CN112694532A - anti-Siglec-15 antibody or antigen binding fragment thereof and application - Google Patents

Abstract

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

Description

anti-Siglec-15 antibody or antigen binding fragment thereof and application

Technical Field

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

Background

The Siglec sialic acid binding Ig-like lectin family is a member of the Ig superfamily, is expressed on various leukocytes, recognizes glycoprotein sialic acid structures expressed on cell surfaces, mediates signal transduction. The Siglec family is the largest group of vertebrate lectins known to recognize sialylated glycans (Varki and Dangata 2006; Crocker et al 2007). Most mammalian Siglecs expressed in the immune system have Immunoreceptor Tyrosine Inhibition Motifs (ITIMs) in the cytoplasmic domains, and these Siglecs have been shown to have negative regulatory effects on cells expressing them (Otipoby et al 1996; Sato et al 1996; Nitschke et al 1997; Mingari et al 2001; Uldanova et al 2001; Nutku et al 2003; von Gunten et al 2005). Recently discovered Siglecs are related to the signal adaptor 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)), which reveals the potential of the subset of Siglecs to activate signaling 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 with an extracellular domain of 244 amino acids, including an Ig-like V-type domain and an Ig-like C2-type domain. Siglec-15 is expressed on cells of the immune system, can recognize sialylated ligands, and can interact with the signal adaptor molecules DNAX activating proteins DAP12 and DAP10 through lysine residues in the transmembrane domain. Siglec-15 is an 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. Biochemical, 2012). Siglec-15 on osteoclast precursors recognizes CD44 on adjacent osteoclast precursors and transduces signals through the DAP12-Syk pathway, cross-talks to the RANK-TRAF6 pathway and enhances 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 the sialic acid-Tn (Neu5 Ac. alpha.2-6 GalNAc. alpha.1) structure, which is found in several tumor-associated antigens, such as MUC-1, and that high expression is associated with poor prognosis (TakashiAngata et al, glycobiology, 2007; Jennifer Munkley, J.International molecular sciences, 2016). Siglec-15 is expressed on Tumor Associated Macrophages (TAMs) of various human tumor tissues, its expression is significantly elevated in macrophage colony stimulating factor-induced M2-like macrophages, and Siglec-15 produces more transforming growth factor-beta (TGF-beta) on sTn positive cells than negative cells, indicating that Siglec-15 recognizes tumor sTn antigens and transduces signals for increased secretion of TGF-beta in TAMs. Recent studies (Jun Wang et al, nature medicine, 2019) showed that Siglec-15 may be an important immune checkpoint expressed only on tumor cells expressing PD-L1 and interacting with unknown receptors expressed on T cells negatively regulating T cell proliferation. In a mouse tumor model, antibodies against Siglec-15 were shown to have anti-tumor efficacy in vitro rescue experiments on T cell proliferation.

Disclosure of Invention

The invention provides antibodies or antigen-binding fragments thereof against Siglec-15, particularly Siglec-15 humanized monoclonal antibodies that include 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 an in vitro experiment. The invention further provides the use of the Siglec-15 antibodies for the treatment of various cancers.

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

in some embodiments, the anti-Siglec-15 antibodies or antigen-binding fragments thereof provided herein comprise: 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 nos: 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 nos: 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, the anti-Siglec-15 antibodies or antigen-binding fragments thereof provided herein comprise: 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:

in some embodiments, the anti-Siglec-15 antibodies or antigen-binding fragments thereof provided by the present invention comprise a sequence of one or more heavy chain complementarity determining region HCDRs selected from the group consisting of (1) HCDR1 comprising or consisting of an amino acid sequence having at least 95% identity to an 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 the group consisting of 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 the group consisting of SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:63, SEQ ID NO: 71; (4) the HCDR of (1), (2) and (3) above which contains one or more amino acid substitutions, deletions or insertions of not more than 5 amino acids.

In some embodiments, the anti-Siglec-15 antibodies or antigen-binding fragments thereof provided herein comprise a sequence of one or more light chain complementarity determining region (LCDR) selected from the group consisting of (1) LCDR1 comprising a heavy chain variable region (LCDR) consisting of SEQ ID NO: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 the group consisting of 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) LCDRs 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, the antibodies or antigen-binding fragments thereof against Siglec-15 provided herein comprise a HCDR1/HCDR2/HCDR3 combination and/or a LCDR1/LCDR2/LCDR3 combination: (1) the HCDR1/HCDR2/HCDR3 combination comprises sequences of one or more sets of heavy chain complementarity determining region HCDRs selected from: HCDR1/HCDR2/HCDR3 are 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:74/SEQ ID NO:85/SEQ ID NO:63, SEQ ID NO: SEQ ID NO:78/SEQ ID NO:63, SEQ ID NO:74/SEQ ID NO:87/SEQ ID NO:63, SEQ ID NO:90/SEQ ID NO:85/SEQ ID NO:63, SEQ ID NO:74/SEQ ID NO:91/SEQ ID NO:63, SEQ ID NO:74/SEQ ID NO:92/SEQ ID NO:63, SEQ ID NO:74/SEQ ID NO:95/SEQ ID NO:63, SEQ ID NO:74/SEQ ID NO:97/SEQ ID NO:63, SEQ ID NO:74/SEQ ID NO:98/SEQ ID NO: 63; (2) LCDR1/LCDR2/LCDR3 in combination comprises one or more sets of light chain complementarity determining regions LCDR1/LCDR2/LCDR3 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:64/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:93/SEQ ID NO:88/SEQ ID NO:96, SEQ ID NO:99/SEQ ID NO:88/SEQ ID NO:89, SEQ ID NO:99/SEQ ID NO:88/SEQ ID NO: 96; (3) substitution, deletion or insertion of one or more amino acid residues of not more than 5 amino acids in the amino acid sequence of the CDRs.

The sequences are shown in the following table:

in some embodiments, the anti-Siglec-15 antibodies or antigen-binding fragments thereof provided herein comprise: a human constant domain which is an 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 anti-Siglec-15 antibody is a murine monoclonal antibody, a chimeric monoclonal antibody, a humanized antibody, a single chain antibody, a light chain constant region optionally being a human kappa chain or lambda chain constant region, a heavy chain constant region optionally being a human antibody IgG1 or constant region of IgG4, and a mutant of a constant region of IgG1 or IgG 4.

