CN112679611B - Humanized CD47 antibody or antigen binding fragment thereof and application - Google Patents

Abstract

The invention provides a humanized CD47 antibody or an antigen binding fragment thereof and application thereof, which have low toxicity and high efficiency, do not generate erythrocyte agglutination in vitro and do not cause erythrocyte clearance. In addition, the humanized CD47 antibody provided by the present invention exhibits very weak levels of low or no binding to platelets and erythrocytes, demonstrating a more specific targeting specificity for CD47+ tumor cells. The humanized CD47 antibody or the antigen binding fragment thereof provided by the invention can effectively block the binding of CD47 and SIRP alpha and activate the phagocytic activity of mediated macrophages on tumor cells.

Description

Humanized CD47 antibody or antigen binding fragment thereof and application

Technical Field

The invention relates to the field of biomedicine, in particular to the technical field of antibodies and the field of immunity, and more particularly relates to a humanized CD47 antibody or an antigen-binding fragment thereof and application thereof.

Background

CD47, also known as Integrin Associated Protein (IAP), is widely expressed on the cell surface and interacts with signal regulatory Protein α (sirpa), thrombospondin (TSP 1) and integrins (integrins) to mediate a series of responses including apoptosis, proliferation, and immunity. CD47 was first identified as a tumor antigen for human ovarian cancer in the 80's 20 th century, and CD47 was subsequently found to be expressed in various types of human tumors, including Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), Acute Lymphocytic Leukemia (ALL), non-hodgkin's lymphoma (NHL), Multiple Myeloma (MM), Bladder Cancer (BC), and other solid tumors. Its expression and activity has been linked to a number of diseases and disorders. It is a widely expressed transmembrane glycoprotein with one immunoglobulin-like domain and five transmembrane regions, which acts as a cellular ligand for sirpa and binds sirpa via the NH 2-terminal V-like domain of signal-regulated protein α (sirpa). Sirpa is expressed primarily in bone marrow cells, including macrophages, granulocytes, Dendritic Cells (DCs), mast cells, and their precursor cells, including hematopoietic stem cells.

CD47 is a "self" marker, representing a "do not eat me" signal. The human body needs 20-30 trillion red blood cells to maintain efficient oxygen transport throughout the body. The life cycle of erythrocytes is short, only 120 days, with 100 billion erythrocytes produced per hour, and numerous aged erythrocytes phagocytosed and cleared by macrophages. However, macrophages are how to differentiate between young and senescent erythrocytes, and the mechanism of attacking only senescent erythrocytes is unknown. Until 2000, Oldenborg et al demonstrated that CD47 is an important "self" marker on the cell surface and an important signal for regulating macrophage phagocytosis. CD47 can bind to macrophage surface SIRP alpha, phosphorylate its ITIM, and subsequently recruit SHP-1 protein, resulting in a cascade of reactions that inhibit phagocytosis by macrophages. Young erythrocytes express higher CD47 releasing the "self-eat me" signal to macrophages, while senescent erythrocytes, CD47, are down-regulated and eventually cleared by macrophages.

Different studies have shown that almost all tumor cells and tissues highly express CD 47. The CD47 highly expressed on the surface of the tumor cell is combined with SIRP alpha on the surface of macrophage to release a 'do not eat me' signal, so that the macrophage in a tumor tissue infiltration area is harmonious with the tumor cell, and the macrophage can inhibit T-cell effect by promoting the proliferation of blood vessels in the tumor to promote the expansion and growth of the tumor cell. Various studies have shown that at the cellular level, the phagocytosis of cancer cells by macrophages can be effectively activated by blocking the correlation between sirpa-CD 47, and that the blocking of the correlation between sirpa-CD 47 by CD47 antibodies in vivo can effectively inhibit tumor growth in mice.

There have been many studies on inhibitors for blocking SIRP α -CD47 signaling pathway, including anti-CD 47 mab H5F9-G1(US9382320B2) from fortysern, mab CC9002 (patent US9045541B2) from Celgene, mab ADI-26630 (patent CN201710759828.9) from dawn organism, mab 13H3/1F8 (patent CN201780003451) from niche organism, etc., and also trilliu hermatrautics, bifunctional fusion protein TTI-621 (patent US20150329616a1) from inc, ALX148 (patent US20160186150a1) from ALX, etc., which are currently in clinical phase i/II. Clinical preliminary results show that the combination of the medicine and the CD20 has surprising curative effect on the relapse of CD20 or the refractory Follicular Lymphoma (FL) and the large B-cell lymphoma (DLBCL), and the combination of the ALX148 and Trastuzumab and Pembrolizumab has certain curative effect on Gastric Cancer (GC), head and neck cancer (HNSCC) and non-small cell lung cancer (NSCLC). CD47 inhibitors (including monoclonal antibodies, SIRP alpha fusion proteins) and multiple antibody inhibitors such as CD20, HER2 and PD-1 are in clinical research progress so far.

The CD47 inhibitor which is developed clinically or preclinically can block the combination of CD47 and SIRPa, promote macrophages to play phagocytosis and simultaneously cause agglutination of red blood cells to different degrees. In addition, the CD47 monoclonal antibodies have high target binding property with erythrocytes and platelets, so that the corresponding antibodies not only greatly reduce the treatment effect, but also introduce drug side effects, and clinically reduce erythrocytes and platelets and other related side effects.

Disclosure of Invention

Aiming at the technical problems, the invention provides a humanized CD47 antibody or an antigen binding fragment thereof and application thereof, wherein the humanized CD47 antibody or the antigen binding fragment thereof has low toxicity and high efficiency, does not cause erythrocyte agglutination in vitro, and does not cause erythrocyte elimination. In addition, the humanized CD47 antibody provided by the present invention exhibits very weak levels of low or no binding to platelets and erythrocytes, demonstrating a more specific targeting specificity for CD47+ tumor cells. The humanized CD47 antibody or the antigen binding fragment thereof provided by the invention can effectively block the binding of CD47 and SIRP alpha and activate the phagocytic activity of mediated macrophages on tumor cells.

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

in some embodiments, the invention provides anti-CD 47 antibodies or antigen-binding fragments thereof (preferably human CD47 antigen-binding fragments) that bind to CD47 or fragments thereof (preferably human CD47 protein).

In some embodiments, the humanized CD47 antibodies or antigen-binding fragments thereof provided herein are selected from the group consisting of: comprising a heavy chain variable region VH sequence having at least 98% identity to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57; the VL sequence has at least 98% identity to an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO: 2, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, seq id NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, seq id NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54.

in some embodiments, the humanized CD47 antibody or antigen binding fragment thereof provided herein comprises: a VH/VL sequence pair comprising the amino acid sequences of VH and VL chains and nucleotide sequences thereof, said VH/VL sequence pair having at least 98% identity to a VH and VL amino acid sequence pair selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 11, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ id no: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38, SEQ id no: 39 and SEQ ID NO: 40, SEQ ID NO: 41 and SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50, SEQ ID NO: 49 and SEQ ID NO: 52, SEQ ID NO: 49 and SEQ ID NO: 54, SEQ ID NO: 51 and SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52, SEQ ID NO: 51 and SEQ ID NO: 54, SEQ ID NO: 53 and SEQ ID NO: 50, SEQ ID NO: 53 and SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 50, SEQ ID NO: 55 and SEQ ID NO: 52, SEQ ID NO: 55 and SEQ ID NO: 54, SEQ ID NO: 56 and SEQ ID NO: 50, SEQ ID NO: 56 and SEQ ID NO: 52, SEQ ID NO: 56 and SEQ ID NO: 54, SEQ ID NO: 57 and SEQ ID NO: 50, SEQ ID NO: 57 and SEQ ID NO: 52, SEQ ID NO: 57 and SEQ ID NO: 54. the sequences are shown in table 1 below:

TABLE 1 VH and VL sequences of an exemplary humanized CD47 antibody of the invention

In some embodiments, the humanized CD47 antibodies or antigen-binding fragments thereof provided herein, when encoding the antibody HCDRs according to Kabat encoding rules, (1) the HCDR1/HCDR2/HCDR3 combination comprises a sequence of one or more sets of heavy chain complementarity determining region HCDRs selected from: HCDR1/HCDR2/HCDR3 are selected from the group consisting of SEQ ID NO 61/SEQ ID NO 62/SEQ ID NO 63, SEQ ID NO 67/SEQ ID NO 68/SEQ ID NO 69, SEQ ID NO 73/SEQ ID NO 74/SEQ ID NO 69, SEQ ID NO 77/SEQ ID NO 78/SEQ ID NO 79. (2) The HCDR of above (1) comprising one or more amino acid substitutions, deletions or insertions of not more than 5 amino acids.

In some embodiments, the humanized CD47 antibody or antigen-binding fragment thereof provided herein, when encoding the antibody LCDRs according to Kabat encoding rules, (1) the LCDR1/LCDR2/LCDR3 combination comprises a sequence of one or more sets of heavy chain complementarity determining regions, LCDRs, selected from: LCDR1/LCDR2/LCDR3 is selected from the group consisting of SEQ ID NO 64/SEQ ID NO 65/SEQ ID NO 66, SEQ ID NO 70/SEQ ID NO 71/SEQ ID NO 72, SEQ ID NO 70/SEQ ID NO 75/SEQ ID NO 76, SEQ ID NO 80/SEQ ID NO 81/SEQ ID NO 82. (2) The LCDR of (1) above which contains one or more amino acid substitutions, deletions or insertions of not more than 5 amino acids.

In some embodiments, the humanized CD47 antibody or antigen-binding fragment thereof provided herein, when the CDRs of the antibody are encoded according to Kabat encoding rules, HCDR1/HCDR2/HCDR3 is paired with LCDR1/LCDR2/LCDR3 as follows (1): SEQ ID NO 61/62/63 and 64/65/66, 67/68/69 and 70/71/72, 74/69 and 70/75/76, 77/78/79 and 80/81/82. The sequences of the CDRs are shown in Table 2 below:

TABLE 2 HCDRs and LCDR sequences of an exemplary humanized CD47 antibody of the invention

In some embodiments, the light chain constant region of the humanized CD47 antibody is optionally a human kappa or lambda chain constant region (SEQ ID NO:58), and the heavy chain constant region is optionally a human IgG1 or IgG 4S 228P mutated constant region (SEQ ID NO:59 or SEQ ID NO: 60).

In some embodiments, the antigen binding fragment is selected from a Fab, Fab '-SH, Fv, scFv, or (Fab') 2 fragment.

The present invention provides nucleic acids encoding the humanized CD47 antibody or antigen-binding fragment thereof described in any of the above. The nucleic acid sequences are shown in table 3:

TABLE 3 nucleic acid sequences encoding the exemplary humanized CD47 antibodies or antigen binding fragments thereof of the present invention

The invention also provides expression vectors comprising the nucleic acids, and host cells comprising the expression vectors. In some embodiments, the host cell is preferably a yeast cell, a mammalian cell such as a chinese hamster ovary cell, a human embryonic kidney cell, or other cell suitable for use in the production of an antibody or antigen binding fragment thereof.

In some embodiments, the invention provides a bispecific antibody. The bispecific antibody comprises two distinct arms, a first arm (arm1) and a second arm (arm2), one arm (arm1) comprising a humanized CD47 antibody or antigen-binding fragment thereof of any of the above that is capable of binding to human CD47, and the other arm (arm2) comprising an additional monoclonal antibody or fusion protein that does not bind to human CD 47. The other arm (arm2) is capable of binding to malignant/immune cells, targeting the following targets: EGFR, HER2, CD47, PD-1, PD-L1, CTLA4, TGF beta, VEGF, HER3, TIGIT, CLAUDIN18.2, CD3, TIM3, LAG3, CD20, CD19, CD33 and other targets.

