HK40012331B - Anti-b7-h4 antibody, antigen-binding fragment thereof and pharmaceutical use thereof - Google Patents

Description

Anti-B7-H4 antibodies, their antigen-binding fragments, and their pharmaceutical uses

Technical Field

This invention relates to an anti-B7-H4 antibody that is immunoreactive to the human B7-H4 receptor, and its antigen-binding fragment, a chimeric antibody comprising the CDR region of the anti-B7-H4 antibody, a humanized antibody, and a pharmaceutical composition comprising the human anti-B7-H4 antibody and its antigen-binding fragment, and its use as an anticancer drug.

Background Technology

Tumor immunotherapy has been a long-standing and ongoing research and development hotspot in the field of cancer treatment, with T-cell tumor immunotherapy occupying a central position. Tumor escape is a major obstacle faced by tumor immunotherapy. While most tumor expression can be recognized by the host's immune system to varying degrees, in many cases, the inefficient activation of effector T cells leads to an insufficient immune response. Therefore, tumor cells utilize their own inhibitory effect on the immune system to promote tumor growth. Tumor immunotherapy fully utilizes and mobilizes cytotoxic T cells and/or other immune cells in cancer patients to kill tumor cells.

Studies of the CD28 receptor and its ligands have led to the characterization of related molecules known as the B7 superfamily. Members of the B7 family are a class of immunoglobulins possessing both an immunoglobulin V-like domain (IgV) and an immunoglobulin C-like domain (IgC). Members include costimulatory factors B7.1 (CD80) and B7.2 (CD86), ligands of inducible costimulatory factors (ICOS-L/B7-H2), programmed death-1 ligand (PD-L1/B7-H1), programmed death-2 ligand (PD-L2/B7-DC), B7-H4, and others.

Human B7-H4 is a type I transmembrane protein composed of 282 amino acids, and its encoding gene is located in the p111 region of chromosome 1 (Choi IH et al., J Immunol. 2003 Nov 1; 171(9): 4650-4). B7-H4 plays a negative regulatory role in T cell immune responses. B7-H4 exerts a wide range of inhibitory effects on the differentiation and development of CD4+ and CD8+ T cells, cell cycle progression, and cytokine production (Sica GL et al., Immunity. 2003 Jun; 18(6): 849-61). No immune cell dysregulation or autoimmune phenomena were observed in B7-H4 gene 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). The receptor and signal transduction pathway of B7-H4 are currently unclear.

Recent studies have found that the B7-H4 protein is highly expressed in various tumor tissues, enabling tumor cells to evade the body's immune system. Targeting the B7-H4 molecule as a tumor therapeutic target provides a novel approach for tumor immunotherapy.

Currently, human B7-H4 is known to be expressed in various cancer cells, including breast cancer, ovarian cancer, lung cancer, cervical cancer, kidney cancer, bladder cancer, and liver cancer. B7-H4 mRNA expression has been found in the spleen, lungs, thymus, liver, skeletal muscle, kidneys, pancreas, testes, and ovaries. At the protein level, low levels of B7-H4 expression are observed in tissues such as the breast (ducts and lobules), fallopian tube epithelium, and endometrial glands. Related studies have also shown that B7-H4 is overexpressed in tumor-associated macrophages (TAMs) (Kryczek, I. et al., J. Exp. Med. 2006, 203(4): 871-881), and macrophages constitute an important component of the tumor microenvironment and can represent up to 50% of tumor mass.

Several multinational pharmaceutical companies are currently developing monoclonal antibodies and/or drug-drug conjugates targeting B7-H4 to enhance the patient's immune system response to tumors and directly kill tumor cells. Related patents include WO2013025779 and US20140322129. Anti-B7-H4 monoclonal antibodies from companies like Medimmune and FivePrime are currently in preclinical development; Genentech's anti-B7-H4 antibody-drug conjugate is also in preclinical development.

This invention provides anti-B7-H4 antibodies with high affinity, high selectivity, and high biological activity for monoclonal antibody immunotherapy of tumors and related applications. It also includes pharmaceuticals, compositions, and methods for treating B7-H4-positive tumors.

Summary of the Invention

This invention provides a B7-H4 antibody or its antigen-binding fragment, comprising:

The antibody light chain variable region comprises at least one LCDR selected from the following sequences:

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

The antibody heavy chain variable region comprises at least one HCDR selected from the following sequences:

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 of the present invention, an anti-B7-H4 antibody or its antigen-binding fragment as described above, wherein the variable region of the antibody heavy chain comprises, respectively, as follows:

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

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

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

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

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

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

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

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

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

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

In a preferred embodiment of the present invention, an anti-B7-H4 antibody or its antigen-binding fragment as described above, wherein the variable region of the antibody light chain comprises, respectively, as follows:

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

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

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

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

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

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

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

LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 64

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

Particularly preferred anti-B7-H4 antibodies or their antigen-binding fragments may be selected from any of the following, comprising one or more CDR region sequences selected from the following or amino acid sequences having at least 95% sequence identity with them:

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

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

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

(4) The antibody light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32 respectively; the antibody heavy chain variable region includes 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 includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40 respectively; the antibody heavy chain variable region includes 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 includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48 respectively; the antibody heavy chain variable region includes 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 includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56 respectively; the antibody heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 51, SEQ ID NO: 52 and SEQ ID NO: 53 respectively.

(8) The antibody light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64 respectively; the antibody heavy chain variable region includes 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 includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72 respectively; the antibody heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 67, SEQ ID NO: 68 and SEQ ID NO: 69 respectively.

(10) The variable region of the antibody light chain includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 respectively; the variable region of the antibody heavy chain includes 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 present invention, a B7-H4 antibody or antigen-binding fragment thereof as described above is provided, wherein the antibody or antigen-binding fragment thereof is a murine antibody or fragment thereof.

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

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

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

In a preferred embodiment of the present invention, a B7-H4 antibody or an antigen-binding fragment thereof as described above, wherein the humanized antibody light chain variable region is selected from light chain variable regions containing the following sequences: SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80 or SEQ ID NO: 82.

In a preferred embodiment of the present invention, a B7-H4 antibody or an antigen-binding fragment thereof as described above, wherein the humanized antibody heavy chain variable region is selected from heavy chain variable regions containing the following sequences: SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79 or SEQ ID NO: 81.

In a preferred embodiment of the present invention, a B7-H4 antibody or its antigen-binding fragment as described above is provided, wherein the variable region of the humanized antibody heavy chain further comprises a heavy chain FR region of human IgG1, IgG2, IgG3 or IgG4 or its variants, preferably comprising a heavy chain FR region of human IgG1, IgG2 or IgG4; more preferably using IgG1 that enhances ADCC toxicity after amino acid mutation.

In a preferred embodiment of the present invention, a B7-H4 antibody or its antigen-binding fragment as described above, wherein the light chain of the humanized antibody is selected from light chains containing the following sequences: SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88 or SEQ ID NO: 90.

In a preferred embodiment of the present invention, a B7-H4 antibody or an antigen-binding fragment thereof as described above, wherein the heavy chain of the humanized antibody is selected from heavy chains containing the following sequences: SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87 or SEQ ID NO: 89.

In a preferred embodiment of the present invention, a B7-H4 antibody or its antigen-binding fragment as described above, wherein the humanized antibody light chain variable region is selected from a light chain variable region containing the following sequence: SEQ ID NO: 76 or SEQ ID NO: 80.

In a preferred embodiment of the present invention, a B7-H4 antibody or its antigen-binding fragment as described above, wherein the humanized antibody heavy chain variable region is selected from a heavy chain variable region containing the following sequence: SEQ ID NO: 75 or SEQ ID NO: 79

In a preferred embodiment of the present invention, a B7-H4 antibody or its antigen-binding fragment as described above, wherein the light chain of the humanized antibody is selected from light chains containing the following sequence: SEQ ID NO: 84 or SEQ ID NO: 88.