In some embodiments, the antibody comprises a monospecific antibody, bispecific antibody or fusion protein, a trispecific antibody or fusion protein, or a 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, TGF β, VEGF, HER3, TIGIT, CLAUDIN18.2, CD3, TIM3, LAG3, and the like.

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

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

Preferably, the invention provides a nucleic acid sequence comprising SEQ ID NO 101, SEQ ID NO: 102. SEQ ID NO: 103. 104 sequence of SEQ ID NO. As shown in the following table:

in some embodiments, the present 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-described expression vector, preferably the host cell is selected from a lactating animal cell.

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

In some embodiments, the anti-Siglec-15 antibodies or antigen-binding fragments thereof provided herein are therapeutically active on Siglec-15 expressing or overexpressing cancer cells, macrophages, or cancers.

In some embodiments, the antibodies or antigen binding fragments thereof against Siglec-15 provided herein can inhibit osteoclastogenesis and be used to treat osteoporosis.

In some embodiments, the present invention provides a method of inhibiting osteoclastogenesis, comprising administering to a subject in need thereof an effective amount of the anti-Siglec-15 antibody or 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 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 or antigen-binding fragments thereof against Siglec-15 provided herein are capable of enhancing an immune response, impeding or preventing tumor growth, inhibiting tumor-mediated immunosuppression, eliminating a tumor, depleting or blocking the activity of tumor-associated macrophages (TAMs) so as to alter the activity thereof, reducing TAM-mediated immunosuppression, reducing or reversing T-cell suppression; can enhance the secretion of IFN gamma and TNF-alpha as cytokines and activate CD4+ T cells or CD + T cells; binding of Siglec-15 to its ligand may be increased and/or Siglec-15 mediated signal transduction may be increased or enhanced. Antibodies modified will also exhibit impaired, enhanced or Fc-free receptor (FcR) binding activity as well as enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) activity.

The invention has the following beneficial effects:

the anti-Siglec 15 antibody is screened from a series of technical routes such as antigen design and synthesis, mouse immunization, cell fusion, hybridoma screening, subclone identification, in-vitro protein binding experiments, affinity identification, epitope identification, cell binding experiments with human/mouse/cynomolgus monkey, T cell-related functional experiments, antibody humanization, antibody affinity maturation, osteoclast differentiation experiments and the like to obtain a mouse antibody (11H5E6, 48B5B1 and 69C4D8) with good biological activity and a series of humanized and affinity matured antibody molecules, wherein the antibody molecules such as L2H1-58-PTM, 14-58-2 and the like show affinity better than that of a control antibody 5G12 in the cell binding experiments and show excellent activity in-vitro T cell proliferation experiments and the like. Has obvious difference with the binding epitope and sequence of the control antibody. Has wide application prospect in the fields of cancer and osteoporosis.

Drawings

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

FIG. 2a shows the results of the detection of antibody titers in an ELISA assay in which mouse sera were bound to human Siglec-15 protein; FIG. 2b shows the results of FACS experiments in which mouse serum was bound to human Siglec-15 protein to detect antibody titers.

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

FIGS. 4a and 4b show the FACS comparative screening results of blank CHOS cells and CHOS cells expressing human siglc-15 for hybridoma cell lines with successful subcloning.

FIGS. 5a, 5b, 5c and 5d show the results of experiments in which 26 subcloned cell lines identified were expressed and the expression supernatants were cell-bound to blank CHOS cells, human Siglec-15 expressing CHOS cells, cynomolgus monkey Siglec-15 expressing CHOS cells and mouse Siglec-15 expressing CHOS cells, respectively.

FIG. 6 shows the results of experiments on the proliferation of recovered CD4/CD 8T cells from antibody supernatants from 26 hybridoma cell lines and 4 blood cell donors.

Fig. 7a and 7b show that 26 hybridoma cell supernatants purified antibodies were subjected to osteoclast differentiation experiments.

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

FIGS. 9a, 9B, and 9C show the 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 to detect the relative binding epitope of a candidate antibody to 5G12 by BLI technique (ForteBio).

Fig. 12a, 12b, and 12c show the results of the thermal stability test using the melting curve.

Figure 13 shows the affinity results of the humanized antibody by Fortebio assay.

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

Figure 15 shows the humanized antibody in recovery of T cell proliferation experimental results.

FIG. 16 shows the results of FACS cell binding of affinity matured antibodies to CHOS cells expressing human Siglec-15.

FIG. 17 shows the results of FACS cell binding of affinity matured antibodies to CHOS cells expressing murine Siglec-15.

FIG. 18 shows the results of FACS cell binding of affinity matured antibodies to CHOS cells expressing cynomolgus Siglec-15.

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

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

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

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

FIG. 23, FIG. 24, FIG. 25 show the results of experiments on the recovery of T cell proliferation by L2H1-58-PTM, 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 methodologies, 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 the 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. "antibodies" (or "Abs") and "immunoglobulins" (or "Igs") are glycoproteins having the same structural features, and antibodies 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 by a covalent disulfide bond (also referred to as a "VH/VL pair"), each heavy and light chain also having a regular arrangement of intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by a plurality of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; 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 the method of its preparation. Monoclonal antibodies or immunologically active fragments thereof can 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 involving the production of monoclonal antibodies in the present invention include in vitro culture production of hybridoma cells or production by DNA recombinant 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 sequences of the variable domains of antibodies vary to a greater degree and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is unevenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the light and heavy chain variable regions, called Complementarity Determining Regions (CDRs) or hypervariable regions. The more highly conserved portions of the variable domains are called the Framework (FR). The variable domains of 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 N-terminus to C-terminus. Most FR regions adopt a β -sheet configuration, connected by three CDRs, forming a loop junction, and in some cases forming part of the β -sheet structure. The CDRs in each chain are held 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, for example, to Kabat et al, immunological protein sequences, fifth edition, national institutes of health, besesda, 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) and complement killing pathway (CDC).