In some embodiments, the invention provides a trispecific antibody or trispecific fusion protein comprising the humanized CD47 antibody or antigen binding fragment described above, the other two ends of the trispecific antibody or trispecific fusion protein targeting tumor associated antigens of tumor cells or immune cell targeting immunoassay targets comprising EGFR, HER2, CD47, PD-1, PD-L1, CTLA4, TGF β, VEGF, HER3, TIGIT, CLAUDIN18.2, CD3, TIM3, LAG3 CD20, CD19, CD33, etc. targets.

In some embodiments, the present invention provides a pharmaceutical composition comprising a humanized CD47 antibody or antigen-binding fragment thereof provided herein or a bispecific antibody comprising a humanized CD47 antibody or antigen-binding fragment thereof described herein as one of the arms, and a pharmaceutically optional pharmaceutically acceptable carrier.

In some embodiments, the invention also provides a use of the humanized CD47 antibody or antigen binding fragment thereof described above in the preparation of a medicament for the treatment of an overexpression of CD47 in a neoplastic disorder.

Preferably, the neoplastic disease includes at least one of hematological tumors, at least one of Acute Myeloid Leukemia (AML), Chronic Myeloid Leukemia (CML), Acute Lymphocytic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), adult T-cell leukemia, Multiple Myeloma (MM), mixed leukemia, non-hodgkin's lymphoma (NHL), and solid tumor disease; the solid tumor is at least one of lymphoma, breast cancer, head and neck cancer, gastric cancer, lung cancer, esophageal cancer, intestinal cancer, ovarian cancer, cervical cancer, liver cancer, renal cancer, pancreatic cancer, bladder cancer, colorectal cancer, glioma, melanoma and other solid tumors.

The invention has the following beneficial effects:

the invention provides a novel humanized CD47 antibody or an antigen binding fragment thereof, the humanized CD47 antibody can specifically block the interaction between a CD47 protein and SIRPa, has corresponding biological functions, and can activate macrophages to mediate phagocytosis on CD47+ cells at different levels. The humanized CD47 antibody showed very weak levels of low or no binding to platelets and erythrocytes, demonstrating a more specific targeting specificity for CD47+ tumor cells. The humanized CD47 antibody is capable of specifically targeting tumor cells in a mixed population of tumor cells with human normal red blood cells or human peripheral blood PBMCs or other normal cells that may express the CD47 molecule. The humanized CD47 antibodies or antigen-binding fragments thereof described herein can also inhibit the growth and/or proliferation of tumors or tumor cells.

Drawings

Figure 1a binding activity of humanized 6E8 antibody to CD47 protein; figure 1B binding activity of humanized 100 and 99B5 antibodies to CD47 protein; figure 1℃ binding activity of humanized 81C1 antibody to CD47 protein; figure 1d. binding activity of humanized 99B5 antibody to CD47 protein.

FIG. 2a. binding activity of humanized 99B5 antibody to jurkat cells, MDA-MB-231 cells, CHO-K1/Human CD47 cells, SHP77 cells; FIG. 2b binding activity of humanized 6E8 antibody to U-87 cells, CHO-K1/cyno CD47 cells, CHO-K1/Human CD47 cells, Raji cells.

FIG. 3a. blocking effect of humanized 99B5 antibody on binding of human CD47 protein to SIRPa; figure 3b. binding blocking effect of humanized 6E8 antibody on human CD47 protein to sirpa.

FIG. 4 blocking effect of humanized 99B5 antibody on human CD47 ligand SIRP alpha interaction with CD47 at cellular level.

FIG. 5a shows the results of erythrocyte agglutination experiments with various concentration gradients of humanized 99B5 antibody; FIG. 5b shows the results of hemagglutination experiments with humanized 100 antibody at various concentration gradients; figure 5c shows the results of agglutination of erythrocytes for each concentration gradient of humanized 6E8 antibody.

FIG. 6a shows the experimental results of the high expression of CD47 in various tumor cells; FIG. 6b shows the experimental results of high expression of CD47 in various tumor cells

FIG. 7a shows the activity of the humanized antibodies h99B5-IgG4-1, h99B5-IgG4-2, h99B5-IgG4-3, h100-IgG4-1, h100-IgG4-4, h100-IgG4-5, h100-IgG4-6, h100-IgG4-7, etc., of CD47 of the present invention in the Jurkat tumor model; FIG. 7B shows the activity of the humanized antibodies h99B5-IgG4-1, h99B5-IgG4-2, h99B5-IgG4-3, h100-IgG4-1, h100-IgG4-2, h100-IgG4-3, h100-IgG4-4, h100-IgG4-5, h100-IgG4-6, h100-IgG4-7, etc., of the CD47 of the present invention in the SHP-77 tumor model.

FIG. 8 shows the results of the experiment that the humanized CD47 antibody has significant ADCC tumor killing activity on Raji cells.

Detailed Description

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

Definition of terms:

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

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

The term "variable" refers to the fact that certain portions of the sequences of the variable domains of antibodies vary strongly and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is unevenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the light and heavy chain variable regions, called Complementarity Determining Regions (CDRs) or hypervariable regions. The more highly conserved portions of the variable domains are called the Framework (FR). The variable domains of natural heavy and light chains each comprise four FR regions, and the two light and heavy chain variable regions typically comprise, from N-terminus to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. Most FR regions adopt a β -sheet configuration, connected by three CDRs, forming a loop junction, and in some cases forming part of the β -sheet structure. The CDRs in each chain are held in close proximity by the FR region and, together with the CDRs from the other chain, contribute to the formation of the antigen binding site of the antibody. Please refer, for example, to Kabat et al, immunological protein sequences, fifth edition, national institutes of health, besesda, maryland (1991). The constant region is not directly involved in binding of an antibody to an antigen, but exhibits various biological effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement killing pathway (CDC).

Antibodies can be classified into 5 classes, distinguished by the amino acid sequence of the antibody heavy chain constant region: IgA, IgD, IgE, IgG, and IgM, and several of these homogeneous classes can be further divided into subclasses, e.g., IgG1, IgG2, IgG3 and IgG1, IgA1, and IgA 2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively. Kappa and lambda can be classified according to the constant region (CL) of the antibody light chain. Within full-length light and heavy chains, the variable and constant regions are typically linked by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids.

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

The term "chimeric antibody" as used herein means that a portion of the heavy and/or light chain (which generally refers to the variable region) is derived from the same or homologous to corresponding sequences in antibodies of a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (which generally refers to the constant region) is derived from an antibody of another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, which have the same or homologous corresponding sequences, so long as they exhibit the desired biological activity. The variable regions of the heavy/light chains of chimeric antibodies, such as those from murine CD47 antibodies, involved in the present invention are grafted to the constant regions of the heavy/light chains of human antibodies by antibody engineering techniques, which exhibit similar biological activities.

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

The term "bispecific antibody" is an artificial antibody containing 2 specific antigen binding sites, which can bridge between a target cell and a functional molecule (cell) to trigger a targeted immune response, and is one of genetically engineered antibodies. Diabodies may be bivalent or bispecific. Diabodies are more fully described in e.g. EP 404,097; WO 1993/01161; hudson et al, nat. med.9: 129-134 (2003); and Hollinger et al, proceedings of the national academy of sciences of the united states (proc.natl.acad.sci.usa) 90: 6444- > 6448 (1993).

The term "trispecific antibody" is an artificial antibody containing three specific antigen binding sites, which can bridge between target cells and functional molecules (cells) to stimulate targeted immune response, and is one of genetically engineered antibodies. The triabody may be trivalent or trispecific.

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

The term "competition" herein in the context of antigen binding proteins that are used to compete for the same epitope (e.g., neutralizing antigen binding proteins or neutralizing antibodies) means competition between the antigen binding proteins as determined by the following assay: in such assays, the antigen binding protein to be detected (e.g., an antibody or immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., CD47 or a fragment thereof).

The term "block" means capable of reduced CD47 signaling in the presence of an antibody of the invention. CD 47-mediated signaling disruption refers to a decrease in CD47 signaling levels in the presence of a CD47 antibody of the invention by an amount greater than or equal to 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99% or 100% below the control level of CD47 (i.e., the level of CD47 signaling in the absence of the antibody), as measured by a variety of standard techniques, such as, by way of non-limiting example, downstream gene activation and/or luciferase reporter assays in response to CD47 activation.

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

The term "host cell" refers to a cell into which an exogenous nucleic acid is introduced, including progeny of such a cell. And can express exogenous nucleic acid in cell or cell membrane or release it to outside of cell.

The term "agglutination" refers to clumping of cells, while the term "hemagglutination" refers to clumping of a particular type of cells (i.e., red blood cells). Therefore, hemagglutination is a type of agglutination.

The humanized CD47 antibody of the present invention can be used in immunoassays in vitro, and the CD47 antibody of the present invention can be coupled to a detectable label such as luciferase, biotinidase, etc., and used in liquid or solid phase for direct or indirect immunoassays such as FACS, IHC, ELISA, etc., including competitive or non-competitive. The CD47 antibody provided by the invention can be conveniently used in a kit, and the CD47 in vivo or in vitro biological fluid or on tissue can be detected by the CD47 antibody provided by the invention. Can be used for any sample containing a detectable amount of CD 47. Samples may include, but are not limited to, liquids such as urine, saliva, cerebrospinal fluid, blood, serum, and the like, or samples may be solid or semi-solid such as tissue, stool, and the like, or may be solid tissue such as those commonly used in histological diagnostics.

The humanized CD47 antibodies provided herein can optionally be formulated with one or more agents that enhance activity, or enhance therapeutic efficacy. The CD47 antibody provided by the present invention can be optionally formulated with one or more drugs including small and large molecule drugs that inhibit tumor growth, preferably various monoclonal antibody drugs that attack tumor cells by T cell recognition, such as rituximab, cetuximab, and trastuzumab.

The humanized CD47 antibodies or pharmaceutical compositions provided herein can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal. Parenteral injection includes intramuscular, intravenous, arterial, intraperitoneal or subcutaneous administration.

The following examples further illustrate the invention, however, it is to be understood that the examples are described by way of illustration and not limitation, and that various modifications may be made by those skilled in the art.

Example 1: production and purification of humanized and control CD47 antibodies

The amino acid sequence and CDR sequence of the humanized CD47 antibody of the present invention are shown in Table 1 and Table 2, and the expression vector of the humanized CD47 antibody is constructed in vitro.

Specifically, the light chain variable region (VL) sequences in Table 1 were constructed into the kappa chain constant region of human antibody light chain (SEQ ID NO:58), and the heavy chain variable region (VH) sequences were constructed into the constant region of human antibody heavy chain, preferably human IgG4(S228P) heavy chain constant region (SEQ ID NO: 60). The following expression vectors for the humanized antibody were constructed: H99B5-IgG4-1, H99B5-IgG4-2, H99B5-IgG4-3, H99B5-IgG4-4, H99B5-IgG4-5, H81C1-IgG4-1, H81C1-IgG4-2, H81C 4-IgG 4-3, H81C 4-IgG 4-4, H81C 4-IgG 4-5, H100-IgG4-1, H100-IgG4-2, H100-IgG4-3, H100-IgG4-4, H100-IgG4-5, H100-IgG4-6, H100-4-7, H100 IgG-4-8, H100-IgG4-9, H100-IgG4-10, H6E 4-4, H4-4, H4-2, H-4-2, H99-IgG4-L2H1, H99-IgG4-L3H1, H99-IgG4-L1H2, H99-IgG4-L2H2, H99-IgG4-L3H2, H99-IgG4-L1H3, H99-IgG4-L2H3, H99-IgG4-L3H3, H99-IgG4-L1H4, H99-IgG4-L2H4, H99-IgG4-L3H4, H99-IgG4-L1H5, H99-IgG4-L2H5, H5-IgG 5-L3H 5, H5-L5-IgG 5, H5-L1H 5-5, H5-IgG 5, H5-5, H5-5H 5, H5-5 and H5-5H 5B 5, H5-5H 5 and H5.

