Monoclonal antibody targeting human CD276 and its application

Abstract

The present invention provides a monoclonal antibody targeting human CD276 and its application. Specifically, the present invention provides an antibody targeting CD276, which has high affinity and high specificity for human CD276. The present invention also provides a cell and a pharmaceutical composition comprising the antibody, as well as a preparation method and application of the antibody.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more specifically to a monoclonal antibody targeting human CD276 and an application thereof. Background Art

[0002] CD276 molecules, also known as B7-H3 molecules, are a recently discovered type I transmembrane glycoprotein that belongs to the B7 immune co-stimulatory and co-inhibitory globulin family. Human B7-H3 molecules have two subtypes, 2Ig-B7-H3 and 4Ig-B7H3, while mouse B7-H3 has only one subtype, 2Ig-B7-H3, which has 88% amino acid sequence homology with human 2Ig-B7-H3 subtype. At present, the function of B7-H3 molecules still needs to be further elucidated, but existing studies have shown that B7-H3 molecules have both immune activation and immunosuppression. On the one hand, B7-H3 molecules can play an immune-suppressive role by reducing the secretion of type I cytokine IFN-γ. Many research results support that B7-H3 has a negative immune regulatory effect in models of graft-versus-host disease, cardiac transplant rejection, airway inflammation, and autoimmune encephalomyelitis; on the other hand, B7-H3 has also been found to play a role in providing immune co-stimulatory signals for T cells in autoimmune diseases in vivo and in vitro.

[0003] Numerous studies have shown that B7-H3 is overexpressed in a variety of human tumors, including leukemia, gastric cancer, breast cancer, non-small cell lung cancer, ovarian cancer, osteosarcoma, neuroblastoma, pancreatic cancer, breast cancer, etc., but it is only limitedly expressed in normal tissues. At the same time, it has also been reported to be expressed in important tumor microenvironment components such as tumor stem cells, tumor stromal cells and tumor angiogenesis. Overexpression of B7-H3 usually reduces the infiltration of tumor-associated lymphocytes in the tumor, accelerates disease progression, and is often closely related to the poor prognosis of cancers such as pancreatic ductal carcinoma, prostate cancer, ovarian cancer, lung cancer and clear cell renal carcinoma.

[0004] Currently, there are still many deficiencies in the research on B7-H3 monoclonal antibodies, and the field needs to develop new B7-H3 monoclonal antibodies and related applications. Summary of the invention

[0005] The purpose of the present invention is to provide a monoclonal antibody targeting human CD276 and application thereof.

[0006] In a first aspect of the present invention, there is provided a heavy chain variable region of an antibody, wherein the heavy chain variable region comprises the following three complementary determining regions CDR:

[0007] CDR1 shown in SEQ ID NO: 1,

[0008] CDR2 shown in SEQ ID NO:2, and

[0009] CDR3 shown in SEQ ID NO:3 or 7.

[0010] In another preferred embodiment, any one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one (such as 1-3, preferably 1-2, and more preferably 1) amino acid and can retain CD276 binding affinity.

[0011] In another preferred example, the heavy chain variable region further includes a human FR region or a mouse FR region.

[0012] In another preferred example, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 8 or 9.

[0013] In another preferred example, the heavy chain variable region has an amino acid sequence shown in SEQ ID NO: 26-30.

[0014] The second aspect of the present invention provides an antibody heavy chain, wherein the heavy chain has the heavy chain variable region as described in the first aspect of the present invention.

[0015] In another preferred embodiment, the heavy chain of the antibody further includes a heavy chain constant region.

[0016] In another preferred embodiment, the heavy chain constant region is of human, mouse or rabbit origin.

[0017] In a third aspect of the present invention, there is provided a light chain variable region of an antibody, wherein the light chain variable region comprises the following three complementary determining regions CDR:

[0018] CDR1' shown in SEQ ID NO:4,

[0019] CDR2' shown in SEQ ID NO:5, and

[0020] CDR3' shown in SEQ ID NO:6.

[0021] In another preferred embodiment, any one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one (such as 1-3, preferably 1-2, and more preferably 1) amino acid and can retain CD276 binding affinity.

[0022] In another preferred example, the light chain variable region further includes a human FR region or a mouse FR region.

[0023] In another preferred example, the light chain variable region has the amino acid sequence shown in SEQ ID NO:10.

[0024] In another preferred example, the light chain variable region has an amino acid sequence shown in SEQ ID NO: 21-25.

[0025] The fourth aspect of the present invention provides a light chain of an antibody, wherein the light chain has the light chain variable region as described in the third aspect of the present invention.

[0026] In another preferred embodiment, the light chain of the antibody further includes a light chain constant region.

[0027] In another preferred embodiment, the light chain constant region is of human, mouse or rabbit origin.

[0028] In a fifth aspect of the present invention, there is provided an antibody targeting CD276, wherein the antibody has:

[0029] (1) the heavy chain variable region as described in the first aspect of the present invention; and / or

[0030] (2) the light chain variable region as described in the third aspect of the present invention;

[0031] Alternatively, the antibody has: the heavy chain as described in the second aspect of the present invention; and / or the light chain as described in the fourth aspect of the present invention.

[0032] In another preferred embodiment, the affinity constant KD (M) of the antibody binding to human CD276 protein (preferably wild type) is (1-15)×10 -8 , preferably (5-12)×10 -8 .

[0033] In another preferred embodiment, the antibody binds to amino acids 31-40 at the N-terminus of human CD276 protein.

[0034] In another preferred embodiment, the antibody is selected from: an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.

[0035] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0036] In another preferred embodiment, the antibody is a monoclonal antibody.

[0037] In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

[0038] In another preferred embodiment, the heavy chain variable region sequence of the antibody is shown as SEQ ID NO: 8 or 9; and / or the light chain variable region sequence of the antibody is shown as SEQ ID NO: 10.

[0039] In another preferred embodiment, the heavy chain variable region sequence of the antibody is shown as SEQ ID NO: 26; and the light chain variable region sequence of the antibody is shown as SEQ ID NO: 21.

[0040] In another preferred embodiment, the heavy chain variable region sequence of the antibody is shown as SEQ ID NO: 29; and the light chain variable region sequence of the antibody is shown as SEQ ID NO: 24.

[0041] In a sixth aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has:

[0042] (i) the heavy chain variable region of the first aspect of the invention, the heavy chain of the second aspect of the invention, the light chain variable region of the third aspect of the invention, the light chain of the fourth aspect of the invention, or the antibody of the fifth aspect of the invention; and

[0043] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0044] In another preferred embodiment, the tag sequence includes a 6His tag.

[0045] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0046] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0047] In the seventh aspect of the present invention, a CAR construct is provided, wherein the scFV segment of the monoclonal antibody antigen binding region of the CAR construct is a binding region that specifically binds to CD276, and the scFv has the heavy chain variable region as described in the first aspect of the present invention and the light chain variable region as described in the third aspect of the present invention.