Antibodies can be classified into 5 classes, distinguished by the amino acid sequence of the antibody heavy chain constant region: IgA, IgD, IgE, IgG, and IgM, and several of these homogeneous classes can be further divided into subclasses, e.g., IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA 2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. Kappa and lambda can be classified according to the constant region (CL) of the antibody light chain. Within full-length light and heavy chains, the variable and constant regions are typically linked 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 the constant heavy chain domain (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 includes 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 by the addition of several residues 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. The Fv fragment is the smallest fragment in the products of the enzymatic analysis of antibodies of the IgG and IgM type. Fv fragment antigen binding region, it is composed of VH and VC region, but they lack CH1 and CL region. VH and VL are held together by non-covalent bonds in the Fv fragment. ScFv are Fv-type fragments that comprise VH and VL regions joined together by a flexible polypeptide chain.

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

The term "humanized antibody" refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a 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 the sequence of a non-human antibody, and all or substantially all of the FRs correspond to the sequence of a human antibody. The humanized antibody of the invention mainly refers to an antibody which is reformed and re-expressed by a mouse monoclonal antibody by gene cloning and DNA recombination technology, most of amino acid sequences (mainly comprising a constant region and a FR region) of the humanized antibody are replaced by human sequences, and a CDR region adopts no change or little mutation, so that the affinity and the specificity of the parent mouse monoclonal antibody are basically kept, the heterogeneity 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 between a target cell and a functional molecule (cell) to trigger a targeted immune response, and is one of genetically engineered antibodies. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404, 097; WO 1993/01161; hudson et al, nat. med.9: 129-134 (2003); and Hollinger et al, proceedings of the national academy of sciences of the united states (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 between target cells and functional molecules (cells) to stimulate targeted immune response, and is one of genetically engineered antibodies. The triabody may be trivalent or trispecific.

The term "epitope" refers to any antigenic determinant on an antigen that binds to the paratope of an antibody. Epitopic determinants generally consist of chemically active surface groups of the molecule, 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 which are self-replicating nucleic acid structures as well as vectors which are incorporated into 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 into which an exogenous nucleic acid is introduced, including progeny of such a cell. And can express exogenous nucleic acid in cell or cell membrane or release it to outside of cell.

The term "cancer" refers to a neoplasm or tumor resulting from abnormal uncontrolled growth of cells. As used herein, cancer specifically includes leukemia and lymphoma. The term "cancer" refers to a disease involving cells that have the potential to metastasize to distant sites and exhibit phenotypic properties different from non-cancerous cells, e.g., colony formation in a three-dimensional matrix such as soft agar or tubular network or reticulated matrix 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 term "immunological", "immunological" or "immune" response is a humoral (antibody-mediated) and/or cellular (mediated by antigen-specific T cells or their secretory products) response that is beneficial for the development of peptides in recipient patients. Such responses may be active responses induced by administration of an immunogen or passive responses induced by administration of antibodies or primed T cells. The cellular immune response is elicited by the presentation of polypeptide epitopes associated with MHC class I or class II molecules to activate antigen-specific CD4+ T helper cells and/or CD8+ cytotoxic T cells. The response may also involve activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, activation or recruitment of 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 contribution of humoral and cellular responses to the protective or therapeutic effect of an immunogen can be distinguished by isolating antibodies and T cells from the immunized syngeneic animal alone and measuring the protective or therapeutic effect in a second subject.

The term "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties. A typical variant of a polypeptide differs 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. The variant 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 a 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 hydrophilicity index in conferring interactive biological function to a polypeptide is generally understood in the art. It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still result in polypeptides having similar biological activity. Each amino acid has been assigned a hydropathic index based on its hydrophobic 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).

It is believed that the relative hydrophilic character of the amino acids determines 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 for another amino acid with a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, amino acids with a hydropathic index within ± 2 are preferably substituted, particularly preferably those within ± 1, even more particularly preferably those within ± 0.5.

Substitutions of like amino acids may also be made on the basis of hydrophilicity, particularly where the resulting biologically functional equivalent polypeptide or peptide is intended for use in immunological embodiments. The following hydrophilicity values have been assigned to amino acid residues: 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 is understood that an amino acid may be substituted for another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent, in particular an immunologically equivalent polypeptide. In such changes, amino acids with 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 the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take into account the various aforementioned characteristics are well known to those skilled in the art and include (original residue: exemplary substitution): (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). Accordingly, embodiments of the present disclosure contemplate functional or biological equivalents of the antibody sequences as described above. In particular, large embodiments may include variants having about 95% or more sequence identity to the antibody sequence.

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

Examples

Example 1 Generation of leader anti-Siglec-15 antibodies

1.1 antigen design and synthesis: the sequence of the extracellular domain of human Siglec-15 protein (amino acids 20-263, https:// www.uniprot.org/blast/: "FVRTKIDTTENLLNTEVHSSPAQRWSMQVPPEVSAEAGDAAVLPCTFTHPHRHYDGPLTAIWRAGEP YAGPQVFRCAAARGSELCQTALSLHGRFRLLGNPRRNDLSLRVERLALADDRRYFCRVEFAGDVHDR YESRHGVRLHVTAAPRIVNISVLPSPAHAFRALCTAEGEPPPALAWSGPALGNSLAAVRSPREGHGHLV TAELPALTHDGRYTCTAANSLGRSEASVYLFRFHGASGAST". DNA encoding the above amino acid sequence was synthesized and ligated with DNA encoding camel FC, and the synthesized gene was constructed into PCDNA3.4 vector. The plasmid was transformed into E.coli TOP10, and a single clone was picked and expanded overnight in LB medium containing 1 ‰ ampicillin. After centrifugation at 10000G for 10 minutes, the plasmid was extracted by alkaline lysis (Biomiga, PD 1511).

When animals are immunized by using the antigen with human Fc connected with Siglec15 extracellular domain, the antigen expression yield is not high, and the animal serum is not easy to convert positive. In order to overcome the limitations of protein immunization and the problem of positive conversion efficiency, camel Fc is connected with Siglec15 extracellular domain, three linear immune peptides, namely NH2-CTFTHPHRHYDGPLT-CONH2, NH2-CRVEFAGDVHDRYESR-CONH2NH2-CWRAGEPYAGPQV-CONH2, are creatively designed aiming at the Siglec15 protein, and one cyclopeptide, namely NH2-CTFTHPHRHYDGPLTC-CONH2, are coupled with KLH protein for auxiliary boosting immunization so as to break through immune barriers.