The light chain variable region (VL) sequence was constructed into the kappa chain constant region of the human antibody light chain (SEQ ID NO:58) and the heavy chain variable region (VH) sequence was constructed into the constant region of the human antibody heavy chain, preferably the human IgG1 heavy chain constant region (SEQ ID NO: 59). The following expression vectors for the humanized antibody were constructed: H99B5-IgG1-1, H99B5-IgG1-2, H99B5-IgG1-3, H99B5-IgG1-4, H99B5-IgG1-5, H81C1-IgG1-1, H81C1-IgG1-2, H81C1-IgG1-3, H81C1-IgG1-4, H81C1-IgG1-5, H100-IgG1-1, H100-IgG1-2, H100-IgG1-3, H100-IgG1-4, H100-IgG1-5, H100-IgG1-6, H100-1-7, H100 IgG-1-8, H100-IgG1-9, H100-IgG1-10, H6E 1-1, H1-1, H1-2, H-1-2, H-1, H-1-2, H-IgG-L2H, H-IgG-L3H, H-IgG-L1H, H-IgG-L2H, H-IgG-L3H, H-IgG-L1H, H-IgG-L2H, H-IgG-L3H, H-IgG-L1H, H-IgG-L2H, H-IgG-L3H, H-IgG-L1H, H-IgG-L2H, H-IgG-L3H, H-IgG-L1H, H-IgG-L2H, H-IgG-L3H.

Adding optimized signal peptide into the N end of the heavy chain/light chain amino acid of the recombinant antibody for secretion expression, performing codon optimization on the amino acid of the recombinant antibody, adding a Kozak sequence GCCGCCACC into the 5' end of the nucleotide, adding EcoRI/HindIII enzyme cutting sites of pcDNA3.4 into the two ends of the nucleotide, and connecting the synthetic gene to a pcDNA3.4 vector after enzyme cutting.

The humanized antibody h5F9-G1(FortySeven) has the same sequence as the antibody "5F 9" in US2015/0183874A1, and h5F9-G1 is the first clinically-entering CD47 antibody inhibitor. The B6H12 sequence is from WO2011143624A 2; the AB6.12(Celgene) sequence was derived from the sequence of the antibody "AB 6.12" in U.S. Pat. No. 5, 9045541. The chimeric antibody 99B5-G1, 81C1-G1, 6E8-G1,100-IgG, 99B5-G4, 81C1-G4, 6E8-G4 and 100-IgG4 is derived from patent CN 111454359A.

The expression vector is amplified and extracted by escherichia coli, endotoxin is removed, and the expression vector is amplified and extracted by the plasmids: Expi-293F cells were transfected at 1. mu.g/ml medium. The Transfection reagent used was Expifactamine 293Transfection Kit (Theromfisher, Lot #: A14524) with a cell density of 25 x 10 at Transfection⁵cells/ml, 16-18h after transfection, adding expression enhancers Enhancer1 and Enhancer2, and collecting cell supernatant 5 days after transfection. Purifying with ProteinA, centrifuging at 4 deg.C at 10000rpm/min for 30min to remove cell debris, balancing 10 column volumes with balancing solution (0.02MPB, 0.15MNaCl, pH7.0), flowing the supernatant through the column at 2ml/min, washing the combined column with balancing solution for 5 column volumes, eluting with eluent (0.02M PB, 0.15MNaCl, pH3.0), and dropping the eluent into a collecting tube containing Tris neutralizing solution (1M, pH9.0). The protein eluate was collected, concentrated and buffer replaced by PBS (HyClone SH30256.01) by ultrafiltration using an ultrafiltration tube (Millipore UFC903096)4000G, and stored at-20 ℃ after SDS-PAGE detection. Removing endotoxin, filtering and sterilizing, and detecting the purity by SDS-PAGE and SEC-HPLC.

Example 2: dose Effect (ELISA) of the humanized CD47 antibody of the invention binding to human CD47 recombinant fusion protein

hCD47-his (Cat # CD7-H5227, Lot # C56P1-737F1-FA) was coated at 1. mu.g/m, the coating buffer selected from PBS (HyClone Lot: AC13298279), 100. mu.l/well, coating at room temperature (25 ℃) for 16-18H, washing the plate with TBST for 2 times, blocking with PBS + 3% BSA, blocking at 200. mu.l/well, blocking at room temperature (25 ℃) for 16-18H, washing the plate with TBST for 1 time, draining, and drying at 37 ℃ for 2 hours. The CD47 antibody and the control antibody of the invention are formulated in 330. mu.l 100. mu.g/ml, with 10. mu.g/ml being the first gradient and a 4-fold gradient dilution being carried out, for example, the second gradient is 80. mu.l of the first gradient added to 240. mu.l of PBS, and so on, for a total of 11 gradient concentrations. Incubate at 37 ℃ for 1 hour. After washing the plate 3 times with the automatic plate washer PBST, 100. mu.l of 1: 20000 dilutions of goat anti-human HRP secondary antibody (abcam Lot #: ab98624) were incubated for 45 min at 37 ℃. After incubation, the plate is washed for 3 times by an automatic plate washing machine, and after the last washing, the residual liquid is completely removed from the ELISA plate to the absorbent paper. Each hole100. mu.l of TMB developing solution was added. The reaction was carried out in the dark for 3 to 5 minutes, and 50. mu.l of 1% H was added to each well₂SO₄The reaction was terminated. Setting the light absorption values of reading by an MD (I3X) microplate reader to be 450nm and 630nm, and storing data after automatically reading the values.

The results show that the exemplary antibody of the invention has good binding activity to human CD47 protein, and that the humanized antibody shows better protein binding activity than the positive control antibody h5F 9-G4.

FIG. 1a shows the superior binding activity of humanized 6E8 antibody to CD47 protein; FIG. 1B shows the superior binding activity of humanized 100 and 99B5 antibodies to CD47 protein; FIG. 1C shows the superior binding activity of humanized 81C1 antibody to CD47 protein; FIG. 1d shows the superior binding activity of the humanized 99B5 antibody to CD47 protein. The results show that the exemplary humanized CD47 antibodies of the present invention all have strong binding ability to CD47 fusion protein.

Example 3: cell binding Activity (FACS) of the humanized CD47 antibody of the present invention and cancer Cell lines expressing human CD 47/cynomolgus monkey and various cancer Cell lines the binding activity of the CD47 antibody of the present invention to cancer Cell lines expressing human or cynomolgus monkey CD47, such as CHO-K1 stable Cell line, Jurkat, Raji, MDA-MB-231, SHP77, U-87 cells, was measured using a flow cytometer (BD FACS Celesta Cell Analyzer).

In this example, 2 stable cell lines (CHO-K1-Cyno-CD47/CHO-K1-Human-CD47) expressing CD47 and 5 cancer cell lines (CHO-K1-Cyno-CD47/CHO-K1-Human-CD47, which are derived from Nanjing King Shirui Biotech Co., Ltd., Jurkat, Raji, MDA-MB-231, SHP77, U-87 (cell Bank of China academy of sciences in Shanghai) were used in total. After the cells listed were digested (suspension cells were not digested), centrifuged at room temperature at 1000rpm for 5mins, the supernatant was discarded, washed with PBS, and the PBS was resuspended in a flow tube with the cell concentration adjusted to 1X 10⁶cells/ml. The volume of PBS in each tube is 250 mu L, the antibody is set to 8 reaction concentrations, 10 mu g/ml, 3 times of gradient dilution is carried out, and after the concentration of the antibody is adjusted, 250 mu L of corresponding protein is added in each tube; after 1h incubation at room temperature, PBS was washed 3 times (1500rpm, 5 mins); mu.L of secondary antibody Goatanti-human IgG/Alexa 647(Bioss Lot: AE041526) was added to each tube of sample, and after incubation for 30mins at room temperature in the absence of light,washing 3 times (1500rpm, 5mins) with 1mL PBS; 500. mu.L of LPBS was added to each tube to resuspend the cells and test them on the machine.

FIG. 2a shows good binding activity of humanized 99B5 antibody to MDA-MB-231 cells, jurkat cells, SHP77, CHO-K1/Human CD47 cells, respectively; FIG. 2b shows good binding activity of humanized 6E8 antibody to U-87 cells, CHO-K1/cyno CD47 cells, CHO-K1/Human CD47, Raji cells. The results show that the exemplary humanized antibodies of CD47 of the present invention all have strong binding ability to cells overexpressing CD 47. Most were at the same level in binding activity of each cell type compared to the positive control antibody h5F 9-G1.

Example 4: the humanized CD47 antibody of the invention blocks dose Effects (ELISA) at the protein level on the interaction of human CD47 ligand SIRP alpha with CD47

Biotin marks SIRP alpha-Fc (Cat #4546-SA-050, R & D System), the antibody to be detected competitively binds CD47 with the SIRP alpha-Fc, and the binding blocking effect of the antibody on the SIRP alpha-Fc and CD47 is detected.

Specifically, CD47(Acro, CD7-H5227-1mg, His tag) was coated at 1. mu.g/ml in 100. mu.l/well in PBS buffer, mixed with different concentrations of the test sample, added to a 96-well plate precoated with CD47 antigen, and incubated at 37 ℃ for 45 minutes. After incubation, the plate is washed for 5 times by an automatic plate washing machine, and the residual liquid is completely removed from the water absorption paper by an enzyme label plate after the last washing. Mu.l of Streptavidin-HRP was added to each well and incubated at 37 ℃ for 45 min. After incubation, the plate is washed for 5 times by an automatic plate washing machine, and the residual liquid is completely removed from the water absorption paper by an enzyme label plate after the last washing. 100. mu.l of TMB developing solution was added to each well. The reaction was carried out for 3 to 5 minutes under exclusion of light, and 50. mu.l of 1% H was added to each well₂SO₄The reaction was terminated. Setting the light absorption values of reading by an MD (I3X) microplate reader to be 450nm and 630nm, and storing data after automatically reading the values. The initial concentration of the antibody to be detected is 10 mu g/ml, and the antibody is diluted by 3 times of gradient, and the total concentration is 12 concentration gradients.

The results show that the exemplary antibodies of the invention can effectively block the binding of human CD47 protein and SIRPa. As shown in fig. 3a, 3b. All of the exemplified humanized antibodies blocked the binding of human CD47 protein to SIRP α with similar or superior effect to the positive control antibody h5F9-G4, or the positive control antibody h5F 9-G4.

Example 5: blocking of human CD47 ligand SIRP alpha interaction with CD47 at the cellular level (FACS) of the humanized CD47 antibodies of the invention

Jurkat cells were centrifuged at room temperature at 1000rpm for 5mins, the supernatant was discarded, washed with PBS, and the PBS was resuspended in a cell flow tube at a cell concentration of 1X 10⁷cells/ml. Biotin-labeled SIRP alpha-Fc (Cat #4546-SA-050, R)&D System), mixed with different concentrations of the test sample, respectively, and added to Jurkat cells (2.5X 10)⁵cells/action). Each reaction system is 250. mu.L, the reaction concentration of the recombinant antibody is 10 gradients, the initial concentration is 300nM, and 3-fold gradient dilution is performed. After incubation for 1h at 4 ℃, PBS was washed 3 times (1500rpm, 5 mins); 100 μ L of Anti-SAiFlour647 (gentript, 3 μ g/mL) was added to each tube, incubated at 4 ℃ in the dark for 30mins, and washed 3 times with 1mL of PBS (1500rpm, 5 mins); 500. mu.L of LPBS was added to each tube to resuspend the cells and test them on the machine.

As shown in fig. 4, the results show that the exemplary humanized 99B5 antibody of the present invention is effective at blocking the interaction of human CD47 ligand sirpa with CD47 at the cellular level.

