RU2792748C2 - Antibody to b7-h4, its antigen-binding fragment and its pharmaceutical use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions is described, including an antibody to B7-H4 or its antigen-binding fragment, a DNA molecule encoding the antibody or its antigen-binding fragment, an expression vector containing a DNA molecule, a host cell for expressing an antibody to B7-H4 or its antigen-binding fragment, a method for producing an antibody including culturing the host cell and isolating the antibody, a pharmaceutical composition for the treatment of B7-H4-mediated diseases or conditions, containing an antibody to B7-H4 or its antigen-binding fragment, a detection reagent designed for immunodetection or determination of B7-H4, a diagnostic tool for diagnosing B7-H4 mediated diseases or conditions; and the use of an anti-B7-H4 antibody or antigen-binding fragment or pharmaceutical composition in the manufacture of a medicament for the treatment or prevention of a B7-H4 mediated disease or condition.

EFFECT: invention expands the arsenal of agents that bind to B7-H4.

23 cl, 4 dwg, 23 tbl, 10 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to an anti-B7-H4 antibody, its antigen-binding fragment having immunoreactivity with respect to the human B7-H4 receptor, chimeric antibodies and humanized antibodies containing CDR regions (complementary determining region - complementarity determining region) of the specified anti-B7-H4 antibody, pharmaceutical compositions containing a human anti-B7-H4 antibody and an antigen-binding fragment thereof, and their use as anti-cancer agents.

PRIOR ART

Tumor immunotherapy represents a “challenging spot” in long-term research and development in the field of cancer treatment and among which T-cell tumor immunotherapy occupies a central position. Tumor escape from immunological surveillance is a huge hurdle facing tumor immunotherapy. Most expressing tumors can be recognized to varying degrees by the host's immune system, but in many cases tumor growth is stimulated by inhibition of the immune system caused by the tumor cells themselves due to an inadequate immune response caused by inefficient activation of effector T cells. Tumor immunotherapy involves the full use and recruitment of killer T cells and/or other cells of the immune system in tumor patients to destroy tumors.

Studies on the CD28 receptor and its ligands have led to the characterization of related molecules known as the B7 superfamily. B7 family members are a class of immunoglobulins with immunoglobulin V-like domain (IgV) and immunoglobulin C-like domain (IgC), whose members include co-stimulatory factors B7.1 (CD80) and B7.2 (CD86), an inducible ligand for stimulus factor (ICOS-L/B7-H2), programmed cell death ligand-1 (PD-L1/B7-H1), programmed cell death ligand-2 (PD-L2/B7-DC), B7-H4 and B7-H4, etc.

Human B7-H4 is a 282 amino acid type I transmembrane protein whose gene is located in the p11.1 region of chromosome 1 (Choi IH et al., J Immunol. 2003 Nov 1; 171(9): 4650-4) . B7-H4 plays a role in the down-regulation of the T-cell immune response. B7-H4 has a strong inhibitory effect on the differentiation, development, cell cycle progression and cytokine production of CD4 ⁺ and CD8 ⁺ T cells (Sica GL et al. Immunity. 2003 Jun; 18(6): 849-61). Immune cell disorders and autoimmune events were not detected in B7-H4 knockout mice (Zhu G et al, Blood. 2009 Feb 19; 113(8): 1759-67; Suh WK et al., Blood. Mol Cell Biol. 2006 Sep;26(17):6403-11). B7-H4 receptors and their signaling pathways are currently still unclear.

Recent studies have found that the B7-H4 protein is highly expressed in various tumor tissues, making it possible for tumor cells to avoid attack by the body's immune system. As a target for tumor therapy, the B7-H4 molecule provides a new method for tumor immunotherapy.

Currently, human B7-H4 is known to be expressed on various cancer cells such as breast cancer, ovarian cancer, lung cancer, cervical cancer, kidney cancer, bladder cancer, and liver cancer. B7-H4 mRNA (messenger RNA) expression has been found in spleen, lung, thymus, liver, skeletal muscle, kidney, pancreas, testis, and ovary. A low level of B7-H4 expression at the protein level was found in tissues such as mammary gland (catheter or lobular), fallopian tube epithelium and endometrial gland. Related studies have also shown that B7-H4 is overexpressed in tumor-associated macrophage (TAM) macrophages (Kryczek, I. et al., J. Exp. Med. 2006, 203(4): 871- 881), along with the fact that macrophages are an important component of the tumor microenvironment and can account for up to 50% of the tumor mass.

Numerous national pharmaceutical companies are currently developing anti-B7-H4 monoclonal antibodies and/or their drug conjugates to improve the patient's own immune response to tumors and achieve direct tumor cell killing. Related patents are, for example, WO2013025779, US20140322129 and the like. Monoclonal antibodies to B7-H4, available from companies such as Medimmune and FivePrime, are currently in the preclinical phase; anti-B7-H4 antibody drug conjugates from Genentech are also in the preclinical phase of development.

The present invention provides anti-B7-H4 antibodies with high affinity, high selectivity and high biological activity for use in tumor monoclonal antibody immunotherapy and related applications. Also provided are drugs, compositions, and methods for treating B7-H4 positive tumors.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides an anti-B7-H4 antibody, or antigen-binding fragment thereof, comprising:

an antibody light chain variable region comprising at least one LCDR (light chain complementarity determining region) selected from the group consisting of:

SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8;

SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16;

SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24;

SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32;

SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40;

SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48;

SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56;

SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64;

SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72; And

an antibody heavy chain variable region comprising at least one HCDR (heavy chain complementarity determining region) selected from the group consisting of:

SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5;

SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13;

SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21;

SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29;

SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37;

SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45;

SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53;

SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61;

SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69;

SEQ ID NO: 73, SEQ ID NO: 74.

In a preferred embodiment, the invention provides an anti-B7-H4 antibody, or antigen-binding fragment thereof, as described above, wherein the heavy chain variable region of said antibody comprises:

HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively;

HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively;

HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively;

HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, respectively;

HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37, respectively;

HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, respectively;

HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53, respectively;

HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, respectively;

HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, respectively; or

HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 21, respectively.

In a preferred embodiment of the invention, there is provided an anti-B7-H4 antibody, or antigen-binding fragment thereof, wherein the light chain variable region of said antibody comprises:

LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively;

LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively;

LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively;

LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, respectively;

LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, respectively;

LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, respectively;

LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, respectively;

LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64, respectively; or

LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, respectively.

A particularly preferred anti-B7-H4 antibody or antigen-binding fragment thereof may be any antibody or fragment thereof selected from the group consisting of the following, containing one or more of the following sequences of CDR regions or sequences showing at least 95% identity with these sequences:

(1) the antibody light chain variable region contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively; and the variable region of the heavy chain of the antibody contains HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively;

(2) the antibody light chain variable region contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; and the heavy chain variable region of the antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13, respectively;

(3) the antibody light chain variable region contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; and the variable region of the heavy chain of the antibody contains HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively;

(4) the variable region of the light chain of the antibody contains LCDR1, LCDR2 and LCDR3, as shown in SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, respectively; and the heavy chain variable region of the antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, respectively;

(5) the antibody light chain variable region contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively; and the heavy chain variable region of the antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, respectively;

(6) the antibody light chain variable region contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48, respectively; and the heavy chain variable region of the antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively;

(7) the antibody light chain variable region contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively; and the heavy chain variable region of the antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively;

(8) the variable region of the light chain of the antibody contains LCDR1, LCDR2 and LCDR3, as shown in SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64, respectively; and the variable region of the heavy chain of the antibody contains HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, respectively;

(9) the antibody light chain variable region contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72, respectively; and the variable region of the heavy chain of the antibody contains HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, respectively; And

(10) the antibody light chain variable region contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively; and the heavy chain variable region of the antibody comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 73, SEQ ID NO: 74, and SEQ ID NO: 21, respectively.

In a preferred embodiment of the invention, an anti-B7-H4 antibody or antigen-binding fragment thereof is provided, as described above, wherein the antibody or antigen-binding fragment thereof is a mouse antibody or fragment thereof.

In a preferred embodiment of the invention, an anti-B7-H4 antibody or antigen-binding fragment thereof is provided, wherein the antibody or antigen-binding fragment thereof is a chimeric antibody or fragment thereof.

In a preferred embodiment of the invention, an anti-B7-H4 antibody or antigen-binding fragment thereof is provided, as described above, wherein the antibody or antigen-binding fragment thereof is a human antibody or fragment thereof.

In a preferred embodiment of the invention, an anti-B7-H4 antibody or antigen-binding fragment thereof is provided, as described above, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or fragment thereof.

In a preferred embodiment, the invention provides an anti-B7-H4 antibody, or antigen-binding fragment thereof, as described above, wherein the light chain variable region of the humanized antibody is a light chain variable region comprising sequence(s) selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, or SEQ ID NO: 82.

In a preferred embodiment, the invention provides an anti-B7-H4 antibody, or antigen-binding fragment thereof, as described above, wherein the heavy chain variable region of the humanized antibody is a heavy chain variable region comprising sequence(s) selected from the group consisting of SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, or SEQ ID NO: 81.

In a preferred embodiment of the invention, there is provided an anti-B7-H4 antibody, or antigen-binding fragment thereof, as described above, wherein the heavy chain variable region of the humanized antibody further comprises FR (framework region) heavy chain region(s) of a human IgG1, IgG2, IgG3, or IgG4 or a variant thereof, preferably contains the heavy chain FR region(s) of human IgG1, IgG2 or IgG4; more preferably contains FR region(s) of the IgG1 heavy chain that have undergone an amino acid mutation to enhance ADCC (antibody-dependent cellular cytotoxicity - antibody-dependent cellular cytotoxicity) toxicity.

In a preferred embodiment, the invention provides an anti-B7-H4 antibody, or antigen-binding fragment thereof, as described above, wherein the humanized antibody light chain is a light chain comprising a sequence selected from the group consisting of SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88 or SEQ ID NO: 90.

In a preferred embodiment, the invention provides an anti-B7-H4 antibody, or antigen-binding fragment thereof, as described above, wherein the heavy chain of the humanized antibody is a heavy chain comprising a sequence selected from the group consisting of SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 or SEQ ID NO: 89.

In a preferred embodiment, the invention provides an anti-B7-H4 antibody, or antigen-binding fragment thereof, as described above, wherein the light chain variable region of the humanized antibody is a light chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 76 or SEQ ID NO: 80.

In a preferred embodiment, the invention provides an anti-B7-H4 antibody, or antigen-binding fragment thereof, as described above, wherein the heavy chain variable region of the humanized antibody is a heavy chain variable region comprising a sequence selected from the group consisting of SEQ ID NO: 75 or SEQ ID NO: 79.

In a preferred embodiment, the invention provides an anti-B7-H4 antibody, or antigen-binding fragment thereof, as described above, wherein the humanized antibody light chain comprises a sequence selected from the group consisting of SEQ ID NO: 84 or SEQ ID NO: 88.

In a preferred embodiment, the invention provides an anti-B7-H4 antibody, or antigen-binding fragment thereof, as described above, wherein the heavy chain of the humanized antibody comprises a sequence selected from the group consisting of SEQ ID NO: 83 or SEQ ID NO: 87.

In a more preferred embodiment of the invention, the humanized antibody is selected from any of the following containing:

(1) a light chain variable region of SEQ ID NO: 76 and a heavy chain variable region of SEQ ID NO: 75;

(2) a light chain variable region of SEQ ID NO: 78 and a heavy chain variable region of SEQ ID NO: 77;

(3) a light chain variable region of SEQ ID NO: 80 and a heavy chain variable region of SEQ ID NO: 79, or

(4) the light chain variable region of SEQ ID NO: 82 and the heavy chain variable region of SEQ ID NO: 81.

In yet another preferred embodiment of the invention, the humanized antibody is selected from any of the following antibodies containing:

(1) a light chain of SEQ ID NO: 84 and a heavy chain of SEQ ID NO: 83;

(2) a light chain of SEQ ID NO: 86 and a heavy chain of SEQ ID NO: 85;

(3) a light chain of SEQ ID NO: 88 and a heavy chain of SEQ ID NO: 87, or

(4) a light chain of SEQ ID NO: 90 and a heavy chain of SEQ ID NO: 89.

An anti-B7-H4 antibody, or antigen-binding fragment thereof, having at least one of the following: (1) binding to a B7-H4 epitope containing amino acids 41-60 of SEQ ID NO: 100; and (2) binding to the B7-H4 epitope containing amino acids 53-59 in SEQ ID NO: 100.

An anti-B7-H4 antibody, or antigen-binding fragment thereof, having at least one of the following: (1) binding to a B7-H4 epitope containing amino acid 53 in SEQ ID NO: 100; (2) binding to the B7-H4 epitope containing amino acid 54 in SEQ ID NO: 100; (3) binding to the B7-H4 epitope containing amino acid 56 in SEQ ID NO: 100; (4) binding to the B7-H4 epitope containing amino acid 57 in SEQ ID NO: 100; (5) binding to the B7-H4 epitope containing amino acid 58 in SEQ ID NO: 100; and (2) binding to the B7-H4 epitope containing amino acid 59 in SEQ ID NO: 100.