[0048] In another preferred embodiment, the chimeric antigen receptor has the following structure:

[0049] L-scFv-H-TM-C-CD3ζ (I)

[0050] In the formula,

[0051] Each "-" is independently a connecting peptide or a peptide bond;

[0052] L is none or a signal peptide sequence;

[0053] scFv is a scFv targeting CD276;

[0054] H is an optional hinge region;

[0055] TM is the transmembrane domain;

[0056] C is a co-stimulatory signal molecule;

[0057] CD3ζ is the CD3ζ cytoplasmic signaling sequence.

[0058] The eighth aspect of the present invention provides a recombinant immune cell, wherein the immune cell expresses an exogenous CAR construct as described in the seventh aspect of the present invention.

[0059] In another preferred embodiment, the immune cells are selected from the following group: NK cells and T cells.

[0060] In another preferred embodiment, the immune cells are from humans or non-human mammals (such as mice).

[0061] The ninth aspect of the present invention provides an antibody-drug conjugate, wherein the antibody-drug conjugate comprises:

[0062] (a) an antibody portion, the antibody portion being selected from the group consisting of a heavy chain variable region as described in the first aspect of the invention, a heavy chain as described in the second aspect of the invention, a light chain variable region as described in the third aspect of the invention, a light chain as described in the fourth aspect of the invention, or an antibody as described in the fifth aspect of the invention, or a combination thereof; and

[0063] (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0064] In another preferred embodiment, the antibody portion and the coupling portion are coupled via a chemical bond or a linker.

[0065] The tenth aspect of the present invention provides a use of an active ingredient, wherein the active ingredient is selected from the following group: the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the immune cell as described in the eighth aspect of the present invention, the antibody-drug conjugate as described in the ninth aspect of the present invention, or a combination thereof, wherein the active ingredient is used for (a) preparing a detection reagent or a kit; and / or (b) preparing a drug for preventing and / or treating a CD276-related disease.

[0066] In another preferred embodiment, the CD276-related diseases include tumors and autoimmune diseases.

[0067] In another preferred embodiment, the tumor is a CD276 positive tumor.

[0068] In another preferred embodiment, the tumor is selected from the following group: a blood tumor, a solid tumor, or a combination thereof.

[0069] In another preferred embodiment, the blood tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.

[0070] In another preferred embodiment, the solid tumor is selected from the following group: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract tumor, thyroid cancer, or a combination thereof.

[0071] In another preferred embodiment, the tumor is gastric cancer, breast cancer, or osteosarcoma.

[0072] In another preferred embodiment, the antibody is in the form of an antibody-drug conjugate (ADC).

[0073] In another preferred embodiment, the detection reagent or kit is used for:

[0074] (1) detecting CD276 protein in a sample; and / or

[0075] (2) detecting endogenous CD276 protein in tumor cells; and / or

[0076] (3) Detect tumor cells expressing CD276 protein.

[0077] In another preferred embodiment, the detection reagent is a detection sheet.

[0078] The eleventh aspect of the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises:

[0079] (i) an active ingredient, wherein the active ingredient is selected from the group consisting of the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the immune cell as described in the eighth aspect of the present invention, the antibody-drug conjugate as described in the ninth aspect of the present invention, or a combination thereof; and

[0080] (ii) a pharmaceutically acceptable carrier.

[0081] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0082] In another preferred embodiment, the pharmaceutical composition is an injection.

[0083] In the twelfth aspect of the present invention, a polynucleotide is provided, wherein the polynucleotide encodes a polypeptide selected from the group consisting of:

[0084] (1) the heavy chain variable region as described in the first aspect of the present invention, the heavy chain as described in the second aspect of the present invention, the light chain variable region as described in the third aspect of the present invention, the light chain as described in the fourth aspect of the present invention, or the antibody as described in the fifth aspect of the present invention; or

[0085] (2) The recombinant protein according to the sixth aspect of the present invention;

[0086] (3) The CAR construct as described in the seventh aspect of the present invention.

[0087] In another preferred embodiment, the nucleotide sequence is shown in SEQ ID NO: 11-20.

[0088] The thirteenth aspect of the present invention provides a vector, wherein the vector contains the polynucleotide as described in the twelfth aspect of the present invention.

[0089] In another preferred embodiment, the vector includes: bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0090] The fourteenth aspect of the present invention provides a genetically engineered host cell, wherein the host cell contains the vector as described in the thirteenth aspect of the present invention or the polynucleotide as described in the twelfth aspect of the present invention is integrated into its genome.

[0091] In a fifteenth aspect of the present invention, there is provided a method for in vitro detection (including diagnostic or non-diagnostic) of CD276 protein in a sample, the method comprising the steps of:

[0092] (1) contacting the sample with the antibody according to the fifth aspect of the present invention or the antibody-drug conjugate according to the ninth aspect of the present invention in vitro;

[0093] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CD276 protein in the sample.

[0094] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0095] In the sixteenth aspect of the present invention, a detection plate is provided, comprising: a substrate (support plate) and a test strip, wherein the test strip contains the antibody as described in the fifth aspect of the present invention or the antibody-drug conjugate as described in the ninth aspect of the present invention.

[0096] A seventeenth aspect of the present invention provides a kit, comprising:

[0097] (1) a first container, wherein the first container contains the antibody according to the fifth aspect of the present invention; and / or

[0098] (2) a second container, wherein the second container contains a secondary antibody against the antibody according to the fifth aspect of the present invention;

[0099] Alternatively, the kit contains the detection plate as described in the sixteenth aspect of the present invention.

[0100] In an eighteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the method comprising:

[0101] (a) culturing the host cell according to the fourteenth aspect of the present invention under conditions suitable for expression;

[0102] (b) isolating a recombinant polypeptide from the culture, wherein the recombinant polypeptide is the antibody according to the fifth aspect of the present invention or the recombinant protein according to the sixth aspect of the present invention.

[0103] In the nineteenth aspect of the present invention, a method for preventing and / or treating CD276-related diseases is provided, the method comprising: administering to a subject in need thereof the antibody as described in the fifth aspect of the present invention, the antibody-drug conjugate of the antibody, or the CAR-T cell expressing the antibody, or a combination thereof.

[0104] In another preferred embodiment, the method further comprises: administering other drugs or treatment methods to a subject in need for combined treatment.

[0105] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 The vector schematic diagrams of the heavy chain expression vector pcDNA3.1-IgG1Fc and the light chain expression vector pcDNA3.1-IgKc are shown.

[0107] Figure 2 The results of ELISA assays of immunized mice are shown.

[0108] Figure 3 ELISA results of hybridoma fusion plates are shown.

[0109] Figure 4 The results of ELISA testing of the first subclone plate are shown.