Example 6: the humanized CD47 antibody of the invention can be used for detecting the activity of promoting erythrocyte agglutination (hemagglutination activity)

The agglutination activity of the CD47 antibody of the present invention and the control antibody on erythrocytes was measured using erythrocytes from healthy persons.

The specific implementation is as follows:

collecting whole blood with anticoagulant such as sodium citrate; putting the whole blood into a 15ml centrifuge tube, supplementing PBS to 15ml, centrifuging at room temperature, centrifuging at 200 Xg for 10mins, and discarding the supernatant; make up RBCs to 15mL with PBS, mix well, centrifuge at room temperature, 1500rpm, 5 mins. Washing for 3 times; after the last wash, the RBCs were adjusted to 2% concentration with PBS (e.g., 49ml PBS in 1ml RBCs); carrying out 2-fold gradient dilution on the recombinant protein and the positive antibody according to 200 mu g/ml, and totaling 15 concentration gradients; using a 96-well round plate, 50. mu.l of recombinant protein or monoclonal antibody at the corresponding concentration and 50. mu.l of RBCs were added to each well, and the reaction results were observed and recorded after incubation at room temperature for 2 hours.

The control antibody h5F9-G4 started to undergo significant erythrocyte aggregation at low concentrations, as shown in FIGS. 5a, 5B, and 5c, with FIG. 5a showing that no substantial erythrocyte aggregation was caused by each concentration gradient of the humanized 99B5 antibody. Figure 5b shows that the humanized 100 antibody did not cause substantial erythrocyte aggregation at each concentration gradient. Figure 5C shows that the humanized 81C1 antibody did not cause substantial erythrocyte aggregation at each concentration gradient. The results show that the exemplary humanized CD47 antibody of the invention did not cause substantial erythrocyte aggregation at each concentration gradient, whereas the agglutination reaction of the control antibody h5F9-G4 was very pronounced. Further observation under the microscope, antibody h5F9-G1 resulted in the aggregation of several erythrocytes. The invention tests that the antibody is safe to hemagglutination of erythrocytes from different healthy people. Therefore, the humanized antibody of the CD47 has lower side effect in treatment than the h5F9G4 antibody and higher safety in clinic. In addition, H99IgG4-1, H99-IgG4-1, H99-G4-1 in the figure correspond to the molecule H99B5-IgG 4-1; h99IgG4-2, h99-IgG4-2 correspond to the molecule h99B5-IgG 4-2; h99IgG4-3, h99-IgG4-3 correspond to the molecule h99B5-IgG 4-3; h99IgG4-L3H3, H99-IgG4-L3H3 corresponds to the molecule H99B5-IgG4-L3H 3; h99IgG4-gen, 99-IgG4-gen corresponds to molecule 99B5-IgG 4; 100IgG4 corresponds to molecule 100-IgG 4; 5F9G4 corresponds to the molecule h5F 9-G4; H100-G4-1 corresponds to H100-G4-1, H100-G4-2 corresponds to H100-G4-2; H100-G4-3 corresponds to H100-G4-3; H100-G4-4 corresponds to H100-G4-4; H100-G4-5 corresponds to H100-G4-5; H100-G4-6 corresponds to H100-G4-; H100-G4-7 corresponds to H100-G4-7; H100-G4-8 corresponds to H100-G4-8; H100-G4-9 corresponds to H100-G4-9; H100-G4-10 corresponds to H100-G4-10; h81-IgG 1-chimeric corresponds to 81C1-IgG 1; h81-IgG1-1 corresponds to h81C1-IgG 1-1; h81-IgG1-2 corresponds to h81C1-IgG 1-2; h81-IgG1-3 corresponds to h81C1-IgG 1-3; h81-IgG1-4 corresponds to h81C1-IgG 1-4; h81-IgG1-5 corresponds to h81C1-IgG 1-5; h81-IgG 4-chimeric corresponds to 81C1-IgG 4; h81-IgG4-2 corresponds to h81C1-IgG 4-2; h81-IgG4-1 corresponds to h81C1-IgG 4-1; h81-IgG4-5 corresponds to h81C1-IgG 4-5.

Example 7: expression level of CD47 in tumor cells

The CD47 expression level of blood tumor and solid tumor cell lines such as Raji, Daudi, Ramous, HL60, MDA-MB231, SHP-77 and the like and RBC is measured by a flow cytometer (BDFACSCELESTaCellAnalyzer). The specific implementation is as follows:

1. the cells were observed under a microscope, rounded and clear, and collected under normal conditions, counted, and centrifuged at 1500rpm for 5 min.

2. The supernatant was discarded, and after each cell was resuspended in PBS (Hyclone, SH30256.01), the cells were added to a 96-well plate at 25X 10/well⁴Cells, 50 μ L per well.

3. At the same time, 10. mu.g/mL and 50. mu.L of the corresponding antibody (h5F9-G1, HIgG) was added to each well. Incubate for 30min at room temperature in the dark.

4. After the incubation was completed, the cells were washed 3 times by centrifugation at 1500rpm for 5min, and Alexa Fluor 647 AffinipurGoat Anti-Human IgG + IgM (H + L) (Jackson, 109605044) was added. Incubate for 30min at room temperature in the dark.

5. After the incubation was completed, the supernatant was discarded after 3 washes by centrifugation at 1500rpm for 5min, 100. mu.L of PBS was added to each well, and the data was analyzed by flow cytometry (Beckman, cytoflex) and plotted using GraphPadprism.

The results show (FIGS. 6a and 6b) that CD47 is highly expressed in hematological tumor cell lines such as Daudi, CCRF-CEM, HL60, Raji, PC-3, Jurkat, etc., and in solid tumor cell lines such as MDA-MB-231, A431, etc. The humanized CD47 antibody has wide application foreground.

Example 8: detection of Effect of the humanized CD47 antibody of the present invention on activation of macrophage phagocytic tumor cells (ADCP)

MDM isolation induction of effector cells: extracting 20 persons of venous blood to separate PBMC, separating monocyte, adding GM-CSF/M-CSF to induce differentiation MDM, and identifying differentiated macrophage MDM by CD11b, CD14, CD45, CD163 and CD206 biomarkers after 1 week; ADCP: removing target cell HL-60 from the incubator, collecting the cells into a 15ml centrifuge tube, centrifuging, discarding the supernatant, resuspending the cells in PBS, counting, staining the target cells with PKH26(SIGMA-ALDRICH), standing at 37 deg.C&5％CO₂Culturing overnight; taking out the target cells from the culture medium the next day, centrifugally collecting the cells, discarding the supernatant, using the complete culture medium for resuspension, and counting; taking target cells, adding complete culture medium, adding cells and sample to be testedPutting the mixture into a corresponding 96-well plate, 50000 cells/well; incubating for 0.5h at room temperature; taking out effector cells (MDM) from a culture medium, collecting the supernatant, adding PBS for washing, adding Accutase for digesting the MDM, removing the MDM from the wall, adding complete culture medium with the same volume for terminating digestion, transferring the cell suspension into a centrifuge tube, and centrifuging the cell supernatant and the digested cells for 10min according to 300 g; MDM cells were added to a 96-well plate corresponding to the target cells by adding a corresponding volume of MDM cells to the complete medium. Effector cells: target cells were incubated at 37 ℃ for 4h at 1: 1; adding Accutase, observing whether the adherent cells are digested under a microscope (about 15min), taking out the cells, transferring the cells to another plate, and adding the removed cell suspension into the corresponding hole again after the cells are completely digested; centrifuging, adding detection antibody CD11b, and incubating at 4 deg.C for 15 min; adding PBS to each hole, centrifuging, removing supernatant, and adding PBS for heavy suspension; detecting by a flow cytometer; and (3) data analysis: % Phagocytosis byMDMS { (PKH26+&CD11b+cells)/AllPKH26+cells}×100％。

FIG. 7a shows that the humanized antibodies h99B5-IgG4-1, h99B5-IgG4-2, h99B5-IgG4-3, h100-IgG4-1, h100-IgG4-4, h100-IgG4-5, h100-IgG4-6, h100-IgG4-7, etc., of CD47 of the present invention all show stronger activity than h5F9-G4 in the Jurkat tumor model. The CD47 humanized antibody of the invention has potential killing and treatment effects on blood tumor over expressing CD 47.

FIG. 7B shows that the humanized antibodies h99B5-IgG4-1, h99B5-IgG4-2, h99B5-IgG4-3, h100-IgG4-1, h100-IgG4-2, h100-IgG4-3, h100-IgG4-4, h100-IgG4-5, h100-IgG4-6, h100-IgG4-7, etc., of the CD47 of the present invention all show stronger activity in the SHP-77 tumor model than h5F 9-G4. The CD47 humanized antibody of the invention has potential killing and treatment effects on solid tumors over-expressing CD 47.

Example 9: ADCC Activity of the humanized antibody of the present invention on tumor cells

The (RBC) ADCC activity of the antibody against tumor cells Raji was examined by Jurkat-Fc γ RIIIa-NFAT-Luciferase cells as effector cells to assess their mediated killing of tumor cells.

The specific implementation is as follows:

1. the target cells were observed under a microscope, and the cells were collected under normal conditions with the cells round and clear, counted, and centrifuged at 1500rpm for 5 min.

2. Discarding the supernatant, resuspending the target cells in 1640+ 0.5% FBS, mixing well, adding a white flat-bottom sterile plate, 1X 10⁴cells/well, 25. mu.l per well.

3. The prepared 4-fold final concentration of the antibody to be tested was added to the plate at 25. mu.l/well, 37 ℃ and 5% CO₂Incubate 30 min.

4. After incubation was complete, Jurkat-Fc γ RIIIa-NFAT-Luciferase cells, 6X 10 cells were added⁴cells/well, 50. mu.l per well. The volume is less than 100 μ l (control well), and is supplemented to 100 μ l with 1640+ 0.5% FBS medium, mixed well, and then 5% CO at 37 deg.C₂Incubate for 6 h.

5. Add 40. mu.l Bio-glo to each well, incubate for 5min in the dark, read chemiluminescence with a microplate reader.

The results in FIG. 8 show that the humanized antibodies, in particular h99B5-IgG4-2 and h99B5-IgG4-3, both had significant ADCC tumor killing activity against Raji cells, whereas the control antibody h5F9 did not have ADCC activity.