In a preferred embodiment of the invention, there is provided an anti-B7-H4 antibody, or an antigen-binding fragment thereof, as described above, wherein the antigen-binding fragment is a Fab, Fv, sFv, F(ab') ₂ , a linear antibody, a single chain antibody, a nanobody, a domain antibody, or a multispecific antibody .

The present invention further provides a DNA sequence encoding an anti-B7-H4 antibody, or antigen-binding fragment thereof, as described above.

The present invention further provides an expression vector containing a DNA sequence as described above.

The invention further provides a host cell that is introduced into or contains an expression vector as described above.

In a preferred embodiment of the invention, the host cell as described above is characterized in that the host cell is a bacterium, preferably Escherichia coli.

In a preferred embodiment of the invention, the host cell as described above is a yeast, preferably Pichia pastoris.

In a preferred embodiment of the invention, the host cell as described above is a mammalian cell, preferably a Chinese Hamster Ovary (CHO) cell or a human embryonic kidney (HEK) 293 cell.

The invention also provides a method for producing an anti-B7-H4 antibody, comprising culturing the host cell as described above, isolating the antibody from the culture, and purifying the antibody.

The invention also provides a multispecific antibody comprising a light chain variable region and a heavy chain variable region as described above.

The invention also provides a single chain antibody comprising a light chain variable region and a heavy chain variable region as described above.

The invention also provides a detection reagent or diagnostic tool comprising an anti-B7-H4 antibody, or antigen-binding fragment thereof, as described above.

The present invention also provides a method for immunodetection or determination of B7-H4, which includes the use of an anti-B7-H4 antibody or antigen-binding fragment of the present invention.

The present invention also provides a method for diagnosing diseases associated with B7-H4 positive cells, which comprises detecting or measuring the level of B7-H4 or B7-H4 positive cells by using an anti-B7-H4 antibody or antigen-binding fragment thereof according to the present invention.

The invention further provides a pharmaceutical composition comprising an anti-B7-H4 antibody, or antigen-binding fragment thereof, as described above, and a pharmaceutically acceptable excipient, diluent, or carrier.

The present invention further provides the use of an anti-B7-H4 antibody, or antigen-binding fragment thereof, as described above, in the manufacture of a medicament for the treatment of a B7-H4 mediated disease or condition; where the disease is preferably cancer; preferably, the disease is a cancer expressing B7-H4; the cancer is most preferably selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer , gallbladder cancer, glioblastoma and melanoma.

The present invention further provides a method for treating and preventing B7-H4 mediated diseases or conditions comprising administering to a subject in need thereof a therapeutically effective amount of an anti-B7-H4 antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the foregoing, wherein the disease is preferably cancer. ; preferably the disease is a cancer expressing B7-H4; the cancer is most preferably selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colon cancer, bladder cancer, esophageal cancer, gallbladder cancer , cervical cancer, glioblastoma and melanoma.

DESCRIPTION OF GRAPHICS

Fig. 1: ELISA (enzyme-linked immunosorbent assay) in vitro antibody binding assay showing that all seven chimeric antibodies have binding activity to purified human B7-H4 antigen, where chimeric antibodies 2F7 and 2F8 have an EC ₅₀ (half maximum effective concentration) about 0.1 nM.

Fig. 2: Indirect ELISA binding assays demonstrating the antigenic epitope to which the hu2F7 anti-B7-H4 antibody binds.

Fig. 3: Pharmacological analysis in mice demonstrating the anti-tumor effect of the anti-B7-H4 hu1C9 antibody.

Fig. 4: Immune function test demonstrating the effect of anti-B7-H4 hu1C9 and hu2G6 antibodies in enhancing immunity (T-cell proliferation).

DETAILED DESCRIPTION OF THE INVENTION

1. Terminology

For an easier understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise specifically indicated elsewhere in this document, all other technical and scientific terms used in this document have the meaning normally implied by one of ordinary skill in the art to which this invention belongs.

As used herein, the one-letter code and the three-letter code for amino acids is as described in J. Biol. Chem, 243, p 3558 (1968).

As used herein, the term "antibody" refers to an immunoglobulin, a four peptide chain structure formed by joining together two identical heavy chains and two identical light chains by an interchain disulfide bond(s) . Different immunoglobulin heavy chain constant regions exhibit different amino acid compositions and sequences, thus representing different antigenicity. Accordingly, immunoglobulins can be classified into five categories or so-called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, their heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. According to its amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds, the same type of Ig can be subdivided into different subcategories, for example, IgG can be subdivided into IgG1, IgG2, IgG3 and IgG4. The light chain can be subdivided into a κ or λ chain, according to different constant regions. Each of the five Igs can have a κ or λ chain.

In the present invention, the light chain variable region of the antibody described herein further comprises a light chain constant region that contains a human or mouse κ, λ chain, or a variant thereof.

In the present invention, the heavy chain variable region of the antibody described herein further comprises a heavy chain constant region that contains human or mouse IgG1, 2, 3, 4, or a variant thereof.

The approximately 110 amino acid sequences adjacent to the N-terminus of heavy chains and light chains of an antibody vary greatly, this region being known as the variable region (region V); the remainder of the amino acid sequence near the C-terminus is relatively stable, known as the constant region (C region). The variable region contains three hypervariable regions (HVR - hypervariable region) and four frame regions (FR - framework region), having a relatively conservative sequence. Three hypervariable regions determine the specificity of an antibody, also known as the complementarity determining region (CDR). Each light chain variable region (LCVR) and each heavy chain variable region (HCVR) consist of three CDRs and four FRs, with the order from N-terminus to C-terminus being: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The three light chain CDRs are LCDR1, LCDR2, and LCDR3; the three heavy chain CDRs are HCDR1, HCDR2 and HCDR3. The number and position of amino acid residues of the CDRs in the VL and VH of an antibody or antigen-binding fragment herein follow the known Kabat numbering criteria and the Kabat or AbM definition criteria (http://bioinf.org.uk/abs/).

The term "antigen-presenting cell" or "APC" is a cell that exhibits on its surface foreign antigens in complex with MHC (major histocompatibility complex - major histocompatibility complex). T-cells recognize such a complex through T-cell receptors (TCR - T cell receptor). Examples of APCs include, but are not limited to, dendritic cells (DC -dendritic cell), peripheral blood mononuclear cells (PBMC), monocytes, B-lymphoblasts, and dendritic cells of monocytic origin (DC). The term "antigen presentation" refers to the process during which APCs take up antigens and allow them to be recognized by T cells, eg as a component of MHC-I/MHC-II conjugates.

The term "B7-H4" refers to a member of the human B7 protein family, also known as CD276, which is a type I transmembrane protein having four Ig-like extracellular domains. B7-H4 is one of the immune checkpoint proteins expressed on the surface of antigen presenting cells or cancer cells, and it has an inhibitory effect on T cell activation. The term "B7-H4" includes any variant or isoform of B7-H4 naturally expressed by cells. The antibodies of the present invention can cross-react with B7-H4 derived from non-human species. Alternatively, the antibodies may be specific for human B7-H4 and may not show cross-reactivity with other species. B7-H4, or any variant or isotype thereof, may be isolated from cells or tissues in which they are naturally expressed, or obtained by genetic engineering using techniques commonly used in the art and the techniques described herein. Preferably, anti-B7-H4 antibodies target human B7-H4 with a normal glycosylation profile.

The term "recombinant human antibody" includes human antibodies produced, expressed, generated, or isolated by genetic engineering, and these techniques and methods used are well known in the art, such as: (1) an antibody isolated from an animal (e.g., a mouse) , transgenic or transchromosomal in relation to the human immunoglobulin gene, or the resulting hybridoma; (2) an antibody isolated from transformed host cells expressing the antibody, such as a transfectoma; (3) an antibody isolated from a combinatorial library of recombinant human antibodies; and (4) an antibody produced, expressed, generated or isolated by splicing human immunoglobulin gene sequences onto other DNA sequences or the like. Such a recombinant human antibody contains a variable region and a constant region by incorporating specific human germline immunoglobulin sequences, germline-encoded genes, as well as subsequent rearrangements and mutations, such as rearrangements and mutations that occur during antibody maturation.

The term "mouse antibody" in the present invention refers to a monoclonal antibody to human B7-H4, obtained in accordance with knowledge and skill in this field. During preparation, the test subject is injected with B7-H4 antigen, and then a hybridoma is isolated expressing an antibody that has the desired sequence or functional characteristics. In a preferred embodiment of the present invention, the mouse anti-B7-H4 antibody, or antigen-binding fragment thereof, further comprises a mouse κ, λ light chain constant region, or a variant thereof, or further comprises a mouse IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, or a variant thereof.

The term "human antibody" includes antibodies having variable and constant regions from human germline immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, mutations introduced by in vitro random or site-directed mutagenesis or by in vivo somatic mutation). However, the term "human antibody" does not include an antibody in which CDR sequences derived from a mammalian germline of another species, such as a mouse germline, have been grafted onto a human framework sequence (i.e., "humanized antibody").

The term "humanized antibody", also known as a CDR-grafted antibody, refers to an antibody generated by grafting murine CDR sequences into a human antibody variable region framework. Humanized antibodies overcome the undesirable strong humoral response caused by chimeric antibodies that carry large amounts of murine protein components. In order to avoid a decrease in activity caused by reduced immunogenicity, the variable region of a human antibody can undergo minimal backmutation while maintaining activity.

The term "chimeric antibody" is an antibody that is formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, and the chimeric antibody can alleviate the immune response elicited by the mouse antibody. To create a chimeric antibody, first a hybridoma secreting a specific mouse monoclonal antibody is created, the variable region gene is cloned from mouse hybridoma cells, then the human antibody constant region gene is cloned as desired, the mouse variable region gene is fused to the human constant region gene to form a chimeric gene, which is then can be inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system. The human antibody constant region is selected from a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, or a variant thereof, preferably a human IgG2 or IgG4 heavy chain constant region, or an IgG1 heavy chain constant region that exhibits increased ADCC (antibody dependent cellular cytotoxicity). ) due to an amino acid mutation.

The term "antigen-binding fragment" refers to antigen-binding fragments of an antibody or antibody analogs, which typically include at least a portion of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, when activity is expressed in moles, antibody fragments retain at least 10% of the binding activity of the parent antibody. Preferably, antibody fragments retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity in in relation to the target. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

The "Fab fragment" consists of CH1 and the light chain and heavy chain variable regions. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain of the molecule.

The "Fc" region contains two heavy chain fragments containing the CH1 and CH2 domains of the antibody. These two heavy chain fragments are held together by two or more disulfide bonds and the hydrophobic interaction of the CH3 domain.

The "Fab' fragment" contains a light chain and a heavy chain portion containing a VH domain, a CH1 domain, and a region between the CH1 and CH2 domains, and therefore an F(ab')2 molecule can be formed by two heavy chains of two Fab'- fragments linked by interchain disulfide bonds.

The "F(ab')2 fragment" contains two light chains and a portion of the heavy chains containing a constant region between the CH1 and CH2 domains, as a result, interchain disulfide bonds are formed between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

The "Fv region" contains the variable regions from both the heavy and light chains, but no constant regions.

The term "multispecific antibody" is used in its broadest sense and encompasses antibodies with multi-epitope specificity. These multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has multi-epitope specificity; antibodies containing two or more VL and VH regions, where each VH-VL unit binds to different targets or to different epitopes of the same target; antibodies containing two or more single variable regions, where each individual variable region binds to different targets or to different epitopes of the same target; full length antibodies; antibody fragments; diatels; bispecific diabodies and tribodies, antibody fragments that have been covalently or non-covalently linked together.

The term "single chain antibody" is a single chain recombinant protein consisting of an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) connected by a peptide linker, and a single chain antibody is the smallest antibody fragment with intact antigen binding sites.

The term "domain antibody fragment" is an immunoglobulin fragment having immunological functions, and it contains only a heavy chain variable region or a light chain variable region. In some instances, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment. The two VH regions of a bivalent domain antibody fragment may target the same or different antigens.

The term "B7-H4 binding" refers to interaction with human B7-H4. The term "antigen-binding site", as used herein, refers to discrete three-dimensional sites on an antigen recognized by the antibody or antigen-binding fragment of the present application.

The term "epitope" refers to sites on an antigen that specifically bind to an immunoglobulin or antibody. An epitope can be formed by contiguous amino acids or by non-contiguous amino acids, and is rendered closed by the tertiary folding of the protein. The epitope formed by adjacent amino acids is usually retained after exposure to denaturing solvents, while the epitope formed by the tertiary fold is usually lost after treatment with denaturing solvents. Epitopes typically include at least 3-15 amino acids in a single spatial conformation. Methods for determining that an epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays, and the like. nuclear magnetic resonance.

The term "specifically binds to", "selectively binds to" as used herein refers to the binding of an antibody to an epitope on a given antigen. Generally, when recombinant human B7-H4 is used as an analyte and an antibody is used as a ligand, the antibody binds to a given antigen with a dissociation equilibrium constant (K _D ) of approximately less than 10 ^-7 M or even less, and the binding affinity of the antibody with a target antigen is at least twice as high as in the case of non-specific antigens (other than the target antigen or closely related antigens) (such as BSA (Bovine Serum Albumin)), as measured on the device using surface plasmon resonance (SPR) techniques response). The term "antibody that recognizes an antigen" may be used interchangeably herein with the term "specific binding antibody".