[0110] Figure 5 The results of FACS analysis of the first subclone are shown.

[0111] Figure 6 The results of the ELISA assay of the second subclone plate are shown.

[0112] Figure 7 The results of FACS analysis of the second subclone are shown.

[0113] Figure 8 The results of FACS detection of chimeric antibodies are shown.

[0114] Fig. 9 The FACS detection results of humanized antibodies are shown.

[0115] Fig.10 The SDS-PAGE results of humanized antibodies T1H1-T1L1 and T2H1-T2L1 are shown.

[0116] Fig.11 The comparison results of humanized antibody T1H1-T1L1 and mouse antibody are shown.

[0117] Fig.12 The results of the comparison between humanized antibody T2H1-T2L1 and mouse antibody are shown.

[0118] Fig.13 Ligand coupling pre-enrichment results are shown.

[0119] Fig.14 Ligand coupling results are shown.

[0120] Fig.15 Affinity assay results for CD276 Chimeric are shown.

[0121] Fig.16 Affinity assay results for CD276T1H1-T1L1 are shown.

[0122] Fig.17 Affinity assay results for CD276T2H1-T2L1 are shown.

[0123] Fig.18 The results of membrane proteome array detection of humanized CD276 antibody (CD276T1H1-T1L1) are shown.

[0124] Fig.19 The flow cytometry results of CD276 antibody binding to different truncated antigens are shown.

[0125] Fig. 20 The flow cytometry results of CD276 antibody binding to antigens with mutations at different sites are shown. DETAILED DESCRIPTION

[0126] After extensive and in-depth research, the inventor unexpectedly discovered for the first time an antibody targeting CD276 with high affinity and high specificity, and prepared a humanized antibody with affinity equivalent to that of a chimeric antibody. Experiments show that the antigenic epitope of the antibody of the present invention is at the 31st to 40th amino acids at the N-terminus of the CD276 protein. The antibody of the present invention specifically binds only to the CD276 protein and does not specifically bind to other membrane proteins, and has very good specificity and safety. On this basis, the present invention was completed.

[0127] the term

[0128] As used herein, the terms "administering" and "treating" refer to the application of an exogenous drug, therapeutic agent, diagnostic agent or composition to an animal, a human, a subject, a cell, a tissue, an organ or a biological fluid. "Administering" and "treating" may refer to treatment, pharmacokinetics, diagnosis, research and experimental methods. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, contact of a fluid with a cell. "Administering" and "treating" also mean in vitro and ex vivo treatment by an agent, a diagnosis, a combination composition or by another cell. "Treatment" when applied to a human, an animal or a research subject refers to therapeutic treatment, prophylactic or preventive measures, research and diagnosis; including contact of CD276 antibodies with a human or animal, a subject, a cell, a tissue, a physiological compartment or a physiological fluid.

[0129] As used herein, the term "treatment" refers to administering an internal or external therapeutic agent to a patient, including any of the CD276 antibodies and compositions thereof of the present invention, wherein the patient has one or more symptoms of a disease, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the amount of the therapeutic agent that is effective in alleviating one or more symptoms of the disease (therapeutically effective amount) is administered to the patient.

[0130] As used herein, the term "optional" or "optionally" means that the event or situation described subsequently may occur but need not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of a specific sequence may have but need not have, and may be 1, 2 or 3.

[0131] Antibody

[0132] As used herein, the term "antibody" refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the heavy chain of immunoglobulin are different, so its antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or different types of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and the heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. IgG represents the most important class of immunoglobulins. Due to differences in chemical structure and biological function, it can be divided into 4 subclasses: IgG1, IgG2, IgG3 and IgG4. The light chain is divided into κ or λ chains by different constant regions. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known to those in the art.

[0133] The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region (C region). The variable region includes three hypervariable regions (HVRs) and four relatively conservative FR regions (FR). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) are composed of three CDR regions and four FR regions, and the order from the amino terminus to the carboxyl terminus is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain, namely the light chain hypervariable regions (LCDRs), refer to LCDR1, LCDR2 and LCDR3; the three CDR regions of the heavy chain, namely the heavy chain hypervariable regions (HCDRs), refer to HCDR1, HCDR2 and HCDR3. The CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the invention conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3) or the numbering rules of Kabat and Chothia (HCDR1) in number and position. The four FR regions in the natural heavy and light chain variable regions are generally in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody. The amino acids that constitute the FR or CDR region can be determined by comparing the amino acid sequences of antibodies of the same type. The constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cellular toxicity of the antibody.

[0134] As used herein, the term "antigen-binding fragment" refers to a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a single Fv fragment that has antigen-binding activity. The Fv antibody contains the antibody heavy chain variable region, the light chain variable region, but no constant region, and is the smallest antibody fragment with all antigen-binding sites. Generally, the Fv antibody also contains a polypeptide linker between the VH and VL domains, and is capable of forming the structure required for antigen binding.

[0135] As used herein, the term "antigenic determinant" refers to a discrete three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present invention.

[0136] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0137] In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be prepared using DNA recombinant techniques well known in the art.

[0138] As used herein, the term "monoclonal antibody" refers to an antibody secreted by a clone from a single cell source. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. The cell may be a eukaryotic, prokaryotic, or phage clone.

[0139] As used herein, the term "chimeric antibody" refers to an antibody molecule that is spliced ​​from the V region gene of a mouse antibody and the C region gene of a human antibody, and then inserted into a vector and transfected into a host cell for expression. It retains the high specificity and affinity of the parent mouse antibody, and enables its human Fc segment to effectively mediate biological effect functions.

[0140] As used herein, the term "humanized antibody" is a variable region modification form of the mouse antibody of the present invention, which has a CDR region derived from (or substantially derived from) a non-human antibody (preferably a mouse monoclonal antibody), and a FR region and a constant region substantially derived from a human antibody sequence; that is, the CDR region sequence of the mouse antibody is grafted onto different types of human germline antibody framework sequences. Because the CDR sequence is responsible for most of the antibody-antigen interaction, recombinant antibodies that mimic the properties of specific naturally occurring antibodies can be expressed by constructing an expression vector.

[0141] In the present invention, the antibodies may be monospecific, bispecific, trispecific, or more multispecific.

[0142] In the present invention, the antibody of the present invention also includes its conservative variants, which refers to a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

[0143] Table A

[0144]

[0145]

[0146] Anti-CD276 antibody

[0147] As used herein, the term "CD276" generally refers to natural or recombinant human CD276, as well as non-human homologs of human CD276.

[0148] The present invention provides a highly specific and high affinity antibody against CD276, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (VH) amino acid sequence, and the light chain comprises a light chain variable region (VL) amino acid sequence.