Sequence listing

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

<120> humanized CD47 antibody or antigen binding fragment thereof and application

<160> 139

<170> SIPOSequenceListing 1.0

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Lys Tyr Pro Ala Gly Gln Gly Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 2

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln His Phe Trp Asp Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

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<213> Artificial Sequence (Artificial Sequence)

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

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Tyr Pro Ala Gly Gln Gly Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

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<213> Artificial Sequence (Artificial Sequence)

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

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Arg Tyr Pro Ala Gly Gln Gly Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 5

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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

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

20 25 30

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

35 40 45

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

50 55 60

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

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Glu Asp Phe Ala Thr Tyr Phe Cys Gln His Phe Trp Asp Thr Pro Trp

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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

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<213> Artificial Sequence (Artificial Sequence)

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

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Arg Tyr Pro Ala Gly Gln Gly Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 7

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

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 Arg Ala Ser Gly Asn Ile His Asn Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln His Phe Trp Asp Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 8

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Tyr Pro Ala Gly Gln Gly Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 9

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

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 Arg Ala Ser Gly Asn Ile His Asn Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Asp Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 10

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

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

1 5 10 15

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

20 25 30

Tyr Ile Asn Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Ala Pro Gly Ser Val Ile Thr His Tyr Asn Glu Met Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Ser Asp Phe Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 11

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 12

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

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

1 5 10 15

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

20 25 30

Tyr Ile Asn Trp Ile Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Ala Pro Gly Ser Val Ile Thr His Tyr Asn Glu Met Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Ser Asp Phe Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 13

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 14

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

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

1 5 10 15

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

20 25 30

Tyr Ile Asn Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Ala Pro Gly Ser Val Ile Thr His Tyr Asn Glu Met Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Ser Asp Phe Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 15

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Ala Pro Gly Ser Val Ile Thr His Tyr Asn Glu Met Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Ser Asp Phe Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 16

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 17

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Ala Pro Gly Ser Val Ile Thr His Tyr Asn Glu Met Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Ser Asp Phe Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 18

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

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 Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

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

35 40 45

Tyr Tyr Thr Ser Ser Leu His Ser 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 Tyr Ser Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 19

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Ala Pro Gly Thr Val Ile Thr His Phe Asn Glu Met Phe

50 55 60

Arg Ala Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Ser Asp Phe Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 20

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

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 Gln Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

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

35 40 45

Tyr Tyr Ser Ser Ser Leu His Ser 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 Tyr Arg Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 21

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Ala Pro Gly Thr Val Ile Thr His Phe Asn Glu Met Phe

50 55 60

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

65 70 75 80

Ile Glu Leu Ser Ser Leu Ser Ser Asp Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Ser Asp Phe Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 22

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Arg Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 23

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Gln Val Gln Leu Val Gln Ser Gly Asp Glu Val Val Glu Ala Ser Val

1 5 10 15

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

20 25 30

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

35 40 45

Ile Ala Pro Gly Thr Val Ile Thr His Phe Asn Glu Met Phe Arg Ala

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser

115

<210> 24

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Arg Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 25

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Ala Pro Gly Thr Val Ile Thr His Phe Asn Glu Met Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Ser Asp Phe Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 26

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Arg Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 27

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Ala Pro Gly Thr Val Ile Thr His Phe Asn Glu Met Phe

50 55 60

Arg Ala Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Ser Asp Phe Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 28

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

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 Ser Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

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

35 40 45

Tyr Tyr Ser Ser Ser Leu His Ser 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 Tyr Arg Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 29

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Ala Pro Gly Thr Val Ile Thr His Phe Asn Glu Met Phe

50 55 60

Arg Ala Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Ser Asp Phe Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 30

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Arg Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 31

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Ala Pro Gly Thr Val Ile Thr His Phe Asn Glu Met Phe

50 55 60

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

65 70 75 80

Ile Glu Leu Ser Ser Leu Ser Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Ser Asp Phe Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 32

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Arg Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 33

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

Gln Val Gln Leu Val Gln Ser Gly Asp Glu Val Val Glu Ala Ser Val

1 5 10 15

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

20 25 30

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

35 40 45

Ile Ala Pro Gly Thr Val Ile Thr His Phe Asn Glu Met Phe Arg Ala

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser

115

<210> 34

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Arg Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 35

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Ala Pro Gly Thr Val Ile Thr His Phe Asn Glu Met Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Ser Asp Phe Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 36

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Arg Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 37

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 37

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Ala Pro Gly Thr Val Ile Thr His Phe Asn Glu Met Phe

50 55 60

Arg Ala Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Ser Asp Phe Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 38

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 38

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 Arg Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Arg Lys Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 39

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 39

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

1 5 10 15

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

20 25 30

Thr Leu His Trp Val Arg Gln Ala Pro Gly Arg Ser Leu Glu Trp Met

35 40 45

Gly Val Val Asn Pro Asn Ile Gly Ala Thr Thr Tyr Asn Gln Met Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser

115

<210> 40

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 40

Glu Ile Val Leu Thr Gln Ser Pro Val Phe Gln Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Asn Asn

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Asp Ser Gln Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ala Gly Thr Tyr Phe Cys Gln Gln Thr Asn Ser Trp Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 41

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 41

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

1 5 10 15

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

20 25 30

Thr Leu His Trp Val Lys Gln Ser Pro Gly Arg Ser Leu Glu Trp Ile

35 40 45

Gly Val Val Asn Pro Asn Ile Gly Ala Thr Thr Tyr Asn Gln Met Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser

115

<210> 42

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 42

Asp Ile Val Leu Thr Gln Ser Pro Val Thr Leu Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Asn Asn

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Asp Ser Gln Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ala Gly Met Tyr Phe Cys Gln Gln Thr Asn Ser Trp Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 43

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 43

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

1 5 10 15

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

20 25 30

Thr Leu His Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Trp Met

35 40 45

Gly Val Val Asn Pro Asn Ile Gly Ala Thr Thr Tyr Asn Gln Met Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser

115

<210> 44

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 44

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

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Asn Asn

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Asp Ser Gln Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ala Gly Thr Tyr Phe Cys Gln Gln Thr Asn Ser Trp Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 45

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 45

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

1 5 10 15

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

20 25 30

Thr Leu His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Val Asn Pro Asn Ile Gly Ala Thr Thr Tyr Asn Gln Met Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser

115

<210> 46

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 46

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 Asn Asn Asn

20 25 30

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

35 40 45

Lys Tyr Asp 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 Gln Thr Asn Ser Trp Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 47

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 47

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

1 5 10 15

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

20 25 30

Thr Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Val Asn Pro Asn Ile Gly Ala Thr Thr Tyr Asn Gln Met Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser

115

<210> 48

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 48

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

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Asn Asn Asn

20 25 30

Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Asp Ser Gln Ser Ile Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Thr Asn Ser Trp Pro Phe

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 49

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 49

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 50

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 50

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 Arg Ala Ser Gly Asn Ile His Asn Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 51

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 51

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

Thr Lys Tyr Pro Ala Gly Gln Gly Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 52

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 52

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 Arg Ala Ser Gly Asn Ile His Asn Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Gly Ser Tyr Phe Cys Gln His Phe Trp Asp Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 53

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 53

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Arg Val Thr Ile Thr Arg 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

Thr Lys Tyr Pro Ala Gly Gln Gly Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 54

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 54

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 Arg Ala Ser Gly Asn Ile His Asn Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 55

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 55

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Asp 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

Thr Lys Tyr Pro Ala Gly Gln Gly Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 56

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 56

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Asp 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

Thr Lys Tyr Pro Ala Gly Gln Gly Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 57

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 57

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Lys Asp Arg Val Thr Ile Thr Arg 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

Thr Lys Tyr Pro Ala Gly Gln Gly Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 58