The term "cross-reactivity" refers to the ability of an antibody of the present invention to bind to B7-H4 originating from different species. For example, an antibody of the present invention that binds to human B7-H4 may also bind to B7-H4 derived from another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (eg SPR and ELISA), or by detecting binding or functional interaction with cells physiologically expressing B7-H4. Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis or flow cytometry.

The terms "inhibition" or "blockade" are used interchangeably and cover both partial and complete inhibition/blockade. Ligand inhibition/blockade preferably reduces the normal level of ligand binding or changes the type of ligand-binding activity, compared to ligand-binding activity in the absence of inhibition or blockade. Inhibition and blockade is also expected to include any measurable decrease in binding affinity where the ligand is contacted with an anti-B7-H4 antibody when compared to binding affinity in the absence of an anti-B7-H4 antibody.

The term "growth inhibition" (eg, in the context of cells) is intended to include any measurable reduction in cell growth.

The terms "inducing immune response" and "enhancing the immune response" are used interchangeably and refer to the immune response to stimulation with a particular antigen (namely, passive or adaptive). In relation to the induction of CDC (complement-dependent cytotoxicity - complement-dependent cytotoxicity) or ADCC, the term "causing" means the stimulation of a specific mechanism for the direct destruction of cells.

As used herein, the term "ADCC", namely antibody-dependent cellular cytotoxicity, refers to cells expressing Fc receptors that directly kill antibody-coated target cells by recognizing the Fc segment of an antibody. ADCC effector function of an antibody can be reduced or eliminated by modification of the IgG Fc segment. Modification refers to mutation(s) in the heavy chain constant region of an antibody.

Methods for preparing and purifying antibodies and antigen-binding fragments are well known in the art and can be found, for example, in the Cold Spring Harbor Antibody Laboratory Technology Manual, chapters 5-8 and 15. For example, mice can be immunized with human B7-H4 or fragments thereof, and the resulting antibodies can then be annealed, purified and sequenced using conventional methods well known in the art. Antigen binding fragments can also be obtained by conventional methods. An antibody or antigen-binding fragment of the present invention can be obtained by introducing one or more human framework regions (FRs) into non-human CDR derivatives using genetic engineering techniques. Human germline FR sequences can be obtained from ImMunoGeneTics (IMGT), on the website http://imgt.cines.fr, or from The Immunoglobulin FactsBook, 2001ISBN012441351.

The engineered antibody or antigen-binding fragment of the present invention may be prepared or purified using conventional methods. For example, cDNA sequences encoding appropriate antibodies can be cloned and recombined into a GS expression vector. The CHO cells can then be stably transfected with a recombinant immunoglobulin expression vector. As a more recommended method, well known in the art, mammalian expression systems can lead to antibody glycosylation, especially at the highly conserved N-terminus in the F _C region. Stable clones can be obtained from the expression of an antibody that specifically binds to a human antigen. Positive clones can be expanded in serum-free culture media to generate antibodies in bioreactors. The culture medium into which the antibody has been secreted can be purified and collected by conventional techniques. The antibody can be filtered and concentrated using standard techniques. Soluble mixtures and aggregates can be effectively removed by standard techniques such as molecular sieve or ion exchange. The resulting product must be frozen immediately, for example at minus 70 °C, or may be lyophilized.

The antibodies of the present invention are referred to as monoclonal antibodies. A monoclonal antibody or mAb, as used herein, refers to an antibody that is derived from a single clone, including, but not limited to, a eukaryotic, prokaryotic, or phage single clone. Monoclonal antibodies or antigen-binding fragments thereof can be produced, for example, by hybridoma-based techniques, genetic engineering techniques, phage display techniques, synthesis techniques (eg, CDR grafting), or other techniques known in the art.

The term "administration" and "treatment" when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to bringing an exogenous pharmaceutical, therapeutic, diagnostic agent or composition into contact with an animal, human, subject, cell, tissue, organ, or biological fluid. The terms "administration" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods. Processing a cell encompasses bringing a reagent into contact with a cell, as well as bringing the reagent into contact with a fluid, where the fluid is in contact with the cell. The terms "administration" and "treatment" also refer to in vitro and ex vivo treatments of, for example, a cell, a reagent, a diagnostic agent, a binding composition, or another cell. The term "treatment", when applied to a human subject, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.

The term "treat" means to administer a therapeutic agent, such as a composition containing any of the binding compounds of the present invention, orally or topically, to a patient suffering from one or more symptoms of a disease for which the agent has a known therapeutic activity. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated, or to cause regression of the symptom(s) or inhibit the progression of such symptom(s) to any clinically measurable degree. The amount of a therapeutic agent that is effective in alleviating any particular symptom of a disease (also referred to as a "therapeutically effective amount") may vary depending on factors such as the course of the disease, the age and weight of the patient, and the ability of the agent to elicit the desired response in the patient. . Whether a symptom of a disease has been alleviated can be assessed by a clinical measurement, commonly used by physicians or other qualified healthcare professionals, to assess the severity or progression status of said symptom. While an embodiment (e.g., method of treatment or product of manufacture) of the present invention may not be effective in alleviating the disease symptom(s) under investigation in each patient, it should alleviate the disease symptom(s) under investigation in a statistically significant number of patients, as determined by any statistical test known in the art, such as Student's t-test, chi-square test, Mann-Whitney U-test, Kruskal-Wallis (H-test), Jonkheer-Terpstra test, and Wilcoxon test .

The term "essentially consisting of" or variations thereof, as used throughout the description of the invention and the claims, means the inclusion of all elements or groups of elements, and optionally other elements that exhibit similar properties or even a property different from the properties of the specified elements. These other elements do not significantly change an existing or new property of a given dosing regimen, method, or composition. As a non-limiting example, a binding compound essentially consisting of the described amino acid sequence may also include one or more amino acids that do not significantly affect the properties of the binding compound.

The term "naturally occurring", when applied to an object in the present invention, refers to the fact that the object can be found in nature. For example, polypeptide sequences or polynucleotide sequences that exist in organisms (including viruses) and have not been artificially modified in the laboratory occur naturally, where said organism can be isolated from natural sources.

The term "effective amount" encompasses an amount sufficient to alleviate or prevent a symptom or sign of a medical condition. An effective amount also means an amount sufficient to enable or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the general health of the patient, the route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

The term "exogenous" refers to a substance that is produced outside the body, cell, or human body, depending on the context. The term "endogenous" refers to substances that are produced in a cell, organism, or human body, depending on the context.

The term "homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two sequences to be compared is occupied by the same monomeric subunit - base or amino acid - for example, if a position in each of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology of two sequences is a function where the number of matching or homologous positions that two sequences share is divided by the number of positions compared and then multiplied by 100. For example, in an optimal sequence alignment, if 6 out of 10 positions in two sequences are the same or homologous , then these two sequences are considered to be 60% homologous. In general, a comparison is made when the maximum percentage of homology is obtained when the two sequences are aligned.

As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably, and all such designations include their progeny. Thus, the words "transformants" and "transformed cells" include the primary cell of interest and cultures derived from it, without reference to the number of passages. It should also be understood that all progeny may not be exactly identical in DNA content due to targeted or random mutations. Also counted are progeny with mutations that are thus screened to exhibit the same function or biological activity as the function or biological activity of the originally transformed cell. Where separate designations are intended, this will be apparent from the context.

The term "optional" or "optional" means that the event or circumstance that follows may occur, but does not necessarily occur, and the description includes an example in which the event or situation will or will not occur. For example, the phrase "optionally containing 1-3 antibody heavy chain variable regions" means that an antibody heavy chain variable region having a particular sequence may be present, but need not be present.

The term "pharmaceutical composition" refers to a mixture containing one or more compounds according to the present invention or its (their) physiologically/pharmaceutically acceptable salt or prodrug together with other chemical components, as well as additional components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the introduction into the body and facilitate the absorption of the active substance, and thus exert a biological effect.

The present invention is further described below in this document with reference to examples; however, the scope of the present invention is not limited to them. In examples of the present invention, where no specific conditions are described, experiments are usually carried out under conventional conditions as described in the Antibodies A Laboratory Manual, Molecular Cloning, Cold Spring Harbor or under the conditions proposed by the producers of raw materials or products. Where the source of the reagents is not specifically given, the reagents are commercially available conventional reagents.

Example 1 Obtaining Antigens and Construction of Stable Cell Lines

The HisFlag-tagged human B7-H4 coding sequence (huB7-H4-HF) and the huFc-tagged human B7-H4 coding sequence (h-B7-H4-Fc) were synthesized by CRO integrated DNA technology (IDT - Integrated DNA Technology) (the template sequence for each of the above recombinant B7-H4 proteins was constructed by the inventors), and cloned into the pTT5 vector (Biovector), respectively. Recombinant B7-H4 proteins were expressed in 293T cells and purified according to Example 2.

Purified proteins were used in the following examples.

huB7-H4-Fc sequence:

FGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKAGSGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 99

huB7-H4-his sequence:

MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKADYKDDDDKGSHHHHHHHH

SEQ ID NO: 100

Purification steps for huB7-H4-his:

Cell-expressed supernatant samples were centrifuged at high speed to remove impurities, buffer exchanged with PBS (phosphate buffered saline), and imidazole was added thereto to a final concentration of 5 mM. The nickel column was equilibrated with a PBS solution containing 5 mM imidazole and washed with 2-5 column volumes. After buffer exchange, supernatant samples were applied to the column. The column was washed with PBS containing 5 mM imidazole until the measured A280 returned to baseline. The column was then washed with PBS plus 10 mM imidazole, non-specifically bound contaminants were removed, and the eluate was collected. The target protein was eluted with PBS containing 300 mM imidazole and the elution peak was collected. The collected eluate was further purified by ion exchange (SP column). Stock solution A: 0.01 M PB, pH 8.0. Mother solution B: Solution A plus 1 M NaCl. To elute the target protein, the PBS-imidazole solution was replaced with Solution A, and the SP column was equilibrated with solution A. And then the samples were applied to the column. Then the column was washed with solution B in a concentration gradient from 0 to 100% with 10 column volumes, and the elution peak was collected. The resulting protein was identified as the desired protein by electrophoresis and aliquoted for use. Received human B7-H4 with HisFlag-tag (hu-B7-H4 his).

Purification steps for huB7-H4-Fc:

Supernatant samples expressed by HEK293 cells were centrifuged at high speed to remove impurities and subjected to buffer exchange with PBS. The Protein A affinity column was equilibrated with 10 mM phosphate buffer and washed with 2-5 column volumes. After buffer exchange, supernatant samples were applied to the column. The column was washed with 25 column volumes of buffer until the measured A280 returned to baseline. The target protein was eluted with 0.8% acetate buffer, pH 3.5, and the elution peak was collected. Aliquots were immediately added with buffer 1 M Tris-Cl, pH 8.0, for neutralization. And then the solution was replaced with PBS through an Amico-15 Millipore filter column. The resulting protein was identified by electrophoresis, peptide mapping and LC (liquid chromatography)-MS (mass spectrometry) and divided into aliquots for use.

Construction of a stable pool of CHO-S cells:

The full length sequence encoding the human or cynomolgus monkey B7-H4 protein (huB7-H4 or cyB7-H4) was synthesized by integrated DNA technology (IDT) (the above recombinant B7-H3 proteins were constructed by the present inventors) and cloned into the constructed pcDNA3 vector. 1, pcDNA3.1/puro (Invitrogen #V79020), respectively. CHO-S cells (ATCC) were cultured in CD-CHO culture medium (Life Technologies, #10743029) until reaching 0.5×10 ⁶ /ml. 10 μg of the vector encoding the huB7H3 or cyB7H3 gene was mixed with 50 μl of LF-LTX (Life Technologies, #A12621) in 1 ml of Opti-MEM medium (Life Technologies, #31985088), incubated at room temperature for 20 minutes, supplemented with culture medium for CHO cells and placed in a CO ₂ incubator for cultivation. After 24 hours, the medium was replaced with fresh medium and 10 μg/ml puromycin was added. Thereafter, the culture medium was changed every 2-3 days, and after 10-12 days, a stable pool of CHO-S cells was obtained by screening.

Example 2: Obtaining Mouse Hybridomas and Antibody Sequences

Animals were immunized with the human huB7-H4-Fc antigen. Five Balb/c mice and five A/J mice (female, 10 weeks old) were used. Immunogen and immunoadjuvant (Complete Freund's Adjuvant Sigma (CFA - Complete Freund's Adjuvant) or incomplete Freund's Adjuvant Sigma (IFA - Incomplete Freund's Adjuvant)) were thoroughly mixed in a 1:1 ratio and emulsified to obtain a stable water-in-oil liquid; The dose for injection was 25 μg/200 μl/mouse.

Day 1 First immunization, CFA Day 21 Second immunization, IFA Day 35 Third immunization, IFA Day 42 Blood collection and detection of serum titer (blood collected after three immunizations) Day 49 Fourth immunization, IFA Day 56 Blood collection and determination of serum titer (blood collected after four immunizations)

Serum titer and ability to bind to cell surface antigens was assessed using sera from immunized mice by indirect ELISA and capture ELISA as described in Example 3. Cell fusion was initiated, depending on the results of the titer analysis (greater than 100,000-fold dilution) . Mice with high serum titer, affinity, and strong FACS (fluorescent-activated cell sorting) binding were subjected to a final immunization and then sacrificed. Spleen cells and SP2/0 myeloma cells were fused and plated to give hybridomas, which were screened by indirect ELISA and capture ELISA to give the target hybridomas. Monoclonal cell lines were created by limiting dilution. CHO-S cells stably expressing B7-H4 were further transfected with the resulting antibody-positive lines. Empty CHO-S cells were used as controls to exclude hybridoma lines of non-specific binding antibodies. Eight hybridoma lines that not only bind to recombinant proteins but also bind to antigens that are expressed by the cells were obtained by flow sorting. Hybridoma cells were harvested in logarithmic growth phase, RNA was isolated with Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript™ Reverse Transcriptase, Takara #2680A). The reverse transcription cDNAs were amplified by PCR (polymerase chain reaction) amplification using the Mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and sequenced, and 8 mouse antibody sequences were ultimately obtained.