[0149] Preferably, the CDR of the heavy chain variable region (VH) is selected from the following group:

[0150] CDR1 shown in SEQ ID NO: 1,

[0151] CDR2 shown in SEQ ID NO:2, and

[0152] CDR3 shown in SEQ ID NO: 3 or 7; and / or

[0153] The CDRs of the light chain variable region (VL) are selected from the group consisting of:

[0154] CDR1' shown in SEQ ID NO:4,

[0155] CDR2' shown in SEQ ID NO:5, and

[0156] CDR3' shown in SEQ ID NO:6.

[0157] Among them, any one of the above-mentioned amino acid sequences also includes a derivative sequence with CD276 binding affinity after adding, deleting, modifying and / or replacing at least one (such as 1-5, 1-3, preferably 1-2, and more preferably 1) amino acid.

[0158] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or replacing at least one amino acid sequence is preferably an amino acid sequence with a homology of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.

[0159] The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, and humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and more preferably fully humanized antibodies.

[0160] The antibody derivatives of the present invention can be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0161] Wherein, the animal is preferably a mammal, such as a mouse.

[0162] The antibody of the present invention may be a murine antibody, a chimeric antibody, a humanized antibody, a CDR-grafted and / or modified antibody targeting human CD276.

[0163] Antibody preparation

[0164] Any method suitable for producing monoclonal antibodies can be used to produce the anti-CD276 antibodies of the present invention. For example, animals can be immunized with CD276 or a fragment thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.

[0165] Any suitable form of CD276 (such as CHO-K1-CD276 recombinant cell line) can be used as an immunogen (antigen) to produce non-human antibodies specific for CD276 and screen the biological activity of the antibodies. The stimulating immunogen can be full-length mature human CD276, including natural homodimers, or peptides containing single / multiple epitopes. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art. The immunogen can be purified from a natural source or produced in genetically modified cells. The DNA encoding the immunogen can be genomic or non-genomic (e.g., cDNA) in origin. The DNA encoding the immunogen can be expressed using a suitable genetic vector, including but not limited to adenoviral vectors, adeno-associated viral vectors, baculoviral vectors, plasmids and non-viral vectors.

[0166] Humanized antibodies can be selected from any class of immunoglobulins, including IgM, IgD, IgG, IgA and IgE. In the present invention, the antibody is an IgG antibody, using the IgG1 subtype. Optimization of the necessary constant domain sequences is easily achieved by screening the antibodies with the biological assays described in the examples below to produce the desired biological activity.

[0167] Likewise, any type of light chain can be used in the compounds and methods herein. Specifically, kappa, lambda chains or variants thereof can be used in the compounds and methods of the invention.

[0168] The sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and the heavy chain can be fused together to form a single-chain antibody.

[0169] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0170] In addition, artificial synthesis methods can also be used to synthesize the relevant sequence, especially when the fragment length is short. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0171] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0172] The host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S, CHO-K1, HEK-293 cells.

[0173] The step of transforming host cells with recombinant DNA described in the present invention can be carried out by techniques well known in the art. The obtained transformants can be cultured by conventional methods, and the transformants express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, conventional culture medium is used to cultivate under suitable conditions.

[0174] Typically, the transformed host cells are cultured under conditions suitable for the expression of the antibodies of the present invention, and then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc., conventional separation and purification means well known to those skilled in the art to obtain the antibodies of the present invention.

[0175] The resulting monoclonal antibodies can be characterized by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0176] Antibody-drug conjugates (ADCs)

[0177] The present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.

[0178] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is coupled to the effector molecule, and preferably chemically coupled. The effector molecule is preferably a drug with therapeutic activity. In addition, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug or a radionuclide.

[0179] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent can be any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent utilizing a carboxyl group can be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (the coupling site is an acylhydrazone).

[0180] Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection agents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0181] Antibodies can be coupled to drugs to form antibody drug conjugates (ADCs). Typically, ADCs contain a linker between the drug and the antibody. The linker can be a degradable or non-degradable linker. Degradable linkers are typically easily degraded in the intracellular environment, such as degradation of the linker at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (such as linkers that hydrolyze when the pH is less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (such as disulfide linkers). Non-degradable linkers typically release drugs under conditions where the antibody is hydrolyzed by proteases.

[0182] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved through the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimide compounds, halogenated amides (e.g., iodinated, brominated or chlorinated); halogenated esters (e.g., iodinated, brominated or chlorinated); halogenated methyl ketones (e.g., iodinated, brominated or chlorinated), benzyl halides (e.g., iodinated, brominated or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, maleimide attached to the antibody via thiosuccinimide.

[0183] The drug can be any cytotoxic, cytostatic or immunosuppressive drug. In an embodiment, a linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group that can form a bond with the linker. In the case where the drug is directly connected to the linker, the drug has a reactive group before being connected to the antibody.

[0184] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc. In the present invention, drug-linkers can be used to form ADCs in a simple step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step method. For example, a cysteine ​​residue reacts with a reactive portion of a linker in a first step, and in a subsequent step, a functional group on the linker reacts with a drug to form an ADC.

[0185] Typically, the functional groups on the linker are selected to react specifically with the appropriate reactive groups on the drug moiety. As a non-limiting example, an azide-based moiety can be used to react specifically with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker by a 1,3-dipolar cycloaddition between the azide and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other attachment strategies, such as those described in Bioconjugation Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that when a complementary pair of reactive functional groups is selected for selective reaction of the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0186] The present invention also provides a method for preparing ADC, which may further comprise: combining an antibody with a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0187] In certain embodiments, the methods of the invention include: combining the antibody with the bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the invention further include: combining the antibody-linker conjugate with the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody via the linker.

[0188] In some embodiments, the antibody drug conjugate ADC has the following molecular formula:

[0189]

[0190] in:

[0191] Ab is antibody,

[0192] LU is the connector;

[0193] D is for medicine;

[0194] And the subscript p is a value selected from 1 to 8.

[0195] Pharmaceutical composition

[0196] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its ADC or corresponding CAR-T cell, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary with the nature of the formulated substance and the condition to be treated. The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0197] The antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0198] The pharmaceutical composition of the present invention can be directly used to bind to CD276 protein molecules, and thus can be used to prevent and treat CD276-related diseases. In addition, other therapeutic agents can also be used simultaneously.

[0199] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

[0200] When using a pharmaceutical composition, a safe and effective amount of the pharmaceutical composition is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 50 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight to about 20 milligrams / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0201] Test Uses and Kits

[0202] The antibodies of the present invention can be used in detection applications, for example, for detecting a sample to provide diagnostic information.

[0203] In the present invention, the sample (specimen) used includes cells, tissue samples and biopsy specimens. The term "biopsy" used in the present invention should include all types of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention can include, for example, tissue samples prepared by endoscopic methods or puncture or needle biopsy of an organ.

[0204] Samples used in the present invention include fixed or preserved cell or tissue samples.

[0205] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be fixed to a detection plate.