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 58

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

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

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

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

65 70 75 80

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

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 59

<211> 330

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 59

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 60

<211> 327

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 60

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

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

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

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

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

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

210 215 220

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

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

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

260 265 270

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

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 61

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 61

Ser Tyr Trp Ile Asn

1 5

<210> 62

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 62

Arg Ile Asp Leu Tyr Asp Ser Glu Thr His Tyr Ser Gln Lys Phe Lys

1 5 10 15

Asp

<210> 63

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 63

Tyr Pro Ala Gly Gln Gly Ala Trp Phe Ala Tyr

1 5 10

<210> 64

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 64

Arg Ala Ser Gly Asn Ile His Asn Tyr Leu Ala

1 5 10

<210> 65

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 65

Asn Ala Lys Thr Leu Ala Asp

1 5

<210> 66

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 66

Gln His Phe Trp Asp Thr Pro Trp Thr

1 5

<210> 67

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 67

Ser Tyr Tyr Ile Asn

1 5

<210> 68

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 68

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

1 5 10 15

Gly

<210> 69

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 69

Ser Asp Phe Tyr Gly Met Asp Tyr

1 5

<210> 70

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 70

Ser Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 71

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 71

Tyr Thr Ser Ser Leu His Ser

1 5

<210> 72

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 72

Gln Gln Tyr Ser Lys Leu Pro Trp Thr

1 5

<210> 73

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 73

Thr Phe Tyr Ile Asn

1 5

<210> 74

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 74

Arg Ile Ala Pro Gly Thr Val Ile Thr His Phe Asn Glu Met Phe Arg

1 5 10 15

Ala

<210> 75

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 75

Tyr Ser Ser Ser Leu His Ser

1 5

<210> 76

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 76

Gln Gln Tyr Arg Lys Leu Pro Trp Thr

1 5

<210> 77

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 77

Glu Tyr Thr Leu His

1 5

<210> 78

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 78

Val Val Asn Pro Asn Ile Gly Ala Thr Thr Tyr Asn Gln Met Phe Lys

1 5 10 15

Gly

<210> 79

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 79

Arg Ala Tyr Leu Asp Tyr

1 5

<210> 80

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 80

Arg Ala Ser Gln Ser Ile Asn Asn Asn Leu His

1 5 10

<210> 81

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 81

Tyr Asp Ser Gln Ser Ile Ser

1 5

<210> 82

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 82

Gln Gln Thr Asn Ser Trp Pro Phe Thr

1 5

<210> 83

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 83

caggtgcagc tggtgcagcc aggagctgag gtggtgaagc ctggcgcctc cgtgaagctg 60

tcttgtaaga cctccgacta caccatcaca tcttattgga tcaactgggt gagacaggct 120

ccaggacagg gactggagtg gatgggaagg atcgacctgt acgatagcga gacccattat 180

tctcagaagt tcaaggacag ggtgaccatg acagtggata agtccaccac aagcacatac 240

ctggagctga ggtctctgcg gtccgacgat accgccgtgt actattgcac aaagtaccca 300

gctggacagg gagcttggtt tgcttattgg ggacagggca ccctggtgac agtgtccgct 360

<210> 84

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 84

gacatccaga tgacacagag cccttccagc ctgagcgcct ctgtgggcga tagggtgacc 60

atcacatgtc gggcctctgg caacatccat aattacctgg cttggtatag acagaagccc 120

ggcaagtccc ctcagctgct ggtgtacaac gccaagaccc tggctgacgg agtgccaagc 180

cgcttttccg gaagcggctc tggcacagat tacaccctga caatctcttc cctgcagcca 240

gaggacttcg ctacctattt ttgccagcac ttctgggata ccccatggac attcggcgga 300

ggaaccaagc tggagatcaa g 321

<210> 85

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 85

caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggagcctc cgtgaagctg 60

tcttgtaaga cctccgacta caccatcaca tcctattgga tcaactgggt gagacaggct 120

cctggacagg gactggagtg gatgggaagg atcgacctgt acgatagcga gacacattat 180

tctcagaagt tcaaggacag ggtgaccatg acagtggata agtccaccac aagcacctac 240

ctggagctga ggtctctgcg gtccgacgat acagccgtgt actattgcgc tagataccca 300

gctggacagg gagcttggtt tgcttattgg ggccagggca ccctggtgac cgtgtccgcc 360

<210> 86

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 86

caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggagcctc cgtgaagctg 60

tcttgtaaga cctccgacta caccatcaca tcctattgga tcaactggat cagacaggct 120

cctggacagg gactggagtg gatgggaagg atcgacctgt acgatagcga gacccattat 180

tctcagaagt tcaaggacag ggtgaccatg acagtggata agtccaccac aagcacatac 240

ctggagctga ggtctctgcg gtccgacgat accgccgtgt actattgcac aagataccca 300

gctggccagg gcgcctggtt tgcttattgg ggccagggca ccctggtgac cgtgtccgcc 360

<210> 87

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 87

gacatccaga tgacacagag cccttccagc ctgagcgcct ctgtgggcga tagggtgacc 60

atcacatgtc gggcctctgg caacatccat aattacctgg cttggtatca gcagaagccc 120

ggcaagtccc ctaagctgct ggtgtacaac gccaagaccc tggctgacgg cgtgccaagc 180

aggttttccg gcagcggctc tggcacagat tacaccctga caatctcttc cctgcagcca 240

gaggacttcg ctacctattt ttgccagcac ttctgggata ccccatggac attcggcgga 300

ggaaccaagc tggagatcaa g 321

<210> 88

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 88

caggtgcagc tggtgcagcc aggagctgag gtgaagaagc ctggagcctc cgtgaagctg 60

tcttgtaaga cctccgacta caccatcaca tcttattgga tcaactggat cagacaggct 120

ccagagcagg gactggagtg gatgggccgc atcgacctgt acgatagcga gacccattat 180

tctcagaagt tcaaggacag ggtgaccatg acagtggata agtccaccac aagcacatac 240

ctggagctga ggtctctgcg gtccgacgat accgccgtgt actattgcac aagatacccc 300

gctggccagg gcgcctggtt tgcttattgg ggacagggca ccctggtgac agtgtccgct 360

<210> 89

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 89

gacatccaga tgacacagag cccttccagc ctgagcgcct ctgtgggcga tagggtgacc 60

atcacatgtc gggcctctgg caacatccat aattacctgg cttggtatca gcagaagccc 120

ggcaagtccc ctcagctgct ggtgtacaac gccaagaccc tggctgacgg cgtgccaagc 180

aggttttccg gcagcggctc tggcacagat tacaccctga caatctcttc cctgcagcca 240

gaggacttcg ctacctattt ttgccagcac ttctgggata ccccatggac attcggcgga 300

ggaaccaagc tggagatcaa g 321

<210> 90

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 90

caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggagcctc cgtgaaggtg 60

tcttgtaagg cctccggcta caccttcaca tcctattgga tcaactgggt gagacaggct 120

cctggacagg gactggagtg gatgggaagg atcgacctgt acgatagcga gacacattat 180

tctcagaagt ttaaggacag ggtgaccatg accacagata cctccacaag caccgcctac 240

atggagctga ggtctctgcg gtccgacgat acagccgtgt actattgcgc tagataccca 300

gctggacagg gagcttggtt tgcttattgg ggccagggca cactggtgac cgtgagcgcc 360

<210> 91

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 91

gacatccaga tgacacagag cccttccagc ctgtccgcct ccgtgggcga tagggtgacc 60

atcacatgtc gggcctctgg caacatccat aattacctgg cttggtatca gcagaagccc 120

ggcaaggccc ctaagctgct gctgtacaac gccaagaccc tggctgacgg agtgccatcc 180

aggttctccg gaagcggctc tggcacagat tataccctga caatctcttc cctgcagcca 240

gaggactttg ccacctacta ttgccagcac ttctgggata ccccctggac atttggcggc 300

ggcaccaagc tggagatcaa g 321

<210> 92

<211> 351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 92

caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggagcttc cgtgaaggtg 60

agctgtaagg cctctggcta caccttcaca agctactata tcaactggat caagcagagg 120

ccaggacagg gactggagtg gatcggaagg atcgctcctg gctctgtgat cacacattac 180

aacgagatgt tcaagggcag ggtgaccctg acagtggaca ccagcacatc taccgtgtac 240

atcgagctgt ccagcctgag atctgaggat accgccgtgt atttctgcgc tcgctccgac 300

ttttacggca tggattattg gggccagggc acatccgtga ccgtgtcttc c 351

<210> 93

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 93

gacatccaga tgacccagac cacatccagc ctgtccgcca gcgtgggcga tagggtgaca 60

atctcttgtt ccgcctccca gggcatctct aactacctga attggtatca gcagaagccc 120

ggcaaggccc ctaagctgct gatctactat acctcttccc tgcattctgg agtgccatcc 180

cggttctctg gatccggaag cggaaccgac tactccacca caatcagctc tctgcagcca 240

gaggatatcg ctacatacta ttgccagcag tatagcaagc tgccctggac ctttggcggc 300

ggcacaaagc tggagatcaa g 321

<210> 94

<211> 351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 94

caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggagcttc cgtgaaggtg 60

agctgtaagg cctctggcta caccttcaca agctactata tcaactggat caggcagagg 120

ccaggacagg gactggagtg gatgggcagg atcgctcctg gctctgtgat cacacattac 180

aacgagatgt tcaagggcag agtgaccctg acagtggaca ccagcacatc taccgtgtac 240

atggagctgt ccagcctgag atctgaggat accgccgtgt atttctgcgc tcgctccgac 300

ttttacggca tggattattg gggccagggc acatccgtga ccgtgtcttc c 351

<210> 95

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 95

gacatccaga tgacccagag cacatccagc ctgtccgcca gcgtgggcga tagggtgacc 60

atcacatgtt ctgcttccca gggcatctct aactacctga attggtatca gcagaagccc 120

ggcaaggccc ctaagctgct gatctactat acctcttccc tgcatagcgg agtgccatct 180

cggttcagcg gatctggatc cggaaccgac tactctacca caatcagctc tctgcagcca 240

gaggatatcg ctacatacta ttgccagcag tattccaagc tgccctggac ctttggcggc 300

ggcacaaagc tggagatcaa g 321

<210> 96

<211> 351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 96

caggtgcagc tggtgcagag cggcgacgat ctggtgaagc caggcgcctc cgtgaagctg 60

agctgtaagg cttctggcta caccttcaca agctactata tcaactggat caagcagaga 120

ccaggacagg gactggagtg gatcggaagg atcgctcctg gatctgtgat cacacattac 180

aacgagatgt tcaagggcag ggtgaccctg acacgggaca ccagcacatc taccgtgtac 240

atcgagctgt ccagcctgag gtctgaggat accgccgtgt atttctgcgc tagatccgac 300

ttttacggca tggattattg gggccagggc acatccgtga ccgtgtcttc c 351

<210> 97

<211> 351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 97

caggtgcagc tggtgcagag cggcgacgag gtggtgaagc caggagcctc cgtgaaggtg 60

agctgtaagg cttctggcta caccttcaca agctactata tcaactgggt gagacaggct 120

ccaggacagg gactggagtg gatgggaagg atcgctcctg gctctgtgat cacacattac 180

aacgagatgt tcaagggcag ggtgaccatg acagtggaca ccagcacatc taccgtgtac 240

atggagctgt ccagcctgag gtctgaggat accgccgtgt atttctgcgc tcggtccgac 300

ttttacggca tggattattg gggccagggc acatccgtga ccgtgtcttc c 351

<210> 98

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 98

gacatccaga tgacccagac cacatccagc ctgtccgcca gcgtgggcga tagggtggcc 60

atcacatgtt ctgcttccca gggcatctct aactacctga attggtatca gcagaagccc 120

ggcaaggctc ctaagctgct gatctactat acctcttccc tgcatagcgg agtgccatct 180

cggttcagcg gatctggatc cggaaccgac tttaccctga caatcagctc tctgcagcca 240

gaggatatcg ccacatacta ttgccagcag tactccaagc tgccctggac ctttggcggc 300