Using thermo Expi293^TMExpression System Kit (A14635) produces antigen. Expi293 cells were recovered, transfected with plasmid using the kit recommendations, and cultured. The culture broth was harvested and centrifuged at 5000G for 10 minutes to remove cells and cell debris. The antigen was purified using protein A/G (50% each, 1:1 mix, Suzhou Bo Chin Biotech; protein A:18-0010-02, protein G: 16-0010-. The microspheres are inverted and mixed evenly, 1ml is filled into a column, and the column is washed by 10 times of column volume of an equilibrium buffer (0.15M sodium chloride, 0.02M PB, pH7.0). Adding the centrifuged culture supernatant, slowly adding into an adsorption column, and repeating twice by gravity flow. After washing with 10 column volumes of equilibration buffer, wash was addedDecuffer (0.15M sodium chloride, 0.1M glycine, 0.02M PB, pH 3.0). Coomassie brilliant blue (10. mu.l per well, 96 well plate) was used to continuously detect protein wash-out, i.e., 10. mu.l was added per well. When Coomassie Brilliant blue became visibly blue, the eluted fractions were collected to no blue. The chromatographic column was passed through 10 column volumes of eluent and 10 column volumes of equilibration fluid and stored in 20% ethanol. The eluted protein was dialyzed against formulation buffer (25 mM sodium citrate, 50 mM sodium chloride, 100 mM arginine, 5% sucrose), assayed for protein concentration by BCA (4.55mg/ml), assayed for protein purity by SDS-PAGE (FIG. 1) and Coomassie blue staining and stored at-20 ℃ until use, designated BPSL 15-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 protein molecular weight prediction.

1.2 mouse immunization and fusion: 6 female Balb/c mice 6-8 weeks old were divided into 2 groups: group a and group B, 3 per group. 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 in the back with 100. mu.g of antigen (BPSL 15-C). Group a mice were first immunized with freund's complete adjuvant. Freund's incomplete adjuvant was used for the second and third immunizations. Group B mice were first immunized with freund's complete adjuvant. The solubility enhancing adjuvant PAP-1 (by SGE Biotech, 20181129) was used at the second and third immunizations. 7 days after the 3 rd immunization, serum was extracted from the tail vein and antibody titer was 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), three days after boosting for hybridoma fusion. Electrofusion of 15C-SGZ-2 and 15C-SGZ-3 splenocytes with SP2/0 cells (splenocytes: SP 2/0: 1) in 5% CO₂Cultured in an incubator (100 96-well plates, 20% FBS HAT medium) and screened by HAT (hypoxanthine aminopterin thymidine) medium.

Mouse sera were tested for antibody titer in combination with human Siglec-15 in ELISA experiments: mu.g/ml human Siglec-15his protein (ACRO SG5-H52H3) was plated overnight in PBS on ELISA plates. The following day the antigen solution was aspirated, washed three times with PBST (0.5 ‰), and then blocked with 200. mu.l of blocking solution (1% BSA in PBS, BSA: Biorfox, 4240GR100) for 2 hours at room temperature. After aspiration of the blocking solution, the serum was washed three times with PBST (0.5 ‰), and 100. mu.l of each mouse serum was added, which was diluted in a gradient with the blocking solution. The mouse serum is diluted by 8 points 4 times with 1 per mill as the initial concentration. After incubation of the samples for 1 hour at room temperature, after washing three times with PBST (0.5 ‰), 100. mu.l of a second antibody (Abcam, ab97040) against horseradish peroxidase conjugated mouse FC dissolved in blocking solution was added and incubated for 1 hour at room temperature. After PBST (0.5 ‰) washing, 100 μ l TMB developing solution (Solabio, PR1200) was added, developing at room temperature for 10min, 50 μ l stop solution (Solabio, C1058) was added to stop the reaction, and OD450 absorbance was measured with a multifunctional microplate reader (FIG. 2 a). FIG. 2a shows the results of the detection of antibody titers in an ELISA assay of mouse sera in combination with human Siglec-15 protein, with 15C-SGZ-2 and 15C-SGZ-3 having the highest titer.

1.3 mouse sera combined with human Siglec-15 to detect antibody titers in FACS experiments: construction of full-length CHOS cells overexpressing human Siglec-15. The synthetic nucleic acid sequence encoding the full length of human Siglec-15 (MEKSIWLLACLAWVLPTGSFVRTKIDTTENLLNTEVHSSPAQRWSMQVPPEVSAEAGDAAVLPCTF THPHRHYDGPLTAIWRAGEPYAGPQVFRCAAARGSELCQTALSLHGRFRLLGNPRRNDLSLRVERLAL ADDRRYFCRVEFAGDVHDRYESRHGVRLHVTAAPRIVNISVLPSPAHAFRALCTAEGEPPPALAWSGP ALGNSLAAVRSPREGHGHLVTAELPALTHDGRYTCTAANSLGRSEASVYLFRFHGASGASTVALLLGA LGFKALLLLGVLAARAARRRPEHLDTPDTPPRSQAQESNYENLSQMNPRSPPATMCSP) was constructed into ORF1 of the PCHO1.0 plasmid, plasmid extracted (by expression of the same antigen), and the plasmid was linearized overnight (total 100. mu.g) with NRUI restriction enzyme (NEB, R0192S) as recommended. The CHOS cells were revived using CD-forti CHO (Gibco, A1148301) +8 mmole glutamine (Gibco, 25030081) + anti-caking agent (Gibco, 0010057AE)120rpm/37 ℃/5% CO₂And (5) shaking culture. And using linearized plasmid and Freestyle MAX reagent (thermo, 16447100) to transfect logarithmic phase CHOS cells (50 ug plasmid/50 ul Freestyle MAX, 1 × 10^6/ml 30ml CHOS cells, 100 nanomolar MTX (SIGMA, A6770-100 MG)/10 ug/ml puromycin (Gibco, A1113803) according to the specified method to select stable cells, i.e. cells transfected for 48 hours are replaced by fresh medium containing the selection pressure, cell density is adjusted to 1 × 10^6/ml, 120rpm/37 ℃/5% CO₂And (5) shaking culture. One week later, cells were examined every other dayActivity rate/density. Maintaining viable cell density less than 2.0 x 10^6/ml, passaging cells at 0.5 x 10^6/ml, and freezing partial cells (growth medium + 10% DMSO) when cell viability reaches more than 95%. And continuously pressurizing and screening the rest cells until the survival rate is more than 99 percent, and freezing and storing the cells again. Mouse sera bound FACS to human Siglec-15: using PBS 1: mouse serum was diluted 500 and 100. mu.l of the diluted serum was incubated with 200000 CHOS cells overexpressing human Siglec-15 for 30 minutes at 4 ℃. After three washes with PBS, cells were incubated with 1: 200 dilution in PBS anti mouse FC fluorescent secondary antibody (eBioscience, 17-4010-82)4 degrees C light-protected incubation for 30 minutes, PBS washing three times, FACS detection of APC fluorescence channel mean-FI value (figure 2 b). FIG. 2b shows that 15C-SGZ-1, 15C-SGZ-2 and 15C-SGZ-3 bind well to CHOS cells.