The heavy and light chain variable region sequences of the mouse mAb 2F7 are as follows:

HCVR 2F7

EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYYMSWVRQTPEKRLEWVAYVSSGGGSTYYSDSVKGRFTISRDNAKNTLYLQMSSLKPEDTAMYYCTRESYSQGNYFDYWGQGTTLTVSS

SEQ ID NO: 1

LCVR 2F7

DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKFASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFSLTFGAGTKLELK

SEQ ID NO: 2

The following CDR sequences are contained:

Name Subsequence SEQID NO: HCDR1 GFTFSNYYMS SEQ ID NO: 3 HCDR2 YVSSGGGSTYYSDSVKG SEQ ID NO: 4 HDDR3 ESYSQGNYFDY SEQ ID NO: 5 LCDR1 RASQSISDYLH SEQ ID NO: 6 LCDR2 FASQSIS SEQ ID NO: 7 LCDR3 QNGHSFSLT SEQ ID NO: 8

The sequences of the variable regions of the M1 heavy and light chains are as follows:

HCVR M1

EIQLQQSGPELVMPGASVKVSCTASGYPFTTYNMYWVKQSHGKSLEWIAYIDPYNGGTSYNQKFKGKATLTVDKSSSTAYMHLNSLTSEDSAVYYCARSGFYDGYYAWYFDVWGAGTTVTVSS

SEQ ID NO: 9

LCVR M1

DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSGGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGAGTKLELK

SEQ ID NO: 10

The following CDR sequences are contained:

Name Subsequence SEQID NO: HCDR1 GYPFTTYNMY SEQ ID NO: 11 HCDR2 YIDPYNGGTSYNQKFKG SEQ ID NO: 12 HDDR3 SGFYDGYYAWYFDV SEQ ID NO: 13 LCDR1 RSSQSLVHSGGNTYLH SEQ ID NO: 14 LCDR2 KVSNRFS SEQ ID NO: 15 LCDR3 SQSTHVPLT SEQ ID NO: 16

The heavy and light chain variable region sequences of the mouse mAb 2F8 are as follows:

HCVR 2F8

QVQLQQPGSVLVRPGASVKLSCKASGYTFNTNSWMNWAKLRPGQGLEWIGGIYPNSGNIEYNEKFKGKATLTVDTSSSTAYMDLTSLTSEDSAVYYCARDSRFSYWGQGTLVTVSA

SEQ ID NO: 17

LCVR 2F8

DIVMTQSHKFMSTSVGDRVSITCKASQDVRTAVAWYQQKPGQSPKLLISSTSYRYTGVPDRFTGSGSGTDFTFIISSVQAEDLAVYYCQQHYSTPLTFGAGTKLELK

SEQ ID NO: 18

The following CDR sequences are contained:

Name Subsequence SEQID NO: HCDR1 GYTFTSWMN SEQ ID NO: 19 HCDR2 GIYPNSGNIEYNEKFKG SEQ ID NO: 20 HDDR3 DSRFSY SEQ ID NO: 21 LCDR1 KASQDVRTAVA SEQ ID NO: 22 LCDR2 STSYRYT SEQ ID NO: 23 LCDR3 QQHYSTPLT SEQ ID NO: 24

The heavy and light chain variable region sequences of the mouse mAb 2F4 are as follows:

HCVR 2F4

EVQLVESGGGLVKPGGSLKLSCAASGLTFSRYAMSWVRQTPEKRLEWVAGISSGGSYTYYSDTVKGRFTISRDNVRNTLYLQMSSLRSEDTAMYYCGREYGRDYWGQGTSVTVSS

SEQ ID NO: 25

LCVR 2F4

DILMTQSPSSMSVSLGDTVSITCHASQGINSNIGWLQQKPGKSFKGLIYHGTNLEDGVPSRFSGSGSGTDYSLTISSLESEDFADYYCVQYAQFPRTFGGGTTLEIK

SEQ ID NO: 26

The following CDR sequences are contained:

Name Subsequence SEQID NO: HCDR1 GLTFSRYAMS SEQ ID NO: 27 HCDR2 GISSGGSYTYYSDTVKG SEQ ID NO: 28 HDDR3 EYGRDY SEQ ID NO: 29 LCDR1 HASQGINSNIG SEQ ID NO: 30 LCDR2 HGTNLED SEQ ID NO: 31 LCDR3 VQYAQFPRT SEQ ID NO: 32

The heavy and light chain variable region sequences of the mouse mAb 2A10 are as follows:

HCVR 2A10

EVQLVESGGGFVKPGGSLKLSCAASGFTFSTFGMSWVRQTPDKRLEWVAGISPGGSYTYYPDTVKGRFTISRDNARNTLYLQMSSLRSEDSAMYYCTRGRSVWGTGTTVSS

SEQ ID NO: 33

LCVR 2A10

DILMTQSPSSMSVSLGDTVSITCHASQDISSNIGWLQQKPGKSFKGLIYHGTTLEDGIPSRFSGSGSGADYSLTISSLESEDFADYYCVQSAQFPWTFGGGTKLEIK

SEQ ID NO: 34

The following CDR sequences are contained:

Name Subsequence SEQID NO: HCDR1 GFTFSTFGMS SEQ ID NO: 35 HCDR2 GISPGGSYTYYPDTVKG SEQ ID NO: 36 HDDR3 GRSV SEQ ID NO: 37 LCDR1 HASQDISSNIG SEQ ID NO: 38 LCDR2 HGTTLED SEQ ID NO: 39 LCDR3 VQSAQFPWT SEQ ID NO: 40

The heavy and light chain variable region sequences of the mouse mAb 2E4 are as follows:

HCVR 2E4

QVQLQQPGSVLVRPGTSVKLSCKASGYTFTSSWMNWVKQRPGQGLEWIGGIYPNRGTTEYNEKFKGKATLTVDTSSSTAFMDLNRLTSEDSAVYYCARDSRFADWGQGTLVTVSA

SEQ ID NO: 41

LCVR 2E4

DIMLTQSHKFMSTSVGDRVSITCKASQDVSAAVAWYQQKPGQSPKLLISSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYNTPLTFGAGTKLELK

SEQ ID NO: 42

The following CDR sequences are contained:

Name Subsequence SEQID NO: HCDR1 GYTFTSSWMN SEQ ID NO: 43 HCDR2 GIYPNRGTTEYNEKFKG SEQ ID NO: 44 HDDR3 DSRFAD SEQ ID NO: 45 LCDR1 KASQDVSAAVA SEQ ID NO: 46 LCDR2 SASYRYT SEQ ID NO: 47 LCDR3 QQHYNTPLT SEQ ID NO: 48

The heavy and light chain variable region sequences of mouse mAb 1E4 are as follows:

HCVR 1E4

EVQLVESGGGLVKPGGSLKLSCAASGFTFSRYAMSWVRQTPEKRLEWVAGISSGGSYTYYPDTLKGRFTVSRDNARNTLYLQMSSLRSEDTAKYFCASQGSNHYFDYWGQGTTLTVSS

SEQ ID NO: 49

LCVR 1E4

DTLMTQSPSSMSVSLGDTVSITCHASQGIHNNIGWLQQKPGKSFKALIYHGTNLEDGVPSRFSGSGSGADYSLIISSLESEDFADYYCVQYAQFPYTFGGGTKLEIK

SEQ ID NO: 50

The following CDR sequences are contained:

Name Subsequence SEQID NO: HCDR1 GFTFSRYAMS SEQ ID NO: 51 HCDR2 GISSGGSYTYYPDTLKG SEQ ID NO: 52 HDDR3 QGSNHYFDY SEQ ID NO: 53 LCDR1 HASQGIHNNIG SEQ ID NO: 54 LCDR2 HGTNLED SEQ ID NO: 55 LCDR3 VQYAQFPYT SEQ ID NO: 56

The heavy and light chain variable region sequences of the mouse mAb 2G6 are as follows:

HCVR 2G6

EVQLVESGGGLVKPGGSLKLSCAASGFTFSRYGMSWVRQTPEKRLEWVAGINGGGSYTYYLDTVKGRFTISRDNSRNTLYLQMSSLRSEDTAMYYCVSQGSNYYFDYWGQGTTLTVSS

SEQ ID NO: 57

LCVR 2G6

DIRMTQSPSSMSVSLGDTVSITCHASQGISSNIGWLQQKPGKSFKALIYHGTNLEDGVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPYTFGGGTKLEIK

SEQ ID NO: 58

The following CDR sequences are contained:

Name Subsequence SEQID NO: HCDR1 GFTFSRYGMS SEQ ID NO: 59 HCDR2 GINGGGSYTYYLDTVKG SEQ ID NO: 60 HDDR3 QGSNYYFDY SEQ ID NO: 61 LCDR1 HASQGISSNIG SEQ ID NO: 62 LCDR2 HGTNLED SEQ ID NO: 63 LCDR3 VQYAQFPYT SEQ ID NO: 64

The heavy and light chain variable region sequences of the mouse mAb 1C9 are as follows:

HCVR 1C9

QVQLQQPGSVLVRPGASVKLSCKASGDTFTTYWMNWVKQRPGQGLEWIGGIYLNSGSSEYNEKFKGKATLSVDTSSSTAYMDLSSLTSEDSAVYYCARDSRFSYWGQGTLVTVSA

SEQ ID NO: 65

LCVR 1C9

DIVMTQSHKFLSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPELLISSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYNTPLTFGAGTQLELK

SEQ ID NO: 66

The following CDR sequences are contained:

Name Subsequence SEQID NO: HCDR1 GDTFTTY SEQ ID NO: 67 HCDR2 YLNSGS SEQ ID NO: 68 HDDR3 DSRFSY SEQ ID NO: 69 LCDR1 KASQDVSTAVA SEQ ID NO: 70 LCDR2 SASYRYT SEQ ID NO: 71 LCDR3 QQHYNTPLT SEQ ID NO: 72

The heavy and light chain variable regions of each mouse mAb were cloned into the human IgG1 heavy chain constant region and the kappa light chain constant region, respectively, and then purified, identified, and tested for activity as described in Example 4.

Example 3 In Vitro Detection of Antibody Binding Activity

(1) In vitro indirect ELISA binding assay:

HuB7-H4 His protein (Sino Biological Inc. cat. no. 10738-H08H) was diluted to 1 μg/ml with PBS, pH 7.4, added to a 96-well high affinity microtiter plate in a volume of 100 μl/ well and incubated at 4°C overnight (16-20 hours). The plate was washed with PBST (PBS containing 0.05% Tween-20, pH 7.4) four times and then 150 µl/well of 3% blocking solution of bovine serum albumin (BSA) diluted in PBST was added and incubated at room temperature for 1 hour to block. After blocking was completed, the blocking solution was discarded and the plate was washed 4 times with PBST buffer.

Antibodies to be tested were diluted with PBST containing 3% BSA in a 10-fold dilution gradient starting at 1 μM for a total of 10 doses. Dilutions were added to the plate at 100 μl/well and incubated at room temperature for 1 hour. After the end of the incubation, the plate was washed 4 times with PBST, 100 µl/well of a secondary antibody of goat to human antibody labeled with HRP (horseradish peroxidase - horseradish peroxidase) (Abeam, cat. No. ab97225), diluted in PBST containing 3% BSA, was added to it, and incubated at room temperature for 1 hour. The plate was washed 4 times with PBST, 100 µl/well of chromogenic substrate TMB (tetramethylbenzidine - tetramethylbenzidine) (Cell Signaling Technology, Cat. No. 7004S) was added to it, incubated at room temperature in the dark for 1 minute, and 100 µl/well was added to it stop solution (Cell Signaling Technology, cat. no. 7002S) to terminate the reaction. Absorbance was read at 450 nm using a microplate reader (BioTek, model Synergy H1) and analyzed. A concentration versus signal value curve was built and the results analyzed as shown in the following table:

Chimeric antibody EC ₅₀ in case of binding to human B7-H4 His antigen (nM) M1 0.063 2A10 0.071 2F4 0.056 2F7 0.17 2F8 0.067 2G6 0.081 1C9 0.068

(2) Competitive ELISA analysis:

HuB7-H4 His protein (Sino Biological Inc. cat. no. 10738-H08H) was diluted to 1 μg/ml with PBS, pH 7.4, added to a 96-well high affinity microtiter plate in a volume of 100 μl/ well and incubated at 4°C overnight (16-20 hours). The plate was washed 4 times with PBST (PBS containing 0.05% Tween-20, pH 7.4), 150 µl/well of 3% blocking solution of bovine serum albumin (BSA) diluted in PBST was added and incubated at room temperature for 1 hour. After blocking was complete, the blocking solution was discarded and the plate was washed 4 times with PBST buffer.

