CN113621068B

CN113621068B - Antibody or antigen binding fragment thereof specifically binding to CD276, and preparation method and application thereof

Info

Publication number: CN113621068B
Application number: CN202111178993.8A
Authority: CN
Inventors: 胡红明; 彭涛; 黄慧
Original assignee: Shanghai Hengrun Dasheng Biotechnology Co ltd
Current assignee: Shanghai Hengrun Dasheng Biotechnology Co ltd
Priority date: 2021-10-11
Filing date: 2021-10-11
Publication date: 2022-01-07
Anticipated expiration: 2041-10-11
Also published as: CN113621068A; WO2023060922A1

Abstract

The invention discloses an antibody or an antigen binding fragment thereof specifically binding to CD276, and a preparation method and application thereof, and relates to the technical field of biological immunotherapy, wherein the antibody has good safety and a treatment effect targeting CD276, and can be used for preparing immune effector cells targeting CD276 and providing a treatment or improvement way for diseases related to CD276 expression.

Description

Antibody or antigen binding fragment thereof specifically binding to CD276, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological immunotherapy, and particularly relates to an antibody or an antigen binding fragment thereof specifically binding to CD276, and a preparation method and application thereof.

Background

CD276, also known as B7-H3, is an important immune checkpoint member of the B7 and CD28 families, and belongs to a type I transmembrane protein, with an extracellular region comprising two identical pairs of immunoglobulin variable and constant regions, a short intracellular region with no clear signaling motifs. The mRNA level expression is wide, but the protein expression is relatively limited to the surfaces of resting non-immune cells such as fibroblasts, endothelial cells, osteoblasts, amniotic fluid stem cells and the like, and induced antigen presenting cells and NK cells.

The research shows that the expression of the CD276 is abnormally increased in various malignant tumors, including brain glioma, pancreatic cancer, ovarian cancer, lung cancer, renal cancer, breast cancer, prostatic cancer, colorectal cancer and other tumors. The adhesion capacity of cell adhesion protein is increased after the expression of CD276 in tumor cells is increased, so that the migration and invasion capacity of the tumor cells are enhanced, the expression level is closely related to poor prognosis and poor clinical outcome of patients, and the expression level is supposed to be involved in immune evasion of tumors. Although the molecular mechanism is not clear, it is a promising target for tumor immunotherapy as a possible immune checkpoint molecule.

Chimeric antigen receptor T cells: (Chimeric Antigen Receptor-T cellCAR-T) is a novel immunotherapeutic approach against tumor cell surface specific antigens. Now, many researchers are developing CAR-T cells for the treatment of solid tumors.

Single chain antibodies as an important component of CAR, murine antibodies are currently traditionally used, and the heterogeneity of murine antibodies causes the human anti-murine antibody response: (Human anti-mouse antibody reactionHAMA), resulting in rapid clearance of CAR-T in the circulatory system, and loss of therapeutic efficacy.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide an antibody or an antigen binding fragment thereof specifically binding to CD276, and a preparation method and application thereof.

The invention is realized by the following steps:

in a first aspect, the embodiments provide an antibody or antigen-binding fragment thereof that specifically binds to CD276, the antibody or antigen-binding fragment thereof comprising at least one of the following heavy chain variable region and light chain variable region;

the heavy chain variable region comprises at least one of HCDR1, HCDR2 and HCDR 3; the sequence of the HCDR1 is shown as SEQ ID NO. 1, 2 or 3, the sequence of the HCDR2 is shown as SEQ ID NO. 4, 5 or 6, and the sequence of the HCDR3 is shown as SEQ ID NO. 7, 8, 9 or 10;

the light chain variable region comprises at least one of LCDR1, LCDR2 and LCDR 3; the sequence of the LCDR1 is shown as SEQ ID NO. 11, 12, 13 or 14, the sequence of the LCDR2 is shown as SEQ ID NO. 15 or 16, and the sequence of the LCDR3 is shown as SEQ ID NO. 17, 18, 19 or 20.

In a second aspect, embodiments of the invention provide an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof that specifically binds CD276 as described in previous embodiments.

In a third aspect, embodiments of the invention provide a vector comprising an isolated nucleic acid as described in the preceding embodiments.

In a fourth aspect, embodiments of the present invention provide a host cell comprising a vector as described in the preceding embodiments.

In a fifth aspect, embodiments of the present invention provide a method for producing an antibody or antigen-binding fragment thereof that specifically binds to CD276, comprising culturing a host cell as described in the previous embodiments.

In a sixth aspect, the present embodiments provide a chimeric antigen receptor CAR against human CD276, said CAR comprising an antigen recognition region, a hinge region, a transmembrane region, and an intracellular region that recognize the CD276 antigen;

the antigen recognition region comprises an antibody or antigen binding fragment thereof that specifically binds to CD276 as described in the previous examples.

In a seventh aspect, embodiments of the invention provide an isolated nucleic acid encoding a chimeric antigen receptor CAR against human CD276 as described in previous embodiments.

In an eighth aspect, embodiments of the invention provide a vector comprising a nucleic acid isolated as in the preceding embodiments.

In a ninth aspect, embodiments of the invention provide a host cell comprising a vector as in the preceding embodiments.

In a tenth aspect, the present invention provides a method for preparing a chimeric antigen receptor CAR against human CD276, which comprises culturing the host cell of the previous embodiment.

In an eleventh aspect, embodiments of the present invention provide an immune effector cell that expresses an antibody or antigen-binding fragment thereof that specifically binds to CD276 as described in the previous embodiments, or a chimeric antigen receptor CAR against human CD276 as described in the previous embodiments.

In a twelfth aspect, embodiments of the present invention provide a method of producing an immune effector cell, comprising infecting an immune effector cell with an isolated nucleic acid as described in the preceding embodiments or a vector as described in the preceding embodiments.

In a thirteenth aspect, embodiments of the present invention provide a pharmaceutical composition comprising: an antibody or antigen-binding fragment thereof that specifically binds CD276 as described in the preceding examples, or an isolated nucleic acid as described in the preceding examples, or a chimeric antigen receptor CAR against human CD276 as described in the preceding examples, an isolated nucleic acid as described in the preceding examples, or a vector as described in the preceding examples, or an immune effector cell as described in the preceding examples.

In a fourteenth aspect, embodiments of the present invention provide the use of an agent selected from the group consisting of: an antibody or antigen-binding fragment thereof that specifically binds to CD276 as described in the preceding examples, an isolated nucleic acid as described in the preceding examples, a vector as described in the preceding examples, a host cell as described in the preceding examples, an antibody or antigen-binding fragment thereof that specifically binds to CD276 as described in the preceding examples, a chimeric antigen receptor CAR for anti-human CD276 as described in the preceding examples, at least one of the isolated nucleic acid described in the preceding examples, the vector described in the preceding examples, the host cell described in the preceding examples, the chimeric antigen receptor CAR against human CD276 prepared by the method for preparing chimeric antigen receptor CAR against human CD276 described in the preceding examples, the immune effector cell described in the preceding examples, and the immune effector cell prepared by the method for preparing immune effector cell described in the preceding examples.

The invention has the following beneficial effects:

the invention provides a fully human antibody for specifically recognizing human CD276, the antibody has good safety and a treatment effect of targeting CD276, and the antibody can be used for preparing immune effector cells of targeting CD276 and provides a treatment or improvement way for diseases related to CD276 expression.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is an SDS electrophoretogram of recombinant human CD276-avi-his antigen protein of example 1;

FIG. 2 is a diagram showing the quality identification of recombinant human CD276-avi-his antigen protein in example 1;

FIG. 3 is the dissociation constants of 1C8, 1H12, 3C1 and 3E5 as determined in example 2 using an Octet K2 instrument;

FIG. 4 is a graph of the binding of CD276 antigen to 1C8, 1H12, 3C1 and 3E5 scFv fragments as determined by ELISA in example 2;

FIG. 5 is a schematic representation of the different cloned CD276 CAR of example 3;

FIG. 6 is the CAR expression positive rate of different cloned CD276 CAR-T cells in example 4;

FIG. 7 is CD107a expression from different cloned CD276 CAR-T cells in example 5;

FIG. 8 is INF γ secretion from various cloned CD276 CAR-T cells in example 5;

FIG. 9 shows IL-2 secretion from different cloned CD276 CAR-T cells of example 5;

FIG. 10 shows the results of the killing experiment of target cells by different cloned CD276 CAR-T cells in example 5.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

First, the present embodiments provide an antibody or antigen-binding fragment thereof that specifically binds to CD276, the antibody or antigen-binding fragment thereof comprising at least one of the following heavy chain variable region and light chain variable region;

The definitions of "antibody" and "immunoglobulin" herein may be mutually equivalent.

The heavy chain variable region and the light chain variable region of an antibody typically include 3 complementarity determining regions CDRs and 4 framework region FRs. The complementarity determining regions are connected by a framework region, and when an antibody is recognized, the FR molecules are curled so that the CDR molecules are close to each other. The complementarity determining region is the binding site of an antibody or antigen-binding fragment to an antigen, and thus, the sequence of the complementarity determining region determines the specificity of the antibody. Herein, "HCDR 1", "HCDR 2", "HCDR 3" are 3 complementarity determining regions of the heavy chain variable region, and "LCDR 1", "LCDR 2" and "LCDR 3" are 3 complementarity determining regions of the light chain variable region.

"antigen-binding fragment" herein refers to a polypeptide fragment having the property of specifically targeting CD276, which contains at least one CDR of an antibody heavy chain variable region and/or a light chain variable region; preferably, it may contain HCDR 1-3 of the heavy chain variable region and/or LCDR 1-3 of the light chain variable region. Antigen-binding fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production by expression from host cells containing the antigen-binding fragment.

Through a series of creative efforts, the invention provides the CD 276-targeted antibody or the antigen binding fragment thereof, the antibody or the antigen binding fragment thereof has good safety and targeting property, can specifically bind to the extracellular domain of the human CD276, and uses a vector containing the coding sequence of the antibody or the antigen binding fragment thereof to infect immune cells, so that immune effector cells with remarkable killing capacity on tumor cells expressing the CD276 can be obtained, and the immune effector cells can be applied to treating or improving diseases related to CD276 expression, thereby laying the foundation for treating CD276 positive tumors.

Without limitation, the sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the antibody or antigen-binding fragment thereof may be randomly selected within the above ranges.

Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region selected from any one of items (a) - (d);

(a) HCDR1 with a sequence shown as SEQ ID NO. 1, HCDR2 with a sequence shown as SEQ ID NO. 4, HCDR3 with a sequence shown as SEQ ID NO. 7, LCDR1 with a sequence shown as SEQ ID NO. 11, LCDR2 with a sequence shown as SEQ ID NO. 15 and LCDR3 with a sequence shown as SEQ ID NO. 17;

(b) HCDR1 with a sequence shown as SEQ ID NO. 2, HCDR2 with a sequence shown as SEQ ID NO. 5, HCDR3 with a sequence shown as SEQ ID NO. 8, LCDR1 with a sequence shown as SEQ ID NO. 12, LCDR2 with a sequence shown as SEQ ID NO. 15 and LCDR3 with a sequence shown as SEQ ID NO. 18;

(c) HCDR1 with a sequence shown as SEQ ID NO. 2, HCDR2 with a sequence shown as SEQ ID NO. 5, HCDR3 with a sequence shown as SEQ ID NO. 9, LCDR1 with a sequence shown as SEQ ID NO. 13, LCDR2 with a sequence shown as SEQ ID NO. 15 and LCDR3 with a sequence shown as SEQ ID NO. 19;

(d) HCDR1 with a sequence shown as SEQ ID NO. 2, HCDR2 with a sequence shown as SEQ ID NO. 5, HCDR3 with a sequence shown as SEQ ID NO. 10, LCDR1 with a sequence shown as SEQ ID NO. 14, LCDR2 with a sequence shown as SEQ ID NO. 16 and LCDR3 with a sequence shown as SEQ ID NO. 20.

Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, and the amino acid sequence of the variable region of the heavy chain is as shown in seq id no: 21. 22, 23 and 24; the amino acid sequence of the light chain variable region is shown as SEQ ID No: 25. 26, 27 and 28.

Optionally, the antibody comprises at least one of a monoclonal antibody, a humanized antibody, a chimeric antibody, a bispecific antibody;

the antigen binding fragment is Fab, F (ab')₂Fd, a single-chain antibody scFv, a disulfide-linked fv (sdFv), or a single-domain antibody.

In alternative embodiments, the antibody or antigen-binding fragment thereof is humanized.

In alternative embodiments, the antibody further comprises an antibody constant region; preferably, the antibody constant region is selected from: a constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

Preferably, the heavy chain constant region of the antibody constant region is selected from the heavy chain constant region of any one of IgG1, IgG2, IgG3, IgG4, preferably IgG 4; the light chain constant region of the antibody constant region is kappa (. kappa.) or lambda (. lamda.), preferably kappa.

The present embodiments also provide an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof that specifically binds CD276 as described in any of the preceding embodiments.

The embodiments also provide a vector comprising the isolated nucleic acid as described in the previous embodiments.

Optionally, the vector is an expression vector or a cloning vector.

Optionally, the vector is a plasmid vector.

Embodiments of the invention also provide a host cell comprising the isolated nucleic acid of the preceding embodiments or the vector of the preceding embodiments.

Optionally, the host cell bank is a prokaryotic cell or a eukaryotic cell. Alternatively, the prokaryotic cell may be an escherichia coli cell, and the eukaryotic cell may be specifically an ovarian cell.

The present embodiments also provide a method for producing an antibody or antigen-binding fragment thereof that specifically binds to CD276, comprising culturing a host cell as described in the previous embodiments.

Specifically, the conditions under which the host cell is cultured are conditions sufficient for the host cell to express an antibody or antigen-binding fragment thereof that specifically binds to CD 276.

The embodiment of the invention also provides a chimeric antigen receptor CAR of anti-human CD276, wherein the CAR comprises an antigen recognition region, a hinge region, a transmembrane region and an intracellular region which can recognize CD276 antigen;

the antigen recognition region comprises an antibody or antigen binding fragment thereof that specifically binds to CD276 as described in any of the preceding examples.

Without limitation, an "antigen recognition region" may be monovalent or multivalent (e.g., bivalent or trivalent). The antigen binding region may be monospecific or multispecific (e.g., bispecific). Bispecific can be against CD276 and another antigen, or against two different epitopes of CD 276. Preferably, the antigen recognition region is a single chain antibody (monovalent or multivalent). The scFv antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are connected through a Linker.

Preferably, the amino acid sequence of the heavy chain variable region of the scFv is set forth in SEQ ID NO:21, the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 25; or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 22, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 26; or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 23, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 27; or the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 24, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 28.

In alternative embodiments, the scFv heavy and light chains are linked in a VH-Linker-VL or VL-Linker-VH. In some embodiments, the Linker sequence may be selected from the existing Linker sequences, and preferably, the Linker sequence may be GGGGSGGGGSGGGGS, or (GGGGS) n, where n is 1 to 6, or GSTSGSGKPGSGEGSTKG.

Preferably, the CAR further comprises a leader signal peptide sequence. In general, a signal peptide is a peptide sequence that targets a polypeptide to a desired site in a cell. In some embodiments, the signal peptide targets the polypeptide to the secretory pathway of the cell, and will allow the polypeptide to integrate and anchor to the lipid bilayer. In some embodiments, the signal peptide is a membrane localization signal peptide. Preferably, the leader peptide sequence is derived from the leader peptide sequence of CD 8; more preferably, the CD8 leader peptide sequence has the amino acid sequence shown in SEQ ID NO. 29.

The "hinge region", "transmembrane region" and "intracellular region" herein may each be selected from the sequences of hinge regions, transmembrane regions and intracellular regions known in the art of CAR-T.

The hinge region of the chimeric antigen receptor is located between the extracellular antigen-binding region and the transmembrane region, is a segment of amino acids that typically exists between two domains of a protein, and can allow for the flexibility of the protein and the movement of the two domains relative to each other. The hinge region may be a hinge region of a naturally occurring protein or a portion thereof. The hinge region of an antibody (such as an IgG, IgA, IgM, IgE, or IgD antibody) may also be used for the chimeric antigen receptors described herein. Non-naturally occurring peptides may also be used as the hinge region of the chimeric antigen receptor described herein. In some embodiments, the hinge region is a peptide linker. Preferably, the hinge region is derived from CD8 a. Preferably, the CD8 a hinge region has the amino acid sequence shown in SEQ ID NO 30.

The transmembrane region of the chimeric antibody receptor may form an alpha helix, a complex of more than one alpha helix, a beta barrel, or any other stable structure capable of spanning the domain of the cellular phospholipid bilayer. The transmembrane region may be of natural or synthetic origin. The transmembrane region may be derived from CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, the alpha, beta or zeta chain of the T cell receptor. Preferably, the transmembrane region is derived from CD8 α. Preferably, the CD8 a transmembrane region has the amino acid sequence shown in SEQ ID NO. 31.

Preferably, the intracellular region of the chimeric antigen receptor comprises a signaling region and/or a costimulatory signaling region. The number of signaling regions and/or co-stimulatory signaling regions may each be one or more.

The intracellular signaling region is responsible for the activation of at least one normal effector function of immune effector cells expressing the chimeric antigen receptor. For example, the effector function of a T cell may be cytolytic activity or helper activity, including secretion of cytokines. While the entire intracellular signaling region can generally be utilized, in many cases, the use of the entire strand is not necessary. For use of a truncated portion of an intracellular signaling region, such a truncated portion may be used in place of the entire strand, so long as it transduces effector function signals. Thus, an intracellular signaling region includes any truncated form of an intracellular signaling region sufficient to transduce an effector function signal. In some embodiments, the signaling region is derived from at least one of CD3 ζ, FcR γ (FCER1G), FcR β (fcepsilon Rib), CD3 γ, CD3 δ, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66 d. Preferably, the intracellular domain is derived from the intracellular domain of human CD3 ζ. Further, the intracellular domain of human CD3 ζ has the amino acid sequence shown in SEQ ID NO: 32.

In addition to stimulation of antigen-specific signals, many immune effector cells also require co-stimulation to promote cell proliferation, differentiation and survival, as well as to activate effector functions of the cells. The "costimulatory signaling region" can be the cytoplasmic portion of the costimulatory molecule. The term "co-stimulatory molecule" refers to an associated binding partner on an immune cell (such as a T cell) that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response by the immune cell, such as, but not limited to, proliferation and survival. The costimulatory signaling region can be derived from the intracellular signaling region of at least one of CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, fcfri, MyD88, and 41 BBL. In some embodiments, the co-stimulatory signaling region is derived from 4-1 BB. In some embodiments, the 4-1BB co-stimulatory signaling region comprises SEQ ID NO: 33, or a pharmaceutically acceptable salt thereof.

Preferably, the nucleotide sequence of the CAR is as shown in any one of SEQ ID NOs 34, 35, 36 and 37.

The embodiments provide an isolated nucleic acid encoding a chimeric antigen receptor CAR against human CD276 as described in any of the preceding embodiments.

The embodiments provide a vector comprising an isolated nucleic acid as described in the preceding examples (a nucleic acid encoding a chimeric antigen receptor CAR against human CD276 as described in any of the preceding examples). The vector may be an expression vector or a cloning vector. In some embodiments, the vector is a viral vector. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex viral vectors and derivatives thereof.

The embodiments provide a host cell comprising a vector as described in any preceding embodiment (a vector containing a nucleic acid encoding a chimeric antigen receptor CAR against human CD276 as described in any preceding embodiment). Alternatively, the host cell may be a prokaryotic cell or a eukaryotic cell. Eukaryotic cells, such as mammalian cells, are preferred.

The embodiment of the invention provides a preparation method of a chimeric antigen receptor CAR of anti-human CD276, which comprises culturing the host cell described in the previous embodiment.

Preferably, the production process is performed under culture conditions sufficient to enable the host cell to express the chimeric antigen receptor CAR against human CD 276.

The embodiments provide an immune effector cell expressing an antibody or antigen-binding fragment thereof that specifically binds to CD276 as described in any of the preceding embodiments, or a chimeric antigen receptor CAR against human CD276 as described in any of the preceding embodiments.

An "immune effector cell" is an immune cell that can perform an immune effector function. In some embodiments, the immune effector cells express at least Fc γ RIII and perform ADCC effector function. Examples of immune effector cells that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMCs), Natural Killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.

Preferably, the immune effector cell is selected from the group consisting of: culturing at least one of differentiated immune cells, T lymphocytes, NK cells, Peripheral Blood Mononuclear Cells (PBMCs) and hematopoietic stem cells from pluripotent stem cells or embryonic stem cells. More preferably, the immune effector cell is a T lymphocyte (homo T cell). In some embodiments, the T cell can be CD4+/CD 8-, CD4-/CD8+, CD4+/CD8+, CD4-/CD8-, or a combination thereof. In some embodiments, the T cells produce IL-2, IFN, and/or TNF when expressing the chimeric antigen receptor and binding to the target cell. In some embodiments, CD8+ T cells lyse antigen-specific target cells when expressing chimeric antigen receptors and binding to the target cells.

Embodiments of the invention provide methods of making immune effector cells as described in any of the preceding embodiments, comprising infecting an immune effector cell with an isolated nucleic acid as described in any of the preceding embodiments or a vector as described in any of the preceding embodiments.

Embodiments of the invention prepare genetically engineered immune effector cells by introducing chimeric antigen receptors into immune effector cells, such as T cells.

It is noted that methods for introducing nucleic acids or vectors into mammalian cells are known in the art, and the vectors can be transferred into immune effector cells by physical, chemical, or biological means. Physical methods for introducing vectors into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Chemical means for introducing nucleic acids or vectors into immune effector cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system for use as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle). Biological methods for introducing nucleic acids or vectors into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, e.g., human, cells. In some embodiments, the transduced or transfected immune effector cells are propagated ex vivo following introduction of the nucleic acid or vector.

In some embodiments, the preparing further comprises further evaluating or screening the transduced or transfected immune effector cells to select engineered immune effector cells.

The embodiment of the invention provides a pharmaceutical composition, which comprises: an antibody or antigen-binding fragment thereof that specifically binds to CD276 as described in any preceding example, an isolated nucleic acid as described in any preceding example, a vector as described in any preceding example, a method of making an antibody or antigen-binding fragment thereof that specifically binds to CD276 as described in any preceding example, at least one of the anti-human CD276 chimeric antigen receptor CAR described in any of the preceding examples, the isolated nucleic acid described in any of the preceding examples, the anti-human CD276 chimeric antigen receptor CAR prepared by the method for preparing an anti-human CD276 chimeric antigen receptor CAR described in any of the preceding examples, the immune effector cell described in any of the preceding examples, and the immune effector cell prepared by the method for preparing an immune effector cell described in any of the preceding examples.