In a preferred embodiment of the present invention, a B7-H4 antibody or its antigen-binding fragment as described above, wherein the heavy chain of the humanized antibody is selected from heavy chains containing the following sequence: SEQ ID NO: 83 or SEQ ID NO: 87.

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

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

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

(3) The light chain variable region of SEQ ID NO: 80 and the 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 a further preferred embodiment of the present invention, the humanized antibody is selected from any of the following:

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

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

(3) The light chain of SEQ ID NO: 88 and the heavy chain of SEQ ID NO: 87;

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

An anti-B7-H4 antibody or its antigen-binding fragment, having at least one of the following characteristics: (1) binding to epitopes of B7-H4 containing amino acids 41-60 of SEQ ID NO: 92; (2) binding to epitopes of B7-H4 containing amino acids 53-59 of SEQ ID NO: 92.

An anti-B7-H4 antibody or its antigen-binding fragment, having at least one of the following characteristics: (1) binding to an epitope of B7-H4 containing amino acid 53 of SEQ ID NO: 92; (2) binding to an epitope of B7-H4 containing amino acid 54 of SEQ ID NO: 92; (3) binding to an epitope of B7-H4 containing amino acid 56 of SEQ ID NO: 92; (4) binding to an epitope of B7-H4 containing amino acid 57 of SEQ ID NO: 92; (5) binding to an epitope of B7-H4 containing amino acid 58 of SEQ ID NO: 92; (6) binding to an epitope of B7-H4 containing amino acid 59 of SEQ ID NO: 92.

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

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

The present invention further provides a host cell that is introduced or contains the expression vector described above.

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

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

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

The present invention also provides a method for producing B7-H4 antibody, comprising: culturing the host cells described above, isolating the antibody from the culture, and purifying the antibody.

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

The present invention also provides a single-chain antibody containing the light chain variable region and the heavy chain variable region as described above.

The present invention also provides a detection reagent or diagnostic reagent containing the B7-H4 antibody or its antigen-binding fragment as described above.

The present invention also provides a method for immunoassay or determination of B7-H4, the method comprising using the B7-H4 antibody or antigen-binding fragment thereof described herein.

The present invention also provides a method for diagnosing diseases associated with B7-H4 positive cells, the method comprising detecting or determining B7-H4 or B7-H4 positive cells using the B7-H4 antibody or its antigen-binding fragment described in this invention.

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

The present invention further provides the use of the anti-B7-H4 antibody or its antigen-binding fragment as described above in the preparation of a medicament for treating B7-H4-mediated diseases or conditions; wherein the disease is preferably cancer; more preferably cancer expressing B7-H4; and the most preferred cancers are breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach 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, the method comprising administering a therapeutically effective amount of an anti-B7-H4 antibody or its antigen-binding fragment, or the pharmaceutical composition thereof, to a desired patient, wherein the disease is preferably cancer; more preferably cancer expressing B7-H4; the most preferred cancers are breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, gallbladder cancer, cervical cancer, glioblastoma, and melanoma.

Attached Figure Description

Figure 1: In vitro ELISA binding assay of antibodies, showing that all seven chimeric antibodies have binding activity to purified human B7-H4 antigen, with the _EC50 of chimeric antibodies 2F7 and 2F8 being approximately 0.1 nM.

Figure 2: Indirect ELISA binding assay showing the antigenic epitope bound by anti-B7-H4 antibody hu2F7.

Figure 3: In vivo efficacy experiment in mice, showing the anti-tumor effect of anti-B7-H4 antibody hu1C9.

Figure 4: Immunological function experiment, showing the immune-enhancing (T cell proliferation) effect of anti-B7-H4 antibodies hu1C9 and hu2G6.

Detailed Implementation

Invention Details

I. Terminology

To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

The amino acid three-letter codes and single-letter codes used in this invention are as described in J. Biol. Chem, 243, p3558 (1968).

The term "antibody" used in this invention refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain differ, thus their antigenicity also differs. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, it can be further divided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ chains or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain.

In this invention, the variable region of the antibody light chain may further include a constant region of the light chain, wherein the constant region of the light chain includes human or mouse κ, λ chains or variants thereof.

In this invention, the antibody heavy chain variable region may further include a heavy chain constant region, wherein the heavy chain constant region comprises human or mouse IgG1, 2, 3, 4 or variants thereof.

The approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains exhibit significant sequence variation, forming the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, forming the constant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). The three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The number and position of CDR amino acid residues in the VL and VH regions of the antibody or antigen-binding fragment described in the invention conform to the known Kabat numbering rules and Kabat or AbM definition rules (http://bioinf.org.uk/abs/).

The term "antigen-presenting cell" or "APC" refers to a cell that displays foreign antigens complexed with MHC on its surface. T cells recognize this complex using T cell receptors (TCRs). Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes, B lymphoblasts, and monocyte-derived dendritic cells (DCs). The term "antigen presentation" refers to the process by which APCs capture antigens and make them recognizable by T cells, for example, as components of MHC-I/MHC-II conjugates.

The term "B7-H4" refers to a member of the human B7 protein family, also known as CD276, a type I transmembrane protein with 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 inhibits the functional activation of T cells. The term "B7-H4" includes any variant or isotype of B7-H4 naturally expressed by cells. The antibodies of the present invention are cross-reactive with B7-H4 derived from non-human species. Alternatively, the antibody may also be specific to human B7-H4 and may not exhibit cross-reactivity with other species. B7-H4 or any variant or isotype thereof may be isolated from cells or tissues that naturally express them, or generated via recombinant techniques using those techniques common in the art and those described herein. Preferably, the anti-B7-H4 antibody targets human B7-H4 with a normal glycosylation pattern.

The term "recombinant human antibody" includes human antibodies prepared, expressed, created, or isolated by recombinant methods, the techniques and methods of which are well known in the art, such as (1) antibodies isolated from transgenic, transchromosomal animals (e.g., mice) or hybridomas prepared therefrom of human immunoglobulin genes; (2) antibodies isolated from host cells transformed to express antibodies, such as transgenic tumors; (3) antibodies isolated from recombinant human antibody libraries; and (4) antibodies prepared, expressed, created, or isolated by methods such as splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations that occur during antibody maturation.

In this invention, the term "mouse antibody" refers to a monoclonal antibody against human B7-H4 prepared according to the knowledge and skills in the art. Preparation involves injecting the test subject with the B7-H4 antigen, followed by isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics. In a preferred embodiment of the invention, the mouse B7-H4 antibody or its antigen-binding fragment may further comprise a light chain constant region of a mouse κ, λ chain, or a variant thereof, or further comprise a heavy chain constant region of mouse IgG1, IgG2, IgG3, or IgG4, or a variant thereof.

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

The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting a mouse CDR sequence into the variable region framework of a human antibody. This overcomes the strong immune response induced by chimeric antibodies that carry a large amount of mouse protein components. To avoid a decrease in activity along with decreased immunogenicity, minimal reverse mutations can be performed on the variable region of the human antibody to maintain its activity.

The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. It can mitigate the immune response induced by murine antibodies. To create a chimeric antibody, a hybridoma that secretes murine-specific monoclonal antibodies is selected. The variable region gene is then cloned from mouse hybridoma cells, followed by the desired human antibody constant region gene. The mouse variable region gene and the human constant region gene are linked to form a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system. The constant region of the human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or their variants, preferably containing the heavy chain constant region of human IgG2 or IgG4. Alternatively, IgG1 with amino acid mutations that enhance ADCC (antibody-dependent cell-mediated cytotoxicity) can be used.

The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and antibody analogues, which typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. The antibody fragment retains at least some of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parent antibody's binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parent antibody's binding affinity 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.