Example 2 subclone screening and characterization

After screening by antigen binding ELISA, 138 clones were selected for FACS binding experimental identification of CHOS cells expressing human siglec-15 (fig. 3). 46 FACS positive clones were subcloned, 30 clones were successfully subcloned and screened by human siglc-15 expressing CHOS cells and naive CHOS cells (see FIGS. 4a and 4b for screening results). 26 subclones were selected for antibody production. The hybridoma supernatants were purified by protein G. FACS binding experiments were performed on blank CHOS cells (fig. 5a), CHOS cells expressing human Siglec-15 (fig. 5B), CHOS cells expressing Cyno Siglec-15 (fig. 5C) and CHOS cells expressing mouse Siglec-15 (fig. 5d), respectively, followed by sequence identification and further characterization by T cell proliferation experiments (fig. 6), osteoclast differentiation experiments (fig. 7a and 7B), Fortebio affinity experiments (fig. 8), screening to obtain 3 lead mouse-derived anti-Siglec-15 antibodies (11H5E6, 30C2B12 and 48B5B 1).

2.1 ELISA, FACS identification of hybridomas: positive clones were identified 10 days after hybridoma cell fusion plating using antigen binding ELISA. The sample is the supernatant of hybridoma supernatant after centrifugation at 5000g for 10 minutes. Methods human Siglec-15 binding ELISA was referenced to mouse serum and the antigen plating concentration was changed to 0.5. mu.g/ml. A total of 138 clones with ELISA signals three times greater than the negative control signal were subjected to FACS binding of human Siglec-15 in the supernatant of 138 clones, as was done with reference mouse serum in combination with human Siglec-15. FACS binding to the experimental sample was 100. mu.l supernatant. FIG. 3 shows that a total of 46 clones with higher signals were selected as FACS positive and subjected to the next subcloning. The clone numbers are respectively: 8H6, 11E3, 11H5, 14H4, 15D1, 16a4, 17A3, 17F5, 23E 5, 27G 5, 30C 5, 32a 5, 37E 5, 40a5, 46a 5, 47C 5, 48a 5, 48B5, 48D 5, 52G 5, 54E 5, 55E 5, 56H 5, 57F 5, 58H 5, 69C 5, 69F 5, 70E 5, 74D 5, 75G 5, 76a 5, 84F 3687 a5, 89F 5, 89G 5, 90H 5, 92B 5, 93B5, 96C 3696C 5, 97B 5, 97H 5, 3699H 5, 3699F 5, 3699.

2.2 subcloning of positive cell lines: the selected 46 cloning wells were subjected to one subcloning. And blowing and mixing the cells in the positive holes uniformly, and counting the number of the living cells by a counting plate. Each clone was sub-clonally plated in1 96-well plate at a density of 0.5 per well in 1640+ 10% FBS + HT (hypoxanthin, Thymidine). 14 days after subcloning, subclone supernatant ELISA assays were performed. A total of 30 subclones of 46 parent clones were successful.

2.3 flow cytometry characterization of subclones: for each parent, 2-3 strong signal subclones were selected for FACS detection, including null cells and CHOS cells overexpressing human SIGEC-15. Methods reference mouse sera were combined with human Siglec-15 in FACS experiments. . The comparative screening results of FIGS. 4a and 4b show that a total of 26 subclones have better binding activity to CHOS cells overexpressing human SIGEC-15.

2.4 production purification of subclone culture supernatant, flow cytometry combined functional validation: the 26 subcloned cell lines selected by the expanded culture were cultured until a considerable number of cells began to die, and the culture supernatant was collected by centrifugation. Antibodies in the supernatant were purified using protein G, the method being referenced to antigen synthesis. CHOS cells over-expressing mouse and cynomolgus monkey Siglec-15 are constructed according to the construction method of over-expressing human Siglec-15 CHOS cells. Flow cytometry binding function validation of null CHOS cells, CHOS overexpressing human Siglec-15, CHOS overexpressing murine Siglec-15, CHOS overexpressing cynomolgus Siglec-15 was performed using purified murine antibodies, the procedure was reference to mouse serum binding FACS with human Siglec-15, samples were changed to gradient diluted purified antibodies. FIGS. 5a, 5b, 5c, and 5d show that the antibody supernatants produced by some of the 26 subcloned cell lines bind well to CHOS cells that overexpress Siglec-15 in murine and cynomolgus monkeys. 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 purified antibody from the supernatant of 26 hybridoma cells was examined in a T cell proliferation assay. Clone 30C2B12 was found to stably recover Siglec-15 inhibited T cell proliferation in experiments with multiple different blood cell donors (fig. 6). The experimental method is as follows:

(1) frozen PBMC (all cells, PB004F) were thawed from liquid nitrogen by rapidly shaking the tubes in a 37 ℃ water bath.