ggcacaaagc tggagatcaa g 321

<210> 99

<211> 351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 99

caggtgcagc tggtgcagag cggagctgag gtgaagaagc caggagcttc cgtgaaggtg 60

agctgtaagg cctctggcta caccttcaca agctactata tcaactgggt gaggcaggct 120

ccaggacagg gactggagtg gatgggcaga atcgctcctg gctctgtgat cacacattac 180

aatgagatgt ttaagggcag ggtgaccatg acacgggaca ccagcacatc taccgtgtat 240

atggagctgt ccagcctgag atctgaggat accgccgtgt actattgcgc tcgctccgac 300

ttttacggca tggattattg gggccagggc acatccgtga ccgtgtcttc c 351

<210> 100

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 100

gacatccaga tgacccagag cccttccagc ctgtccgcca gcgtgggcga tagggtgacc 60

atcacatgtt ctgcttccca gggcatctcc aactacctga attggtatca gcagaagccc 120

ggcaaggccc ctaagctgct gatctactat acatcttccc tgcatagcgg cgtgccatct 180

cggttcagcg gatctggatc cggaaccgac ttcaccttta caatcagctc tctgcagcca 240

gaggatatcg ctacatacta ttgccagcag tacagcaagc tgccctggac ctttggcggc 300

ggcacaaagc tggagatcaa g 321

<210> 101

<211> 344

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 101

agctggtgca gagcggagct gaggtgaaga agccaggagc ctccgtgaag gtgtcttgta 60

aggcctccgg ctacaccttc accaccttct acatcaactg ggtgaggcag gctccaggac 120

agggactgga gtggatggga aggatcgctc ctggcaccgt gatcacacat ttcaatgaga 180

tgtttagggc ccgggtgacc atgacaagag acacctctat ctccacagct tacatggagc 240

tgagcagact gcgctctgac gatacagccg tgtactattg cgctcgctct gacttttacg 300

gcatggatta ttggggccag ggcacctccg tgacagtgtc cagc 344

<210> 102

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 102

gacatccaga tgacccagtc tccttccagc ctgagcgcct ctgtgggcga tagggtgacc 60

atcacatgtc aggcttccca gggcatcagc aactacctga attggtatca gcagaagccc 120

ggcaaggccc ctaagctgct gatctactat tcttccagcc tgcattctgg agtgccatcc 180

aggttctccg gaagcggatc tggaaccgac ttcaccttta caatctcttc cctgcagcca 240

gaggatatcg ctacatacta ttgccagcag tacaggaagc tgccatggac ctttggcgga 300

ggcacaaagc tggagatcaa g 321

<210> 103

<211> 344

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 103

agctggtgca gagcggcgac gaggtggtgg agccaggagc ctccgtgaag gtgagctgta 60

aggcttctgg ctacaccttc accaccttct acatcaactg ggtgaggcag aggccaggac 120

agggactgga gtggatggga aggatcgctc ctggcaccgt gatcacacat ttcaatgaga 180

tgtttagagc ccgcgtgacc ctgacagtgg atacctccag ctctacagct tacatcgagc 240

tgtccagcct gtcttccgac gatacagccg tgtatttctg cgctagatct gacttttacg 300

gcatggatta ttggggccag ggcacctccg tgacagtgag ctct 344

<210> 104

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 104

gacatcgtgc tgacccagtc tccatccagc ctgagcgcct ctgtgggcga tagggtgacc 60

atcacatgtt ccgctagcca gggcatcagc aactacctga attggtatca gcagaagcca 120

aacggcacaa tcaagctgct gatctactat tcttccagcc tgcattctgg agtgccatcc 180

aggttctctg gatccggaag cggaaccgac tttaccctga caatctcttc cctgcagccc 240

gaggatatcg ccacatacta ttgccagcag taccggaagc tgccttggac ctttggcggc 300

ggcacaaagc tggagatcaa g 321

<210> 105

<211> 338

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 105

agctggtgca gtccggcgac gaggtggtgg aggcctctgt gaaggtgtcc tgtaaggcct 60

ccggctacac cttcaccacc ttctacatca actgggtgag gcagaggcca ggacagggac 120

tggagtggat gggaaggatc gctcctggca ccgtgatcac acatttcaat gagatgttta 180

gagcccgcgc taccatgaca gtggacacct ccatcagcac agcttacatc cagctgtcca 240

gcctgtcttc cgaggataca gccgtgtatt tctgcgctag aagcgacttt tacggcatgg 300

attattgggg ccagggcacc tctgtgacag tgagctct 338

<210> 106

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 106

gacatcgtgc tgacccagtc cccatccagc ctgtctgcct ccgtgggcga tagggtggcc 60

atcacatgtc aggctagcca gggcatctct aactacctga attggtatca gcagaagcca 120

aacggcacca tcaagctgct gatctactat tcttccagcc tgcattccgg agtgccaagc 180

aggttcagcg gatctggatc cggaaccgac tacaccctga caatctctaa tctgcagccc 240

gaggatatcg ctacatacta ttgccagcag tatcggaagc tgccttggac ctttggcggc 300

ggcacaaagc tggagatcaa g 321

<210> 107

<211> 344

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 107

agctggtgca gagcggcgac gaggtggtgg agccaggagc ctccgtgaag gtgagctgta 60

aggcttctgg ctacaccttc accaccttct acatcaactg ggtgaggcag aggccaggac 120

agggactgga gtggatggga aggatcgctc ctggcaccgt gatcacacat ttcaatgaga 180

tgtttagagc caaggctacc atgacagtgg ataccagcat ctctacagct tacatggagc 240

tgtccagcct gagatctgac gatacagccg tgtactattg cgctcgctcc gacttttacg 300

gcatggatta ttggggccag ggcacctctg tgacagtgtc ttcc 344

<210> 108

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 108

gacatcgtgc tgacccagtc cccatccagc ctgtctgcct ccgtgggcga tagggtggcc 60

atcacatgtc aggctagcca gggcatctct aactacctga attggtatca gcagaagcca 120

aacggcacca tcaagctgct gatctactat tcttccagcc tgcattccgg agtgccaagc 180

aggttcagcg gatctggatc cggaaccgac tacaccctga caatctctaa tctgcagccc 240

gaggatatcg ctacatacta ttgccagcag tatcggaagc tgccttggac ctttggcggc 300

ggcacaaagc tggagatcaa g 321

<210> 109

<211> 344

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 109

agctggtgca gtccggcgac gaggtgaagg agccaggagc ctctgtgaag gtgtcctgta 60

aggcctccgg ctacaccttc accaccttct acatcaactg gatcagacag gctccaggac 120

agggactgga gtggatggga aggatcgctc ctggcaccgt gatcacacat ttcaatgaga 180

tgtttagggc ccgggtgacc atgacaaggg atacctccat cagcacagct tacatggagc 240

tgtccagcct gtcttccgac gatacagccg tgtatttctg cgctagaagc gacttttacg 300

gcatggatta ttggggccag ggcacctctg tgacagtgag ctct 344

<210> 110

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 110

gacatcgtga tgacccagtc tccttccagc ctgagcgcct ctgtgggcga tagggtgacc 60

atcacatgtt ccgctagcca gggcatcagc aactacctga attggtatca gcagaagccc 120

aacggcgccc ctaagctgct gatctactat tcttccagcc tgcattctgg cgtgccatcc 180

aggttctctg gatccggaag cggaaccgac ttcaccttta caatctcttc cctgcagcca 240

gaggatatcg ctacatacta ttgccagcag tacaggaagc tgccatggac ctttggcgga 300

ggcacaaagc tggagatcaa g 321

<210> 111

<211> 344

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 111

agctggtgca gagcggagct gaggtgaaga agccaggagc ttccgtgaag gtgagctgta 60

aggcctctgg ctacaccttc accaccttct acatcaactg ggtgaggcag gctccaggac 120

agggactgga gtggatgggc agaatcgctc ctggcaccgt gatcacacat ttcaatgaga 180

tgtttagggc tcgggtgacc atgacaaggg acaccagcat ctctacagtg tacatggagc 240

tgtccagcct gagatctgag gatacagccg tgtactattg cgctcgctcc gacttttacg 300

gcatggatta ttggggccag ggcacctctg tgacagtgtc ttcc 344

<210> 112

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 112

gacatccaga tgacccagtc tccttccagc ctgagcgcct ctgtgggcga tagggtgacc 60

atcacatgtc gggcttccca gggcatcagc aactacctga attggtatca gcagaagccc 120

ggcaaggccc ctaagctgct gatctactat tcttccagcc tgcattctgg cgtgccatcc 180

agattctccg gcagcggctc tggcaccgac tttaccctga caatctcttc cctgcagcca 240

gaggatgtgg ctacatacta ttgccagaag tacaggaagc tgccatggac ctttggcgga 300

ggcacaaagc tggagatcaa g 321

<210> 113

<211> 344

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 113

agctggtgca gagcggcgac gaggtggtgg agccaggagc ctccgtgaag gtgagctgta 60

aggcttctgg ctacaccttc accaccttct acatcaactg ggtgaggcag aggccaggac 120

agggactgga gtggatggga aggatcgctc ctggcaccgt gatcacacat ttcaatgaga 180

tgtttagagc tcgcgtgacc ctgacagtgg acacctccag ctctacagtg tacatcgagc 240

tgtccagcct gtcttccgag gatacagccg tgtatttctg cgctagatct gacttttacg 300

gcatggatta ttggggccag ggcacctccg tgacagtgag ctct 344

<210> 114

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 114

gacatcgtgc tgacccagtc tccatccagc ctgagcgcct ctgtgggcga tagggtgacc 60

atcacatgtc gggcttccca gggcatcagc aactacctga attggtatca gcagaagcca 120

aacggcacaa tcaagctgct gatctactat tcttccagcc tgcattctgg agtgccatcc 180

agattctccg gaagcggatc tggcaccgac tttaccctga caatctcttc cctgcagccc 240

gaggatgtgg ccacatacta ttgccagaag taccgcaagc tgccttggac ctttggcggc 300

ggcacaaagc tggagatcaa g 321

<210> 115

<211> 338

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 115

agctggtgca gtccggcgac gaggtggtgg aggcctctgt gaaggtgtcc tgtaaggcct 60

ccggctacac cttcaccacc ttctacatca actgggtgag gcagaggcca ggacagggac 120

tggagtggat gggaaggatc gctcctggca ccgtgatcac acatttcaat gagatgttta 180

gagcccgcgc taccatgaca gtggacacct ccatcagcac agtgtacatc cagctgtcca 240

gcctgtcttc cgaggataca gccgtgtatt tctgcgctag aagcgacttt tacggcatgg 300

attattgggg ccagggcacc tctgtgacag tgagctct 338

<210> 116

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 116

gacatcgtgc tgacccagtc cccatccagc ctgtctgcct ccgtgggcga tagggtggcc 60

atcacatgtc gggctagcca gggcatctct aactacctga attggtatca gcagaagcca 120

aacggcacca tcaagctgct gatctactat tcttccagcc tgcattccgg cgtgcccagc 180

agattcagcg gatctggatc cggaaccgac tacaccctga caatctctaa tctgcagccc 240

gaggatgtgg ctacatacta ttgccagaag tatcgcaagc tgccttggac ctttggcggc 300

ggcacaaagc tggagatcaa g 321

<210> 117

<211> 344

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 117

agctggtgca gagcggcgac gaggtggtgg agccaggagc ctccgtgaag gtgagctgta 60

aggcttctgg ctacaccttc accaccttct acatcaactg ggtgaggcag aggccaggac 120

agggactgga gtggatggga aggatcgctc ctggcaccgt gatcacacat ttcaatgaga 180

tgtttagagc caaggctacc atgacagtgg acaccagcat ctctacagtg tacatggagc 240

tgtccagcct gagatctgag gatacagccg tgtactattg cgctcgctcc gacttttacg 300

gcatggatta ttggggccag ggcacctctg tgacagtgtc ttcc 344

<210> 118

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 118

gacatcgtga tgacccagtc cccatccagc ctgtctgcct ccgtgggcga tagggtggcc 60

atcacatgtc gggctagcca gggcatctct aactacctga attggtatca gcagaagcca 120

aacggcacca tcaagctgct gatctactat tcttccagcc tgcattccgg cgtgcccagc 180

agattcagcg gatctggatc cggaaccgac tacaccctga caatctctaa tctgcagccc 240

gaggatgtgg ctacatacta ttgccagaag tatcgcaagc tgccttggac ctttggcggc 300

ggcacaaagc tggagatcaa g 321

<210> 119

<211> 344

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 119

agctggtgca gtccggcgac gaggtgaagg agccaggagc ctctgtgaag gtgtcctgta 60

aggcctccgg ctacaccttc accaccttct acatcaactg gatcagacag gctccaggac 120

agggactgga gtggatggga aggatcgctc ctggcaccgt gatcacacat ttcaatgaga 180

tgtttagggc ccgggtgacc atgacaaggg acacctccat cagcacagtg tacatggagc 240

tgtccagcct gtcttccgag gatacagccg tgtatttctg cgctagaagc gacttttacg 300

gcatggatta ttggggccag ggcacctctg tgacagtgag ctct 344

<210> 120

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 120

gacatcgtga tgacccagtc tccttccagc ctgagcgcct ctgtgggcga tagggtgacc 60

atcacatgtc gggcttccca gggcatcagc aactacctga attggtatca gcagaagccc 120

aacggcgccc ctaagctgct gatctactat tcttccagcc tgcattctgg cgtgccatcc 180

agattctccg gcagcggctc tggcaccgac tttaccctga caatctcttc cctgcagcca 240

gaggatgtgg ctacatacta ttgccagaag tacaggaagc tgccatggac ctttggcgga 300

ggcacaaagc tggagatcaa g 321

<210> 121

<211> 345

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 121

caggtgcagc tggtgcagtc aggagcagaa gtggtgaaac ccggagccag cgtgaaggtg 60

tcttgcaagg ccagcggcta caccttcacc gagtacacac tgcattgggt ccggcaggct 120

ccaggaagat ctctcgagtg gatgggcgtg gtgaacccta acatcggcgc caccacctac 180

aaccagatgt tcaagggcaa ggtcaccatg acagtggaca ccagcaccag caccgtgtac 240

atggagctga gcagcctgag aagcgaggac acagccgtgt actattgcgc caggagagcc 300

tacctggact attggggcca gggcacaacc ctgaccgtgt ctagc 345

<210> 122

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 122

gagatcgtgc tgacccagag cccagtgttc cagtcagtga cccccaagga gaaggtcacc 60

atcacttgcc gggccagcca gagcatcaac aacaacctgc attggtacca gcagaagccc 120

gaccagagcc ctaagctgct gatcaagtac gatagccaga gcatcagcgg cgtgccttct 180

agattcagcg gaagcggcag cggcacagat ttcaccctga ccatcaacag cctggaggca 240

gaggacgcag gcacctactt ttgccagcag accaactctt ggcccttcac cttcggcagc 300

ggcaccaagc tggagatcaa g 321

<210> 123

<211> 345

