr Phe Thr Ser Tyr Trp Val Asn

1 5 10

<210> 78

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 78

Met Ile His Pro Ser Asp Ser Tyr Thr Arg

1 5 10

<210> 79

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 79

Ala Ala Ser Asn Leu Glu Pro

1 5

<210> 80

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 80

Ser Ile His Pro Ser Asp Ser Glu Thr Arg

1 5 10

<210> 81

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 81

Arg Ala Ser Gln Ser Val Tyr Tyr Asn Gly Ile Ser Tyr Met Asp

1 5 10 15

<210> 82

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 82

Leu Ala Ser Asn Leu Glu Ser

1 5

<210> 83

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 83

Ala Ile His Pro Ser Asp Ser Phe Thr Arg

1 5 10

<210> 84

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 84

Ala Ala Ser Ser Leu Glu Ser

1 5

<210> 85

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 85

Met Ile His Pro Ser Asp Ser Phe Thr Arg

1 5 10

<210> 86

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 86

Gly Tyr Thr Phe Thr Ser Tyr Trp Met Val

1 5 10

<210> 87

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 87

Met Ile His Pro Ser Ser Ser Phe Thr Arg

1 5 10

<210> 88

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 88

His Ala Ser Asn Leu Glu Ser

1 5

<210> 89

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 89

Gln Gln Ser Ile Gly Thr Pro Trp Thr

1 5

<210> 90

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 90

Ala Tyr Ser Phe Ile Ser Tyr Trp Met Asn

1 5 10

<210> 91

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 91

Met Ile His Pro Ser Ser Ser Glu Thr Arg

1 5 10

<210> 92

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 92

Met Ile His Pro Ser Glu Ser Phe Thr Arg

1 5 10

<210> 93

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 93

Arg Ala Ser Gln Ser Val Asp Tyr Gln Gly Ile Ser Tyr Met Asp

1 5 10 15

<210> 94

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 94

Gln Gln Ser Ile Gly Glu Pro Trp Thr

1 5

<210> 95

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 95

Met Ile His Pro Ser Asn Ser Phe Thr Arg

1 5 10

<210> 96

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 96

Gln Gln Ser Ile Gly Asn Pro Trp Thr

1 5

<210> 97

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 97

Met Ile His Pro Ser Thr Ser Phe Thr Arg

1 5 10

<210> 98

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 98

Met Ile His Pro Ser Gly Ser Phe Thr Arg

1 5 10

<210> 99

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 99

Arg Ala Ser Gln Ser Val Asp Tyr Asp Gly Ile Ser Tyr Met Asp

1 5 10 15

<210> 100

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 100

Arg Ala Ser Gln Ser Val Asp Tyr Ser Gly Ile Ser Tyr Met Asp

1 5 10 15

<210> 101

<211> 1404

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 101

atgaccaggc tgacagtgct ggctctgctg gccggactgc tggcttcttc tagagctcag 60

gtgcagctgg tgcagtcagg agcagaagtg aagaagcccg gagccagcgt gaaagtgtct 120

tgcaaggcca gcggctacac cttcatcagc tactggatga attgggtccg gcaggctcca 180

ggacagggac tcgagtggat tggcatgatc caccccagct caagcttcac aaggctgaac 240

cagaagttca aggacaaggc caccctgacc gtggacaaga gcaccagcac cgtgtacatg 300

gagctgagca gcctgaggag cgaggatacc gccgtgtact attgcgcccg gaactacccc 360

tacttcccca tggacttttg gggccaggga acaagcgtga ccgtgtctag cgctagcacc 420

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660

aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 720

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 780

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 960

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 1140

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1320

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1380

ctctccctgt ctccgggtaa atga 1404

<210> 102

<211> 714

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 102

atgaccaggc tgacagtgct ggctctgctg gccggactgc tggcttcttc tagagctgac 60

atccagatga cccagagccc cagctctctg agcgcttcag tgggccagag agtgacaatc 120

acttgcagag ccagccagag cgtggactac agcggcatca gctacatgga ttggtaccag 180

cagaagccag gcaaggctcc taagctgctg atctaccacg cctctaacct ggagagcgga 240

gtgcctagca gattcagcgg cagcggaagc ggaaccgact tcaccctgac catcagctct 300

ctgcagccag aggacttcgc cacctactac tgccagcaga gcatcggcac tccttggaca 360

ttcggcggcg gcacaaagct ggagatcaag cgtacggtgg ctgcaccatc tgtcttcatc 420

ttcccgccat ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 480

aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt 540

aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc 600

accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc 660

catcagggcc tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg ttag 714

<210> 103

<211> 1404