A "Fab fragment" consists of a light chain, a heavy chain (CH1), and a variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

The “Fc” region contains two heavy chain segments, including the CH1 and CH2 domains of the antibody. The two heavy chain segments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain.

The “Fab’ fragment” contains a light chain and a heavy chain portion containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, thereby enabling the formation of interchain disulfide bonds between the two heavy chains of two Fab’ fragments to form the F(ab’)2 molecule.

The “F(ab’)2 segment” contains two light chains and two heavy chains containing portions of a constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab’)2 segment consists of two Fab’ segments held together by disulfide bonds between the two heavy chains.

The “Fv region” contains variable regions from both the heavy and light chains, but lacks constant regions.

The term "multispecific antibody," used in its broadest sense, encompasses antibodies that exhibit 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 exhibits multi-epitope specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments covalently or non-covalently linked together, etc.

The term "single-chain antibody" refers to a single-chain recombinant protein composed of the heavy chain variable region (VH) and light chain variable region (VL) of an antibody linked by a linker peptide. It is the smallest antibody fragment with a complete antigen-binding site.

The term "domain antibody fragment" refers to an immunoglobulin fragment with immunological function containing only the heavy chain variable region or the light chain variable region. In some cases, two or more VH regions are covalently linked to peptide linkers to form a bivalent domain antibody fragment. The two VH regions of a bivalent domain antibody fragment can target the same or different antigens.

The term "binding to B7-H4" in this invention refers to a site capable of interacting with human B7-H4. The term "antigen binding site" in this invention refers to a discontinuous three-dimensional spatial site on the antigen that is recognized by the antibody or antigen-binding fragment of this invention.

The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed from adjacent amino acids or from non-adjacent amino acids arranged side-by-side through the ternary folding of a protein. Epitopes formed from adjacent amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3-15 amino acids in a distinctive spatial conformation. Methods for determining which epitopes bind to a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

As used herein, the terms "specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, when recombinant human B7-H4 is used as the analyte and an antibody as the ligand, and the assay is performed in an instrument using surface plasmon resonance (SPR) technology, the antibody binds to the predetermined antigen with an equilibrium dissociation constant (K_{_D}) of approximately less than 10 ^{^-7} M or even smaller, and its affinity for the predetermined antigen is at least twice that for nonspecific antigens other than the predetermined antigen or closely related antigens (such as BSA). The term "antibody that recognizes an antigen" may be used interchangeably with the term "antibody that specifically binds."

The term "cross-reactivity" refers to the ability of the antibodies of this invention to bind to B7-H4 from different species. For example, the antibodies of this invention that bind to human B7-H4 can also bind to B7-H4 from another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays (e.g., SPR and ELISA), or 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” and “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of a ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable reduction in ligand binding affinity upon contact with an anti-B7-H4 antibody compared to ligands not contacted with an anti-B7-H4 antibody.

The term “growth inhibition” (e.g., involving cells) is intended to include any measurable reduction in cell growth.

The terms “inducing an immune response” and “enhancing an immune response” are used interchangeably and refer to the stimulation of an immune response by a specific antigen (i.e., passive or adaptive). The term “inducing” in relation to induced CDC or ADCC refers to the stimulation of a specific direct cell-killing mechanism.

The "ADCC" mentioned in this invention, which stands for antibody-dependent cell-mediated cytotoxicity, refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors through recognition of the Fc fragment of antibodies. The ADCC effector function of antibodies can be enhanced, reduced, or eliminated by modifying the Fc fragment of IgG. This modification refers to mutation in the constant region of the antibody's heavy chain.

Methods for producing and purifying antibodies and antigen-binding fragments are well-known and readily available in the prior art, such as in Cold Spring Harbor's Guide to Antibody Experimentation, Chapters 5-8 and 15. For example, mice can be immunized with human B7-H4 or fragments thereof, and the resulting antibodies can be renatured, purified, and sequenced amino acids using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in this invention utilize genetic engineering methods to add one or more human FR regions to non-human CDR regions. Human FR germline sequences are available from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr, or from the journal Immunoglobulins, 2001 ISBN012441351.

The engineered antibodies or antigen-binding fragments of this invention can be prepared and purified using conventional methods. The corresponding antibody cDNA sequence can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminus of the FC region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. The culture medium secreting antibodies can be purified and collected using conventional techniques. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

The antibody of this invention refers to a monoclonal antibody. The monoclonal antibody (mAb) described in this invention refers to an antibody obtained from a single clonal cell line, and the cell line is not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using techniques such as hybridoma technology, recombinant technology, phage display technology, synthetic technology (such as CDR-grafting), or other existing technologies.

When applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, "giving" and "treatment" refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Giving" and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Giving" and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. When applied to humans, veterinary, or research subjects, "treatment" refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

"Treatment" means administering an oral or topical therapeutic agent, such as a composition comprising any of the compounds of the present invention, to a patient who has one or more symptoms of a disease, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more symptoms of a disease, whether by inducing the regression of such symptoms or inhibiting their progression to any clinically measured extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical testing method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although embodiments of the present invention (e.g., treatment methods or articles) may be ineffective in alleviating the symptoms of the target disease present in every patient, they should alleviate the symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

The term "consistently composed of" or variations thereof, used throughout the specification and claims, means that all said elements or groups of elements are included, and optionally other elements that are similar to or different in nature from said elements, which do not significantly alter or introduce new properties to the specified dosing regimen, method, or composition. As a non-limiting example, a binding compound consisting essentially of the mentioned 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" as used in this invention refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in organisms (including viruses) that can be isolated from natural sources and has not been intentionally modified artificially in a laboratory is considered naturally occurring.

An "effective dose" includes a dose sufficient to improve or prevent the symptoms or condition of a medical condition. An effective dose also means a dose sufficient to allow or facilitate diagnosis. The effective dose for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective dose can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

"Exogenous" refers to substances produced outside an organism, cell, or human body, depending on the context. "Endogenous" refers to substances produced within a cell, organism, or human body, depending on the context.

"Homology" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. Two compared sequences are homologous at positions occupied by the same bases or amino acid monomer subunits; for example, if every position in two DNA molecules is occupied by adenine. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, sequences are compared when the highest percentage of homology is obtained.

The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include their progeny. Therefore, the words “transformation” and “transformed cell” include primary test cells and cultures derived from them, regardless of the number of transfections. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in terms of DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells. Where different names are used, the context will be clear.

"Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur. For example, "optionally contains 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may, but do not have to, be present.

"Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically/pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically/pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

The following examples are used to further describe the present invention, but these examples are not intended to limit the scope of the invention. Experimental methods in the embodiments of the present invention that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents that do not specify a specific source are commercially available, conventional reagents.

Example 1: Antigen preparation and construction of stable cell lines

The human B7-H4 (huB7-H4-HF) sequence encoding the HisFlag tag and the human B7-H4 (h-B7-H4-Fc) sequence encoding the huFc tag were synthesized by Integrated DNA Technology (IDT), a CRO company (all B7-H4 recombinant proteins were designed using the template sequences of this invention) and cloned into the pTT5 vector (Biovector). After expression in 293T cells, the recombinant B7-H4 protein was purified according to Example 2.