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 123

caggtgcagc tggtgcagtc aggagcagaa gtggtgaaac ccggagccag cgtgaaggtg 60

tcttgcaagg ccagcggcta caccttcacc gagtacaccc tgcattgggt gaagcagagc 120

ccaggaagaa gcctcgagtg gatcggagtg gtgaacccta acatcggcgc caccacctac 180

aaccagatgt tcaagggcaa ggtcaccatg acagtggaca ccagcaccag caccgtgtac 240

atggagctga gcagcctgag aagcgaggac acagccgtgt actattgcgc caggagagcc 300

tacctggact attggggcca gggcacaacc ctgaccgtgt ctagc 345

<210> 124

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 124

gacatcgtgc tgacccagtc tccagtgacc ctgagcgtga cacccaagga gaaggtcacc 60

atcacttgcc gggccagcca gagcatcaac aacaacctgc attggtacca gcagaagccc 120

gaccagagcc ctaagctgct gatcaagtac gatagccaga gcatcagcgg cgtgccttct 180

agattcagcg gaagcggcag cggcacagat ttcaccctga ccatcaacag cctggagacc 240

gaggacgccg gcatgtactt ttgccagcag accaactctt ggcccttcac cttcggcagc 300

ggcaccaagc tggagatcaa g 321

<210> 125

<211> 345

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 125

caggtgcagc tggtgcagtc aggagcagaa gtgaagaagc ccggagccag cgtgaaagtg 60

tcttgcaagg ccagcggcta caccttcacc gagtacacac tgcattgggt ccggcaggct 120

ccaggacagt ctctcgagtg gatgggcgtg gtgaacccta acatcggcgc caccacctac 180

aaccagatgt tcaagggccg cgtgaccatg accgtggata ccagcaccag caccgtgtac 240

atggagctga gcagcctgag aagcgaggac acagccgtgt actattgcgc caggagagcc 300

tacctggact attggggcca gggcacaacc ctgaccgtgt ctagc 345

<210> 126

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 126

gacatcgtgc tgacccagag cccagtgttc cagtcagtga cccccaagga gaaggtcacc 60

atcacttgcc gggccagcca gagcatcaac aacaacctgc attggtacca gcagaagccc 120

gaccagagcc ctaagctgct gatcaagtac gatagccaga gcatcagcgg cgtgccttct 180

agattcagcg gaagcggcag cggcacagat ttcaccctga ccatcaacag cctggaggca 240

gaggacgcag gcacctactt ttgccagcag accaactctt ggcccttcac cttcggcagc 300

ggcaccaagc tggagatcaa g 321

<210> 127

<211> 345

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 127

gaagtgcagc tgcagcagag cggaccagaa ctggtgaagc caggagccag cgtgaagatg 60

tcttgcaagg ccagcggcta caccttcacc gagtacaccc tgcattgggt gaagcagaag 120

ccaggacagg gactcgagtg gatcggagtg gtgaacccta acatcggcgc caccacctac 180

aaccagatgt tcaagggcaa ggccaccctg accagcgata agagcagcag caccgcctac 240

atggagctgt ctagcctgac cagcgaggac tcagccgtgt actattgcgc caggagagcc 300

tacctggact attggggcca gggcacaacc ctgaccgtgt ctagc 345

<210> 128

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 128

gacatcgtga tgacccagag cccagccaca ctgagcgtga caccaggcga cagagtgtct 60

ctgtcttgca gggccagcca gagcatcaac aacaacctgc attggtaccg gcagaagagc 120

cacgagagcc ctcggctgct gatcaagtac gacagccaga gcatcagcgg catcccttct 180

agattcagcg gcagcggcag cggaagcgat ttcaccctga gcatcaacag cgtggagcca 240

gaggacgtgg gcgtgtacta ttgccagcag accaactctt ggcccttcac cttcggcagc 300

ggcaccaagc tggagatcaa g 321

<210> 129

<211> 345

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 129

caggtgcagc tggtgcagtc aggagcagaa gtgaagaagc ccggagccag cgtgaaagtg 60

tcttgcaagg ccagcggcta caccttcacc gagtacacac tgcattgggt ccggcaggct 120

ccaggacagg gactcgagtg gatgggagtg gtgaacccta acatcggcgc caccacctac 180

aaccagatgt tcaagggccg cgtgaccatg accagagaca ccagcaccag caccgtgtac 240

atggagctga gcagcctgag aagcgaggac acagccgtgt actattgcgc caggagagcc 300

tacctggact attggggcca gggcacaacc ctgaccgtgt ctagc 345

<210> 130

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 130

gagatcgtgc tgacccagag cccagacttc cagtcagtga cccccaagga gaaggtcacc 60

atcacttgcc gggccagcca gagcatcaac aacaacctgc attggtacca gcagaagccc 120

gaccagagcc ctaagctgct gatcaagtac gatagccaga gcatcagcgg cgtgccttct 180

agattcagcg gaagcggcag cggcacagat ttcaccctga ccatcaacag cctggaggca 240

gaggacgcag ccacctacta ttgccagcag accaactctt ggcccttcac cttcggcagc 300

ggcaccaagc tggagatcaa g 321

<210> 131

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 131

caggtgcagc tggtgcagtc tggagctgag gtgaagaagc caggagcttc cgtgaaggtg 60

agctgcaagg cctctgacta caccatcaca tcctattgga tcaactgggt gagacaggct 120

cctggacagc gcctggagtg gatgggaagg atcgacctgt acgattccga gacccactat 180

agccagaagt tcaaggacag ggtgaccatc acacgggata ccagcgcctc tacagcttac 240

atggagctgt ccagcctgag aagcgaggat acagccgtgt actattgtgc taggtaccca 300

gctggacagg gagcttggtt tgcttattgg ggccagggca ccctggtgac agtgtcttcc 360

<210> 132

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 132

gacatccaga tgacacagtc cccttccagc ctgagcgcct ctgtgggcga tagggtgacc 60

atcacatgcc gggccagcgg caacatccac aattacctgg cttggtatca gcagaagccc 120

ggcaaggtgc ctaagctgct gatctacaac gccaagaccc tggctgacgg cgtgccatct 180

aggttctccg gcagcggctc tggcacagac ttcaccctga caatctcttc cctgcagcca 240

gaggacgtgg ctacctacta ttgtcagcat ttctgggata ccccctggac atttggcggc 300

ggcaccaagg tggagatcaa g 321

<210> 133

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 133

caggtgcagc tggtgcagtc tggagctgag gtgaagaagc caggagcttc cgtgaaggtg 60

agctgcaagg cctctgacta caccatcaca tcctattgga tcaactggat caggcaggct 120

cctggacagg gactggagtg gatcggaagg atcgacctgt acgattccga gacccactat 180

agccagaagt tcaaggacag agtgaccatc acagtggata caagcgcctc tgccgcttac 240

atggagctgt ccagcctgcg cagcgaggat accgccgtgt actattgtac aaagtaccca 300

gctggccagg gcgcctggtt tgcttattgg ggccagggca ccctggtgac agtgtcttcc 360

<210> 134

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 134

gacatccaga tgacccagtc cccttccagc ctgagcgcct ctgtgggcga tagggtgacc 60

atcacatgcc gggccagcgg caacatccac aattacctgg cttggtatag acagaagccc 120

ggcaaggtgc ctaagctgct ggtgtacaac gccaagaccc tggctgacgg cgtgccatct 180

cgcttttccg gcagcggctc tggcacagat ttcaccctga caatctcttc cctgcagcca 240

gaggacttcg gctcctattt ttgtcagcat ttctgggata ccccctggac atttggcggc 300

ggcacaaagg tggagatcaa g 321

<210> 135

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 135

caggtgcagc tggtgcagtc tggagctgag gtgaagaagc caggagcttc cgtgaaggtg 60

agctgcaagg cctctgacta caccatcaca tcctattgga tcaactggat cagacaggct 120

cctggacagg gactggagtg gatcggaagg atcgacctgt acgattccga gacccactat 180

agccagaagt tcaaggacag ggtgaccatc acacgggata ccagcgcctc tacagcttac 240

atggagctgt ccagcctgag gagcgaggat accgccgtgt actattgtac aaagtaccca 300

gctggccagg gcgcctggtt tgcttattgg ggccagggca ccctggtgac agtgtcttcc 360

<210> 136

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 136

gacatccaga tgacacagtc cccttccagc ctgagcgcct ctgtgggcga tagggtgacc 60

atcacatgcc gggccagcgg caacatccac aattacctgg cttggtatag acagaagccc 120

ggcaaggtgc ctaagctgct ggtgtacaac gccaagaccc tggctgacgg agtgccatct 180

cgcttctccg gaagcggctc tggcacagac ttcaccctga caatctcttc cctgcagcca 240

gaggacgtgg ctacctattt ctgtcagcat ttttgggata ccccatggac attcggcgga 300

ggaaccaagg tggagatcaa g 321

<210> 137

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 137

caggtgcagc tggtgcagtc tggagctgag gtgaagaagc caggagcttc cgtgaaggtg 60

agctgcaagg cctctgacta caccatcaca tcctattgga tcaactggat caggcaggct 120

cctggacagc ggctggagtg gatcggcaga atcgacctgt acgattccga gacccactat 180

agccagaagt tcaaggaccg cgtgaccatc acagtggata ccagcgcctc tacagcttac 240

atggagctgt ccagcctgag gagcgaggat accgccgtgt actattgtac aaagtaccca 300

gctggccagg gcgcctggtt tgcttattgg ggccagggca ccctggtgac agtgtcttcc 360

<210> 138

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 138

caggtgcagc tggtgcagtc tggagctgag gtgaagaagc caggagcttc cgtgaaggtg 60

agctgcaagg cctctgacta caccatcaca tcctattgga tcaactgggt gaggcaggct 120

cctggacagc ggctggagtg gatcggcaga atcgacctgt acgattccga gacccactat 180

agccagaagt tcaaggaccg cgtgaccatc acagtggata ccagcgcctc tacagcttac 240

atggagctgt ccagcctgag gagcgaggat accgccgtgt actattgtac aaagtaccca 300

gctggccagg gcgcctggtt tgcttattgg ggccagggca ccctggtgac agtgtcttcc 360

<210> 139

<211> 360

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 139

caggtgcagc tggtgcagtc tggagctgag gtgaagaagc caggagcttc cgtgaaggtg 60

agctgcaagg cctctgacta caccatcaca tcctattgga tcaactgggt gagacaggct 120

cctggacagc gcctggagtg gatcggaagg atcgacctgt acgattccga gacccactat 180

agccagaagt tcaaggacag ggtgaccatc acacgggata ccagcgcctc tacagcttac 240

atggagctgt ccagcctgag aagcgaggat accgccgtgt actattgtac aaagtaccca 300

gctggccagg gcgcctggtt tgcttattgg ggccagggca ccctggtgac agtgtcttcc 360

Claims (11)

1. A humanized CD47 antibody or antigen-binding fragment thereof, characterized in that the VH and VL sequence pair is SEQ ID NO: 1 and SEQ ID NO: 2, when the antibody CDRs are encoded according to Kabat encoding rules, HCDR1, HCDR2, HCDR3 are paired with LCDR1, LCDR2, LCDR3 as follows: 61, 62 and 63 are the same as 64, 65 and 66.

2. The humanized CD47 antibody or antigen-binding fragment thereof according to claim 1, wherein the humanized CD47 antibody has a light chain constant region optionally a human kappa chain or lambda chain constant region and a heavy chain constant region optionally a human antibody IgG1 or IgG 4S 228P mutated constant region.

3. The humanized CD47 antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is selected from a Fab, Fab '-SH, Fv, scFv, or (Fab') 2 fragment.

4. A nucleic acid encoding the humanized CD47 antibody or antigen-binding fragment thereof of any one of claims 1-3; the nucleic acid sequence is selected from the group consisting of SEQ ID NO 83 and SEQ ID NO 84.

5. An expression vector comprising the nucleic acid of claim 4.

6. A host cell comprising the expression vector of claim 5; the host cells include yeast cells, chinese hamster ovary cells, and human embryonic kidney cells.

7. A bispecific antibody comprising two different arms, one of which comprises the humanized CD47 antibody or antigen-binding fragment thereof of any one of claims 1-3 capable of binding to human CD47, the other arm comprising a target for targeted binding to malignant/immune cells comprising: EGFR, HER2, CD47, PD-1, PD-L1, CTLA4, TGF β, VEGF, HER3, TIGIT, CLAUDIN18.2, CD3, TIM3, LAG3, CD20, CD19, CD 33.

8. A trispecific antibody/fusion protein comprising the humanized CD47 antibody or antigen binding fragment thereof of any one of claims 1-3, the other two ends of the trispecific antibody/fusion protein targeting tumor associated antigens of tumor cells or immune cell targeting immunoassay targets comprising EGFR, HER2, CD47, PD-1, PD-L1, CTLA4, TGF β, VEGF, HER3, TIGIT, CLAUDIN18.2, CD3, TIM3, LAG3 CD20, CD19, CD 33.

9. A pharmaceutical composition comprising the humanized CD47 antibody or antigen-binding fragment thereof of any one of claims 1-3 or a bispecific antibody comprising the humanized CD47 antibody or antigen-binding fragment thereof of any one of claims 1-3 as one arm, and a pharmaceutically optional pharmaceutically acceptable carrier.

10. Use of the humanized CD47 antibody or antigen-binding fragment thereof of any one of claims 1-3 in the preparation of a medicament for the treatment of an overexpression of CD47 in a neoplastic disorder.

11. The use of claim 10, wherein the neoplastic condition comprises at least one of a hematological neoplasm, which is at least one of acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, adult T-cell leukemia, multiple myeloma, mixed leukemia, non-hodgkin's lymphoma; the solid tumor is at least one of lymphoma, breast cancer, head and neck cancer, gastric cancer, lung cancer, esophageal cancer, intestinal cancer, ovarian cancer, cervical cancer, liver cancer, renal cancer, pancreatic cancer, bladder cancer, colorectal cancer, glioma, and melanoma.

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