In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

Pharmaceutical compositions can be prepared by mixing the active agent with the desired purity, optionally with a pharmaceutically acceptable carrier, in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are non-toxic to recipients at the dosages and concentrations employed and may include at least one of buffers, antioxidants, preservatives, isotonicity agents, stabilizers, and surfactants. Furthermore, in order for pharmaceutical compositions to be useful for in vivo administration, they must be sterile. The pharmaceutical composition may be sterilized by filtration through a sterile filtration membrane.

In some embodiments, the pharmaceutical composition may contain: a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressive agent, a growth inhibitory agent, and at least one additive of an active agent required for the particular indication to be treated. The specific addition amount of the additive can be adjusted according to actual needs.

Embodiments of the invention also provide the use of an agent selected from the group consisting of: the antibody or antigen-binding fragment thereof that specifically binds to CD276 of any of the preceding examples, the isolated nucleic acid of any of the preceding examples, the vector of any of the preceding examples, the host cell of any of the preceding examples, at least one of the antibody or antigen-binding fragment thereof specifically binding to CD276, the chimeric antigen receptor CAR against human CD276, the immune effector cell, and the immune effector cell, which are prepared by the method for preparing the immune effector cell, according to any of the preceding embodiments.

Preferably, the treatment or amelioration of cancer refers to an agent capable of stimulating or enhancing immune function in a cancer patient.

Preferably, the cancer is a cancer associated with CD276 expression.

Herein, "cancer associated with CD276 expression" refers to a disease caused directly or indirectly by an abnormal expression of CD276, and generally refers to a disease caused by an overexpression of CD 276.

Preferably, the cancer is selected from at least one of brain glioma, kidney cancer, ovarian cancer, lung cancer, stomach cancer, liver cancer, intestinal cancer, prostate cancer, pancreatic cancer and mesothelial cancer.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1: the construction of recombinant human CD276 protein expression vector and eukaryotic expression.

Synthesis of gene sequence of 29 th to 466 th amino acid interval of CD276 and construction of expression vector of protein.

The amino acid sequence from 29 th to 466 th of CD276 (uniprot access No: Q5ZPR 3-1) is introduced into an online codon optimization tool (http:// www.jcat.de/# opennewwindow) to obtain a nucleic acid sequence after codon optimization, and then a gene sequence is obtained by chemical synthesis, and meanwhile, the nucleic acid sequences of avi-tag and 6 × his-tag are added to the 3' end of the gene sequence, and the fusion gene sequence encodes the amino acid sequence shown in SEQ ID NO. 39 and the nucleotide sequence shown in SEQ ID NO. 40. The spliced product is cloned into pCDNA3.1 (Thermo) by using a TaKaRa seamless cloning kit through molecular cloning to obtain an expression vector.

22. Expression, purification and activity identification of the recombinant human CD276 protein.

After 293T cells (ATCC) were transfected with the obtained expression vector for 5 days, culture supernatants were collected and recombinant human CD276 protein was purified using AKTA explorer 100 (GE). Due to glycosylation modification, the size of the recombinant human CD276 protein was approximately 65 kilodaltons as shown in FIG. 1, as shown by Coomassie blue staining after electrophoresis on reduced SDS-PAGE.

The recombinant human CD276 protein is subjected to ELISA activity identification by using a commercial CD276 murine antibody: recombinant human CD276 protein was coated in ELISA plates at 2-fold gradient dilution, the first well was coated with 200ng, diluted 2-fold sequentially (100 uL/well), incubated overnight at 4 ℃, washed 3 times the next day with 200uL PBST, and blocked for 1 hour with 1% BSA/PBS. Blocking solution was removed, and murine anti-human CD276 antibody (100 uL per well) diluted 1:4000 was added and incubated at 37 ℃ for 1 hour. After washing 3 times with 200uL PBST, a 1:4000 dilution of horseradish peroxidase-labeled goat anti-mouse IgG1 antibody (100 uL per well) was added and incubated at 37 ℃ for 1 hour. After washing 200uL PBST for 3 times, 100uL TMB developing solution was added, development was carried out at 37 ℃ for 10 minutes, 100uL 1M hydrochloric acid was added to terminate the development, and OD450 was read, and the analysis results are shown in FIG. 2 and Table 1.

TABLE 1 analytical results

	CD276
		EC50（ng/μL）	15.12
Rsquare	0.9946

Example 2: preparation of human anti-human CD276 antibody.

1. Constructing a phage display library of human natural antibodies.

A two-step method is adopted to construct a phage display library of the human natural antibody, namely, the light chain gene and the heavy chain gene of the human natural antibody are connected into a phage display vector in two steps.

Human immunoglobulin kappa chain gene was PCR-amplified using human immunoglobulin kappa chain variable region forward primer (VK-mix-F) and light chain constant region reverse primer (CK-mix-R) and human PBMC cDNA as template, and PCR reaction conditions were as follows: pre-denaturation at 98 ℃ for 1 min, then temperature cycling, denaturation at 98 ℃ for 30 sec, annealing at 58 ℃ for 30 sec, extension at 72 ℃ for 1 min, cycling for 30 times, and final extension at 72 ℃ for 10 min. After the PCR product was electrophoresed through 1% agarose gel, a kappa chain gene fragment of about 750bp was recovered using a gel recovery kit (Promega). The kappa chain gene and the phage display vector pcomb3Z are subjected to double enzyme digestion by NheI and SalI DNA endonuclease (NEB), and the kappa chain gene subjected to enzyme digestion is directly recovered by using a gel recovery kit. The digested pcomb3Z vector was subjected to 1% agarose gel electrophoresis, and then a 5200bp vector fragment was recovered using a gel recovery kit (Promega). The kappa chain gene was ligated into pcomb3Z vector using T4 DNA ligase kit (Invitrogen), and the ligation product was desalted using PCR recovery kit. The desalted ligation product was shock-transformed into TG1 electroporation competent cells to give kappa libraries, which were amplified overnight and the next day kappa library plasmids were extracted using a plasmid Mass extraction kit (MN).

Human immunoglobulin IgM Fd (VH-CH 1) gene was PCR-amplified using human immunoglobulin heavy chain variable region forward primer (VH-mix-F) and IgM CH1 constant region reverse primer (IgM-CH 1-R) using human PBMC cDNA as a template, and the PCR reaction conditions were the same as described above. After the PCR product was electrophoresed through 1% agarose gel, an Fd gene fragment of about 750bp was recovered using a gel recovery kit (Promega). And carrying out single enzyme digestion on the IgM Fd gene fragment and the kappa chain library plasmid by using SfiI DNA endonuclease, and directly recovering the IgM Fd gene subjected to enzyme digestion by using a glue recovery kit. And the digested kappa chain library is subjected to 1% agarose gel electrophoresis, and then a 4000bp vector fragment is recovered by using a gel recovery kit (Promega). The IgM Fd gene was ligated to the kappa chain library using T4 DNA ligase kit (Invitrogen), and the ligation product was desalted using PCR recovery kit. Electrically shocking the desalted connecting product to transform TG1 into competent cell to obtain human natural antibody Fab phage display library, overnight amplifying the electrically shocked and transformed antibody library, centrifuging to collect library thallus, and storing at the final concentration of 20% glycerin-80 deg.

Inoculating frozen partial human natural antibody phage display library into 2YT culture set at 0.1OD density, culturing the bacterial liquid at 37 deg.c and 220rpm for about 1.5 hr to reach 0.6OD density, adding M13KO7 phage in 20 times the number of bacteria, standing for 30 min to infect, and culturing at 30 deg.c and 220rpm overnight. The next day, the bacterial solution was centrifuged at 10000 XG, the culture supernatant was collected, 1/4 volumes of PEG/NaCl solution (20% PEG8000,2.5M NaCl) was added to the culture supernatant, and the mixture was mixed in ice bath for 1 hour. After the ice bath is finished, centrifuging at 8000 XG for 10 minutes, collecting the precipitate, and re-dissolving the precipitate by PBS to obtain the human natural antibody phage display library.

2. The CD276 antibodies were screened.

2.1 recombinant human CD276 protein was coupled to streptavidin magnetic beads.

And performing biotin modification on avi-tag of the recombinant human CD276 protein by using a biotinylation kit (Yijinyi) according to the kit specification to obtain the biotinylated CD276 protein. 10ug of the above biotin-modified recombinant protein was added to 100uL streptavidin magnetic beads (DynaBeads 280) washed 3 times with PBS, and coupled on a rotary shaker at 18 rpm for 30 minutes at room temperature, followed by 3 washes with PBS.

2.2 blocking the phage library and magnetic beads.

0.5mL of 1% BSA/PBS was added to 0.5mL of the phage library and placed on a rotary shaker at 18 rpm and spin blocked at room temperature for 1 hour, and the phage was Input 1. At the same time, 100uL of uncoupled protein DynaBeads 280 was washed 3 times with PBS, and 1mL of 1% BSA/PBS was added, followed by incubation for 1 hour with rotation under the above conditions. To the CD276 coupled beads, 1mL of 1% BSA/PBS was added and spin blocked for 1 hour under the above conditions.

2.3 negative panning.

To remove the antibodies that interacted with the magnetic beads, a negative panning was necessary. The BSA blocked phage library was mixed with magnetic beads not coupled with antigen, and incubated for 1 hour with rotation under the above conditions. After incubation, the phage-magnetic bead mixture was placed on a magnetic frame, and after the beads were attached to the wall, the supernatant was transferred to a new EP tube.

2.4 Positive panning.

And adding the closed magnetic beads coupled with the CD276 protein into the phage supernatant subjected to negative panning for positive panning, and performing rotary incubation for 1 hour at room temperature according to the conditions. After incubation, the beads were washed with 1mL of PBST (0.1% Tween-20 in PBS), and the washing was repeated 10 times. After washing, 1mL of 100mM glycine (pH 2.0) was added, and the mixture was placed on a rotary shaker at a speed of 18 rpm and subjected to rotary elution for 10 minutes. After the elution is finished, the EP tube is placed on a magnetic frame, and the eluent is transferred to a new EP tube after the magnetic beads are attached to the wall. To the eluate was added 0.2mL of 1M Tris-HCl solution (pH 8.0) for neutralization. The neutralized eluate was added to 30mL of TG1 bacterial solution with OD600 of about 0.6, allowed to stand for 30 minutes, then M13KO7 phage was added in an amount of 20 times the number of cells, allowed to stand for 30 minutes, finally 100mL of 2YT medium and final concentrations of 100ug/mL of ampicillin and kanamycin were added, and cultured overnight at 30 ℃ and 220 rpm. The next day, the phage were harvested as described above for the phage library, at which time the resulting phage was Input 2.

2.5 repeat positive panning.

The panning procedure described above was repeated 2 times, i.e., Input2 was subjected to the next negative panning and positive panning to obtain Input 3. The difference is that after the eluent obtained by panning with Input3 infects TG1, 10uL of bacterial liquid is taken for gradient dilution without adding M13KO7, 100uL of bacterial liquid with three dilution gradients of 103, 104 and 105 is taken to coat a 2YT/amp plate, and cultured overnight at 30 ℃; the remaining bacterial suspension was cultured overnight at 30 ℃ and 220 rpm.