The purified protein can be used in the experiments described in the following examples.

huB7-H4-Fc sequence:

huB7-H4-his sequence:

purification steps for huB7-H4-his:

Cell expression supernatant samples were centrifuged at high speed to remove impurities, and the buffer was replaced with PBS. Imidazole was added to a final concentration of 5 mM. The nickel column was equilibrated with PBS containing 5 mM imidazole and washed 2-5 column volumes. The supernatant sample was then loaded onto the column. The column was washed with PBS containing 5 mM imidazole until the _A280 reading dropped to baseline. The column was then washed with PBS + 10 mM imidazole to remove non-specifically bound proteins, and the eluent was collected. The target protein was then eluted with PBS containing 300 mM imidazole, and the elution peaks were collected. The collected eluent was further purified using an ion exchange column (SP column). Solution A: 0.01 M PB, pH 8.0. Solution B: Solution A + 1 M NaCl. The target protein was first eluted with the imidazole-containing PBS solution and then replaced with Solution A. The SP column was equilibrated with Solution A, and the sample was loaded. Solution B was used in a concentration gradient of 0-100%, and eluted 10 column volumes. Each elution peak was collected. The obtained protein was identified by electrophoresis and then dispensed for use. Human B7-H4 tagged with HisFlag (hu-B7-H4 his) was obtained. huB7-H4-Fc purification steps:

The supernatant sample from HEK293 cells was centrifuged at high speed to remove impurities, and the buffer was replaced with PBS. The Protein A affinity column was equilibrated with 10 mM phosphate buffer and washed 2-5 column volumes. The replaced supernatant sample was loaded onto the column. The column was washed with 25 column volumes of buffer until the A ₂₈₀ reading dropped to baseline. The target protein was then eluted with 0.8% acetate buffer at pH 3.5, and the elution peak was collected. After aliquoting, the protein was immediately neutralized with 1 M Tris-Cl pH 8.0 buffer, and then replaced with PBS using a Millipore's Amico-15 filter. The obtained protein was identified by electrophoresis, peptide mapping, and LC-MS before being aliquoted and used.

Construction of a stable CHO-S cell pool:

The full-length sequences encoding human or cynomolgus monkey B7-H4 proteins (huB7-H4 or cyB7-H4) were synthesized by Integrated DNA Technology (IDT) (all B7-H3 recombinant proteins were designed using template sequences from this invention) and cloned into the modified pcDNA3.1 vector, pcDNA3.1/puro (Invitrogen #V79020). CHO-S (ATCC) cells were cultured in CD-CHO medium (Life Technologies, #10743029) to a density of 0.5 x ^10⁶ /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, and then added to the CHO cell culture medium and incubated in a CO₂ incubator. After 24 hours, the medium was replaced with fresh medium and 10 μg/ml puromycin was added. The culture medium was then replaced every 2-3 days, and after 10-12 days of screening, a stable CHO-S cell pool was obtained.

Example 2: Obtaining mouse hybridoma and antibody sequences

Animals were immunized with human antigen huB7-H4-Fc. Five Balb/c mice and five A/J mice, female and 10 weeks old, were used. The immunogen and immune adjuvant were thoroughly mixed and emulsified in a 1:1 ratio to prepare a stable water-in-oil liquid. The injection dose was 25 μg/200 μL/mouse.

Day 1 First immunization, with Freund's adjuvant. Day 21 Second immunization, incomplete Freund's adjuvant. Day 35 The third immunization was administered with an incomplete Freund's adjuvant. Day 42 Blood collection and serum titer testing (3-immunoassay) Day 49 Fourth immunization, with incomplete Freund's adjuvant. Day 56 Blood collection and serum titer testing (4 immune blood tests)

Serum from immunized mice was assessed for titer and binding capacity to cell surface antigens using indirect ELISA and capture ELISA methods as described in Example 3. Cell fusion was initiated based on titer (greater than 100,000-fold dilution). Mice with high serum titer, affinity, and FACS binding were selected for a final immunization, followed by sacrifice. Spleen cells were fused with SP2/0 myeloma cells and plated to obtain hybridomas. Target hybridomas were screened using indirect ELISA and capture ELISA, and monoclonal cell lines were established using limiting dilution. Positive antibody strains were further screened using CHO-S cells stably expressing B7-H4, compared with blank CHO-S cells to exclude non-specific antibody-binding hybridoma strains. Flow cytometry was used to select 8 hybridomas that bound both recombinant proteins and cell-expressed antigens. Hybridoma cells in logarithmic growth phase were collected, and RNA was extracted using Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript ^™ Reverse Transcriptase, Takara#2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326Rev.B 0503) and then sequenced to obtain the sequences of 8 mouse antibodies.

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

It contains the following CDR sequences:

name sequence serial number HCDR1 GFTFSNYYMS SEQ ID NO: 3 HCDR2 YVSSGGGSTYYSDSVKG SEQ ID NO: 4 HCDR3 ESYSQGNYFDY SEQ ID NO: 5 LCDR1 RASQSISDYLH SEQ ID NO: 6 LCDR2 FASQSIS SEQ ID NO: 7 LCDR3 QNGHSFSLT SEQ ID NO: 8

The heavy and light chain variable region sequences of M1 are as follows:

It contains the following CDR sequences:

name sequence serial number HCDR1 GYPFTTYNMY SEQ ID NO: 11 HCDR2 YIDPYNGGTSYNQKFKG SEQ ID NO: 12 HCDR3 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 mouse monoclonal antibody 2F8 are as follows:

It contains the following CDR sequences:

name sequence serial number HCDR1 GYTFTNSWMN SEQ ID NO: 19 HCDR2 GIYPNSGNIEYNEKFKG SEQ ID NO: 20 HCDR3 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 mouse monoclonal antibody 2F4 are as follows:

It contains the following CDR sequences:

name sequence serial number HCDR1 GLTFSRYAMS SEQ ID NO: 27 HCDR2 GISSGGSYTYYSDTVKG SEQ ID NO: 28 HCDR3 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 mouse monoclonal antibody 2A10 are as follows:

It contains the following CDR sequences:

name sequence serial number HCDR1 GFTFSTFGMS SEQ ID NO: 35 HCDR2 GISPGGSYTYYPDTVKG SEQ ID NO: 36 HCDR3 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 mouse monoclonal antibody 2E4 are as follows:

It contains the following CDR sequences:

name sequence serial number HCDR1 GYTFTSSWMN SEQ ID NO: 43 HCDR2 GIYPNRGTTEYNEKFKG SEQ ID NO: 44 HCDR3 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 monoclonal antibody 1E4 are as follows:

It contains the following CDR sequences:

name sequence serial number HCDR1 GFTFSRYAMS SEQ ID NO: 51 HCDR2 GISSGGSYTYYPDTLKG SEQ ID NO: 52 HCDR3 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 mouse monoclonal antibody 2G6 are as follows:

It contains the following CDR sequences:

name sequence serial number HCDR1 GFTFSRYGMS SEQ ID NO: 59 HCDR2 GINGGGSYTYYLDTVKG SEQ ID NO: 60 HCDR3 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 mouse monoclonal antibody 1C9 are as follows:

                                                       

It contains the following CDR sequences:

name sequence serial number HCDR1 GDTFTTY SEQ ID NO: 67 HCDR2 YLNSGS SEQ ID NO: 68 HCDR3 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 antibody were cloned into the constant regions of the human IgG1 heavy chain and κ light chain, respectively, and purified, identified, and subjected to relevant activity tests as described in Example 4.

Example 3: Method for detecting the in vitro binding activity of antibodies

(1) In vitro indirect ELISA binding assay:

Dilute huB7-H4His protein (Sino Biological Inc., cat#10738-H08H) to 1 μg/ml using PBS (pH 7.4), and add 100 μl/well to each well of a 96-well high-affinity microplate. Incubate overnight (16-20 hours) at 4°C. Wash the plate four times with PBST (pH 7.4 PBS containing 0.05% Tween-20), then add 150 μl/well of 3% bovine serum albumin (BSA) blocking buffer diluted with PBST and incubate at room temperature for 1 hour. After blocking, discard the blocking buffer and wash the plate four times with PBST buffer.