[0206] The main advantages of the present invention include:

[0207] (a) A new murine monoclonal antibody targeting human CD276 was developed.

[0208] (b) A humanized antibody sequence targeting human CD276 monoclonal antibody was developed.

[0209] (c) The bispecific antibodies, CAR-T cells, or antibody-drug conjugates constructed using humanized antibody sequences developed by the present invention can reduce drug rejection reactions in the body and further improve the therapeutic effect compared to drugs constructed using mouse antibody sequences.

[0210] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0211] General Materials and Methods:

[0212] 1. The main reagents used in the examples are as follows:

[0213] PBS (Gibco, CAT#14190-250)

[0214] DMEM (Gibco, CAT#41965-062)

[0215] F12K(Gibco,CAT#21127030)

[0216] RPMI1640 (Gibco, CAT#61870044)

[0217] FBS (Gibco, CAT#10099-141)

[0218] Genomic DNA purification kit (Thermo, CAT#K0512)

[0219] T4 DNA ligase (Takara, CAT#2011B)

[0220] B7-H3 (CD276) protein, human, recombinant (His Tag), (Sinobiological, Cat#11188-H08H)

[0221] PrimeScript TM 1 st Strand cDNA Synthesis Kit (Takara, Cat#6110A)

[0222] Trizol RNA extraction reagent (Thermo, CAT#15596018)

[0223] Q5 PCR Cloning Kit (NEB, Cat#E0555S)

[0224] AxyPrep DNA Gel Extraction Kit (Axygen, Cat# AP-GX-250G)

[0225] BamHI restriction endonuclease (NEB, CAT#R3136M)

[0226] KpnI restriction endonuclease (NEB, CAT#R3142M)

[0227] TOP10 competent cells (prepared by iCarTab)

[0228] pcDNA3.1-IgG1Fc vector (made by iCarTab)

[0229] pcDNA3.1-IgKc vector (made by iCarTab)

[0230] PE-Anti-human-IgG(Biolegend,CAT#409304)

[0231] PE-Anti-mouse IgG(MultiSciences,CAT#70-GAM0041)

[0232] FreeStyle TM 293Expression Medium(Thermo,CAT#12338018)

[0233] LVtransm transfection reagent (iCarTab, Cat#LVTran100)

[0234] Buffer: 10×HBS-EP (GE, LOT: BCBV2312)

[0235] Amino coupling kit (GE, LOT: 2067793)

[0236] pH 5.5 acetate buffer (GE, LOT: 20278961601)

[0237] pH 1.5 glycine buffer (GE, LOT: 10240798)

[0238] S series CM5 chip (GE, LOT:10255694)

[0239] 2. The main equipment used in the embodiment is as follows:

[0240] Ordinary optical inverted microscope

[0241] Tabletop centrifuge Thermo ST41

[0242] High-speed centrifuge Thermo RC6+

[0243] Biological Safety Cabinets

[0244] Roche 480 Fluorescence Quantitative PCR Instrument

[0245] Thermo Attune Nxt flow cytometer

[0246] Thermo 3111CO2 Incubator

[0247] BiaCore T200

[0248] 3. The main methods involved in the embodiments are as follows:

[0249] 3.1 Hybridoma Screening

[0250] 3.1.1 Immunization of mice

[0251] Five Balb / c mice were prepared. The CHO-K1-CD276 recombinant cell line was fully washed with saline for five times and then injected intraperitoneally. Each mouse was injected with 5×10 6 The recombinant cell lines were injected once every 14 days. Ten days after the third intraperitoneal injection, 100uL of peripheral blood was collected through the tail vein of the mice for immune titer detection. The mice with the best immune titer were selected and 5×10 6 Three days after immunization, the spleen of mice was isolated for hybridoma fusion.

[0252] 3.1.2 Immune titer detection

[0253] 1) Take the mouse out of the cage, disinfect the mouse's tail with a 75% medical alcohol cotton ball, and use a 5mm blood collection needle to puncture a small wound on the mouse's tail;

[0254] 2) Collect blood drops using capillary glass blood collection tubes (100uL of plasma needs to be prepared);

[0255] 3) After collecting blood, use a dry sterile cotton ball to gently press the blood collection point to stop bleeding, and then return the mouse to the cage for observation;

[0256] 4) Place the centrifuge tube containing the blood sample in a 37°C incubator for 1 hour; then transfer the blood sample to 4°C overnight.

[0257] 5) Separate the serum from the blood clot and transfer it to a new sterile centrifuge tube, and centrifuge at 4°C, 10,000 x g for 10 min;

[0258] 6) Transfer the serum to a new sterile centrifuge tube and detect the immune titer using ELISA.

[0259] 3.1.3 ELISA test

[0260] 1) Dilute CD276-His recombinant protein with sterile PBS to a final concentration of 1ug / mL. Take a new 96-well plate and add 100uL / well to coat overnight at 4℃.

[0261] 2) Remove the antigen coating solution and wash three times with PBST (containing 0.5% Tween).

[0262] 3) Add 200uL / well of 3% BSA and block at 37°C for 2 hours;

[0263] 4) After removing the blocking buffer, wash the plate three times with PBST;

[0264] 5) Add 100ul hybridoma supernatant and incubate at room temperature for 1 hour. The control wells are PBS;

[0265] 6) Remove the liquid in the wells and wash three times with PBST;

[0266] 7) Add 100uL HRP-mouse IgG (1:10000 dilution) and incubate at room temperature for 1 hour;

[0267] 8) After removing the liquid in the wells, wash the plate three times with PBST;

[0268] 9) Add 100uL / well TMB colorimetric solution;

[0269] 10) Incubate at room temperature in the dark for 15 minutes;

[0270] 11) Add 50uL / well stop solution;

[0271] 12) Use a microplate reader to read the OD value in the well.

[0272] 3.1.4 Hybridoma fusion

[0273] 1) The mouse with the best immune titer was killed by cervical dislocation, and the spleen of the mouse was obtained under sterile conditions. B cell single cell suspension was prepared and mixed with non-antibody secreting SP2 / 0 myeloma cells at a ratio of 1:1, and cell fusion was performed using the Bio-rad GenePulser electroporation system.

[0274] 2) After electrofusion, all cells were immediately suspended in complete culture medium (DMEM, 20% FBS and HAT) and seeded into 96-well plates.

[0275] 3) About 10 days after fusion, the medium was changed to HT medium. After two days of culture, the supernatant was collected to detect specific antibodies.

[0276] 3.1.5 Hybridoma Screening

[0277] Take out 120uL of culture supernatant from each well of the 96-well plate, and add fresh HT culture medium to each well. Incubate the culture supernatant with the ELISA plate pre-coated with the target antigen, and identify according to the standard ELISA operation steps. Select the wells with higher OD values ​​for the second and third rounds of subcloning. After each round of subcloning, refer to the same steps for ELISA identification until a single clone is formed.