2.6 ELISA screening for positive antibodies.

Randomly picking TG1 from the above plate with toothpick, placing into a deep-well plate containing 600uL 2YT/amp, coating a gas-permeable membrane on the deep-well plate, culturing at 37 deg.C and 220rpm for 3 hr, removing the gas-permeable membrane, adding IPTG with final concentration of 1mM into the well, and culturing at 30 deg.C and 220rpm overnight. The ELISA plate was coated with 100ng of recombinant human CD276 protein per well. The next day, the deep-well plate was centrifuged at 4000rpm for 10 minutes, the medium in the well was removed and the pellet of the cells was retained, 100uL of TES solution (20% sucrose, 0.1mM EDTA, 50mM Tris-HCl, pH 8.0) was added to each well, the cells were resuspended by shaking, then ice-washed for 30 minutes, 200uL of ultrapure water was added and mixed by shaking, and the supernatant in the deep-well plate was centrifuged at 4000rpm for 10 minutes, at which time the antibody-containing periplasmic cavity extract was obtained. The ELISA plates were washed three times with plate washers, then 200uL of 1% BSA/PBS was added and blocked at 37 ℃ for 1 hour. Removing blocking solution from ELISA plate, adding 100uL of the above periplasmic cavity extract, incubating at 37 deg.C for 1 hr, washing with plate washing machine for 3 times, adding HRP-conjugated-protein-L (horse radish peroxidase labeled protein L) solution, incubating at 37 deg.C for 1 hr, washing with plate washing machine for 3 times, adding 100uL of TMB developing solution, developing at 37 deg.C for 10 min, and adding 100uL of 1M hydrochloric acid to stop. And (3) reading the OD450 value by using a microplate reader, and carrying out Sanger sequencing on the clone with the reading value being 3 times higher than the background value to obtain the gene sequence of the antibody.

2.7 verification of positive clones.

Based on the sequencing results, clones with large differences in the amino acid sequence of antibody CDR3 were selected for re-inoculation and induction overnight, and again verified for the ability of the selected clones to bind CD276 by ELISA as described above. Finally, four antibody sequences of 1C8, 1H12, 3C1 and 3E5 are obtained.

1C8 is as set forth in SEQ ID NO:21, the amino acid sequence of the light chain variable region sequence is shown as SEQ ID NO: shown at 25.

The heavy chain variable region amino acid sequence of 1H12 is set forth in SEQ ID NO:22, the amino acid sequence of the light chain variable region sequence is shown as SEQ ID NO: shown at 26.

The amino acid sequence of the heavy chain variable region of 3C1 is set forth in SEQ ID NO:23, the amino acid sequence of the light chain variable region sequence is shown as SEQ ID NO: as shown at 27.

The heavy chain variable region amino acid sequence of 3E5 is set forth in SEQ ID NO:24, the amino acid sequence of the light chain variable region sequence is shown as SEQ ID NO: shown at 28.

3. Off-rate constants koff.

The extracts of the cloned periplasmic space selected in the above procedure to bind to the recombinant human CD276 protein were analyzed using an Octet K2 molecular interaction analyzer. SA probe immobilization was performed using 200ul of 100nM biotinylated recombinant human CD276 protein, 1nM in immobilization height. Dissociation constants were determined and ranked using periplasmic cavity extract as the analyte, and the results are shown in fig. 3 and table 2.

TABLE 2 dissociation constants

Name (R)	Response	Kdis（1/s）	KdisError	DissocR^2
					3C1	0.8149	7.40E-03	1.32E-04	0.987189
1H12	0.4629	1.02E-02	1.90E-04	0.979583
					3E5	0.4776	7.39E-03	9.03E-05	0.993932
1C8	0.2436	1.50E-02	3.87E-04	0.948326

4. The binding curves of recombinant human CD276 protein with 1C8, 1H12, 3C1 and 3E5 scFv fragments were determined by ELISA.

1C8, 1H12, 3C1 and 3E5 scFv-Fc fusion antibodies are respectively constructed and purified in eukaryotic cells by transient expression, and the combination of the recombinant human CD276 protein and the 4 scFv fragments is detected by ELISA (enzyme-linked immuno sorbent assay) which is specifically operated as follows: 100 ng/well of recombinant human CD276 protein prepared above was added to the microplate and coated overnight at 4 ℃. PBS wash three times, add 1% BSA/PBS, 200 uL/well, 37 degrees C blocking for 1 hours. After washing the plate with 100ul PBS, a gradient of 4 scFv proteins was added and binding was performed for 1 hour at 37 ℃. PBST was washed three times and 100ul of HRP-goat anti-human IgG (Fab specific) diluted 1:5000 was added for binding at 37 ℃ for 1 hour. PBST was washed three times, 100uL/well TMB developing solution was added, color development was performed at 37 ℃ for 10 minutes, 100uL/well ELISA stop solution was added, and OD450 value was read by a microplate reader, the results are shown in FIG. 4 and Table 3.

TABLE 3 test results

	1C8	1H12	3C1	3E5
					EC50	51.58	32.19	15.12	32.14

Example 3: stock retroviral solutions containing anti-human CD276 chimeric antigen receptor elements were prepared.

1. Preparation of chimeric antigen receptors targeting the human CD276 antigen.

A chimeric antigen receptor sequence containing scFv of a single-chain antibody against human CD276 antigen, a hinge region, a transmembrane region and an intracellular signal segment is genetically synthesized or cloned, and the structure is shown in FIG. 5. The chimeric antigen receptors were named 1C8-BBz, 1H12-BBz, 3C1-BBz and 3E5-BBz, respectively, according to the difference of loading scFv and intracellular signal, and the nucleotide sequences thereof were shown as SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37, respectively. Meanwhile, the scFv of the known CD276 murine antibody (clone number: 376.96) reported in the literature was selected to construct a chimeric antigen receptor as a control, which was named 376.96-BBz, the nucleotide sequence of which is shown in SEQ ID NO:38, and the sequence information specifically refers to Table 4.

The retroviral plasmid expressing the chimeric antigen receptor cloned by 376.96, 1C8, 1H12, 3C1 and 3E5 is constructed by taking the retroviral vector MSGV as a framework vector. Selecting a clone with correct sequencing, inoculating a bacterium solution into 300 ml of LB culture medium, shaking the bacterium overnight, and completing large-scale plasmid extraction according to the instruction of a NucleoBondXtra Maxi EF kit.

2. And (4) packaging the retrovirus.

Packaging of the retrovirus with cationic polymer pei (polyplus) the procedure is as follows: diluting PEI and the retrovirus packaging plasmid (virus master plasmid, Gag-pol, 10A 1) with serum-free DMEM, respectively; adding PEI/DMEM into the plasmid/DMEM mixture, vortex, shaking and mixing uniformly, and standing for 15 minutes at room temperature; plasmid-PEI complexes were added to pre-plated 293T cells. Changing the solution 16h after transfection, collecting the virus supernatant after 48h, filtering by a 0.45um filter, subpackaging the stock solution by a 15mL centrifuge tube, and storing at-80 ℃ for later use.