Dilute the antibody to be tested with PBST containing 3% BSA, starting at 1 μM, in a 10-fold gradient, 10 doses, and add 100 μl/well to the microplate. Incubate at room temperature for 1 hour. After incubation, wash the plate 4 times with PBST, add 100 μl/well of HRP-labeled goat anti-human secondary antibody (Abcam, cat#ab97225) diluted with PBST containing 3% BSA, and incubate at room temperature for 1 hour. After washing the plate 4 times with PBST, add 100 μl/well of TMB chromogenic substrate (Cell Signaling Technology, cat#7004S), incubate at room temperature in the dark for 1 minute, and stop the reaction by adding 100 μl/well of Stop Solution (Cell Signaling Technology, cat#7002S). Read the absorbance at 450 nm using a microplate reader (BioTek, model Synergy H1) and analyze the data. The results of the concentration signal curve analysis are shown in the table below:

(2) Competitive ELISA assay:

Dilute huB7-H4His protein (Sino Biological Inc., cat#10738-H08H) to 1 μg/ml using PBS (pH 7.4). Add 100 μl/well to each well of a 96-well high-affinity microplate and incubate overnight (16-20 hours) at 4°C. Wash the plate four times with PBST (pH 7.4 PBS containing 0.05% Tween-20). Then, add 150 μl/well of 3% bovine serum albumin (BSA) blocking buffer diluted with PBST and incubate at room temperature for 1 hour to block. After blocking, discard the blocking buffer and wash the plate four times with PBST buffer.

Prepare a 0.1 nM reference chimeric antibody using PBST containing 3% BSA. Use this antibody as a diluent to dilute the mouse antibody to be tested to 100 nM as a starting point, in a 10-fold gradient, for 10 doses. Add 100 μL of the diluted antibody to each well of the ELISA plate and incubate at room temperature for 1 hour. After incubation, wash the plate four times with PBST, add 100 μL of HRP-labeled goat anti-human secondary antibody (Abcam, cat#ab97225) diluted in PBST containing 3% BSA to each well, and incubate at room temperature for 1 hour. After washing the plate four times with PBST, add 100 μl/well of TMB chromogenic substrate (Cell Signaling Technology, cat#7004S), incubate at room temperature in the dark for 1 minute, and then add 100 μl/well of Stop Solution (Cell Signaling Technology, cat#7002S) to terminate the reaction. Read the absorbance at 450 nm using a BioTek Synergy H1 microplate reader and analyze the data. Competitive inhibition rate = ((Reference antibody absorbance - Competitive antibody absorbance) / Abv antibody absorbance) * 100.

(3) In vitro capture ELISA binding assay:

Dilute goat anti-mouse IgG secondary antibody (Jackson Immuno Research, CAT#115-006-071) to a concentration of 2 μg/ml using PBS buffer (pH 7.4), and add 100 μl/well to each well of a 96-well microplate. Incubate at 37°C for 2 hours. After washing once with PBST, add 200 μl/well of blocking buffer (5% skim milk, Bright Dairy skim milk powder) diluted with PBST. Incubate at 37°C for 2 hours or overnight at 4°C (16-18 hours) for blocking. After blocking, discard the blocking buffer and wash the plate four times with PBST.

Dilute the mouse serum or purified recombinant antibody to different concentrations using sample diluent (PBST of 2.5% skim milk) containing 5% NHS. After incubating at room temperature for 40 minutes, add 100 μL/well to each well of the plate and incubate at 37°C for 40 minutes. After incubation, wash the plate four times with PBST, add 100 μL/well of biotinylated huB7-H4-his (Sino Biotech #10738-H08H) protein solution diluted with sample diluent, and incubate at 37°C for 40 minutes. After incubation, wash the plate four times with PBST, add 100 μL/well of HRP-labeled streptavidin (Jackson Immuno Research, CAT #016-030-084) diluted with PBST, and incubate at 37°C for 40 minutes. After washing the plate four times with PBST, add 100 μl/well of TMB chromogenic substrate (Huzhou Yingchuang Biotechnology Co., Ltd.), incubate at room temperature in the dark for 10-15 min, and stop the reaction by adding 50 μl/well of 1M _{H2SO4 .} Read the absorbance at 450 nm using a microplate reader (Beijing Pulang New Technology Co., Ltd., model DNM-9602) and analyze the data.

(4) In vitro cell binding experiment:

Collect well-cultured, stably transfected CHO-huB7-H4 cells or SK-BR3 cells, adjust cell density, and seed them into 96-well U-plates at 1–2 x 10⁵ cells per well. Centrifuge at 1200 g for 5 min, discard the supernatant, add 100 μL of serially diluted antibody solution or mouse immune serum, and incubate at 4°C for 60 min. Centrifuge at 1200 g for 5 min, discard the supernatant, wash cells twice with PBS, add 100 μL of fluorescently labeled secondary antibody (PE-GAM or PE-GAH) per well, and incubate at 4°C for 60 min. Centrifuge at 1200 g for 5 min, discard the supernatant. Wash cells twice with PBS, resuspend in PBS, and detect signal using a flow cytometer, then analyze the concentration curve results.

Example 4: Construction and expression of anti-B7-H4 recombinant chimeric antibody

The variable regions (VH) and (VL) of the heavy chain and light chain of each mouse antibody in this invention are subjected to site-directed amino acid mutations in the FR (framework region). Based on different combinations of amino acid mutations, different humanized antibody heavy and light chains are designed. Transfecting cells with plasmids containing different combinations of light and heavy chains can produce humanized antibodies.

The heavy chain vector is designed as follows: signal peptide + mutated heavy chain variable region sequence + human IgG1 constant region sequence.

The light chain vector is designed as follows: signal peptide + mutated light chain variable region sequence + human Kappa constant region sequence.

The above sequences were inserted into the pCEP4 vector. Expression vectors were synthesized according to the above design. After obtaining the vector plasmids, the plasmids were extracted and sent for sequencing verification. The verified plasmids were transfected into human 293F cells using PEI and cultured continuously. 293F cells were cultured in serum-free medium (Shanghai OPMI Biotechnology, OPM-293CD03) to the 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 IREduced Serum Medium (GIBCO, 31985-070), mixed well, and then 200 μg of PEI was added. The mixture was incubated at RT for 15 min and then added to 50 mL of cells. Cell culture conditions: 5% _CO2 , 37℃, 125 rpm/min. During culture, feed was added on days 1 and 3 until cell viability was below 70%. The cell supernatant was collected, centrifuged, and filtered. The centrifuged and filtered cell culture medium was loaded onto an antibody purification affinity column, washed with phosphate buffer, eluted with glycine hydrochloride buffer (pH 2.70, 1M Gly-HCl), neutralized with 1M Tris hydrochloride at pH 9.0, and dialyzed with phosphate buffer to finally obtain the purified chimeric antibodies.

Example 5: In vitro binding affinity and kinetics experiments:

The Biacore method is a recognized objective method for detecting the affinity and binding kinetics between proteins. The affinity and binding kinetics of the B7-H4 antibody to be tested in this invention were characterized using a Biacore T200 (GE) analyzer.

Using a kit provided by Biacore, the anti-B7-H4 antibody of this invention was covalently linked to the CM5(GE) chip using the NHS standard amino-coupled method. Then:

a) Inject 50 nM human huB7-H4-his protein (Sinochem #10738-H08H) diluted in the same buffer at a flow rate of 10 μL/min. After injection, regenerate the protein using the regeneration reagent provided in the kit. Track antigen-antibody binding kinetics for 3 minutes and dissociation kinetics for 10 minutes. Analyze the data using GE's BIAevaluation software with a 1:1 (Langmuir) binding model. The estimated single-point kd (koff) data for each murine antibody using this method are shown in the table below.

b) Human huB7-H4-his protein diluted in the same buffer at a series of concentration gradients was injected in each subsequent cycle at a flow rate of 10 μL/min. After each injection, the protein was regenerated using the regeneration reagent provided in the kit. Antigen-antibody binding kinetics were monitored for 3 minutes, and dissociation kinetics for 10 minutes. The data were analyzed using GE's BIAevaluation software with a 1:1 (Langmuir) binding model. The chimeric antibody ka (kon), kd (koff), and _KD values determined by this method are shown in the table below.