(2) The cells were added to 10mL of pre-warmed T cell culture medium (AIM-V + 5% human AB serum; AIM-V: Gibco, 12055091). Human AB serum: GEMINI, 100-. The cells were resuspended in 15mL of T cell media (Invitrogen, 18047019) containing 0.01% DNase I, placed in a T75 flask and incubated at 37 ℃ for 2 hours.

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

(4) Add 5. mu.g/mL Siglec-15(ACRO, SG5-H5253) to the wells. anti-Siglec-15 antibody was then added to the wells at 45. mu.g/mL. Cells were cultured at 37 ℃ for 72 h.

(5) After 72 hours, the 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 activated with APC-Cy ^TM7 mouse anti-human CD3(BD, 557832)/BV421 mouse anti-human CD4(BD, 562842)/APC mouse anti-human CD8(BD, 555369) staining.

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

2.6 osteoclast differentiation assay: the 26 hybridoma cell supernatants were purified for osteoclast differentiation. FIGS. 7a and 7B show that clones 11H5E6/48B5B1 and 69C4D8 were found to reduce TRAP enzyme activity in the supernatant and reduce osteoclast differentiation, and clone 48B5B1 was more pronounced but weaker than the positive control. For experimental methods reference is made to the patent issued by the company Next cure: WO 2018057735. The experimental method is as follows: PBMC were recovered from liquid nitrogen in fresh medium and monocytes were purified according to the recommended method of the kit (gentle, 130-096-537). Cells were plated at a density of 10 ten thousand cells per well in 96-well flat-bottom cell culture plates for a total of three parallel wells. The cell culture medium is alpha-MEM (Gibco, 32571-. After 3 hours incubation at 37 ℃ for adherence, an anti-human Siglec-15 antibody (containing the positive control antibody 25E9 from Alethia, U.S. Pat. No. US8741289) was added at a final concentration of 50. mu.g/ml. After three days of culture, the fresh medium containing the stimulating factor was replaced. After seven days of culture, the supernatant was transferred and centrifuged to remove cells. Adherent monocytes/osteoclasts were assayed for TRAP enzyme activity in the supernatant (FIG. 7a) following the kit recommendations TRAP staining (B-Bridge, PMC-AK04F-COS, FIG. 7B). And (4) taking a photograph and staining the photograph by using a microscope photo-set camera, and detecting the OD540 light absorption value by using a multifunctional microplate reader.

2.7 antibody affinity assay: the affinity of the candidate antibody in different screening states was tested by 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 100nM his tag (Kactus, SIG-HM415) in solution. The association and dissociation rates were measured and K-on/K-off and KD calculated by fitting the signal curves. FIG. 8 shows that both 11H5E6/48B5B1 and 69C4D8 antibodies have higher affinity for antigen.

2.8 molecular epitope identification of lead chimeric antibodies: relative binding of the candidate antibody to 5G12 was detected by BLI technique (Fortebio). For example, 100nM 5G12-IgG4 antibody was loaded onto the AHC sensor; siglec-15his protein (Kactus, SIG-HM415) was associated with the 5G12 antibody. The sensor is then blocked by the human Fc containing protein, and another antibody is associated with his antigen. If the secondary antibody is capable of binding to the antigen, the secondary antibody is on the other box. If not, both antibodies are placed on the same box. The results in FIG. 11 show that the three antibody molecules 11H5E6, 30C2B12, 48B5B1 are not in the same epitope as 5G12, 11H5E6 and 30C2B12 have the same epitope

2.9 thermostability assay of lead chimeric antibodies: materials and methods: the melting curve was detected using a Rogowski optical cycler 480 real-time PCR instrument. Briefly, 45 μ L of 5 μ M protein was added in triplicate to a PCR tube (or plate). Fresh 200 XSSYPRO orange solution (from 5000 stock, 5000. times. concentration DMSO solution; Life Technologies S-6650) was prepared in the buffer of choice. To each sample was added 5. mu.L of 200 XSSYPRO orange solution and mixed well. The sample was held at 25 ℃ for 30s and readings taken; raised by 0.5 ℃, raised from 25 ℃ to 99 ℃ and then returned to 25 ℃. Tm was measured using melting curves (fig. 12a, 12b, 12c), and the Tm1 for the 3 chimeric antibodies were: 11H5E6 was 60.5 ℃, 30C2B12 was 62.5 ℃, 48B5B1 was 62 ℃, and thermal stability was good.

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

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

3.1.1 Gene cloning: the sequences of antibody heavy/light chains were obtained using Rapid Amplification of CDNA Ends (RACE). The method specifically comprises the following steps: (1) the hybridoma cells were expanded to 80% by blowing from the flask, centrifuged at 1500rpm for 5 minutes, and 200. mu.l PBS was used to resuspend the cells. (2) Add 800. mu.l Trizol lysate, shake vigorously, and let stand at 4 ℃ for 10 minutes. (3) After adding 250. mu.l of chloroform, the mixture was vigorously shaken for 5 minutes and allowed to stand at 4 ℃ for 5 minutes. (4) centrifuging the sample at 12000rpm at 4 ℃ for 15 minutes, adding the supernatant to 600. mu.l of isopropanol, gently mixing the supernatant and the isopropanol, and standing the mixture at 4 ℃ for 10 minutes. (5) After centrifugation at 12000rpm/4 ℃ for 10 minutes, the supernatant was carefully decanted. Adding 800 μ l of pre-cooled 75% ethanol, washing, removing ethanol, and oven drying at 72 deg.C. (6) cDNA was obtained using a reverse transcription kit. (7) The cDNA is PCR amplified with degenerate primers that capture the variable region of the heavy chain or the variable region of the light chain. (8) constructing a T vector by using the PCR amplification product, transforming TOP10 competent cells, and picking and sequencing a single clone. Three single clones were sequenced in total: 11H5E6/30C2B12/48B5B1, resulting in the corresponding VH and VL sequences.