TABLE 4 sequence information

Numbering	Sequence of	Description of the invention
			SEQ ID NO：1	GFTFGTYA	VH-CDR1-1
SEQ ID NO：2	GFTFSSYA	VH-CDR1-2
			SEQ ID NO：3	GGTFSRFA	VH-CDR1-3
SEQ ID NO：4	IDGGGSTT	VH-CDR2-1
			SEQ ID NO：5	ISGSGGST	VH-CDR2-2
SEQ ID NO：6	IIPIFGTA	VH-CDR2-3
			SEQ ID NO：7	TRATGGTKHDY	VH-CDR3-1
SEQ ID NO：8	AIGIHYGFDY	VH-CDR3-2
			SEQ ID NO：9	ASISSPDDY	VH-CDR3-3
SEQ ID NO：10	ARGHSGSYTLDY	VH-CDR3-4
			SEQ ID NO：11	QSVGIY	VK-CDR1-1
SEQ ID NO：12	QSISFY	VK-CDR1-2
			SEQ ID NO：13	QGIGTF	VK-CDR1-3
SEQ ID NO：14	QSVSSY	VK-CDR1-4
			SEQ ID NO：15	AAS	VK-CDR2-1
SEQ ID NO：16	DAF	VK-CDR2-2
			SEQ ID NO：17	QQSLSAPLT	VK-CDR3-1
SEQ ID NO：18	QQSYSTPPWT	VK-CDR3-2
			SEQ ID NO：19	QQTYGNPPWT	VK-CDR3-3
SEQ ID NO：20	LHRSNWPPLFT	VK-CDR3-4
			SEQ ID NO：21	QVQLVESGGGLVQPGGSLRLSCAASGFTFGTYAMSWVRQAPGKGLEWV SRIDGGGSTTNYADSVKGRFTISRDNAKNALYLQMDSLRAEDTAMYYC TRATGGTKHDYWGQGTLVTVSS	1C8-VH
SEQ ID NO：22	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV SAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRADDTAVYYC AIGIHYGFDYWGQGTLVTVSS	1H12-VH
			SEQ ID NO：23	EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV SAISGSGGSTYYADSVKGRFTISRDNSKNSLYLQMNSLGAEDTAVYYC ASISSPDDYWGQGTLVTVSS	3C1-VH
SEQ ID NO：24	QVQLVQSGAEVKKPGSSVKVSCKASGGTFSRFAISWVRQAPGQGLEWM GGIIPIFGTASYAQKFQGRATITADESTSTAYMELSSLRSEDTAVYFC ARGHSGSYTLDYWGQGTLVTVSS	3E5-VH
			SEQ ID NO：25	DVVMTQSPSSLSASVGDRVTITCRASQSVGIYLNWYQQKPGKAPKLLI YAASRLQSGVPSRFGGSGSGTDFTLTISSLQPEDSATYYCQQSLSAPL TFGGGTKVEIK	1C8-VK
SEQ ID NO：26	DIQMTQSPSSLSASVGDRVTITCRASQSISFYLNWYQQKPGKAPKLLI YAASSLQSGVPSRFGGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPP WTFGQGTKVEIK	1H12-VK
			SEQ ID NO：27	DIQMTQSPSSLSASVGDRVTISCRASQGIGTFLNWYQQKPGKVPKLLI HAASSLQGGVPSRFSGSGSGTDFTLTINSLQREDLATYYCQQTYGNPP WTFGQGTKVEIK	3C1-VK
SEQ ID NO：28	QTPATLSLSPGERATLSCRASQSAGSQSVSSYLAWYQQKPGQAPRLLI NDAFNRAPGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCLHRSNWPP LFTFGGGTKVEIK	3E5-VK
			SEQ ID NO：29	MALPVTALLLPLALLLHAARP	CD8 alpha signal peptide
SEQ ID NO：30	TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY	CD8 alpha hinge region
			SEQ ID NO：31	IWAPLAGTCGVLLLSLVITLYC	CD8 alpha transmembrane domain
SEQ ID NO：32	VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPR	CD3z cytoplasmic domain
			SEQ ID NO：33	RFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELR	41BB cytoplasmic region
SEQ ID NO：34	atggctctgcctgtgaccgccctgctgctgcctctggctctgctgct gcacgccgctcggcctgatgttgtgatgacacagtctccatcctccc tgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagt cagagtgttggcatctatttaaattggtatcagcagaaaccagggaa agcccctaagctcctgatctatgctgcatccagattgcaaagtgggg tcccatcaaggttcggtggcagtggatctgggacagatttcactctc accatcagcagtctgcaacctgaagattctgcaacttactactgtca acagagtctcagtgcccctctcactttcggcggagggaccaaggtgg agatcaagcgaggcggcggcggcagtggtggtggtggtagtggagga ggaggaagtcaggtgcagctggtggagtccgggggaggcttggtaca gcctggggggtccctgagactctcctgtgcagcctctggattcacct ttggcacctatgccatgagctgggtccgccaggctccagggaagggg ctggagtgggtctcacgtattgatggtggtgggagcactacaaacta cgcggactccgtgaagggccgattcaccatctccagagacaacgcca agaacgcgctgtatctgcaaatggacagtctgagagccgaggacacg gctatgtattattgtacaagagcaacaggtggaacaaagcatgacta ctggggccagggaaccctggtcaccgtctcctcaactacaactccag cacccagaccccctacacctgctccaactatcgcaagtcagcccctg tcactgcgccctgaagcctgtcgccctgctgccgggggagctgtgca tactcggggactggactttgcctgtgatatctacatctgggcgccct tggccgggacttgtggggtccttctcctgtcactggttatcaccctt tactgcaggttcagtgtcgtgaagagaggccggaagaagctgctgta catcttcaagcagcctttcatgaggcccgtgcagactacccaggagg aagatggatgcagctgtagattccctgaagaggaggaaggaggctgt gagctgagagtgaagttctcccgaagcgcagatgccccagcctatca gcagggacagaatcagctgtacaacgagctgaacctgggaagacggg aggaatacgatgtgctggacaaaaggcggggcagagatcctgagatg ggcggcaaaccaagacggaagaacccccaggaaggtctgtataatga gctgcagaaagacaagatggctgaggcctactcagaaatcgggatga agggcgaaagaaggagaggaaaaggccacgacggactgtaccagggg ctgagtacagcaacaaaagacacctatgacgctctgcacatgcaggc tctgccaccaagatag	1C8-BBz
			SEQ ID NO：35	atggctctgcctgtgaccgccctgctgctgcctctggctctgctgct gcacgccgctcggcctgacatccagatgacccagtctccatcctccc tgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagt cagagcattagcttctatttaaattggtatcagcagaaaccagggaa agcccctaagctcctgatctatgctgcatccagtttgcaaagtgggg tcccatcaaggttcggtggcagtggatctgggacagacttcaccctc accatcagcagcctgcagcctgaagattttgcaacttactactgtca acagagttacagtacccctccgtggacgttcggccaagggaccaagg tggaaatcaaacgaggcggcggcggcagtggtggtggtggtagtgga ggaggaggaagtgaggtgcagctggtggagtctgggggaggcttggt acagcctggggggtccctgagactctcctgtgcagcctctggattca cctttagcagctatgccatgagctgggtccgccaggctccagggaag gggctggagtgggtctcagctattagtggtagtggtggtagcacata ctacgcagactccgtgaagggccggttcaccatctccagagacaatt ccaagaacacgctgtatctgcaaatgaacagcctgagagccgacgac acggccgtatattactgtgcgattgggatccattacggctttgacta ctggggccagggaaccctggtcaccgtctcttcaactacaactccag cacccagaccccctacacctgctccaactatcgcaagtcagcccctg tcactgcgccctgaagcctgtcgccctgctgccgggggagctgtgca tactcggggactggactttgcctgtgatatctacatctgggcgccct tggccgggacttgtggggtccttctcctgtcactggttatcaccctt tactgcaggttcagtgtcgtgaagagaggccggaagaagctgctgta catcttcaagcagcctttcatgaggcccgtgcagactacccaggagg aagatggatgcagctgtagattccctgaagaggaggaaggaggctgt gagctgagagtgaagttctcccgaagcgcagatgccccagcctatca gcagggacagaatcagctgtacaacgagctgaacctgggaagacggg aggaatacgatgtgctggacaaaaggcggggcagagatcctgagatg ggcggcaaaccaagacggaagaacccccaggaaggtctgtataatga gctgcagaaagacaagatggctgaggcctactcagaaatcgggatga agggcgaaagaaggagaggaaaaggccacgacggactgtaccagggg ctgagtacagcaacaaaagacacctatgacgctctgcacatgcaggc tctgccaccaagatag	1H12-BBz
SEQ ID NO：36	atggctctgcctgtgaccgccctgctgctgcctctggctctgctgct gcacgccgctcggcctgacatccagatgacccagtctccatcctccc tgtctgcatctgttggagacagagtcaccatcagttgccgggcaagt cagggtattggtacttttttaaattggtaccagcagaagccagggaa agtccccaaacttctgatccatgctgcttccagtttgcaaggtgggg tcccatcaaggttcagtgggagtggatctgggacagatttcactctc accatcaacagcctgcaacgtgaagatcttgctacatactactgtca gcagacgtatggcaaccctccgtggacgttcggccaagggaccaagg tggagatcaaacgaggcggcggcggcagtggtggtggtggtagtgga ggaggaggaagtgaggtgcagctggtggagtctgggggaggcttggt acagcctggggggtccctgagactctcctgtgcagcctctggattca cctttagcagctatgccatgagctgggtccgccaggctccagggaag gggctggagtgggtctcagctattagtggtagtggtggtagcacata ctacgcagactccgtgaagggccggttcaccatctccagagacaatt ccaagaactcactgtatctgcaaatgaacagcctgggagccgaggac acggctgtgtattactgtgcatcaatctcctcccccgatgactactg gggccagggaaccctggtcaccgtctcctcaactacaactccagcac ccagaccccctacacctgctccaactatcgcaagtcagcccctgtca ctgcgccctgaagcctgtcgccctgctgccgggggagctgtgcatac tcggggactggactttgcctgtgatatctacatctgggcgcccttgg ccgggacttgtggggtccttctcctgtcactggttatcaccctttac tgcaggttcagtgtcgtgaagagaggccggaagaagctgctgtacat cttcaagcagcctttcatgaggcccgtgcagactacccaggaggaag atggatgcagctgtagattccctgaagaggaggaaggaggctgtgag ctgagagtgaagttctcccgaagcgcagatgccccagcctatcagca gggacagaatcagctgtacaacgagctgaacctgggaagacgggagg aatacgatgtgctggacaaaaggcggggcagagatcctgagatgggc ggcaaaccaagacggaagaacccccaggaaggtctgtataatgagct gcagaaagacaagatggctgaggcctactcagaaatcgggatgaagg gcgaaagaaggagaggaaaaggccacgacggactgtaccaggggctg agtacagcaacaaaagacacctatgacgctctgcacatgcaggctct gccaccaagatag	3C1-BBz
			SEQ ID NO：37	atggctctgcctgtgaccgccctgctgctgcctctggctctgctgct gcacgccgctcggcctgatattgtgatgacccagactccagccaccc tgtctttgtctccaggggaaagagccaccctctcctgcagggccagt cagagcgctggcagtcaaagtgttagcagctacttagcctggtacca gcagaaacctggccaggctcccaggctcctcatcaatgatgcattca acagggcccctggcatcccagccaggttcagtggcagtgggtctggg acagacttcactctcaccatcagcagcctagagcctgaagattttgc agtttattactgtctgcaccgtagcaactggcctcctttgttcactt tcggcggggggaccaaggtggaaatcaaacgaggcggcggcggcagt ggtggtggtggtagtggaggaggaggaagtcaggtgcagctggtgca gtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcct gcaaggcttctggaggcaccttcagcagatttgctatcagctgggtg cgacaggccccgggacaagggcttgagtggatgggagggatcatccc tatctttggaacagcaagctacgcacagaagttccagggcagagcca cgattaccgcggacgaatccacgagcacagcctacatggagctgagc agcctgagatctgaggacacggccgtgtatttctgtgcgagggggca tagtgggagctacacccttgactactggggccagggaaccctggtca ccgtctcctcaactacaactccagcacccagaccccctacacctgct ccaactatcgcaagtcagcccctgtcactgcgccctgaagcctgtcg ccctgctgccgggggagctgtgcatactcggggactggactttgcct gtgatatctacatctgggcgcccttggccgggacttgtggggtcctt ctcctgtcactggttatcaccctttactgcaggttcagtgtcgtgaa gagaggccggaagaagctgctgtacatcttcaagcagcctttcatga ggcccgtgcagactacccaggaggaagatggatgcagctgtagattc cctgaagaggaggaaggaggctgtgagctgagagtgaagttctcccg aagcgcagatgccccagcctatcagcagggacagaatcagctgtaca acgagctgaacctgggaagacgggaggaatacgatgtgctggacaaa aggcggggcagagatcctgagatgggcggcaaaccaagacggaagaa cccccaggaaggtctgtataatgagctgcagaaagacaagatggctg aggcctactcagaaatcgggatgaagggcgaaagaaggagaggaaaa ggccacgacggactgtaccaggggctgagtacagcaacaaaagacac ctatgacgctctgcacatgcaggctctgccaccaagatag	3E5-BBz
SEQ ID NO：38	atggctctgcctgtgaccgccctgctgctgcctctggctctgctgct gcacgccgctcggcctgatatcgtgatgacccagagccacaagttca tgagcaccagcatcggcgccagagtgagcatcacctgcaaggccagc caggacgtgaggaccgccgtggcctggtaccagcagaagcctggcca gagccccaagctgctgatctacagcgccagctacagatacaccggcg tgcccgacagattcaccggcagcggcagcggcaccgacttcaccttc accatcagcagcgtgcaggccgaggacctggccgtgtactactgcca gcagcactacggcacccctccctggaccttcggcggcggcaccaagc tggagatcaagggcggaggcggctccggcggcggcggctccggcggc ggcggctccgaggtgcagctggtggagagcggcggcggcctggtgaa gcccggcggcagcctgaagctgagctgcgaggccagcagattcacct tcagcagctacgccatgagctgggtgaggcagacccccgagaagaga ctggagtgggtggccgccatcagcggcggcggcaggtacacctacta ccccgacagcatgaagggcaggttcaccatcagcagggacaacgcca agaacttcctgtacctgcagatgagctccctgagaagcgaggacacc gccatgtactactgcgccagacactacgacggctacctggactactg gggccagggcaccaccctgaccgtgagcagcaccaggactacaactc cagcacccagaccccctacacctgctccaactatcgcaagtcagccc ctgtcactgcgccctgaagcctgtcgccctgctgccgggggagctgt gcatactcggggactggactttgcctgtgatatctacatctgggcgc ccttggccgggacttgtggggtccttctcctgtcactggttatcacc ctttactgcaggttcagtgtcgtgaagagaggccggaagaagctgct gtacatcttcaagcagcctttcatgaggcccgtgcagactacccagg aggaagatggatgcagctgtagattccctgaagaggaggaaggaggc tgtgagctgagagtgaagttctcccgaagcgcagatgccccagccta tcagcagggacagaatcagctgtacaacgagctgaacctgggaagac gggaggaatacgatgtgctggacaaaaggcggggcagagatcctgag atgggcggcaaaccaagacggaagaacccccaggaaggtctgtataa tgagctgcagaaagacaagatggctgaggcctactcagaaatcggga tgaagggcgaaagaaggagaggaaaaggccacgacggactgtaccag gggctgagtacagcaacaaaagacacctatgacgctctgcacatgca ggctctgccaccaagatag	376.96-BBz
			SEQ ID NO：39	LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLV HSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFV SIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYP EAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSC LVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRC SFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPD LLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPS MTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQ MANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPM TFPASGLNDIFEAQKIEWHEHHHHHH	Recombinant CD276 egg White amino acid sequence
SEQ ID NO：40	ctggaggtgcaggtgcccgaggaccccgtggtggccctggtgggcac cgacgccaccctgtgctgcagcttcagccccgagcccggcttcagcc tggcccagctgaacctgatctggcagctgaccgacaccaagcagctg gtgcacagcttcgccgagggccaggaccagggcagcgcctacgccaa ccgcaccgccctgttccccgacctgctggcccagggcaacgccagcc tgcgcctgcagcgcgtgcgcgtggccgacgagggcagcttcacctgc ttcgtgagcatccgcgacttcggcagcgccgccgtgagcctgcaggt ggccgccccctacagcaagcccagcatgaccctggagcccaacaagg acctgcgccccggcgacaccgtgaccatcacctgcagcagctaccag ggctaccccgaggccgaggtgttctggcaggacggccagggcgtgcc cctgaccggcaacgtgaccaccagccagatggccaacgagcagggcc tgttcgacgtgcacagcatcctgcgcgtggtgctgggcgccaacggc acctacagctgcctggtgcgcaaccccgtgctgcagcaggacgccca cagcagcgtgaccatcaccccccagcgcagccccaccggcgccgtgg aggtgcaggtgcccgaggaccccgtggtggccctggtgggcaccgac gccaccctgcgctgcagcttcagccccgagcccggcttcagcctggc ccagctgaacctgatctggcagctgaccgacaccaagcagctggtgc acagcttcaccgagggccgcgaccagggcagcgcctacgccaaccgc accgccctgttccccgacctgctggcccagggcaacgccagcctgcg cctgcagcgcgtgcgcgtggccgacgagggcagcttcacctgcttcg tgagcatccgcgacttcggcagcgccgccgtgagcctgcaggtggcc gccccctacagcaagcccagcatgaccctggagcccaacaaggacct gcgccccggcgacaccgtgaccatcacctgcagcagctaccgcggct accccgaggccgaggtgttctggcaggacggccagggcgtgcccctg accggcaacgtgaccaccagccagatggccaacgagcagggcctgtt cgacgtgcacagcgtgctgcgcgtggtgctgggcgccaacggcacct acagctgcctggtgcgcaaccccgtgctgcagcaggacgcccacggc agcgtgaccatcaccggccagcccatgaccttccccgctagcggtct gaacgacatcttcgaggctcagaaaatcgaatggcacgaacatcatc accatcaccat	Recombinant CD276 egg White nucleic acid sequence