Example 6: Mouse Antibody Humanization Experiment

The humanization of murine anti-human B7-H4 monoclonal antibodies is performed using methods disclosed in many publications in the art. In short, by replacing the parental (murine antibody) constant domain with a human constant domain, and selecting human antibody sequences based on the homology between the murine and human antibodies, this invention humanizes murine candidate molecules 2F7, 2F8, 2G6, and 1C9.

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

The CDR regions of murine antibodies 2F7, 2F8, 2G6, and 1C9 were transplanted into their respective humanized templates. Specifically, the HCDR1 region (GYTFTNSWMN, SEQ ID NO: 19) and HCDR2 region (GIYPNSGNIEYNEKFKG, SEQ ID NO: 20) of 2F8 were mutated to GYTFTSSWMN (SEQ ID NO: 73) and GIYPNRGNIEYNEKFKG (SEQ ID NO: 74), respectively, to remove potential deamidation unstable sites. The humanized variable regions were replaced and then 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 murine antibodies, reverse mutations were performed on embedded residues, residues that directly interact with the CDR regions, and residues that significantly affect the conformation of VL and VH. The chemically unstable amino acid residues in the CDR regions were optimized. Antibodies composed of humanized light and heavy chain variable region sequences were designed and tested.

Based on expression testing and comparison of the number of reverse mutations, the final humanized hu2F7, hu2F8, hu2G6 and hu1C9 antibody molecules were selected. Their respective heavy chain and light chain variable region sequences are shown in SEQ ID NO: 75-82, and their respective heavy chain and light chain sequences are shown in SEQ ID NO: 83-90.

Based on the amino acid sequences of the light and heavy chains of the humanized antibodies described above, cDNA fragments were synthesized and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). HEK293 cells (Life Technologies Cat. No. 11625019) were transfected with the expression vector and transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) at a 1:2 ratio and incubated in a _CO2 incubator for 4-5 days. The expressed antibodies were recovered by centrifugation and purified according to the method in Example 4 to obtain the humanized antibody proteins hu2F7 and hu2F8 of this invention.

Example 7: Assay of Humanized Antibody Activity

The following experiments were performed in vitro to determine the humanized antibodies against hu2F7 and hu2F8:

1. In vitro cell binding experiment:

Cultured MX-1 cells were collected and, after adjusting cell density with PBS at pH 7.4, were seeded into 96-well V-plates at 1 x 10⁵ cells per well. Centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, and 100 μl of serially diluted chimeric antibody solution (diluted in PBS containing 0.5% BSA, starting at 1 μM, 3-fold gradient, 10 doses) was added to each well. The mixture was incubated at 4°C with shaking for 1 hour. Centrifuged again at 2000 rpm for 5 minutes, the supernatant was discarded, and the cells were washed twice with PBS. Then, 100 μl of FITC-labeled goat anti-human secondary antibody (Abcam, cat#ab97224) diluted in PBS containing 0.5% BSA was added to each well, the mixture was incubated at 4°C with shaking for 30 minutes, and the cells were centrifuged again at 2000 rpm for 5 minutes, the supernatant was discarded, and the cells were washed twice with PBS and resuspended in PBS. The signal was detected using a flow cytometer (BECKMAN COULTER, model DxFLEX), and the results were analyzed using a concentration curve. The results are shown in the figure. Humanized antibodies 2F7, 2F8, 2G6 and 1C9 all bound positively to MX-1 cells that highly expressed B7-H4.

Antibody name <![CDATA[FACS Binding EC₅₀(nM)<!-- 24 -->]]> hu2F7 7.32

Antibody name <![CDATA[FACS Binding EC₅₀(nM) with MX-1 cells]]> hu2F8 7.26

Antibody name <![CDATA[FACS Binding EC₅₀(nM) with MX-1 cells]]> hu2G6 14.3

Antibody name <![CDATA[FACS Binding EC₅₀(nM) with MX-1 cells]]> hu1C9 7.25

2. Affinity kinetics experiment (method and steps are the same as in Example 5), the results are shown in the table below. Humanized antibodies hu2F7, hu2F8, hu2G6 and hu1C9 all showed strong affinity for human B7-H4 antigen protein.

Example 8: Assay of humanized antibody binding epitopes

The method and steps were the same as in 3(1) in vitro indirect ELISA binding experiment. B7-H4 of SEQ ID NO: 92 was decomposed into an antigen fragment of 20 amino acids in length. In vitro indirect ELISA binding experiment revealed that antigen fragment P12 (amino acid sequence: TVASAGNIGEDGILSCTFEP) specifically bound to humanized anti-B7-H4 antibody hu2F7, as shown in Figure 2. To further confirm the antibody binding epitope, an alanine scan was performed on antigen fragment P12, i.e., each individual amino acid in P12 was mutated to alanine. In vitro indirect ELISA binding experiment revealed that the mutation in the amino acid sequence: ILSCTFE significantly weakened the binding between the antibody and the antigen fragment, indicating that the antibody binding epitope was located in the amino acid sequence: TVASAGNIGEDGILSCTFEP, specifically in the amino acid sequence: ILSCTFE (SEQ ID NO: 109). The results are shown in the table below:

In vitro indirect ELISA binding assays revealed that the antigen fragment P12 (amino acid sequence: TVASAGNIGEDGILSCTFEP) specifically binds to the humanized anti-B7-H4 antibody hu1C9, as shown in the table below. To further confirm the antibody binding epitope, an alanine scan was performed on the antigen fragment P12, involving mutation of individual amino acids in P12 to alanine. In vitro indirect ELISA binding assays showed that mutations in the amino acid sequence: LSCTF significantly weakened the binding between the antibody and the antigen fragment, indicating that the antibody binding epitope is located in the amino acid sequence: LSCTF (SEQ ID NO: 110) of the amino acid sequence: TVASAGNIGEDGILSCTFEP. The results are shown in the table below.

antigen fragment sequence SEQ ID NO: Affinity EC50 (nM) TVASAGNIGEDGILSCTFEP 101 271.7 TVASAGNIGEDGIASCTFEP 103 466.9 TVASAGNIGEDGILSATFEP 104 292.3 TVASAGNIGEDGILSCTAEP 106 487.9

In vitro indirect ELISA binding assays revealed that the antigen fragment P12 (amino acid sequence: TVASAGNIGEDGILSCTFEP) specifically binds to the humanized anti-B7-H4 antibody hu2G6, as shown in the table below. To further confirm the antibody binding epitope, an alanine scan was performed on the antigen fragment P12, involving mutation of individual amino acids in P12 to alanine. In vitro indirect ELISA binding assays showed that mutations in the amino acid sequence:ILSCTFEP significantly weakened the binding between the antibody and the antigen fragment, indicating that the antibody binding epitope is located in the amino acid sequence:ILSCTFEP (SEQ ID NO: 111) of the amino acid sequence: TVASAGNIGEDGILSCTFEP. The results are shown in the table below.

antigen fragment sequence SEQ ID 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: Anti-tumor effect of humanized antibodies

MC38 tumor cells were implanted into mice with the human B7-H4 gene introduced via gene editing technology. Once the tumors grew to an average size of 100 ^mm³ , the mice were injected every 3 days with either the control or humanized B7-H4 antibody hu1C9 (10 mg/kg or 30 mg/kg). Observation of tumor size revealed that hu1C9 significantly inhibited tumor growth and exhibited anti-tumor effects. Specific results are shown in Figure 3 and the table below.