A 0.1 nM reference chimeric antibody was prepared with PBST containing 3% BSA and used as a dilution solution for the mouse antibody to be tested, producing a 10-fold dilution gradient starting at 100 nM for a total of 10 doses. Diluted antibodies were added to the plate at 100 μl/well and incubated for 1 hour at room temperature. After the end of the incubation, the plate was washed 4 times with PBST and 100 µl/well HRP labeled goat anti-human secondary antibody (Abcam, cat# ab97225) diluted in PBST containing 3% BSA was added and incubated for 1 hour at room temperature . The plate was washed 4 times with PBST, and then 100 μl/well of TMB chromogenic substrate (Cell Signaling Technology, cat. no. 7004S) was added and incubated at room temperature for 1 minute in the dark. 100 μl/well of stop solution (Cell Signaling Technology, cat. no. 7002S) was added to terminate the reaction, and absorbance was read at 450 nm using a microplate reader (BioTek, model Synergy H1). Data analysis was carried out. The degree of competitive inhibition = ((absorption in the case of a reference antibody - absorption in the case of a competitive antibody)/absorption in the case of Abv antibody) * 100.

(3) In vitro capture ELISA binding assay:

Goat r-mouse IgG secondary antibody (Jackson Immuno Research, cat. no. 115-006-071) was diluted to 2 µg/ml with PBS buffer, pH 7.4, added to a 96-well microtiter plate at a volume of 100 µl/well and incubated in an incubator at 37°C for 2 hours. The plate was washed once with PBST, a blocking solution containing 5% skimmed milk (Bright Dairy, Skimmed Milk Powder) diluted in PBST at 200 µl/well was added to it and incubated at 37°C for 2 hours or at 4 °C during the night (16-18 hours) for blocking. After blocking was completed, the blocking solution was discarded and the plate was washed 4 times with PBST.

Mouse sera or purified recombinant antibodies to be tested were diluted to various concentrations with a sample dilution solution containing 5% NHS (PBST with 2.5% skim milk), incubated for 40 minutes at room temperature, added to the plate in an amount of 100 µl /well and incubated in an incubator for 40 minutes at 37°C. After the end of incubation, the plate was washed 4 times with PBST, 100 µl/well of biotinylated huB7-H4-his protein solution (Sino Biological #10738-H08H) diluted in sample dilution solution was added to it, and incubated at 37 °C for 40 minutes. After the end of the incubation, the plate was washed 4 times with PBST, 100 µl/well of HRP-labeled streptavidin (Jackson Immuno Research, cat. no. 016-030-084) diluted in PBST was added to it, and incubated at 37°C for 40 minutes. After washing the plate 4 times with PBST, 100 µl/well of TMB chromogenic substrate (InnoReagents Biotechnology Co., Ltd.) was added, incubated at room temperature for 10-15 min in the dark, 50 µl/well of 1M H ₂ SO ₄ was added to stop reactions. Absorbance was read at 450 nm using a microplate reader (Beijing Perlong New Technology Co., Ltd., Model DNM-9602) and data analysis was performed.

(4) In vitro cell binding assay:

Cultured SK-BR3 cells or cells stably transfected with CHO-huB7-H4 were harvested and then plated in a 96-well U-bottomed plate at a cell density of 1×10 ⁵ to 2×10 ⁵ cells per well. The supernatant was removed by centrifugation at 1200 g for 5 min, 100 μl of serially diluted solutions of antibodies or mouse immune sera were added and incubated at 4°C for 60 min, and the supernatant was removed by centrifugation at 1200 g for 5 min. The cells were washed twice with PBS, a fluorescently labeled secondary antibody (PE-GAM or PE-GAH) was added to them in an amount of 100 µl per well, incubated for 60 min at 4°C, and centrifuged at 1200 g for 5 min to remove the supernatant. Cells were washed twice with PBS and then resuspended in PBS. Signals were detected using a flow cytometer and a concentration curve was plotted and the results analyzed.

Example 4: Construction and expression of recombinant chimeric antibodies to B7-H4

Site-directed amino acid mutations were performed in the FR region(s) (framework regions) of the heavy chain variable region (VH) and the light chain variable region (VL) for each mouse antibody of the present invention. Different heavy and light chain humanized antibodies were designed according to different combinations of amino acid mutations. Cells transfected with plasmids of various combinations of heavy and light chains have been used to obtain humanized antibodies.

The heavy chain vector was constructed as follows: signal peptide plus mutant heavy chain variable region sequence plus human IgG1 constant region sequence.

The light chain vector was constructed as follows: signal peptide plus mutant light chain variable region sequence plus human kappa constant region sequence.

The above sequences were inserted into the pCEP4 vector, respectively. Expression vectors were synthesized according to the above construct, the resulting vector plasmids were extracted with a maxi isolation kit, and verified by sequencing. The verified plasmids were transfected into human 293F cells with PEI (Polyethylenimine) and cultured continuously. 293F cells were cultured in serum-free medium (Shanghai opmbiosciences, OPM-293CD03) to logarithmic growth phase for cell transfection. 21.4 μg of humanized antibody light chain plasmid and 23.6 μg of humanized antibody heavy chain plasmid were dissolved in 10 ml of Opti-MEM® I reconstituted serum medium (GIBCO, 31985-070), mixed well, then 200 μg of PEI, well mixed, incubated for 15 min at RT (room temperature) was added to 50 ml of cells. Cell culture conditions were as follows: 5% CO ₂ , 37°C, 125 rpm. During the culture period, medium was replenished on day 1 and day 3 until cell viability was less than 70%. The cell supernatant was collected and centrifuged for filtration. After centrifugation, the cell culture was applied to an affinity chromatography column to purify the antibodies. Purified chimeric antibodies were finally obtained after washing the column with phosphate buffer, eluting glycine with hydrochloride buffer (0.1 M Gly-HCl, pH 2.7), neutralizing with 1 M Tris-HCl, pH 9.0, and dialyzing against phosphate buffer. .

Example 5 Binding Affinity and In Vitro Kinetics Analysis

The Biacore method is recognized as a method for objectively detecting affinity and kinetics in proteins. The affinity and binding kinetics of the anti-B7-H4 antibodies of the invention to be tested were analyzed by Biacore T200 (GE).

The anti-B7-H4 antibody of the present invention to be tested was covalently bound to a CM5 (GE) chip by a standard NHS amino binding method using a kit provided by Biacore. Then,

a) 50 nM human huB7-H4-his protein (Sino Biological #10738-H08H) diluted with the same buffer was loaded at a flow rate of 10 μl/min, and the chip was regenerated with the regenerating reagent provided in the kit. Antigen-antibody binding kinetics were recorded over 3 minutes and dissociation kinetics were recorded over 10 minutes. The data obtained was analyzed by the BIAevaluation GE software using a 1:1 binding model (Langmuir). The kd (koff) data for each mouse antibody assessed by this method is shown in the following table.

mouse antibody antigen Degree of dissociation
k _d (1/s) 2F7 huB7-H4-his 2.53E-03 2F8 6.87E-05 M1 3.98E-05 2A10 3.31E-04 2G6 8.91E-05 2F4 1.66E-03 2E4 5.97E-05 1E4 2.93E-04

b) A concentration series of human huB7-H4-his protein diluted in the same buffer was respectively loaded at a flow rate of 10 μl/min and the chip was regenerated with the regenerating reagent provided in the kit. Antigen-antibody binding kinetics were recorded over 3 minutes and dissociation kinetics were recorded over 10 minutes. The data obtained was analyzed by the BIAevaluation GE software using a 1:1 binding model (Langmuir). The ka(kon), kd(koff) and K _D values for each chimeric antibody estimated by this method are shown in the following table:

Chimeric antibody antigen Degree of association, k _a (1/M*s) degree of dissociation,
k _d (1/s) affinity,
K _D ch-2F7 huB7-H4-his 4.417E+04 3.038E-03 68.8 nM ch-2F8 1.126E+06 1.136E-04 119 pM ch-M1 6.743E+05 9.411E-05 140 pM ch-2F4 1.040E+06 2.593E-03 2.5 nM

Example 6 Humanization of Mouse Antibodies

Humanization of mouse monoclonal antibodies to human B7-H4 was performed as disclosed in many literature sources in this field. Briefly, the parental (mouse antibody) constant domain was replaced with a human constant domain, and human antibody sequences were selected based on homology between mouse antibody and human antibody. In the present invention, murine candidate molecules 2F7, 2F8, 2G6 and 1C9 were humanized.

Based on the typical structure of the VH/VL CDR of the resulting mouse antibody, the heavy and light chain variable region sequences were compared with a human germline antibody database to obtain a human germline template with high homology.

The CDR region of mouse antibodies 2F7, 2F8, 2G6 and 1C9 were grafted onto the selected appropriate humanized template. In the case of 2F8, the HCDR1 region (GYTFTNSWMN, SEQ ID NO: 19) and the HCDR2 region (GIYPNSGNIEYNEKFKG, SEQ ID NO: 20) were replaced with GYTFTSSWMN (SEQ ID NO: 73) and GIYPNRGNIEYNEKFKG (SEQ ID NO: 74), respectively, to remove possible unstable deacetylation sites. The humanized variable regions were replaced and recombined with the IgG constant region (preferably IgG1 for the heavy chain and κ for the light chain). Then, based on the three-dimensional structure of the mouse antibody, backmutations were performed on the included residues, residues that interacted directly with CDRs, and residues that had an important effect on VL and VH conformation. Chemically unstable amino acid residues in the CDR regions were optimized. Received and detected antibodies derived from the combination of humanized sequences of the variable regions of the light and heavy chains.

The humanized hu2F7, hu2F8, hu2G6, and hu1C9 antibody molecules were ultimately selected by expression testing and backmutation comparison, and the respective heavy and light chain variable region sequences are shown in SEQ ID NOs: 75-82, and the respective heavy and light chain sequences are shown. in SEQ ID NO: 83-90.

HCVR hu2F7

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWVAYVSSGGGSTYYSDSVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCTRESYSQGNYFDYWGQGTTVTVSS

SEQ ID NO: 75

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWVAYVSSGGGSTYYSDSVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCARESYSQGNYFDYWGQGTTVTVSS

SEQ ID NO: 91

LCVR hu2F7

EIVMTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQSPRLLIKFASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFSLTFGQGTKLEIK

SEQ ID NO: 76

EIVLTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQAPRLLIYFASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFSLTFGQGTKLEIK

SEQ ID NO: 92

EIVLTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQAPRLLIKFASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFSLTFGQGTKLEIK

SEQ ID NO: 93

EIVMTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQAPRLLIKFASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFSLTFGQGTKLEIK

SEQ ID NO: 94

HCVR hu2F8

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSSWMNWVRQAPGQRLEWMGGIYPNRGNIEYNEKFKGRVTLTVDTSASTAYMELSSLRSEDTAVYYCARDSRFSYWGQGTLVTVSS

SEQ ID NO: 77

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSSWMNWVRQAPGQRLEWMGGIYPNRGNIEYNEKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCARDSRFSYWGQGTLVTVSS

SEQ ID NO: 95

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSSWMNWVRQAPGQGLEWMGGIYPNRGNIEYNEKFKGRVTLTVDTSASTAYMELSSLRSEDTAVYYCARDSRFSYWGQGTLVTVSS

SEQ ID NO: 96

LCVR hu2F8

DIQMTQSPSSLSASVGDRVTITCKASQDVRTAVAWYQQKPGKAPKLLISSTSYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPLTFGGGTKVEIK

SEQ ID NO: 78

DIVMTQSPSSLSASVGDRVTITCKASQDVRTAVAWYQQKPGKAPKLLISSTSYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPLTFGGGTKVEIK

SEQ ID NO: 97

DIVMTQSPSSLSASVGDRVTITCKASQDVRTAVAWYQQKPGKSPKLLISSTSYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPLTFGGGTKVEIK

SEQ ID NO: 98

HCVR hu2G6

EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVRQAPGKGLEWVSGINGGGSYTYYLDTVKGRFTISRDNARNTLYLQMSSLRAEDTAVYYCVSQGSNYYFDYWGQGTLVTVSS

SEQ ID NO: 79

LCVR hu2G6

DIRMTQSPSSLSASVGDRVTITCHASQGISSNIGWLQQKPGKAPKALIYHGTNLEDGVPSRFSGSGSGADYTLTISSLQPEDFATYYCVQYAQFPYTFGGGTKVEIK

SEQ ID NO: 80

HCVR hu1C9

EVQLVQSGAEVKKPGASVKVSCKASGDTFTTYWMNWVRQAPGQRLEWMGGIYLNRGSSEYNEKFKGRVTLTVDTSASTAYMELSSLRSEDTAVYYCARDSRFSYWGQGTLVTVSS

SEQ ID NO: 81

LCVR hu1C9

DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLISSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYNTPLTFGGGTKVEIK

SEQ ID NO: 82

HC hu2F7

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWVAYVSSGGGSTYYSDSVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCTRESYSQGNYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 83

LChu2F7

EIVMTQSPATLSPGERATLSCRASQSISDYLHWYQQKPGQSPRLLIKFASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFSLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACENSFTHQGL

SEQ ID NO: 84

HC hu2F8

EVQLVQSGAEVKKPGASVKVSCKASGYTFTSSWMNWVRQAPGQRLEWMGGIYPNRGNIEYNEKFKGRVTLTVDTSASTAYMELSSLRSEDTAVYYCARDSRFSYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 85

LC hu2F8

DIQMTQSPSSLSASVGDRVTITCKASQDVRTAVAWYQQKPGKAPKLLISSTSYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKTLSKPVNGSFTHQ

SEQ ID NO: 86

HC hu2G6

EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVRQAPGKGLEWVSGINGGGSYTYYLDTVKGRFTISRDNARNTLYLQMSSLRAEDTAVYYCVSQGSNYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 87

LC hu2G6

DIRMTQSPSSLSASVGDRVTITCHASQGISSNIGWLQQKPGKAPKALIYHGTNLEDGVPSRFSGSGSGADYTLTISSLQPEDFATYYCVQYAQFPYTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLECPVRGSFTHNACEVQ

SEQ ID NO: 88

HC hu1C9

EVQLVQSGAEVKKPGASVKVSCKASGDTFTTYWMNWVRQAPGQRLEWMGGIYLNRGSSEYNEKFKGRVTLTVDTSASTAYMELSSLRSEDTAVYYCARDSRFSYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 89

LC hu1C9

DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLISSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYNTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKTLSKPVNGSFTHQ

SEQ ID NO: 90

cDNA fragments were synthesized based on the amino acid sequences of the humanized antibody light and heavy chains mentioned above and inserted into pcDNA3.1 expression vectors (Life Technologies, cat. no. V790-20). Expression vectors and transfection reagent PEI (Polysciences, Inc., cat. no. 23966) were transfected into HEK293 cells (Life Technologies, cat. no. 11625019) at a ratio of 1:2 and incubated in a CO ₂ incubator for 4-5 days. Expressed antibodies were isolated by centrifugation, and antibody purification was performed according to the method of Example 4 to obtain humanized hu2F7 and hu2F8 antibody proteins of the present invention.