Example 4: preparation of CD276 CAR-T cells and CAR-positivity assay

PBMC isolation and activation.

Collection logPeripheral blood of the volunteer, PBMC obtained by separating with Ficcol separating medium, and adjusting cell density to 1X10 with 5% AB serum X-VIVO (LONZA) containing medium⁶and/mL. TC-coated 6-well plates were incubated with 1ml of coating solution containing 50ng/ml of anti-human CD3 antibody (Beijing Hokkaiyuan) and 50ng/ml of CD28 antibody (Beijing Hokkaiyuan) at 37 ℃ for 2 hours in advance, and the coating solution was removed before use. The cells were inoculated at 1 ml/well into antibody-coated 6-well plates, and then 100IU/ml of IL2 (Beijing Erlu) was added to stimulate virus infection after 48 hours of culture.

2. And (5) infecting and culturing virus stock solution.

The activated T cells were adjusted to 5X10⁵PermL, 1mL of T cells and 1mL of virus stock were added to a 24-well plate, 1ul of polybrene per well, 32 ℃, 2500rpm, and centrifuged for 1.5 h. The supernatant was discarded and 1ml of T cell medium (containing IL-2100 IU/ml) was added to each well. The plates were incubated at 37 ℃ in 5% CO₂Culturing in an incubator. And transferring to a 6-well plate 24h after infection, observing the density of the cells every day, and timely supplementing a T cell culture solution containing IL-2100 IU/ml to maintain the density of the T cells at about 1x106/ml so as to expand the cells.

CAR positive rate detection.

Retroviral infected T lymphocytes tested CAR positive rates 72h after viral infection. 1X10 of the chimeric antigen receptor group containing 376.96, 1C8, 1H12, 3C1 and 3E5 clones and the negative uninfected control group NT were taken⁶The cells were centrifuged to remove the medium and 100ul of the resuspended in a flow-up tube (BD) after one PBS wash of the cells. Biotin-labeled CD276 antigen (1: 400) was added and incubated for 30 minutes at four degrees. After one PBS wash, the cells were incubated for 30 minutes at four degrees in the dark with the recommended ratio of the second antibody Brilliant Violet 421 streptavidin (BioLegend). After one cell wash with PBS, 200ul PBS was resuspended and tested on the machine, and the CAR-T positive rate flow analysis results are shown in FIG. 6.

Example 3: based on anti-human CD276 CAR-T cell functional analysis.

1. Anti-human CD276 CAR-T cell CD107a expression assay.

Separate grouping of CAR-T cells and NT cells containing different antibody clones with targets of different groupsThe cells (CD 276 positive expression kidney cancer cell line ACHN, lung cancer cell line A549 and ovarian cancer cell line SKOV 3) have an effective-to-target ratio of 1:1 (both the effective cells and the target cells are 3x 10)⁵Individually), flow-assayed for CD107a expression to assess the degranulation response of CAR-T cells following stimulation by target cells. Mixing effector cells and target cells at 37 deg.C and 5% CO₂After the incubation in the incubator for 4 hours, the ratio of the number of cells expressing CD107a to the number of CD3+ cells in each group of samples was measured by flow. The results of flow analysis of CD107a expression are shown in figure 7.

2. Anti-human CD276 CAR-T cell cytokine secretion ability assay.

CAR-T cells and NT cells containing different antibody clones are respectively incubated with target cells of different groups (cell lines ACHN, A549 and SKOV3 positively expressed by CD 276) for 24 hours according to an effect-target ratio of 1:1 (1 x105 effector cells and target cells respectively), supernatants are collected, and the secretion conditions of IFN-gamma and IL-2 are detected by an ELISA (enzyme-linked immunosorbent assay) method. IFN-gamma detection adopts BD IFN-gamma and IL-2 kit detection, and the experimental steps are carried out according to the product instruction. The results of IFN-. gamma.secretion measurements are shown in FIG. 8. The results of IL-2 secretion are shown in FIG. 9.

3. Anti-human CD276 CAR-T cytotoxicity assay.

CAR-T killing toxicity experiments CAR-T cell function in vitro was assessed by testing the killing effect of CAR-T cells on target cells in vitro. T cells were co-cultured with CD276 positive target cells, ACHN-LUC-GFP, A549-LUC-GFP and SKOV3-LUC-GFP, which stably expressed firefly luciferase, and negative target cells, MOLM13-LUC-GFP, which were different effective target ratios (20: 1, 10:1, 5:1, and 2.5:1, respectively, based on 3X104 target cells), while setting a negative control group (NT) in which target cells and untransfected CAR element T cells were mixed. After overnight incubation, luciferase reaction substrate was added to the culture system, the fluorescence value was measured, and the killing efficiency was calculated by the following formula: killing efficiency = (1-experimental well fluorescence/control well fluorescence) x 100%. The experimental groupings and the analysis results are shown in fig. 10.

The results show that the CAR-T cells targeting CD276 provided by the invention have strong immune function as the control murine CAR, and show excellent CD107a expression, IFN-gamma and IL-2 secretion and specific killing function on target cells. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Shanghai Hengrunheng Dasheng Biotech Co., Ltd

<120> antibody or antigen binding fragment thereof specifically binding to CD276, and preparation method and application thereof

<160> 40

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

<213> Artificial sequence

<400> 1

Gly Phe Thr Phe Gly Thr Tyr Ala

1 5

<210> 2

<211> 8

<212> PRT

<213> Artificial sequence

<400> 2

Gly Phe Thr Phe Ser Ser Tyr Ala

1 5

<210> 3

<211> 8

<212> PRT

<213> Artificial sequence

<400> 3

Gly Gly Thr Phe Ser Arg Phe Ala

1 5

<210> 4

<211> 8

<212> PRT

<213> Artificial sequence

<400> 4

Ile Asp Gly Gly Gly Ser Thr Thr

1 5

<210> 5

<211> 8

<212> PRT

<213> Artificial sequence

<400> 5

Ile Ser Gly Ser Gly Gly Ser Thr

1 5

<210> 6

<211> 8

<212> PRT

<213> Artificial sequence

<400> 6

Ile Ile Pro Ile Phe Gly Thr Ala

1 5

<210> 7

<211> 11

<212> PRT

<213> Artificial sequence

<400> 7

Thr Arg Ala Thr Gly Gly Thr Lys His Asp Tyr

1 5 10

<210> 8

<211> 10

<212> PRT

<213> Artificial sequence

<400> 8

Ala Ile Gly Ile His Tyr Gly Phe Asp Tyr

1 5 10

<210> 9

<211> 9

<212> PRT

<213> Artificial sequence

<400> 9

Ala Ser Ile Ser Ser Pro Asp Asp Tyr

1 5

<210> 10

<211> 12

<212> PRT

<213> Artificial sequence

<400> 10

Ala Arg Gly His Ser Gly Ser Tyr Thr Leu Asp Tyr

1 5 10

<210> 11

<211> 6

<212> PRT

<213> Artificial sequence

<400> 11

Gln Ser Val Gly Ile Tyr

1 5

<210> 12

<211> 6

<212> PRT

<213> Artificial sequence

<400> 12

Gln Ser Ile Ser Phe Tyr

1 5

<210> 13

<211> 6

<212> PRT

<213> Artificial sequence

<400> 13

Gln Gly Ile Gly Thr Phe

1 5

<210> 14

<211> 6

<212> PRT

<213> Artificial sequence

<400> 14

Gln Ser Val Ser Ser Tyr

1 5

<210> 15

<211> 3

<212> PRT

<213> Artificial sequence

<400> 15

Ala Ala Ser

1

<210> 16

<211> 3

<212> PRT

<213> Artificial sequence

<400> 16

Asp Ala Phe

1

<210> 17

<211> 9

<212> PRT

<213> Artificial sequence

<400> 17

Gln Gln Ser Leu Ser Ala Pro Leu Thr

1 5

<210> 18

<211> 10

<212> PRT

<213> Artificial sequence

<400> 18

Gln Gln Ser Tyr Ser Thr Pro Pro Trp Thr

1 5 10

<210> 19

<211> 10

<212> PRT

<213> Artificial sequence

<400> 19

Gln Gln Thr Tyr Gly Asn Pro Pro Trp Thr

1 5 10

<210> 20

<211> 11

<212> PRT

<213> Artificial sequence

<400> 20

Leu His Arg Ser Asn Trp Pro Pro Leu Phe Thr

1 5 10

<210> 21

<211> 118

<212> PRT

<213> Artificial sequence

<400> 21

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Arg Ala Thr Gly Gly Thr Lys His Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 22

<211> 117

<212> PRT

<213> Artificial sequence

<400> 22

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Ile Gly Ile His Tyr Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 23

<211> 116

<212> PRT

<213> Artificial sequence

<400> 23

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Val Ser Ser

115

<210> 24

<211> 119

<212> PRT

<213> Artificial sequence

<400> 24

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Gln Gly Arg Ala Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala 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 Gly His Ser Gly Ser Tyr Thr Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 25

<211> 107

<212> PRT

<213> Artificial sequence

<400> 25

Asp Val 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 Ser Val Gly Ile Tyr

20 25 30

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

35 40 45

Tyr Ala Ala Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Gly 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 Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Leu Ser Ala Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 26

<211> 108

<212> PRT

<213> Artificial sequence

<400> 26

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 Ser Ile Ser Phe Tyr

20 25 30

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

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Gly 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 Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 27