Example 10: The effect of humanized antibodies on enhancing immunity

The anti-tumor effect of anti-B7-H4 may be mediated by enhancing tumor immunity. To verify this mechanism, CD4-positive T cells were extracted from human peripheral blood, and different concentrations of anti-B7-H4 antibodies, including humanized antibodies hu2G6 and hu1C9, were added to the cultured T cells. The specific results are shown in Figure 4 and the table below. Humanized antibodies hu2G6 and hu1C9 can increase T cell proliferation, thus achieving an anti-tumor effect by enhancing tumor immunity.

Sequence List

<110> Jiangsu Hansoh Pharmaceutical Group Co., Ltd.

Shanghai Hansoh Biomedical Technology Co., Ltd.

Changzhou Hengbang Pharmaceutical Co., Ltd.

<120> Anti-B7-H4 antibody, its antigen-binding fragment and its pharmaceutical uses

<130> 719011CPCT

<160>  111

<170> SIPOSequenceListing 1.0

<210>  1

<211> 120

<212> PRT

<213> Mouse (Mus musculus)

<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 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> PRT

<213> Mouse (Mus musculus)

<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> PRT

<213> Mouse (Mus musculus)

<400> 3

Gly Phe Thr Phe Ser Asn Tyr Tyr Met Ser

1 5 10

<210> 4

<211> 17

<212> PRT

<213> Mouse (Mus musculus)

<400>  4

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

1 5 10 15

Gly

<210> 5

<211>  11

<212> PRT

<213> Mouse (Mus musculus)

<400> 5

Glu Ser Tyr Ser Gln Gly Asn Tyr Phe Asp Tyr

1 5 10

<210> 6

<211> 11

<212> PRT

<213> Mouse (Mus musculus)

<400> 6

Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His

1 5 10

<210> 7

<211> 7

<212> PRT

<213> Mouse (Mus musculus)

<400> 7

Phe Ala Ser Gln Ser Ile Ser

1 5

<210> 8

<211>  9

<212> PRT

<213> Mouse (Mus musculus)

<400> 8

Gln Asn Gly His Ser Phe Ser Leu Thr

1 5

<210> 9

<211> 123

<212> PRT

<213> Mouse (Mus musculus)

<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 Thr Val Thr Val Ser Ser

115 120

<210> 10

<211>  112

<212> PRT

<213> Mouse (Mus musculus)

<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> PRT

<213> Mouse (Mus musculus)

<400> 11

Gly Tyr Pro Phe Thr Thr Tyr Asn Met Tyr

1 5 10

<210> 12

<211> 17

<212> PRT

<213> Mouse (Mus musculus)

<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> PRT

<213> Mouse (Mus musculus)

<400> 13

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

1 5 10

<210> 14

<211> 16

<212> PRT

<213> Mouse (Mus musculus)

<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> PRT

<213> Mouse (Mus musculus)

<400> 15

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 16

<211>  9

<212> PRT

<213> Mouse (Mus musculus)

<400> 16

Ser Gln Ser Thr His Val Pro Leu Thr

1 5

<210> 17

<211>  115

<212> PRT

<213> Mouse (Mus musculus)

<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> PRT

<213> Mouse (Mus musculus)

<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> PRT

<213> Mouse (Mus musculus)

<400> 19

Gly Tyr Thr Phe Thr Asn Ser Trp Met Asn

1 5 10

<210>  20

<211> 17

<212> PRT

<213> Mouse (Mus musculus)

<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> PRT

<213> Mouse (Mus musculus)

<400>  21

Asp Ser Arg Phe Ser Tyr

1 5

<210>  22

<211> 11

<212> PRT

<213> Mouse (Mus musculus)

<400>  22

Lys Ala Ser Gln Asp Val Arg Thr Ala Val Ala

1 5 10

<210> 23

<211> 7

<212> PRT

<213> Mouse (Mus musculus)

<400> 23

Ser Thr Ser Tyr Arg Tyr Thr

1 5

<210>  24

<211>  9

<212> PRT

<213> Mouse (Mus musculus)

<400>  24

Gln Gln His Tyr Ser Thr Pro Leu Thr

1 5

<210> 25

<211>  115

<212> PRT

<213> Mouse (Mus musculus)

<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 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> PRT

<213> Mouse (Mus musculus)

<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> PRT

<213> Mouse (Mus musculus)

<400> 27

Gly Leu Thr Phe Ser Arg Tyr Ala Met Ser

1 5 10

<210>  28

<211> 17

<212> PRT

<213> Mouse (Mus musculus)

<400> 28

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

1 5 10 15

Gly

<210>  29

<211> 6

<212> PRT

<213> Mouse (Mus musculus)

<400>  29

Glu Tyr Gly Arg Asp Tyr

1 5

<210> 30

<211>  11

<212> PRT

<213> Mouse (Mus musculus)

<400> 30

His Ala Ser Gln Gly Ile Asn Ser Asn Ile Gly

1 5 10

<210> 31

<211> 7

<212> PRT

<213> Mouse (Mus musculus)

<400> 31

His Gly Thr Asn Leu Glu Asp

1 5

<210> 32

<211>  9

<212> PRT

<213> Mouse (Mus musculus)

<400> 32

Val Gln Tyr Ala Gln Phe Pro Arg Thr

1 5

<210> 33

<211> 113

<212> PRT

<213> Mouse (Mus musculus)

<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 Val Thr Val Ser

100 105 110

Ser

<210> 34

<211> 107

<212> PRT

<213> Mouse (Mus musculus)

<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> PRT

<213> Mouse (Mus musculus)

<400> 35

Gly Phe Thr Phe Ser Thr Phe Gly Met Ser

1 5 10

<210> 36

<211> 17

<212> PRT

<213> Mouse (Mus musculus)

<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> PRT

<213> Mouse (Mus musculus)

<400> 37

Gly Arg Ser Val

1

<210> 38

<211> 11

<212> PRT

<213> Mouse (Mus musculus)

<400> 38

His Ala Ser Gln Asp Ile Ser Ser Asn Ile Gly

1 5 10

<210> 39

<211> 7

<212> PRT

<213> Mouse (Mus musculus)

<400> 39

His Gly Thr Thr Leu Glu Asp

1 5

<210> 40

<211>  9

<212> PRT

<213> Mouse (Mus musculus)

<400>  40

Val Gln Ser Ala Gln Phe Pro Trp Thr

1 5

<210> 41

<211>  115

<212> PRT

<213> Mouse (Mus musculus)

<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 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> PRT

<213> Mouse (Mus musculus)

<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> PRT

<213> Mouse (Mus musculus)

<400>  43

Gly Tyr Thr Phe Thr Ser Ser Trp Met Asn

1 5 10

<210> 44

<211> 17

<212> PRT

<213> Mouse (Mus musculus)

<400>  44

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

1 5 10 15

Gly

<210> 45

<211> 6

<212> PRT

<213> Mouse (Mus musculus)

<400>  45

Asp Ser Arg Phe Ala Asp

1 5

<210> 46

<211>  11

<212> PRT

<213> Mouse (Mus musculus)

<400>  46

Lys Ala Ser Gln Asp Val Ser Ala Ala Val Ala

1 5 10

<210> 47

<211> 7

<212> PRT

<213> Mouse (Mus musculus)

<400>  47

Ser Ala Ser Tyr Arg Tyr Thr

1 5

<210> 48

<211>  9

<212> PRT

<213> Mouse (Mus musculus)

<220>

<221> Structural Domain

<222>  (1)..(10)

<223> Mutated HCDR1 region of 2F8

<400>  48

Gln Gln His Tyr Asn Thr Pro Leu Thr

1 5

<210> 49

<211>  118

<212> PRT

<213> Mouse (Mus musculus)

<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 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 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> PRT