[0278] 3.1.6 Flow cytometry detection of hybridoma clones

[0279] 1) The K562-CD276 cell line was revived from liquid nitrogen and the cell state was adjusted to the logarithmic growth phase using RPMI1640 and 10% FBS complete medium.

[0280] 2) K562-CD276 cell line was divided into several portions, with the number of cells in each portion being 5×10 5 For each cell, 100uL of hybridoma supernatant was used to incubate the target cells, mixed thoroughly, and incubated at room temperature for 1 hour.

[0281] 3) Centrifuge at 800 x g for 5 minutes at room temperature, remove the supernatant containing antibodies, and wash the cells three times with PBS.

[0282] 4) Add 0.5uL PE-labeled Anti-mouse IgG, mix thoroughly, and incubate at room temperature in the dark for 30 minutes;

[0283] 5) Centrifuge at 800 x g for 5 minutes at room temperature, remove the supernatant containing the secondary antibody, and wash the cells three times with PBS;

[0284] 6) Resuspend the cells in 500uL PBS and perform flow cytometry analysis.

[0285] 3.2 Hybridoma Sequencing

[0286] Take 5×10 6 The hybridoma cells finally obtained by screening were lysed by Trizol, and the total RNA of the hybridoma cells was extracted according to the standard method.

[0287] After total RNA was reverse transcribed into cDNA samples using a reverse transcription kit, the heavy and light chain variable regions of the antibody were amplified by PCR using hybridoma sequencing primers, and then TA cloning was performed, and the fragments obtained by PCR were subcloned into the pMD-19T vector. After blue-white screening, 10 clones were selected for each chain for sequencing. The antibody sequences finally obtained were analyzed.

[0288] 3.3 Mouse Antibody Humanization

[0289] 3.3.1 Humanized antibody sequence design

[0290] Humanization design was performed based on the amino acid sequence information of the heavy and light chains of the target antibody obtained through the above sequencing, keeping the CDR region sequence of the original antibody unchanged. Based on the results of germline alignment and antibody structure simulation, different humanized antibody templates were selected for the heavy and light chains, respectively, and back mutations were performed in the framework region after humanization to design candidate humanized antibody sequences.

[0291] 3.3.2 Humanized antibody gene synthesis and expression vector construction

[0292] The heavy chain and light chain of the humanized antibody designed above were synthesized separately, and the heavy chain was subcloned into the pcDNA3.1-IgG1Fc expression vector, and the light chain was subcloned into the pcDNA3.1-IgKc expression vector (the vector schematic diagram is shown in Figure 1 After the vector was verified by sequencing, the Qiagen plasmid extraction kit was used to prepare endotoxin-free plasmid.

[0293] 3.3.3 Humanized Antibody Expression and Purification

[0294] 1) Take out the LVTransm transfection reagent and antibody expression vector from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down. Take out the PBS or HBSS buffer and warm it to room temperature. Take 2mL PBS to one well of a 6-well plate, add 2μg pcDNA3.1-IgG1Fc and 2μg pcDNA3.1-IgKc respectively, mix thoroughly by pipetting up and down, then add 12μLLVTransm, immediately mix thoroughly by pipetting up and down, and let stand at room temperature for 10 minutes.

[0295] 2) Add the above DNA / LVTransm complex to 1.5 mL of 293F-SVP16 cells and shake gently to mix thoroughly. Place the cells in a 37°C, 5% CO2 incubator at 130 RPM for 6-8 hours, then add 1.5 mL of fresh FreeStyle TM 293 expression medium and put the cells back into the incubator for further culture.

[0296] 3) After continuous cultivation for 3 days, the culture supernatant was collected by centrifugation, filtered with a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube for flow cytometry detection.

[0297] 3.3.4 Flow cytometry identification of binding between humanized antibodies and target proteins

[0298] 1) CHO-K1 empty cells and CHO-K1-CD276 recombinant cell lines were revived from liquid nitrogen, and the cells were adjusted to the logarithmic growth phase using F12K and 10% FBS complete culture medium.

[0299] 2) Divide the two types of cells into several portions, with the number of cells in each portion being 5×10 5 Each antibody needs to be incubated with CHO-K1 empty cells and CHO-K1-CD276 recombinant cell lines, mixed thoroughly, and incubated at room temperature for 1 hour.

[0300] 3) Centrifuge at 800 x g for 5 minutes at room temperature, remove the supernatant containing antibodies, and wash the cells three times with PBS;

[0301] 4) Add 2uL PE-labeled Anti-human IgG, mix thoroughly, and incubate at room temperature in the dark for 30 minutes;

[0302] 5) Centrifuge at 800 x g for 5 minutes at room temperature, remove the supernatant containing the secondary antibody, and wash the cells three times with PBS;

[0303] 6) Resuspend the cells in 500uL PBS and perform flow cytometry analysis.

[0304] 3.3.5 Humanized Antibody Affinity Testing

[0305] The CD276-His recombinant protein was fixed on a CM5 chip using 10 mM acetate coupling buffer, and the humanized antibody and human-mouse chimeric antibody prepared above that were positive for flow cytometry identification were used as the mobile phase to detect the binding ability of the antibody before and after humanization with the target protein CD276.

[0306] Example 1 ELISA test of mouse immune titer

[0307] Mice were immunized according to the above method, and serum was isolated from the tail vein of all immunized mice. Figure 2 The dilution gradient shown was used for limiting dilution and ELISA test was performed on 96-well plates pre-coated with antigen.

[0308] The results are as follows Figure 2 As shown, according to the experimental results, mouse No. 81 was selected for shock immunization and subsequent hybridoma fusion.

[0309] Example 2 Fusion plate ELISA detection

[0310] Take 100uL of supernatant from the well plate after hybridoma fusion, and perform ELISA detection using a 96-well plate pre-coated with the target antigen.

[0311] The ELISA test results of each fusion plate are as follows Figure 3 shown.

[0312] According to the OD value detected by ELISA, the best clones were selected for the next round of subcloning. The specific clones selected included B4 of plate 1, D4 of plate 4, E9 of plate 5, F4 of plate 6, C11 of plate 8, and G5 of plate 10.

[0313] Example 3: First subcloning

[0314] Take 100uL of supernatant from the well plate after the first subcloning, and perform ELISA detection using a 96-well plate pre-coated with the target antigen.

[0315] The results of the first subclone ELISA test were as follows Figure 4 As shown, plate 1-1 corresponds to the first subcloning result of plate 1B4 in Example 1, plate 4-1 corresponds to the first subcloning result of plate 4D4 in Example 1, and the remaining markers are named in a similar manner.

[0316] According to the results of the first subclone ELISA test, clones with high OD values ​​and good growth were selected for FACS verification and the second subcloning along with the K562-CD276 cell line.