<211> 108

<212> PRT

<213> Artificial sequence

<400> 27

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 Ser Cys Arg Ala Ser Gln Gly Ile Gly Thr Phe

20 25 30

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

35 40 45

His Ala Ala Ser Ser Leu Gln Gly 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 Gln Arg

65 70 75 80

Glu Asp Leu Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Gly Asn Pro Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 28

<211> 109

<212> PRT

<213> Artificial sequence

<400> 28

Gln Thr Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu

1 5 10 15

Ser Cys Arg Ala Ser Gln Ser Ala Gly Ser Gln Ser Val Ser Ser Tyr

20 25 30

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

35 40 45

Asn Asp Ala Phe Asn Arg Ala Pro Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Leu His Arg Ser Asn Trp Pro Pro

85 90 95

Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 29

<211> 21

<212> PRT

<213> Artificial sequence

<400> 29

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 30

<211> 47

<212> PRT

<213> Artificial sequence

<400> 30

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

35 40 45

<210> 31

<211> 22

<212> PRT

<213> Artificial sequence

<400> 31

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

1 5 10 15

Val Ile Thr Leu Tyr Cys

20

<210> 32

<211> 111

<212> PRT

<213> Artificial sequence

<400> 32

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

1 5 10 15

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

20 25 30

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

35 40 45

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

50 55 60

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

65 70 75 80

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

85 90 95

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 33

<211> 48

<212> PRT

<213> Artificial sequence

<400> 33

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

1 5 10 15

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

20 25 30

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

35 40 45

<210> 34

<211> 1473

<212> DNA

<213> Artificial sequence

<400> 34

atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60

cctgatgttg tgatgacaca gtctccatcc tccctgtctg catctgtagg agacagagtc 120

accatcactt gccgggcaag tcagagtgtt ggcatctatt taaattggta tcagcagaaa 180

ccagggaaag cccctaagct cctgatctat gctgcatcca gattgcaaag tggggtccca 240

tcaaggttcg gtggcagtgg atctgggaca gatttcactc tcaccatcag cagtctgcaa 300

cctgaagatt ctgcaactta ctactgtcaa cagagtctca gtgcccctct cactttcggc 360

ggagggacca aggtggagat caagcgaggc ggcggcggca gtggtggtgg tggtagtgga 420

ggaggaggaa gtcaggtgca gctggtggag tccgggggag gcttggtaca gcctgggggg 480

tccctgagac tctcctgtgc agcctctgga ttcacctttg gcacctatgc catgagctgg 540

gtccgccagg ctccagggaa ggggctggag tgggtctcac gtattgatgg tggtgggagc 600

actacaaact acgcggactc cgtgaagggc cgattcacca tctccagaga caacgccaag 660

aacgcgctgt atctgcaaat ggacagtctg agagccgagg acacggctat gtattattgt 720

acaagagcaa caggtggaac aaagcatgac tactggggcc agggaaccct ggtcaccgtc 780

tcctcaacta caactccagc acccagaccc cctacacctg ctccaactat cgcaagtcag 840

cccctgtcac tgcgccctga agcctgtcgc cctgctgccg ggggagctgt gcatactcgg 900

ggactggact ttgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 960

cttctcctgt cactggttat caccctttac tgcaggttca gtgtcgtgaa gagaggccgg 1020

aagaagctgc tgtacatctt caagcagcct ttcatgaggc ccgtgcagac tacccaggag 1080

gaagatggat gcagctgtag attccctgaa gaggaggaag gaggctgtga gctgagagtg 1140

aagttctccc gaagcgcaga tgccccagcc tatcagcagg gacagaatca gctgtacaac 1200

gagctgaacc tgggaagacg ggaggaatac gatgtgctgg acaaaaggcg gggcagagat 1260

cctgagatgg gcggcaaacc aagacggaag aacccccagg aaggtctgta taatgagctg 1320

cagaaagaca agatggctga ggcctactca gaaatcggga tgaagggcga aagaaggaga 1380

ggaaaaggcc acgacggact gtaccagggg ctgagtacag caacaaaaga cacctatgac 1440

gctctgcaca tgcaggctct gccaccaaga tag 1473

<210> 35

<211> 1473

<212> DNA

<213> Artificial sequence

<400> 35

atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60

cctgacatcc agatgaccca gtctccatcc tccctgtctg catctgtagg agacagagtc 120

accatcactt gccgggcaag tcagagcatt agcttctatt taaattggta tcagcagaaa 180

ccagggaaag cccctaagct cctgatctat gctgcatcca gtttgcaaag tggggtccca 240

tcaaggttcg gtggcagtgg atctgggaca gacttcaccc tcaccatcag cagcctgcag 300

cctgaagatt ttgcaactta ctactgtcaa cagagttaca gtacccctcc gtggacgttc 360

ggccaaggga ccaaggtgga aatcaaacga ggcggcggcg gcagtggtgg tggtggtagt 420

ggaggaggag gaagtgaggt gcagctggtg gagtctgggg gaggcttggt acagcctggg 480

gggtccctga gactctcctg tgcagcctct ggattcacct ttagcagcta tgccatgagc 540

tgggtccgcc aggctccagg gaaggggctg gagtgggtct cagctattag tggtagtggt 600

ggtagcacat actacgcaga ctccgtgaag ggccggttca ccatctccag agacaattcc 660

aagaacacgc tgtatctgca aatgaacagc ctgagagccg acgacacggc cgtatattac 720

tgtgcgattg ggatccatta cggctttgac tactggggcc agggaaccct ggtcaccgtc 780

tcttcaacta caactccagc acccagaccc cctacacctg ctccaactat cgcaagtcag 840

cccctgtcac tgcgccctga agcctgtcgc cctgctgccg ggggagctgt gcatactcgg 900

ggactggact ttgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 960

cttctcctgt cactggttat caccctttac tgcaggttca gtgtcgtgaa gagaggccgg 1020

aagaagctgc tgtacatctt caagcagcct ttcatgaggc ccgtgcagac tacccaggag 1080

gaagatggat gcagctgtag attccctgaa gaggaggaag gaggctgtga gctgagagtg 1140

aagttctccc gaagcgcaga tgccccagcc tatcagcagg gacagaatca gctgtacaac 1200

gagctgaacc tgggaagacg ggaggaatac gatgtgctgg acaaaaggcg gggcagagat 1260

cctgagatgg gcggcaaacc aagacggaag aacccccagg aaggtctgta taatgagctg 1320

cagaaagaca agatggctga ggcctactca gaaatcggga tgaagggcga aagaaggaga 1380

ggaaaaggcc acgacggact gtaccagggg ctgagtacag caacaaaaga cacctatgac 1440

gctctgcaca tgcaggctct gccaccaaga tag 1473

<210> 36

<211> 1470

<212> DNA

<213> Artificial sequence

<400> 36

atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60

cctgacatcc agatgaccca gtctccatcc tccctgtctg catctgttgg agacagagtc 120

accatcagtt gccgggcaag tcagggtatt ggtacttttt taaattggta ccagcagaag 180

ccagggaaag tccccaaact tctgatccat gctgcttcca gtttgcaagg tggggtccca 240

tcaaggttca gtgggagtgg atctgggaca gatttcactc tcaccatcaa cagcctgcaa 300

cgtgaagatc ttgctacata ctactgtcag cagacgtatg gcaaccctcc gtggacgttc 360

ggccaaggga ccaaggtgga gatcaaacga ggcggcggcg gcagtggtgg tggtggtagt 420

ggaggaggag gaagtgaggt gcagctggtg gagtctgggg gaggcttggt acagcctggg 480

gggtccctga gactctcctg tgcagcctct ggattcacct ttagcagcta tgccatgagc 540

tgggtccgcc aggctccagg gaaggggctg gagtgggtct cagctattag tggtagtggt 600

ggtagcacat actacgcaga ctccgtgaag ggccggttca ccatctccag agacaattcc 660

aagaactcac tgtatctgca aatgaacagc ctgggagccg aggacacggc tgtgtattac 720

tgtgcatcaa tctcctcccc cgatgactac tggggccagg gaaccctggt caccgtctcc 780

tcaactacaa ctccagcacc cagaccccct acacctgctc caactatcgc aagtcagccc 840

ctgtcactgc gccctgaagc ctgtcgccct gctgccgggg gagctgtgca tactcgggga 900

ctggactttg cctgtgatat ctacatctgg gcgcccttgg ccgggacttg tggggtcctt 960

ctcctgtcac tggttatcac cctttactgc aggttcagtg tcgtgaagag aggccggaag 1020

aagctgctgt acatcttcaa gcagcctttc atgaggcccg tgcagactac ccaggaggaa 1080

gatggatgca gctgtagatt ccctgaagag gaggaaggag gctgtgagct gagagtgaag 1140

ttctcccgaa gcgcagatgc cccagcctat cagcagggac agaatcagct gtacaacgag 1200

ctgaacctgg gaagacggga ggaatacgat gtgctggaca aaaggcgggg cagagatcct 1260

gagatgggcg gcaaaccaag acggaagaac ccccaggaag gtctgtataa tgagctgcag 1320

aaagacaaga tggctgaggc ctactcagaa atcgggatga agggcgaaag aaggagagga 1380

aaaggccacg acggactgta ccaggggctg agtacagcaa caaaagacac ctatgacgct 1440

ctgcacatgc aggctctgcc accaagatag 1470

<210> 37

<211> 1497

<212> DNA

<213> Artificial sequence

<400> 37

atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60

cctgatattg tgatgaccca gactccagcc accctgtctt tgtctccagg ggaaagagcc 120

accctctcct gcagggccag tcagagcgct ggcagtcaaa gtgttagcag ctacttagcc 180

tggtaccagc agaaacctgg ccaggctccc aggctcctca tcaatgatgc attcaacagg 240

gcccctggca tcccagccag gttcagtggc agtgggtctg ggacagactt cactctcacc 300

atcagcagcc tagagcctga agattttgca gtttattact gtctgcaccg tagcaactgg 360

cctcctttgt tcactttcgg cggggggacc aaggtggaaa tcaaacgagg cggcggcggc 420

agtggtggtg gtggtagtgg aggaggagga agtcaggtgc agctggtgca gtctggggct 480

gaggtgaaga agcctgggtc ctcggtgaag gtctcctgca aggcttctgg aggcaccttc 540

agcagatttg ctatcagctg ggtgcgacag gccccgggac aagggcttga gtggatggga 600

gggatcatcc ctatctttgg aacagcaagc tacgcacaga agttccaggg cagagccacg 660

attaccgcgg acgaatccac gagcacagcc tacatggagc tgagcagcct gagatctgag 720

gacacggccg tgtatttctg tgcgaggggg catagtggga gctacaccct tgactactgg 780

ggccagggaa ccctggtcac cgtctcctca actacaactc cagcacccag accccctaca 840

cctgctccaa ctatcgcaag tcagcccctg tcactgcgcc ctgaagcctg tcgccctgct 900

gccgggggag ctgtgcatac tcggggactg gactttgcct gtgatatcta catctgggcg 960

cccttggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccct ttactgcagg 1020

ttcagtgtcg tgaagagagg ccggaagaag ctgctgtaca tcttcaagca gcctttcatg 1080

aggcccgtgc agactaccca ggaggaagat ggatgcagct gtagattccc tgaagaggag 1140

gaaggaggct gtgagctgag agtgaagttc tcccgaagcg cagatgcccc agcctatcag 1200

cagggacaga atcagctgta caacgagctg aacctgggaa gacgggagga atacgatgtg 1260

ctggacaaaa ggcggggcag agatcctgag atgggcggca aaccaagacg gaagaacccc 1320

caggaaggtc tgtataatga gctgcagaaa gacaagatgg ctgaggccta ctcagaaatc 1380

gggatgaagg gcgaaagaag gagaggaaaa ggccacgacg gactgtacca ggggctgagt 1440

acagcaacaa aagacaccta tgacgctctg cacatgcagg ctctgccacc aagatag 1497

<210> 38

<211> 1476

<212> DNA

<213> Artificial sequence

<400> 38

atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60

cctgatatcg tgatgaccca gagccacaag ttcatgagca ccagcatcgg cgccagagtg 120

agcatcacct gcaaggccag ccaggacgtg aggaccgccg tggcctggta ccagcagaag 180

cctggccaga gccccaagct gctgatctac agcgccagct acagatacac cggcgtgccc 240

gacagattca ccggcagcgg cagcggcacc gacttcacct tcaccatcag cagcgtgcag 300

gccgaggacc tggccgtgta ctactgccag cagcactacg gcacccctcc ctggaccttc 360

ggcggcggca ccaagctgga gatcaagggc ggaggcggct ccggcggcgg cggctccggc 420

ggcggcggct ccgaggtgca gctggtggag agcggcggcg gcctggtgaa gcccggcggc 480

agcctgaagc tgagctgcga ggccagcaga ttcaccttca gcagctacgc catgagctgg 540

gtgaggcaga cccccgagaa gagactggag tgggtggccg ccatcagcgg cggcggcagg 600

tacacctact accccgacag catgaagggc aggttcacca tcagcaggga caacgccaag 660

aacttcctgt acctgcagat gagctccctg agaagcgagg acaccgccat gtactactgc 720

gccagacact acgacggcta cctggactac tggggccagg gcaccaccct gaccgtgagc 780

agcaccagga ctacaactcc agcacccaga ccccctacac ctgctccaac tatcgcaagt 840