<213> Mouse (Mus musculus)

<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> PRT

<213> Mouse (Mus musculus)

<400> 51

Gly Phe Thr Phe Ser Arg Tyr Ala Met Ser

1 5 10

<210> 52

<211> 17

<212> PRT

<213> Mouse (Mus musculus)

<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> PRT

<213> Mouse (Mus musculus)

<400> 53

Gln Gly Ser Asn His Tyr Phe Asp Tyr

1 5

<210> 54

<211> 11

<212> PRT

<213> Mouse (Mus musculus)

<400> 54

His Ala Ser Gln Gly Ile His Asn Asn Ile Gly

1 5 10

<210> 55

<211> 7

<212> PRT

<213> Mouse (Mus musculus)

<400> 55

His Gly Thr Asn Leu Glu Asp

1 5

<210> 56

<211>  9

<212> PRT

<213> Mouse (Mus musculus)

<400> 56

Val Gln Tyr Ala Gln Phe Pro Tyr Thr

1 5

<210> 57

<211>  118

<212> PRT

<213> Mouse (Mus musculus)

<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> PRT

<213> Mouse (Mus musculus)

<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> PRT

<213> Mouse (Mus musculus)

<400> 59

Gly Phe Thr Phe Ser Arg Tyr Gly Met Ser

1 5 10

<210> 60

<211> 17

<212> PRT

<213> Mouse (Mus musculus)

<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> PRT

<213> Mouse (Mus musculus)

<400> 61

Gln Gly Ser Asn Tyr Tyr Phe Asp Tyr

1 5

<210> 62

<211> 11

<212> PRT

<213> Mouse (Mus musculus)

<400> 62

His Ala Ser Gln Gly Ile Ser Ser Asn Ile Gly

1 5 10

<210> 63

<211> 7

<212> PRT

<213> Mouse (Mus musculus)

<400> 63

His Gly Thr Asn Leu Glu Asp

1 5

<210> 64

<211>  9

<212> PRT

<213> Mouse (Mus musculus)

<400> 64

Val Gln Tyr Ala Gln Phe Pro Tyr Thr

1 5

<210> 65

<211>  115

<212> PRT

<213> Mouse (Mus musculus)

<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 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> PRT

<213> Mouse (Mus musculus)

<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> PRT

<213> Mouse (Mus musculus)

<400> 67

Gly Asp Thr Phe Thr Thr Tyr

1 5

<210> 68

<211> 6

<212> PRT

<213> Mouse (Mus musculus)

<400> 68

Tyr Leu Asn Ser Gly Ser

1 5

<210> 69

<211> 6

<212> PRT

<213> Mouse (Mus musculus)

<400> 69

Asp Ser Arg Phe Ser Tyr

1 5

<210> 70

<211>  11

<212> PRT

<213> Mouse (Mus musculus)

<400> 70

Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210> 71

<211> 7

<212> PRT

<213> Mouse (Mus musculus)

<400> 71

Ser Ala Ser Tyr Arg Tyr Thr

1 5

<210> 72

<211>  9

<212> PRT

<213> Mouse (Mus musculus)

<400> 72

Gln Gln His Tyr Asn Thr Pro Leu Thr

1 5

<210> 73

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<221> Structural Domain

<222>  (1)..(10)

<223> HCDR1 of humanized 2F8

<400> 73

Gly Tyr Thr Phe Thr Ser Ser Trp Met Asn

1 5 10

<210> 74

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<221> Structural Domain

<222>  (1)..(17)

<223> Humanized 2F8's HCDR2

<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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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 Thr Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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 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 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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 Thr Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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> PRT

<213> Artificial Sequence

<220>

<221> Structural 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> PRT

<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 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 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> PRT

<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 His

275

<210> 101

<211>  20

<212> PRT

<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> PRT

<213> Artificial Sequence

<220>

<221> Variant

<222>  (1)..(20)

<223> Variants of antigen fragment P12

<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> PRT

<213> Artificial Sequence

<220>

<221> Variant

<222>  (1)..(20)

<223> Variants of antigen fragment P12

<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> PRT

<213> Artificial Sequence

<220>

<221> Variant

<222>  (1)..(20)

<223> Variants of antigen fragment P12

<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> PRT

<213> Artificial Sequence

<220>

<221> Variant

<222>  (1)..(20)

<223> Variants of antigen fragment P12

<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> PRT

<213> Artificial Sequence

<220>

<221> Variant

<222>  (1)..(20)

<223> Variants of antigen fragment P12

<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> PRT

<213> Artificial Sequence

<220>

<221> Variant

<222>  (1)..(20)

<223> Variants of antigen fragment P12

<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> PRT

<213> Artificial Sequence

<220>

<221> Variant

<222>  (1)..(20)

<223> Variants of antigen fragment P12

<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> PRT

<213> Homo sapiens

<400> 109

Ile Leu Ser Cys Thr Phe Glu

1 5

<210>  110

<211> 5

<212> PRT

<213> Homo sapiens

<400> 110

Leu Ser Cys Thr Phe

1 5

<210>  111

<211> 8

<212> PRT

<213> Homo sapiens

<400> 111

Ile Leu Ser Cys Thr Phe Glu Pro

1 5

Claims (16)

1. An anti-B7-H4 antibody or its antigen-binding fragment, comprising: The antibody light chain variable region and the antibody heavy chain variable region, wherein the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 respectively; and the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 respectively. 2. The B7-H4 antibody or its antigen-binding fragment as described in claim 1, wherein the antibody or its antigen-binding fragment is selected from murine antibodies or their fragments, chimeric antibodies or their fragments, human antibodies or their fragments, and humanized antibodies or their fragments. 3. The B7-H4 antibody or its antigen-binding fragment as claimed in claim 2, wherein the heavy chain variable region of the humanized antibody further comprises a heavy chain FR region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof. 4. The B7-H4 antibody or its antigen-binding fragment as described in claim 3, wherein the heavy chain variable region of the humanized antibody further comprises a human IgG1, IgG2 or IgG4 heavy chain FR region. 5. The B7-H4 antibody or its antigen-binding fragment as claimed in claim 4, wherein the heavy chain variable region of the humanized antibody further comprises IgG1 that enhances ADCC toxicity through amino acid mutation. 6. The B7-H4 antibody or its antigen-binding fragment as described in claim 2, wherein the humanized antibody comprises: the light chain variable region of SEQ ID NO:76 and the heavy chain variable region of SEQ ID NO:75. 7. The B7-H4 antibody or its antigen-binding fragment as described in claim 2, wherein the humanized antibody comprises: the light chain of SEQ ID NO:84 and the heavy chain of SEQ ID NO:83. 8. A DNA sequence encoding an antibody or antigen-binding fragment as described in any one of claims 1-7. 9. An expression vector containing the DNA sequence as described in claim 8. 10. A host cell, wherein the expression vector as described in claim 9 is introduced or contains the expression vector described in claim 9. 11. The host cell as described in claim 10, characterized in that, The host cell is a bacterium, yeast, or mammalian cell. 12. The host cell as described in claim 11, characterized in that, The host cells are Escherichia coli, Pichia pastoris, Chinese hamster ovary (CHO) cells, or human embryonic kidney (HEK) 293 cells. 13. Methods for producing antibodies, including: The host cells of claims 10-12 are cultured, and antibodies are isolated from the culture and purified. 14. A pharmaceutical composition comprising an anti-B7-H4 antibody or an antigen-binding fragment thereof as described in any one of claims 1-7, and a pharmaceutically acceptable excipient, diluent, or carrier. 15. A detection reagent comprising the anti-B7-H4 antibody or its antigen-binding fragment as described in any one of claims 1-7. 16. A diagnostic agent comprising the anti-B7-H4 antibody of any one of claims 1-7 or an antigen-binding fragment thereof.