[0317] FACS test results Figure 5 Shown

[0318] Example 4 Second subcloning

[0319] Take 100uL of supernatant from the well plate after the second subcloning, and use a 96-well plate pre-coated with the target antigen for ELISA detection. The results are as follows: Figure 6 As shown, plate 1-2 corresponds to the second subcloning result of plate 1B4 in Example 1, plate 4-2 corresponds to the second subcloning result of plate 4D4 in Example 1, and the remaining markers are named in a similar manner.

[0320] According to the results of the second subclone ELISA test, clones with high OD values ​​and good growth were selected for FACS verification with K562-CD276 cell line. The results are as follows Figure 7 shown.

[0321] Example 5 Hybridoma Sequencing

[0322] The clones with good FACS identification results were selected for amplification, lysed with TRIZOL reagent, RNA was extracted and reverse transcribed into cDNA, and the heavy chain and light chain variable regions corresponding to the hybridoma antibody were sequenced using hybridoma sequencing primers. The CDR region and framework region were analyzed, and two different heavy chain variable regions (VH, VH-7) and one light chain variable region (VL) were obtained. The specific sequences are as follows.

[0323] Amino acid sequence of the heavy chain variable region VH: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0324] EVTLVESGGGLVKPGGSLKLSCAAS GFTFSTY VMSWVRQTPEKRLEWVASI LSGGT SFYADNVKGRFTISRENARNILYLQMSSLKSEDTAMYYCVR RYGHDRYYAMDY WGQGTSVTVSS (SEQ ID NO.: 8)

[0325] Among them, the underlined amino acid sequence is the CDR region, which specifically includes:

[0326] VH CDR1:GTFFSTY(SEQ ID NO.:1)

[0327] VH CDR2: LSGGT (SEQ ID NO.: 2)

[0328] VH CDR3:RYGHDRYYAMDY(SEQ ID NO.:7)

[0329] Amino acid sequence of heavy chain variable region VH-7: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0330] EVTLVESGGGLVKPGGSLKLSCAAS GFTFSTY VMSWVRQTPEKRLEWVASI LSGGT SFYADNVKGRFTISRENARNILYLQMSSLKSEDTAMYYCVR RYGHDHYYAMDY WGQGTSVTVSS (SEQ ID NO.: 9)

[0331] Among them, the underlined amino acid sequence is the CDR region, which specifically includes:

[0332] VH-7 CDR1:GTFFSTY(SEQ ID NO.:1)

[0333] VH-7 CDR2: LSGGT(SEQ ID NO.:2)

[0334] VH-7 CDR3:RYGHDHYYAMDY(SEQ ID NO.:3)

[0335] Amino acid sequence of light chain variable region VL: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

[0336] QIVLTQSPANMSASLGERVTMTC AASSSVSSSYLH WYQQKPGSSPKLWIY STSNLAS GVPGRFNGSGSGTSYSLTISSMEAEDAATYYCH QUR FGGGTKLEIK (SEQ ID NO.: 10)

[0337] Among them, the underlined amino acid sequence is the CDR region, which specifically includes:

[0338] VL CDR1:AASSSVSSSYLH(SEQ ID NO.:4)

[0339] VL CDR2:STSNLAS(SEQ ID NO.:5)

[0340] VL CDR3:QYHRSPWT(SEQ ID NO.:6)

[0341] According to the sequencing results, the sequences of VH-7 and VH were compared and it was found that they only had one amino acid difference in CDR3, and the specific amino acid has been marked in bold in the sequence.

[0342] Example 6 Construction and FACS verification of chimeric antibody expression vector

[0343] Since hybridoma sequencing obtained two different heavy chain variable regions, a chimeric expression vector was constructed and antibodies were expressed for verification. The heavy chain variable region obtained by hybridoma sequencing was subcloned into the pcDNA3.1-IgG1Fc expression vector, and the light chain variable region was subcloned into the pcDNA3.1-IgKc expression vector. The constructed light chain and heavy chain expression vectors were combined in pairs. 293F cells were transiently co-transfected, and the expressed supernatant was collected for FACS detection 72 hours after transfection.

[0344] FACS test results Figure 8 shown.

[0345] According to the flow cytometry results, it was determined that the VH-7 heavy chain variable region shown in SEQ ID NO.: 9 and the VL light chain variable region shown in SEQ ID NO.: 10 were used for humanization design.

[0346] Example 7 Flow cytometric detection of humanized antibodies

[0347] The humanized antibody sequences were designed according to the general methods, and a total of 5 humanized heavy chain variable regions and 5 humanized light chain variable regions were designed.

[0348] Among them, the nucleic acid sequence information of the humanized candidate antibody is as follows:

[0349] >T1L1

[0350] GAGATCGTGCTGACACAGTCTCCCGCCACACTGTCACTGTCTCCAGGCGAAAGAGCCACACTGAGCTGCGCTGCCTCTAGCTCTGTGTCCAGCAGCTACCTGCACTGGTATCAGCAGAAGCCTGGACTGGCCCCTCGGCTGCTGATCTACAGCACAAGCAATCT GGCCAGCGGCATCCCGATAGATTTTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACAATCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACCACAGAAGCCCCTGGACATTTGGCGGAGGCACCAAGGTGGAAATCAAG(SEQ ID NO.:11)

[0351] >T1L2

[0352] GAGATCGTGCTGACACAGTCTCCCGCCACACTGTCACTGTCTCCAGGCGAAAGAGCCACACTGAGCTGCGCTGCCTCTAGCTCTGTGTCCAGCAGCTACCTGCACTGGTATCAGCAGAAGCCTGGCAGCGCTCCCAGACTGCTGATCTACAGCACAAGCAATCTGGCCAGCGGCATCCCCGATAGATTTTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACAATCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACCACAGAAGCCCCTGGACATTTGGCGGAGGCACCAAGGTGGAAATCAAG(SEQ ID NO.:12)

[0353] >T1L3

[0354] GAGATCGTGCTGACACAGTCTCCCGCCACACTGTCACTGTCTCCAGGCGAAAGAGCCACACTGAGCTGCGCTGCCTCTAGCTCTGTGTCCAGCAGCTACCTGCACTGGTATCAGCAGAAGCCTGGCAGCGCTCCCAGACTGCTGATCTACAGCACAAGCAATCTGGCCAGCGGCATCCCCGATAGATTTTCCGGCTCTGGCTCCGGCACCGACTTCACCCTGACAATCTCTAGACTGGAAGCCGAGGACTTCGCCGTGTACTACTGCCAGCAGTACCACAGAAGCCCCTGGACATTTGGCGGAGGCACCAAGGTGGAAATCAAG(SEQ ID NO.:13)