Example 7: Determination of the activity of humanized antibodies

The following in vitro assays were performed on humanized hu2F7 and hu2F8 antibodies:

1. In vitro cell binding assay:

Cultured MX-1 cells were harvested, cell density adjusted with PBS, pH 7.4, then plated in a 96-well V-bottomed plate with 1×10 ⁵ cells per well and centrifuged at 2000 rpm for 5 minutes to removal of the supernatant. Add 100 µl of serially diluted chimeric antibody solution (diluted with 0.5% BSA in PBS to give a gradient of 3-fold dilutions starting at 1 µM for a total of 10 doses) to each well, mix and incubate for 1 hour at 4 °C when shaking; the culture was centrifuged at 2000 rpm for 5 minutes to remove the supernatant, and the cells were washed twice with PBS, and 100 μl of FITC-labeled goat anti-human secondary antibody (Abeam, Cat No. ab97224) diluted 0.5 % BSA in PBS, mixed well and incubated for 30 minutes at 4°C on a shaker. Centrifugation was performed at 2000 rpm for 5 minutes and the supernatant was removed. Cells were washed twice with PBS and resuspended in PBS. Signals were detected using a flow cytometer (BECKMANCOULTER, model DxFLEX) and a concentration curve was plotted and the results analyzed. As shown in the table and figure, the humanized antibodies 2F7, 2F8, 2G6 and 1C9 show positive binding to MX-1 cells, on which B7-H4 was expressed at a high level.

Name of the antibody FACS, EC ₅₀ (nM) when bound to MX-1 cell hu2F7 7.32

Name of the antibody FACS, EC ₅₀ (nM) when bound to MX-1 cell hu2F8 7.26

Name of the antibody FACS, EC ₅₀ (nM) when bound to MX-1 cell hu2G6 14.3

Name of the antibody FACS, EC ₅₀ (nM) when bound to MX-1 cell hu1C9 7.25

2. Kinetic affinity analysis (method procedures were the same as those described in Example 5). The results are shown in the table below. All humanized hu2F7, hu2F8, hu2G6 and hu1C9 antibodies show strong affinity for the human B7-H4 antigen protein.

Antibody Antigen Degree of association, k _a (1/M*s) degree of dissociation,
k _d (1/s) affinity,
_KD hu2F7 hu-B7-H4-his 3.29e+05 2.49e-04 7.57e-10 hu2F8 4.47e+05 1.12e-04 2.50e-10 hu2G6 7.41e+05 1.00e-05 1.35e-11 hu1C9 3.35e+05 1.00e-05 2.98e-11

Example 8 Determination of Binding Epitopes Recognized by Humanized Antibodies

The method procedures were the same as described in Example 3 (1): indirect in vitro ELISA binding assay. B7-H4 SEQ ID NO: 100 was degraded into antigen fragments of 20 amino acids in length, and the P12 antigen fragment (amino acid sequence: TVASAGNIGEDGILSCTFEP) was found to specifically bind the humanized anti-B7-H4 antibody hu2F7, in an indirect ELISA binding assay in vitro. The results are shown in FIG. 2. To further confirm the epitope to which the antibody binds, an alanine scan was performed on the P12 antigen fragment, that is, each individual amino acid located in P12 was mutated to alanine, respectively. In an indirect in vitro ELISA binding assay, mutations in a portion of the ILSCTFE amino acid sequence were found to significantly impair antibody binding to an antigen fragment, demonstrating that the antibody binding epitope was located in a portion of the ILSCTFE amino acid sequence (SEQ ID NO: 109) contained in the amino acid sequence TVASAGNIGEDGILSCTFEP. The results are shown in the following table:

Sequence of an antigen fragment SEQID NO: Affinity EC50 (nM) TVASAGNIGEDGILSCTFEP 101 117.9 TVASAGNIGEDGALSCTFEP 102 770.6 TVASAGNIGEDGIASCTFEP 103 1476 TVASAGNIGEDGILSATFEP 104 2006 TVASAGNIGEDGILSCAFEP 105 462.8 TVASAGNIGEDGILSCTAEP 106 986.5 TVASAGNIGEDGILSCTFAP 107 662.5

In an indirect in vitro ELISA binding assay, it was found that the P12 antigen fragment (amino acid sequence: TVASAGNIGEDGILSCTFEP) was specifically bound by the humanized anti-B7-H4 antibody, hu1C9. The results are shown in the table below. To further confirm the epitope to which the antibody binds, an alanine scan was performed on the P12 antigen fragment, that is, each individual amino acid located in P12 was mutated to alanine, respectively. In an indirect in vitro ELISA binding assay, mutations in a portion of the amino acid sequence of LSCTF were found to significantly attenuate binding of the antibody to the antigen fragment, demonstrating that the antibody binding epitope was located in the portion of the amino acid sequence of LSCTF (SEQ ID NO: 110) contained in the amino acid sequence TVASAGNIGEDGILSCTFEP. The results are shown in the following table:

Sequence of an antigen fragment SEQID NO: Affinity EC50 (nM) TVASAGNIGEDGILSCTFEP 101 271.7 TVASAGNIGEDGIASCTFEP 103 466.9 TVASAGNIGEDGILSATFEP 104 292.3 TVASAGNIGEDGILSCTAEP 106 487.9

In an indirect in vitro ELISA binding assay, it was found that the P12 antigen fragment (amino acid sequence: TVASAGNIGEDGILSCTFEP) was specifically bound by the humanized anti-B7-H4 antibody, hu2G6. The results are shown in the table below. To further confirm the epitope to which the antibody binds, an alanine scan was performed on the P12 antigen fragment, that is, each individual amino acid located in P12 was mutated to alanine, respectively. In an indirect in vitro ELISA binding assay, mutations in a portion of the amino acid sequence of ILSCTFEP were found to significantly attenuate the binding of the antibody to the antigen fragment, demonstrating that the antibody binding epitope was located in the portion of the amino acid sequence of ILSCTFEP (SEQ ID NO: 111) contained in the amino acid sequence TVASAGNIGEDGILSCTFEP. The results are shown in the following table:

Sequence of an antigen fragment SEQID NO: Affinity EC50 (nM) TVASAGNIGEDGILSCTFEP 101 792.6 TVASAGNIGEDGALSCTFEP 102 2626 TVASAGNIGEDGIASCTFEP 103 3158 TVASAGNIGEDGILSATFEP 104 3480 TVASAGNIGEDGILSCAFEP 105 2289 TVASAGNIGEDGILSCTAEP 106 2600 TVASAGNIGEDGILSCTFEA 108 2372

Example 9: Antitumor activity of humanized antibodies

MC38 tumor cells were implanted in mice that were injected with the human B7-H4 gene through genome editing technology. When the tumor reached an average of 100 mm ³ mice were injected with control or humanized antibody against B7-H4 hu1C9 (10 mg/kg or 30 mg/kg) every 3 days. By monitoring tumor size, hu1C9 was found to have a significant effect on tumor growth inhibition and antitumor effect. Specific results are shown in Fig. 3 and in the following table:

Tumor volume (mean, mm ^® ) Day 0 Day 3 Day 6 Day 9 Day 12 Day 15 Day 18 Blank control group 136.2979 290.2921 526.7519 820.9497 1689.954 3625.686 5913.385 1C9 (10 mg/kg) 146.5273 231.8861 404.833 794.0959 1519.589 1717.092 2326.687 1C9 (30 mg/kg) 139.3148 256.9988 437.9834 702.0555 1190.775 2008.451 2744.415

Example 10: Impact of Humanized Antibodies on Immune Enhancement

The antitumor effect of an anti-B7-H4 antibody may be mediated by an immune-enhancing effect against tumors. To test the validity of this mechanism, CD4-positive T cells were isolated from human peripheral blood. Various concentrations of anti-B7-H4 antibodies, including humanized hu2G6 and hu1C9 antibody, were added to cultured T cells. Specific results are shown in Fig. 4 and below in the table. The humanized hu2G6 antibody and the humanized hu1C9 antibody have an effect on enhancing T cell proliferation, and can achieve antitumor activity through immune enhancing effects against tumors.

T cell proliferation (percent, mean) dosage 1C9 2G6 Blank control group 0.1 (µg/ml) 62.49425 53.6423 Minus 12.7833 0.3 (µg/ml) 76.83938 76.88089 Minus 4.12145 3 (mcg/ml) 86.96902 85.86148 Minus 4.39787

--->

SEQUENCE LIST

<110> JIANGSU HENSOH PHARMACEUTICAL GROUP CO., LTD.

SHANGHAI HENSOH BIOMEDICAL CO., LTD.

<120> ANTIBODY TO B7-H4, ITS ANTIGEN-BINDING FRAGMENT AND ITS

PHARMACEUTICAL APPLICATION

<130> 719011CPCT

<160> 111

<170> SIPOSequenceListing 1.0

<210> 1

<211> 120

<212> PROTEIN

<213> House mouse

<400> 1

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

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

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

35 40 45

Ala Tyr Val Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Ser Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Thr Arg Glu Ser Tyr Ser Gln Gly Asn Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 2

<211> 107

<212> PROTEIN

<213> House mouse

<400> 2

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

1 5 10 15

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

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Ser Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 3

<211> 10

<212> PROTEIN

<213> House mouse

<400> 3

Gly Phe Thr Phe Ser Asn Tyr Tyr Met Ser

1 5 10

<210> 4

<211> 17

<212> PROTEIN

<213> House mouse

<400> 4

Tyr Val Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Ser Asp Ser Val Lys

1 5 10 15

gly

<210> 5

<211> 11

<212> PROTEIN

<213> House mouse

<400> 5

Glu Ser Tyr Ser Gln Gly Asn Tyr Phe Asp Tyr

1 5 10

<210> 6

<211> 11

<212> PROTEIN

<213> House mouse

<400> 6

Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His

1 5 10

<210> 7

<211> 7

<212> PROTEIN

<213> House mouse

<400> 7

Phe Ala Ser Gln Ser Ile Ser

15

<210> 8

<211> 9

<212> PROTEIN

<213> House mouse

<400> 8

Gln Asn Gly His Ser Phe Ser Leu Thr

15

<210> 9

<211> 123

<212> PROTEIN

<213> House mouse

<400> 9

Glu Ile Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Met Pro Gly Ala

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 10

<211> 112

<212> PROTEIN

<213> House mouse

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

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

100 105 110

<210> 11

<211> 10

<212> PROTEIN

<213> House mouse

<400> 11

Gly Tyr Pro Phe Thr Thr Tyr Asn Met Tyr

1 5 10

<210> 12

<211> 17

<212> PROTEIN

<213> House mouse

<400> 12

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

1 5 10 15

gly

<210> 13

<211> 14

<212> PROTEIN

<213> House mouse

<400> 13

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

1 5 10

<210> 14

<211> 16

<212> PROTEIN

<213> House mouse

<400> 14

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

1 5 10 15

<210> 15

<211> 7

<212> PROTEIN

<213> House mouse

<400> 15

Lys Val Ser Asn Arg Phe Ser

15

<210> 16

<211> 9

<212> PROTEIN

<213> House mouse

<400> 16

Ser Gln Ser Thr His Val Pro Leu Thr

15

<210> 17

<211> 115

<212> PROTEIN

<213> House mouse

<400> 17

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ala

115

<210> 18

<211> 107

<212> PROTEIN

<213> House mouse

<400> 18

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

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

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Ile Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 19