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 the PCDNA3.4 vector containing the IgG4 heavy chain constant region sequence by PCR, restriction enzyme, to construct the complete heavy chain. The DNA sequence of the light chain variable region was optimized and synthesized, and this sequence was ligated by PCR, enzymatic cleavage, into the PCDNA3.4 vector containing the KAPPA light chain constant region, to construct the complete light chain. The two plasmids were transformed into TOP10 competent cells and the plasmids were extracted in bulk (methods referenced for antigen synthesis).

3.1.3 expression: the CHOS cells were transfected with the large amount of the extracted seed light chain plasmid, and cultured for 7 days with the addition of a feed.

3.1.4 purification: the cells cultured as described above were harvested, the corresponding antibody was purified using protein A, and the purity was checked by SEC/SDS-PAGE (methods referred to antigen synthesis).

3.1.5 chimeric antibody flow cytometry in vitro binding Activity identification: the chimeric antibody and the negative control antibody are taken to carry out a binding experiment with CHOS cells over expressing human/cynomolgus monkey/mouse Siglec-15, and the results of the FACS experiment combining mouse serum and human Siglec-15 are shown in a figure 9a, a figure 9B and a figure 9C, and show that the three chimeric antibodies of 11H5E6, 48B5B1 and 69C4D8 respectively have higher binding activity with the CHOS cells expressing human Siglec-15, the CHOS cells expressing cynomolgus monkey Siglec-15 and the CHOS cells expressing mouse Siglec-15. 3.1.6T cell proliferation assay of chimeric antibodies: the chimeric antibody and the negative control antibody are taken to carry out T cell proliferation experiments, the method refers to hybridoma supernatant purified antibody T cell proliferation experiments, the results are shown in figures 10a and 10B, and the titer of the 30C2B12 chimeric antibody in different donors in the T cell proliferation recovery experiments is superior to that of the control 5G 12.

3.2 humanization of lead anti-Siglec-15 antibodies

3.2.1 molecular design:

humanization of lead candidate 30C2B12 used CDR grafting and back mutation. Application of CDRs numbering convention definitionFRs and CDRs regions. IGBLAST searches were performed for VL and VH of the 30C2B12 sequence, respectively, based on the antibody sequence database. According to the search results, 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 from the mouse 30C2B12 sequence were grafted into VL and VH templates, respectively, and homology modeling was performed using the Modeller open source program. Comparing mouse source sequences of the 3D model 30C2B123D model, and selecting

As a Grafted 3D model.

Transplanted 3D models were energy minimized by NAMD open source procedure and few residues were mutated back to mouse-derived residues according to the rules of three-dimensional structure, physicochemical properties and energy minimization. The sequence of the above models was optimized in terms of isoelectric point, PTMs, aggregability, developability, immunogenicity, etc.

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

3.2.3 antibody characterization: the affinity of the humanized molecules was tested using fortebio and figure 13 shows that the antibodies after humanization all retain consistent binding activity with the murine molecule.

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

3.2.5T cell proliferation assay for humanized antibodies: the purified antibody and the negative control antibody are taken to carry out a T cell proliferation experiment, the method refers to a hybridoma supernatant purified antibody T cell proliferation experiment, the result is shown in figure 15, and most of the daughter molecules show the functional activity equivalent to that of the parent molecule 30C2B 12.

3.3 affinity maturation of humanized anti-Siglec-15 antibodies

3.3.1 antibody affinity maturation using phage display technology with L2H1 as the parent molecule: (1) preparing materials: namely, detection of antigen antibody (ELISA/SDS-PAGE). (2) Gene synthesis: based on hamster codon optimized sequences, primers were designed for insertion into the expression vector. (3) Female parent verification: and (3) verifying the constructed maternal sequence and the activity ELISA of the Fab antibody. (4) Constructing an antibody engineering library: through a single-point saturation mutation method, 8 mutation libraries are constructed through four mutation schemes and two frameworks of LCDR1+ LCDR3+ HCDR3/LCDR2+ HCDR1+ HCDR2/LCDR3/HCDR3 respectively, and the quality and antibody diversity analysis of the libraries is verified. (5) Antibody sea-selection, namely screening phage capable of being combined with the antibody combined on the immune tube by a multi-round panning method based on the immune tube, and detecting the affinity characteristics of the phage ELISA and prokaryotic expression supernatant ELISA. The total number of clones with better affinity activity is 38 after sequencing. (6) According to the primary screening ELISA signal and prokaryotic expression supernatant ELSIA EC50 and whether the sequence has PTM site, more preferably 8 clones are constructed into IgG 4S 228P constant region to be full-field antibody. The 8 cloned plasmids were transfected into CHOS cells and purified by protein A to obtain antibodies.

3.3.2 flow cytometry in vitro binding Activity identification of antibodies after affinity maturation: the purified antibodies and negative control antibody were used for binding experiments with CHOS cells overexpressing human/cynomolgus monkey/mouse Siglec-15, and the results of FACS experiments combining mouse serum and human Siglec-15 were shown in FIG. 16, FIG. 17, and FIG. 18. The result shows that the affinity matured antibody shows better binding activity in the CHOS cell binding experiment of over-expressing human/cynomolgus monkey/mouse Siglec-15.

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

Example 4 Gene clone construction and expression purification of candidate antibody

4.1 clone construction: respectively number the

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

4.2 expression purification: the plasmids are transformed into TOP10 competent cells, and a large amount of plasmids are extracted, transfected into expi-293 cells according to the method recommended by a kit, cultured and fermented. The antibody was purified using a protein a adsorption column.

Example 5 Activity identification and functional assay of optimized antibodies

5.1 flow cytometry in vitro binding Activity identification of the optimized antibodies: the purified antibody and the negative control antibody were used for binding experiments with CHOS cells overexpressing human/cynomolgus monkey/mouse Siglec-15. The antibody was diluted 3-fold for 12 points at 100nM initial concentration. (methods reference mouse sera in combination with human Siglec-15 FACS experiments). The results in fig. 20, 21, 22 show that the antibody molecules after affinity maturation have a higher affinity in binding experiments with CHOS cells overexpressing human/cynomolgus monkey/mouse Siglec-15 than before unaffinity maturation, even exceeding the positive control antibody 5G12 molecule.