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 103

atgggctgga cttggatctt catcctgatc ctgagcgtga ccaccggagt gcattctcag 60

gtgcagctgg tgcagccagg agcagaagtg aagaagcccg gcgccagcgt gaaagtgtct 120

tgcaaggcca gcgcctacag cttcatcagc tactggatga attgggtccg gcaggctcca 180

ggacagggcc tcgagtggat gggtatgatt cacccttctg acagcttcac aagactggcc 240

cagaagttcc agggcagggc taccctgaca gtggacaaga gcaccagcac cgcctacatg 300

gagctgtcta gccctagaag cgaggacacc gcagtgtact attgcgcccg gaactacccc 360

tacttcccca tggacttttg gggccaggga acaacagtga cagtgtccag cgctagcacc 420

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660

aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 720

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 780

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 960

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 1140

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1320

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1380

ctctccctgt ctccgggtaa atga 1404

<210> 104

<211> 723

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 104

atggggctgc aggtgcaggt catcagcttc ctgctgatca gcgtgaccgt gatcatgagc 60

agaggcgaca tcgtgctgac ccagtctcca gatagcctgg cagtgtctct gggcgagaga 120

gccaccatca attgcagggc ctctcagagc gtggactaca gcggcatcag ctacatggat 180

tggttccagc agaaaccagg ccagcctcct aagctgctga tctaccacgc cagcaacctg 240

gagagcggca tcccagacag attcagcgga agcggaagcg gcaccgactt caccctgacc 300

atctcttctc tgcaggccga agacgtggcc gtgtactatt gccagcagag catcggcacc 360

ccttggacct ttggcggcgg aacaaagctg gagatcaagc gtacggtggc tgcaccatct 420

gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 480

ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 540

caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 600

ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 660

gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 720

tag 723

Claims (12)

1. An antibody or antigen binding fragment thereof against Siglec-15, comprising: a VH/VL sequence pair selected from the group of VH and VL amino acid sequence pairs consisting of: SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:32 and SEQ ID NO:11, SEQ ID NO:36 and SEQ ID NO:11, SEQ ID NO:37 and SEQ ID NO:38, seq ID NO:39 and SEQ ID NO:38, seq ID NO:40 and SEQ ID NO:38, a step of carrying out the process;

the combination of HCDR1/HCDR2/HCDR3 and LCDR1/LCDR2/LCDR3 is selected from the group consisting of:

61/62/63 and 64/65/66, 74/63 and 85/88/89, 74/91/100/88/89.

2. The anti-Siglec-15 antibody or antigen-binding fragment thereof of claim 1, comprising: a human constant domain which is IgA, igD, igE, igG or IgM domain.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody against Siglec-15 is a murine monoclonal antibody, a chimeric monoclonal antibody, a humanized antibody, a single chain antibody, a light chain constant region thereof, optionally a human antibody kappa chain or lambda chain constant region, a heavy chain constant region thereof, optionally a human antibody IgG1 or IgG4 constant region, or a mutant of an IgG1 or IgG4 constant region.

4. The anti-Siglec-15 antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises a monospecific antibody, a bispecific antibody, a trispecific antibody.

5. The antibody or antigen binding fragment thereof of claim 4, wherein the other end of the bispecific antibody targets a tumor-associated antigen of a tumor cell or targets an immunoassay target of an immune cell comprising EGFR, HER2, CD47, PD-1, PD-L1, CTLA4, tgfβ, VEGF, HER3, TIGIT, CLAUDIN18.2, CD3, TIM3, LAG3.

6. A nucleic acid encoding the anti-Siglec-15 antibody or antigen-binding fragment thereof of any one of claims 1-5.

7. A nucleic acid according to claim 6, comprising SEQ ID NO 101, SEQ ID NO: 102. SEQ ID NO: 103. SEQ ID NO. 104 sequence.

8. An expression vector comprising the nucleic acid sequence of claim 6.

9. A host cell comprising the expression vector of claim 8.

10. A pharmaceutical composition comprising the anti-Siglec-15 antibody or antigen-binding fragment thereof of any one of claims 1-5 and a pharmaceutically acceptable carrier.

11. Use of an antibody or antigen binding fragment thereof against Siglec-15 according to any one of claims 1-5 for the preparation of a medicament for the treatment of cancer cells, macrophages or cancer expressing or overexpressing Siglec-15.

12. Use of an antibody or antigen-binding fragment thereof against Siglec-15 according to any one of claims 1-5, or a pharmaceutical composition according to claim 11, for the preparation of a medicament for the treatment of a tumor selected from melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer and rectal cancer.

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