<211> 10

<212> PROTEIN

<213> House mouse

<400> 19

Gly Tyr Thr Phe Thr Asn Ser Trp Met Asn

1 5 10

<210> 20

<211> 17

<212> PROTEIN

<213> House mouse

<400> 20

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

1 5 10 15

gly

<210> 21

<211> 6

<212> PROTEIN

<213> House mouse

<400> 21

Asp Ser Arg Phe Ser Tyr

15

<210> 22

<211> 11

<212> PROTEIN

<213> House mouse

<400> 22

Lys Ala Ser Gln Asp Val Arg Thr Ala Val Ala

1 5 10

<210> 23

<211> 7

<212> PROTEIN

<213> House mouse

<400> 23

Ser Thr Ser Tyr Arg Tyr Thr

15

<210> 24

<211> 9

<212> PROTEIN

<213> House mouse

<400> 24

Gln Gln His Tyr Ser Thr Pro Leu Thr

15

<210> 25

<211> 115

<212> PROTEIN

<213> House mouse

<400> 25

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

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser Arg Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Gly Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Ser Asp Thr Val

50 55 60

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

65 70 75 80

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

85 90 95

Gly Arg Glu Tyr Gly Arg Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr

100 105 110

Val Ser Ser

115

<210> 26

<211> 107

<212> PROTEIN

<213> House mouse

<400> 26

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr His Gly Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln Tyr Ala Gln Phe Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Thr Leu Glu Ile Lys

100 105

<210> 27

<211> 10

<212> PROTEIN

<213> House mouse

<400> 27

Gly Leu Thr Phe Ser Arg Tyr Ala Met Ser

1 5 10

<210> 28

<211> 17

<212> PROTEIN

<213> House mouse

<400> 28

Gly Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Ser Asp Thr Val Lys

1 5 10 15

gly

<210> 29

<211> 6

<212> PROTEIN

<213> House mouse

<400> 29

Glu Tyr Gly Arg Asp Tyr

15

<210> 30

<211> 11

<212> PROTEIN

<213> House mouse

<400> 30

His Ala Ser Gln Gly Ile Asn Ser Asn Ile Gly

1 5 10

<210> 31

<211> 7

<212> PROTEIN

<213> House mouse

<400> 31

His Gly Thr Asn Leu Glu Asp

15

<210> 32

<211> 9

<212> PROTEIN

<213> House mouse

<400> 32

Val Gln Tyr Ala Gln Phe Pro Arg Thr

15

<210> 33

<211> 113

<212> PROTEIN

<213> House mouse

<400> 33

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

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val

35 40 45

Ala Gly Ile Ser Pro Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Thr Arg Gly Arg Ser Val Trp Gly Thr Gly Thr Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 34

<211> 107

<212> PROTEIN

<213> House mouse

<400> 34

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

1 5 10 15

Asp Thr Val Ser Ile Thr Cys His Ala Ser Gln Asp Ile Ser Ser Asn

20 25 30

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

35 40 45

Tyr His Gly Thr Thr Leu Glu Asp Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln Ser Ala Gln Phe Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 35

<211> 10

<212> PROTEIN

<213> House mouse

<400> 35

Gly Phe Thr Phe Ser Thr Phe Gly Met Ser

1 5 10

<210> 36

<211> 17

<212> PROTEIN

<213> House mouse

<400> 36

Gly Ile Ser Pro Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Val Lys

1 5 10 15

gly

<210> 37

<211> 4

<212> PROTEIN

<213> House mouse

<400> 37

Gly Arg Ser Val

1

<210> 38

<211> 11

<212> PROTEIN

<213> House mouse

<400> 38

His Ala Ser Gln Asp Ile Ser Ser Asn Ile Gly

1 5 10

<210> 39

<211> 7

<212> PROTEIN

<213> House mouse

<400> 39

His Gly Thr Thr Leu Glu Asp

15

<210> 40

<211> 9

<212> PROTEIN

<213> House mouse

<400> 40

Val Gln Ser Ala Gln Phe Pro Trp Thr

15

<210> 41

<211> 115

<212> PROTEIN

<213> House mouse

<400> 41

Gln Val Gln Leu Gln Gln Pro Gly Ser Val Leu Val Arg Pro Gly Thr

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Asp Ser Arg Phe Ala Asp Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala

115

<210> 42

<211> 107

<212> PROTEIN

<213> House mouse

<400> 42

Asp Ile Met Leu Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

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

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Asn Thr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 43

<211> 10

<212> PROTEIN

<213> House mouse

<400> 43

Gly Tyr Thr Phe Thr Ser Ser Trp Met Asn

1 5 10

<210> 44

<211> 17

<212> PROTEIN

<213> House mouse

<400> 44

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

1 5 10 15

gly

<210> 45

<211> 6

<212> PROTEIN

<213> House mouse

<400> 45

Asp Ser Arg Phe Ala Asp

15

<210> 46

<211> 11

<212> PROTEIN

<213> House mouse

<400> 46

Lys Ala Ser Gln Asp Val Ser Ala Ala Val Ala

1 5 10

<210> 47

<211> 7

<212> PROTEIN

<213> House mouse

<400> 47

Ser Ala Ser Tyr Arg Tyr Thr

15

<210> 48

<211> 9

<212> PROTEIN

<213> House mouse

<220>

<221> DOMAIN

<222> (1)..(10)

<223> mutant region HCDR1 2F8

<400> 48

Gln Gln His Tyr Asn Thr Pro Leu Thr

15

<210> 49

<211> 118

<212> PROTEIN

<213> House mouse

<400> 49

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

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Gly Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Leu

50 55 60

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

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Lys Tyr Phe Cys

85 90 95

Ala Ser Gln Gly Ser Asn His Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 50

<211> 107

<212> PROTEIN

<213> House mouse

<400> 50

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

1 5 10 15

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

20 25 30

Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Phe Lys Ala Leu Ile

35 40 45

Tyr His Gly Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ala Asp Tyr Ser Leu Ile Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln Tyr Ala Gln Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 51

<211> 10

<212> PROTEIN

<213> House mouse

<400> 51

Gly Phe Thr Phe Ser Arg Tyr Ala Met Ser

1 5 10

<210> 52

<211> 17

<212> PROTEIN

<213> House mouse

<400> 52

Gly Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Leu Lys

1 5 10 15

gly

<210> 53

<211> 9

<212> PROTEIN

<213> House mouse

<400> 53

Gln Gly Ser Asn His Tyr Phe Asp Tyr

15

<210> 54

<211> 11

<212> PROTEIN

<213> House mouse

<400> 54

His Ala Ser Gln Gly Ile His Asn Asn Ile Gly

1 5 10

<210> 55

<211> 7

<212> PROTEIN

<213> House mouse

<400> 55

His Gly Thr Asn Leu Glu Asp

15

<210> 56

<211> 9

<212> PROTEIN

<213> House mouse

<400> 56

Val Gln Tyr Ala Gln Phe Pro Tyr Thr

15

<210> 57

<211> 118

<212> PROTEIN

<213> House mouse

<400> 57

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

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Gly Ile Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Leu Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Val Ser Gln Gly Ser Asn Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 58

<211> 107

<212> PROTEIN

<213> House mouse

<400> 58

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

1 5 10 15

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

20 25 30

Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Phe Lys Ala Leu Ile

35 40 45

Tyr His Gly Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln Tyr Ala Gln Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 59

<211> 10

<212> PROTEIN

<213> House mouse

<400> 59

Gly Phe Thr Phe Ser Arg Tyr Gly Met Ser

1 5 10

<210> 60

<211> 17

<212> PROTEIN

<213> House mouse

<400> 60

Gly Ile Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Leu Asp Thr Val Lys

1 5 10 15

gly

<210> 61

<211> 9

<212> PROTEIN

<213> House mouse

<400> 61

Gln Gly Ser Asn Tyr Tyr Phe Asp Tyr

15

<210> 62

<211> 11

<212> PROTEIN

<213> House mouse

<400> 62

His Ala Ser Gln Gly Ile Ser Ser Asn Ile Gly

1 5 10

<210> 63

<211> 7

<212> PROTEIN

<213> House mouse

<400> 63

His Gly Thr Asn Leu Glu Asp

15

<210> 64

<211> 9

<212> PROTEIN

<213> House mouse

<400> 64

Val Gln Tyr Ala Gln Phe Pro Tyr Thr

15

<210> 65

<211> 115

<212> PROTEIN

<213> House mouse

<400> 65

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Gly Ile Tyr Leu Asn Ser Gly Ser Ser Glu Tyr Asn Glu Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ala

115

<210> 66

<211> 107

<212> PROTEIN

<213> House mouse

<400> 66

Asp Ile Val Met Thr Gln Ser His Lys Phe Leu Ser Thr Ser Val Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln His Tyr Asn Thr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Gln Leu Glu Leu Lys

100 105

<210> 67

<211> 7

<212> PROTEIN

<213> House mouse

<400> 67

Gly Asp Thr Phe Thr Thr Tyr

15

<210> 68

<211> 6

<212> PROTEIN

<213> House mouse

<400> 68

Tyr Leu Asn Ser Gly Ser

15

<210> 69

<211> 6

<212> PROTEIN

<213> House mouse

<400> 69

Asp Ser Arg Phe Ser Tyr

15

<210> 70

<211> 11

<212> PROTEIN

<213> House mouse

<400> 70

Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210> 71

<211> 7

<212> PROTEIN

<213> House mouse

<400> 71

Ser Ala Ser Tyr Arg Tyr Thr

15

<210> 72

<211> 9

<212> PROTEIN

<213> House mouse

<400> 72

Gln Gln His Tyr Asn Thr Pro Leu Thr

15

<210> 73

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(10)

<223> humanized HCDR1 2F8

<400> 73

Gly Tyr Thr Phe Thr Ser Ser Trp Met Asn

1 5 10

<210> 74

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(17)

<223> humanized HCDR2 2F8

<400> 74

Gly Ile Tyr Pro Asn Arg Gly Asn Ile Glu Tyr Asn Glu Lys Phe Lys

1 5 10 15

gly

<210> 75

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(120)

<223> hu2F7 HCVR

<400> 75

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

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

35 40 45

Ala Tyr Val Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Ser Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Thr Arg Glu Ser Tyr Ser Gln Gly Asn Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 76

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(107)

<223> hu2F7 LCVR

<400> 76

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

1 5 10 15

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

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile

35 40 45

Lys Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 77

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(115)

<223> hu2F8 HCVR

<400> 77

Glu 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 Ser

20 25 30

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

35 40 45

Gly Gly Ile Tyr Pro Asn Arg Gly Asn Ile Glu Tyr Asn Glu Lys Phe

50 55 60

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

100 105 110

Val Ser Ser

115

<210> 78

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(107)

<223> hu2F8 LCVR

<400> 78

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

1 5 10 15

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

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 79

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(118)

<223> hu2G6 HCVR

<400> 79

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

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

35 40 45

Ser Gly Ile Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Leu Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Val Ser Gln Gly Ser Asn Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 80

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(107)

<223> hu2G6 LCVR

<400> 80

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Gly Ile Ser Ser Asn

20 25 30

Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile

35 40 45

Tyr His Gly Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Tyr Ala Gln Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 81

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(115)

<223> hu1C9 HCVR

<400> 81

Glu 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 Asp Thr Phe Thr Thr Tyr

20 25 30

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

35 40 45

Gly Gly Ile Tyr Leu Asn Arg Gly Ser Ser Glu Tyr Asn Glu Lys Phe

50 55 60

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

100 105 110

Val Ser Ser

115

<210> 82

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(107)

<223> hu1C9 LCVR

<400> 82

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

1 5 10 15

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

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Ser Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Asn Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 83

<211> 450

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(450)

<223> hu2F7 HC

<400> 83

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

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

35 40 45

Ala Tyr Val Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Ser Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Thr Arg Glu Ser Tyr Ser Gln Gly Asn Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

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

435 440 445

Gly Lys

450

<210> 84

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(214)

<223> hu2F7 LC

<400> 84

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

1 5 10 15

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

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile

35 40 45

Lys Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

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

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

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

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 85

<211> 445

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(445)

<223> hu2F8 HC

<400> 85

Glu 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 Ser

20 25 30

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

35 40 45

Gly Gly Ile Tyr Pro Asn Arg Gly Asn Ile Glu Tyr Asn Glu Lys Phe

50 55 60

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

100 105 110

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

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

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

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 86

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(214)

<223> hu2F8 LC

<400> 86

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

1 5 10 15

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

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

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

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

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

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 87

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(448)

<223> hu2G6 HC

<400> 87

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

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

35 40 45

Ser Gly Ile Asn Gly Gly Gly Ser Tyr Thr Tyr Tyr Leu Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Val Ser Gln Gly Ser Asn Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

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

435 440 445

<210> 88

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(214)

<223> hu2G6 LC

<400> 88

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Gly Ile Ser Ser Asn

20 25 30

Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile

35 40 45

Tyr His Gly Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Tyr Ala Gln Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

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

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

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

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 89

<211> 445

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(445)

<223> hu1C9 HC

<400> 89

Glu 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 Asp Thr Phe Thr Thr Tyr

20 25 30

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

35 40 45

Gly Gly Ile Tyr Leu Asn Arg Gly Ser Ser Glu Tyr Asn Glu Lys Phe

50 55 60

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

100 105 110

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

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

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

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 90

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(214)

<223> hu1C9 LC

<400> 90

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

1 5 10 15

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

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Ser Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln His Tyr Asn Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

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

115 120 125

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

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

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

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 91

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(120)

<223> hu2F7 HCVR

<400> 91

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

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

35 40 45

Ala Tyr Val Ser Ser Gly Gly Gly Ser Thr Tyr Tyr Ser Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 92