5.2T cell proliferation assay of the optimized antibodies: the purified antibody and the negative control antibody are taken to carry out a T cell proliferation experiment (the method refers to a hybridoma supernatant purified antibody T cell proliferation experiment). Experiments were performed using PBMCs from three different donors in total. Wherein blank is blank control, namely a group without CD3 stimulation; the CD3 group was added with a CD3 plate as a positive control; the CD3+ Siglec-15 group inhibited T cell proliferation by adding CD3 plate-coated stimulation and adding Siglec-15 protein. 5G12-IgG1 is a positive control, unrelated IgG1 is a negative control, and the functions of the candidate antibodies L2H1-58-PTM and 14-58-2 are detected, and the results are shown in FIG. 23, FIG. 24 and FIG. 25, which show that the molecules L2H1-58-PTM and 14-58-2 have higher in vitro functional activity.

Sequence listing

<110> times Dada pharmaceutical industries (Suzhou) Co., Ltd

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

<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 (16)

1. An antibody or antigen-binding fragment thereof against Siglec-15, comprising: a heavy chain variable region VH sequence and a light chain variable region VL sequence, the 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; the VL sequence has at least 95% identity to an amino acid sequence selected from the group consisting of seq id nos: 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.

2. the anti-Siglec-15 antibody or antigen-binding fragment thereof according to claim 1, comprising: 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.

3. the anti-Siglec-15 antibody or antigen-binding fragment thereof according to claim 1, comprising 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 an 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 the group consisting of 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) the HCDR of (1), (2) and (3) above which contains one or more amino acid substitutions, deletions or insertions of not more than 5 amino acids.

4. The anti-Siglec-15 antibody or antigen-binding fragment thereof according to claim 1, comprising the sequence of one or more light chain complementarity determining regions (LCDRs) selected from the group consisting of (1) LCDR1 comprising a heavy chain variable region (LCDR) consisting of SEQ ID NO: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 the group consisting of 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) LCDRs of (1), (2) and (3) above containing one or more amino acid substitutions, deletions or insertions of not more than 5 amino acids.

5. The anti-Siglec-15 antibody or antigen-binding fragment thereof according to claim 1, comprising a HCDR1/HCDR2/HCDR3 combination and/or a LCDR1/LCDR2/LCDR3 combination: (1) the HCDR1/HCDR2/HCDR3 combination comprises sequences of one or more sets of heavy chain complementarity determining region HCDRs selected from: SEQ ID NO 49/50/51, 55/56/57, 61/62/63, 67/68/63, 69/70/71, 74/75/63, 77/78/63, 74/80/83/63, SEQ ID NO 74/SEQ ID NO 85/SEQ ID NO 63, SEQ ID NO 78/SEQ ID NO 63, SEQ ID NO 74/SEQ ID NO 87/SEQ ID NO 63, SEQ ID NO 90/SEQ ID NO 85/SEQ ID NO 63, SEQ ID NO 74/SEQ ID NO 91/SEQ ID NO 63, SEQ ID NO 74/SEQ ID NO 92/SEQ ID NO 63, SEQ ID NO 74/SEQ ID NO 95/SEQ ID NO 63, SEQ ID NO 74/SEQ ID NO 97/SEQ ID NO 63, SEQ ID NO 74/SEQ ID NO 98/SEQ ID NO 63; (2) LCDR1/LCDR2/LCDR3 in combination comprises one or more sets of light chain complementarity determining region LCDR sequences selected from 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:64/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:93/SEQ ID NO:88/SEQ ID NO:96, SEQ ID NO:99/SEQ ID NO:88/SEQ ID NO:89, SEQ ID NO:99/SEQ ID NO:88/SEQ ID NO: 96; (3) substitution, deletion or insertion of one or more amino acid residues of not more than 5 amino acids in the amino acid sequence of the CDRs.

6. The anti-Siglec-15 antibody or antigen-binding fragment thereof according to claim 1, comprising: a human constant domain that is an IgA, IgD, IgE, IgG, or IgM domain; preferably, the human IgG constant domain is an IgG1, IgG2, IgG3 or IgG4 domain.

7. The anti-Siglec-15 antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-Siglec-15 antibody is a murine monoclonal antibody, a chimeric monoclonal antibody, a humanized antibody, a single chain antibody, a light chain constant region optionally being a human kappa chain or lambda chain constant region, a heavy chain constant region optionally being a human antibody IgG1 or a constant region of IgG4, and a mutant of a constant region of IgG1 or IgG 4.

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

9. The anti-Siglec-15 antibody or antigen-binding fragment thereof according to claim 8, wherein the bispecific antibody or fusion protein targets tumor-associated antigens of tumor cells or immune cell-targeted immunoassay targets at the other end, and the trispecific antibody or fusion protein targets tumor-associated antigens of tumor cells or immune cell-targeted immunoassay targets at the other end, the immunoassay targets comprising EGFR, HER2, CD47, PD-1, PD-L1, CTLA4, TGF β, VEGF, HER3, TIGIT, CLAUDIN18.2, CD3, TIM3, LAG 3.

10. A nucleic acid sequence encoding the anti-Siglec-15 antibody or antigen-binding fragment thereof of any one of claims 1-9.

11. The nucleic acid sequence of claim 10, comprising SEQ ID NO 101, SEQ ID NO: 102. SEQ ID NO: 103. 104 sequence of SEQ ID NO.

12. An expression vector comprising the nucleic acid sequence of claim 10.

13. A host cell comprising the expression vector of claim 12; preferably, the host cell is selected from mammalian cells.

14. A pharmaceutical composition comprising the anti-Siglec-15 antibody or antigen-binding fragment thereof of any one of claims 1-9 and a pharmaceutically-optional pharmaceutically-acceptable carrier.

15. Use of an anti-Siglec-15 antibody or antigen-binding fragment thereof according to any one of claims 1-9 for the preparation of a medicament for the treatment of Siglec-15 expressing or overexpressing cancer cells, macrophages or cancer and for the preparation of a medicament for the treatment of osteoporosis.

16. Use of the anti-Siglec-15 antibody or antigen-binding fragment thereof according to any one of claims 1-9, or the pharmaceutical composition according to claim 14, for 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.

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