cagcccctgt cactgcgccc tgaagcctgt cgccctgctg ccgggggagc tgtgcatact 900

cggggactgg actttgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 960

gtccttctcc tgtcactggt tatcaccctt tactgcaggt tcagtgtcgt gaagagaggc 1020

cggaagaagc tgctgtacat cttcaagcag cctttcatga ggcccgtgca gactacccag 1080

gaggaagatg gatgcagctg tagattccct gaagaggagg aaggaggctg tgagctgaga 1140

gtgaagttct cccgaagcgc agatgcccca gcctatcagc agggacagaa tcagctgtac 1200

aacgagctga acctgggaag acgggaggaa tacgatgtgc tggacaaaag gcggggcaga 1260

gatcctgaga tgggcggcaa accaagacgg aagaaccccc aggaaggtct gtataatgag 1320

ctgcagaaag acaagatggc tgaggcctac tcagaaatcg ggatgaaggg cgaaagaagg 1380

agaggaaaag gccacgacgg actgtaccag gggctgagta cagcaacaaa agacacctat 1440

gacgctctgc acatgcaggc tctgccacca agatag 1476

<210> 39

<211> 458

<212> PRT

<213> Artificial sequence

<400> 39

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

1 5 10 15

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

20 25 30

Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val

35 40 45

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

50 55 60

Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg

65 70 75 80

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

85 90 95

Ser Ile Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala

100 105 110

Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg

115 120 125

Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro

130 135 140

Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly

145 150 155 160

Asn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val

165 170 175

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

180 185 190

Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr

195 200 205

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

210 215 220

Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg Cys

225 230 235 240

Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile

245 250 255

Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly

260 265 270

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

275 280 285

Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val

290 295 300

Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly

305 310 315 320

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

325 330 335

Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr

340 345 350

Ile Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe Trp

355 360 365

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

370 375 380

Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Val Leu Arg Val

385 390 395 400

Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val

405 410 415

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

420 425 430

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

435 440 445

Glu Trp His Glu His His His His His His

450 455

<210> 40

<211> 1374

<212> DNA

<213> Artificial sequence

<400> 40

ctggaggtgc aggtgcccga ggaccccgtg gtggccctgg tgggcaccga cgccaccctg 60

tgctgcagct tcagccccga gcccggcttc agcctggccc agctgaacct gatctggcag 120

ctgaccgaca ccaagcagct ggtgcacagc ttcgccgagg gccaggacca gggcagcgcc 180

tacgccaacc gcaccgccct gttccccgac ctgctggccc agggcaacgc cagcctgcgc 240

ctgcagcgcg tgcgcgtggc cgacgagggc agcttcacct gcttcgtgag catccgcgac 300

ttcggcagcg ccgccgtgag cctgcaggtg gccgccccct acagcaagcc cagcatgacc 360

ctggagccca acaaggacct gcgccccggc gacaccgtga ccatcacctg cagcagctac 420

cagggctacc ccgaggccga ggtgttctgg caggacggcc agggcgtgcc cctgaccggc 480

aacgtgacca ccagccagat ggccaacgag cagggcctgt tcgacgtgca cagcatcctg 540

cgcgtggtgc tgggcgccaa cggcacctac agctgcctgg tgcgcaaccc cgtgctgcag 600

caggacgccc acagcagcgt gaccatcacc ccccagcgca gccccaccgg cgccgtggag 660

gtgcaggtgc ccgaggaccc cgtggtggcc ctggtgggca ccgacgccac cctgcgctgc 720

agcttcagcc ccgagcccgg cttcagcctg gcccagctga acctgatctg gcagctgacc 780

gacaccaagc agctggtgca cagcttcacc gagggccgcg accagggcag cgcctacgcc 840

aaccgcaccg ccctgttccc cgacctgctg gcccagggca acgccagcct gcgcctgcag 900

cgcgtgcgcg tggccgacga gggcagcttc acctgcttcg tgagcatccg cgacttcggc 960

agcgccgccg tgagcctgca ggtggccgcc ccctacagca agcccagcat gaccctggag 1020

cccaacaagg acctgcgccc cggcgacacc gtgaccatca cctgcagcag ctaccgcggc 1080

taccccgagg ccgaggtgtt ctggcaggac ggccagggcg tgcccctgac cggcaacgtg 1140

accaccagcc agatggccaa cgagcagggc ctgttcgacg tgcacagcgt gctgcgcgtg 1200

gtgctgggcg ccaacggcac ctacagctgc ctggtgcgca accccgtgct gcagcaggac 1260

gcccacggca gcgtgaccat caccggccag cccatgacct tccccgctag cggtctgaac 1320

gacatcttcg aggctcagaa aatcgaatgg cacgaacatc atcaccatca ccat 1374

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to CD276, comprising at least the following heavy chain variable region and light chain variable region;

the CDR sequence of the heavy chain variable region and the CDR sequence of the light chain variable region are selected from any one of the items (a) - (d);

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No: 21. 22, 23 and 24; the amino acid sequence of the light chain variable region is shown as SEQ ID No: 25. 26, 27 and 28;

when the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No:21, the amino acid sequence of the light chain variable region is shown as SEQ ID No:25 is shown;

when the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No:22, the amino acid sequence of the light chain variable region is shown as SEQ ID No:26 is shown;

when the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No:23, the amino acid sequence of the light chain variable region is shown as SEQ ID No:27 is shown;

when the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No:24, the amino acid sequence of the light chain variable region is shown as SEQ ID No: shown at 28.

3. The antibody or antigen-binding fragment thereof that specifically binds to CD276 of claim 1 or 2, wherein the antibody comprises at least one of a monoclonal antibody, a humanized antibody, a chimeric antibody, a bispecific antibody;

the antigen binding fragment is Fab and F (ab)’)、F(ab’)₂Fd, a single-chain antibody scFv, a disulfide-linked fv (sdFv), or a single-domain antibody.

4. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-3 that specifically binds to CD 276.

5. A vector comprising the isolated nucleic acid of claim 4.

6. A host cell comprising the isolated nucleic acid of claim 4 or the vector of claim 5.

7. A method of producing an antibody or antigen-binding fragment thereof that specifically binds to CD276, comprising culturing the host cell of claim 6.

8. A chimeric antigen receptor CAR against human CD276, comprising an antigen recognition region, a hinge region, a transmembrane region, and an intracellular region that recognize the CD276 antigen;

the antigen recognition region comprises an antibody or antigen binding fragment thereof of any one of claims 1-3 that specifically binds to CD 276.

9. The anti-human CD276 chimeric antigen receptor CAR of claim 8, wherein said CAR further comprises a leader signal peptide sequence.

10. The anti-human CD276 chimeric antigen receptor CAR according to claim 8, wherein said intracellular domain comprises: a signaling region and/or a co-stimulatory signaling region.

11. The anti-human CD276 chimeric antigen receptor CAR according to any one of claims 8 to 10, wherein the nucleotide sequence of the CAR is as shown in any one of SEQ ID NOs 34, 35, 36 and 37.

12. An isolated nucleic acid encoding the chimeric antigen receptor CAR against human CD276 of any one of claims 8 to 10.

13. A vector comprising the isolated nucleic acid of claim 12.

14. A host cell comprising the vector of claim 13.

15. A method for producing a chimeric antigen receptor CAR against human CD276, comprising culturing the host cell of claim 14.

16. An immune effector cell expressing the antibody or antigen-binding fragment thereof specifically binding to CD276 of any one of claims 1 to 3 or the chimeric antigen receptor CAR against human CD276 of any one of claims 8 to 11.

17. The immune effector cell of claim 16, wherein the immune effector cell is selected from the group consisting of: differentiated immune cells are cultured from pluripotent stem cells or embryonic stem cells.

18. The immune effector cell of claim 16, wherein the immune effector cell is selected from at least one of a T lymphocyte, an NK cell, a peripheral blood mononuclear cell.

19. The method of producing an immune effector cell according to claim 16, comprising: infecting an immune effector cell with the isolated nucleic acid of claim 12 or the vector of claim 13.

20. A pharmaceutical composition, comprising: the antibody or antigen-binding fragment thereof of any one of claims 1-3 that specifically binds to CD276, or the isolated nucleic acid of claim 4, or the chimeric antigen receptor CAR of any one of claims 8-11 that is directed against human CD276, or the isolated nucleic acid of claim 12, or the immune effector cell of any one of claims 16-18.

21. Use of an agent for the manufacture of a medicament for the treatment or amelioration of cancer, wherein the agent is selected from the group consisting of: the antibody or antigen-binding fragment thereof specifically binding to CD276 of any one of claims 1 to 3, the isolated nucleic acid of claim 4, the vector of claim 5, the host cell of claim 6, the host cell of claim 7, the antibody or antigen-binding fragment thereof specifically binding to CD276 produced by the method of producing the antibody or antigen-binding fragment thereof specifically binding to CD276, the chimeric antigen receptor CAR against human CD276 of any one of claims 8 to 11, the isolated nucleic acid of claim 12, the vector of claim 13, the host cell of claim 14, the chimeric antigen receptor CAR against human CD276 produced by the method of producing the chimeric antigen receptor CAR against human CD276 of claim 15, the immune effector cell of any one of claims 16 to 18, and the immune effector cell produced by the method of producing the immune effector cell of claim 19 At least one of;

the cancer is a cancer associated with CD276 expression.

22. Use of an agent according to claim 21 in the manufacture of a medicament for the treatment or amelioration of cancer selected from at least one of brain glioma, renal cancer, ovarian cancer, lung cancer, gastric cancer, liver cancer, intestinal cancer, prostate cancer and pancreatic cancer.