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(107)

<223> hu2F7 LCVR

<400> 92

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

1 5 10 15

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

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 93

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(107)

<223> hu2F7 LCVR

<400> 93

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

1 5 10 15

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

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Lys Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 94

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(107)

<223> hu2F7 LCVR

<400> 94

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

1 5 10 15

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

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Lys Phe Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

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

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 95

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(115)

<223> hu2F8 HCVR

<400> 95

Glu 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 Ser

20 25 30

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

35 40 45

Gly Gly Ile Tyr Pro Asn Arg Gly Asn Ile Glu Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Val Asp Thr Ser Ala 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 Asp Ser Arg Phe Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 96

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(115)

<223> hu2F8 HCVR

<400> 96

Glu 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 Ser

20 25 30

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

35 40 45

Gly Gly Ile Tyr Pro Asn Arg Gly Asn Ile Glu Tyr Asn Glu Lys Phe

50 55 60

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

100 105 110

Val Ser Ser

115

<210> 97

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(107)

<223> hu2F8 LCVR

<400> 97

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

1 5 10 15

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

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 98

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<222> (1)..(107)

<223> hu2F8 LCVR

<400> 98

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

1 5 10 15

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

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 99

<211> 463

<212> PROTEIN

<213> Artificial sequence

<220>

<221> PEPTIDE

<222> (1)..(463)

<223> huB7-H4-Fc

<400> 99

Phe Gly Ile Ser Gly Arg His Ser Ile Thr Val Thr Thr Val Ala Ser

1 5 10 15

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

20 25 30

Asp Ile Lys Leu Ser Asp Ile Val Ile Gln Trp Leu Lys Glu Gly Val

35 40 45

Leu Gly Leu Val His Glu Phe Lys Glu Gly Lys Asp Glu Leu Ser Glu

50 55 60

Gln Asp Glu Met Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val

65 70 75 80

Ile Val Gly Asn Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp

85 90 95

Ala Gly Thr Tyr Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn

100 105 110

Ala Asn Leu Glu Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Val Asn

115 120 125

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

130 135 140

Trp Phe Pro Gln Pro Thr Val Val Trp Ala Ser Gln Val Asp Gln Gly

145 150 155 160

Ala Asn Phe Ser Glu Val Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu

165 170 175

Asn Val Thr Met Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn

180 185 190

Asn Thr Tyr Ser Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly

195 200 205

Asp Ile Lys Val Thr Glu Ser Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln

210 215 220

Leu Leu Asn Ser Lys Ala Gly Ser Gly Gly Gly Gly Asp Lys Thr His

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

<210> 100

<211> 276

<212> PROTEIN

<213> Artificial sequence

<220>

<221> PEPTIDE

<222> (1)..(276)

<223> huB7-H4-his

<400> 100

Met Ala Ser Leu Gly Gln Ile Leu Phe Trp Ser Ile Ile Ser Ile Ile

1 5 10 15

Ile Ile Leu Ala Gly Ala Ile Ala Leu Ile Ile Gly Phe Gly Ile Ser

20 25 30

Gly Arg His Ser Ile Thr Val Thr Thr Val Ala Ser Ala Gly Asn Ile

35 40 45

Gly Glu Asp Gly Ile Leu Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu

50 55 60

Ser Asp Ile Val Ile Gln Trp Leu Lys Glu Gly Val Leu Gly Leu Val

65 70 75 80

His Glu Phe Lys Glu Gly Lys Asp Glu Leu Ser Glu Gln Asp Glu Met

85 90 95

Phe Arg Gly Arg Thr Ala Val Phe Ala Asp Gln Val Ile Val Gly Asn

100 105 110

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

115 120 125

Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn Ala Asn Leu Glu

130 135 140

Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Val Asn Val Asp Tyr Asn

145 150 155 160

Ala Ser Ser Glu Thr Leu Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln

165 170 175

Pro Thr Val Val Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser

180 185 190

Glu Val Ser Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met

195 200 205

Lys Val Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser

210 215 220

Cys Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val

225 230 235 240

Thr Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln Leu Leu Asn Ser

245 250 255

Lys Ala Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser His His His His

260 265 270

His His His His

275

<210> 101

<211> 20

<212> PROTEIN

<213> Homo sapiens

<400> 101

Thr Val Ala Ser Ala Gly Asn Ile Gly Glu Asp Gly Ile Leu Ser Cys

1 5 10 15

Thr Phe Glu Pro

20

<210> 102

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<221> OPTION

<222> (1)..(20)

<223> P12 antigen fragment variant

<400> 102

Thr Val Ala Ser Ala Gly Asn Ile Gly Glu Asp Gly Ala Leu Ser Cys

1 5 10 15

Thr Phe Glu Pro

20

<210> 103

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<221> OPTION

<222> (1)..(20)

<223> P12 antigen fragment variant

<400> 103

Thr Val Ala Ser Ala Gly Asn Ile Gly Glu Asp Gly Ile Ala Ser Cys

1 5 10 15

Thr Phe Glu Pro

20

<210> 104

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<221> OPTION

<222> (1)..(20)

<223> P12 antigen fragment variant

<400> 104

Thr Val Ala Ser Ala Gly Asn Ile Gly Glu Asp Gly Ile Leu Ser Ala

1 5 10 15

Thr Phe Glu Pro

20

<210> 105

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<221> OPTION

<222> (1)..(20)

<223> P12 antigen fragment variant

<400> 105

Thr Val Ala Ser Ala Gly Asn Ile Gly Glu Asp Gly Ile Leu Ser Cys

1 5 10 15

Ala Phe Glu Pro

20

<210> 106

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<221> OPTION

<222> (1)..(20)

<223> P12 antigen fragment variant

<400> 106

Thr Val Ala Ser Ala Gly Asn Ile Gly Glu Asp Gly Ile Leu Ser Cys

1 5 10 15

Thr Ala Glu Pro

20

<210> 107

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<221> OPTION

<222> (1)..(20)

<223> P12 antigen fragment variant

<400> 107

Thr Val Ala Ser Ala Gly Asn Ile Gly Glu Asp Gly Ile Leu Ser Cys

1 5 10 15

Thr Phe Ala Pro

20

<210> 108

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<221> OPTION

<222> (1)..(20)

<223> P12 antigen fragment variant

<400> 108

Thr Val Ala Ser Ala Gly Asn Ile Gly Glu Asp Gly Ile Leu Ser Cys

1 5 10 15

Thr Phe Glu Ala

20

<210> 109

<211> 7

<212> PROTEIN

<213> Homo sapiens

<400> 109

Ile Leu Ser Cys Thr Phe Glu

15

<210> 110

<211> 5

<212> PROTEIN

<213> Homo sapiens

<400> 110

Leu Ser Cys Thr Phe

15

<210> 111

<211> 8

<212> PROTEIN

<213> Homo sapiens

<400> 111

Ile Leu Ser Cys Thr Phe Glu Pro

15

<---

Claims (42)

1. An antibody to B7-H4 or its antigen-binding fragment, containing: an antibody light chain variable region and an antibody heavy chain variable region, where 1) the light chain variable region of said antibody contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; and the variable region of the heavy chain of this antibody contains HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively; 2) the light chain variable region of said antibody contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; and the variable region of the heavy chain of this antibody contains HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively; 3) the light chain variable region of said antibody contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; and the variable region of the heavy chain of this antibody contains HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively; 4) the light chain variable region of said antibody contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, respectively; and the variable region of the heavy chain of this antibody contains HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, respectively; 5) the light chain variable region of said antibody contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, respectively; and the variable region of the heavy chain of this antibody contains HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37, respectively; 6) the light chain variable region of said antibody contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, respectively; and the variable region of the heavy chain of this antibody contains HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO: 43, SEQ ID NO: 44 and SEQ ID NO: 45, respectively; 7) the light chain variable region of said antibody contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, respectively; and the variable region of the heavy chain of this antibody contains HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53, respectively; 8) the light chain variable region of said antibody contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64, respectively; and the variable region of the heavy chain of this antibody contains HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61, respectively; 9) the light chain variable region of said antibody contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, respectively; and the variable region of the heavy chain of this antibody contains HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69, respectively; or 10) the light chain variable region of said antibody contains LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; and the variable region of the heavy chain of this antibody contains HCDR1, HCDR2 and HCDR3, as shown in SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 21, respectively. 2. An anti-B7-H4 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a mouse antibody or fragment thereof, a chimeric antibody or fragment thereof, a human antibody or fragment thereof, and a humanized antibody or fragment thereof. fragment. 3. An anti-B7-H4 antibody or antigen-binding fragment thereof according to claim 2, wherein the humanized antibody heavy chain variable region contains human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region(s) or a variant thereof, preferably contains a constant( s) heavy chain region(s) of human IgG1, IgG2 or IgG4; more preferably contains IgG1 heavy chain constant region(s) that has undergone an amino acid mutation to enhance ADCC (antibody dependent cellular cytotoxicity) toxicity. 4. An anti-B7-H4 antibody or antigen-binding fragment thereof according to claim 2, wherein the light chain variable region of the humanized antibody is a light chain variable region comprising a sequence selected from the group consisting of: SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80 or SEQ ID NO: 82, preferably SEQ ID NO: 76 or SEQ ID NO: 80. 5. An anti-B7-H4 antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain variable region of the humanized antibody is a heavy chain variable region comprising a sequence selected from the group consisting of: SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79 or SEQ ID NO: 81, preferably SEQ ID NO: 75 or SEQ ID NO: 79. 6. An anti-B7-H4 antibody or antigen-binding fragment thereof according to claim 2, wherein the light chain of the humanized antibody is a light chain containing a sequence selected from the group consisting of: SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88 or SEQ ID NO: 90, preferably SEQ ID NO: 84 or SEQ ID NO: 88. 7. An anti-B7-H4 antibody or antigen-binding fragment thereof according to claim 2, wherein the heavy chain of the humanized antibody is a heavy chain containing a sequence selected from the group consisting of: SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 or SEQ ID NO: 89, preferably SEQ ID NO: 83 or SEQ ID NO: 87. 8. An anti-B7-H4 antibody or antigen-binding fragment thereof according to claim 2, wherein the humanized antibody contains: (1) a light chain variable region of SEQ ID NO: 76 and a heavy chain variable region of SEQ ID NO: 75; (2) a light chain variable region of SEQ ID NO: 78 and a heavy chain variable region of SEQ ID NO: 77; (3) a light chain variable region of SEQ ID NO: 80 and a heavy chain variable region of SEQ ID NO: 79; or (4) the light chain variable region of SEQ ID NO: 82 and the heavy chain variable region of SEQ ID NO: 81. 9. An anti-B7-H4 antibody or antigen-binding fragment thereof according to claim 2, wherein the humanized antibody contains: (1) a light chain of SEQ ID NO: 84 and a heavy chain of SEQ ID NO: 83; (2) a light chain of SEQ ID NO: 86 and a heavy chain of SEQ ID NO: 85; (3) a light chain of SEQ ID NO: 88 and a heavy chain of SEQ ID NO: 87; or (4) a light chain of SEQ ID NO: 90 and a heavy chain of SEQ ID NO: 89. 10. Antibody to B7-H4 or antigennegative fragment according to any one of paragraphs. 2-9, where the humanized antibody has the following characteristic: binding to an epitope containing amino acids 41-60 in SEQ ID NO: 100 B7-H4. 11. An anti-B7-H4 antibody or antigen-binding fragment thereof according to claim 10, wherein the humanized antibody has the following characteristic: binding to an epitope containing amino acids 53-59 in SEQ ID NO: 100 B7-H4. 12. A DNA molecule encoding an antibody or antigen-binding fragment according to any one of paragraphs. 1-11. 13. An expression vector containing a DNA molecule according to claim 12. 14. A host cell for expressing an anti-B7-H4 antibody or antigen-binding fragment thereof, into which or which contains the expression vector of claim 13. 15. A host cell according to claim 14, which is a bacterium, preferably Escherichia coli. 16. A host cell according to claim 14, which is a yeast, preferably Pichia pastoris. 17. The host cell of claim 14, which is a mammalian cell, preferably a Chinese hamster ovary (CHO) cell or a human embryonic kidney (HEK) 293 cell. 18. The method of obtaining antibodies, including culturing the host cell according to paragraphs. 14-17, isolation of the antibody from the culture and purification of the antibody. 19. Pharmaceutical composition for the treatment of B7-H4-mediated diseases or conditions, containing an antibody to B7-H4 or antigennegative fragment according to any one of paragraphs. 1-11 and a pharmaceutically acceptable excipient, diluent or carrier. 20. Reagent for detection, intended for immunodetection or determination of B7-H4, containing an antibody to B7-H4 or its antigen-binding fragment according to any one of paragraphs. 1-11. 21. Diagnostic tool for the diagnosis of B7-H4-mediated diseases or conditions, containing an antibody to B7-H4 or antigennegative fragment according to any one of paragraphs. 1-11. 22. The use of antibodies to B7-H4 or antigennegative fragment according to any one of paragraphs. 1-11 or a pharmaceutical composition according to claim 19 in the manufacture of a medicament for the treatment or prevention of a B7-H4 mediated disease or condition. 23. Use according to claim 22, where the disease is cancer; preferably the disease is a cancer expressing B7-H4; the cancer is most preferably selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer , gallbladder cancer, glioblastoma and melanoma.

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