[0355] >T2L1

[0356] GACATCCAGATGACACAGAGCCCCAGCACACTGTCTGCCAGCGTGGGAGACAGAGTGACCATTACATGTGCCGCCAGCAGCAGCGTGTCCAGCTCTTACCTGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACAGCACCAGCAATCTGGCCAGCGGCGTGCCAAGCAGATTTTCTGGCTCTGGCAGCGGCACCGAGTTCACCCTGACCATCTCTAGCCTGCAGCCTGACGACTTCGCCACCTACTACTGCCAGCAGTACCACAGAAGCCCCTGGACATTTGGCCAGGGCACCAAGCTGGAAATCAAG(SEQ ID NO.:14)

[0357] >T2L2

[0358] GACATCCAGATGACACAGAGCCCCAGCACACTGTCTGCCAGCGTGGGAGACAGAGTGACCATTACATGTGCCGCCAGCAGCAGCGTGTCCAGCTCTTACCTGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACAGCACCAGCAATCTGGCCAGCGGCGTGCCAAGCAGATTTTCTGGCTCTGGCAGCGGCACCGAGTTCACCCTGACCATCTCTAGCCTGCAGCCTGATGATGCCGCCACCTACTACTGCCAGCAGTACCACAGAAGCCCCTGGACATTTGGCCAGGGCACCAAGCTGGAAATCAAG(SEQ ID NO.:15)

[0359] >T1H1

[0360] GAGGTGCAGCTGGTTGAATCTGGCGGCGGACTGATTCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCACCTACGTGATGAGCTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCAGCATCCTGAGCGGCGGCACAAGCTTCTACGCCGACAATGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCAGAAGATACGGCCACGACCACTACTACGCCATGGACTATTGGGGCCAGGGCACCCTGGTTACCGTTAGCTCT(SEQ ID NO.:16)

[0361] >T1H2

[0362] GAGGTGCAGCTGGTTGAATCTGGCGGCGGACTGATTCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCACCTACGTGATGAGCTGGGTCCGACAGACACCTGGCAAAGGCCTGGAATGGGTGTCCAGCATTCTGAGCGGCGGCACCAGCTTCTACGCCGACAATGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCAGAAGATACGGCCACGACCACTACTACGCCATGGACTATTGGGGCCAGGGCACCCTGGTTACCGTTAGCTCT(SEQ ID NO.:17)

[0363] >T1H3

[0364] GAGGTGCAGCTGGTTGAATCTGGCGGCGGACTGATTCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCACCTACGTGATGAGCTGGGTCCGACAGACACCTGGCAAAGGCCTGGAATGGGTGTCCAGCATTCTGAGCGGCGGCACCAGCTTCTACGCCGACAATGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCATGTACTACTGCGCCAGAAGATACGGCCACGACCACTACTACGCCATGGATTATTGGGGCCAGGGCACCCTGGTCACCGTTTCTTCT(SEQ ID NO.:18)

[0365] >T2H1

[0366] GAGGTGCAGCTGGTTGAATCTGGTGGCGGCGTTGTCAGACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCACCTACGTGATGAGCTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAATGGGTGTCCAGCATCCTGAGCGGCGGCACAAGCTTCTACGCCGACAATGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCCTGTATCACTGCGCCAGAAGATACGGCCACGACCACTACTACGCCATGGACTATTGGGGCCAGGGCACCACCGTGACAGTCTCTTCT(SEQ ID NO.:19)

[0367] >T2H2

[0368] GAGGTGCAGCTGGTTGAATCTGGTGGCGGCGTTGTCAGACCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTTAGCACCTACGTGATGAGCTGGGTCCGACAGACACCTGGCAAAGGCCTGGAATGGGTGTCCAGCATTCTGAGCGGCGGCACCAGCTTCTACGCCGACAATGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACTCCCTGAGAGCCGAGGACACAGCCCTGTATCACTGCGCCAGAAGATACGGCCACGACCACTACTACGCCATGGACTATTGGGGCCAGGGCACCACCGTGACAGTCTCTTCT(SEQ ID NO.:20)

[0369] The amino acid sequence information of the humanized candidate antibody is as follows:

[0370] >T1L1

[0371] EIVLTQSPATLSLSPGERATLSCAASSSVSSSYLHWYQQKPGLAPRLLIYSTSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYHRSPWTFGGGTKVEIK(SEQ ID NO.:21)

[0372] >T1L2

[0373] EIVLTQSPATLSLSPGERATLSCAASSSVSSSYLHWYQQKPGSAPRLLIYSTSNLASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYHRSPWTFGGGTKVEIK(SEQ ID NO.:22)

[0374] >T1L3

[0375] EIVLTQSPATLSLSPGERATLSCAASSSVSSSYLHWYQQKPGSAPRLLIYSTSNLASGIPDRFSGSGSGTDFTLTISRLEAEDFAVYYCQQYHRSPWTFGGGTKVEIK(SEQ ID NO.:23)

[0376] >T2L1

[0377] DIQMTQSPSTLSASVGDRVTITCAASSSVSSSYLHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYHRSPWTFGQGTKLEIK(SEQ ID NO.:24)

[0378] >T2L2

[0379] DIQMTQSPSTLSASVGDRVTITCAASSSVSSSYLHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPDDAATYYCQQYHRSPWTFGQGTKLEIK(SEQ ID NO.:25)

[0380] >T1H1

[0381] EVQLVESGGGLIQPGGSLRLSCAASGFTFSTYVMSWVRQAPGKGLEWVSSILSGGTSFYADNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRYGHDHYYAMDYWGQGTLVTVSS(SEQ ID NO.:26)

[0382] >T1H2

[0383] EVQLVESGGGLIQPGGSLRLSCAASGFTFSTYVMSWVRQTPGKGLEWVSSILSGGTSFYADNVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRYGHDHYYAMDYWGQGTLVTVSS(SEQ ID NO.:27)

[0384] >T1H3

[0385] EVQLVESGGGLIQPGGSLRLSCAASGFTFSTYVMSWVRQTPGKGLEWVSSILSGGTSFYADNVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARRYGHDHYYAMDYWGQGTLVTVSS(SEQ ID NO.:28)

[0386] >T2H1

[0387] EVQLVESGGGVVRPGGSLRLSCAASGFTFSTYVMSWVRQAPGKGLEWVSSILSGGTSFYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTALYHCARRYGHDHYYAMDYWGQGTTVTVSS(SEQ ID NO.:29)

[0388] >T2H2

[0389] EVQLVESGGGVVRPGGSLRLSCAASGFTFSTYVMSWVRQTPGKGLEWVSSILSGGTSFYADNVKGRFTISRDNAKNSLYLQMNSLRAEDTALYHCARRYGHDHYYAMDYWGQGTTVTVSS(SEQ ID NO.:30)

[0390] After transient transfection of the humanized antibody expression vector into 293F cells, the culture supernatant was collected and flow cytometry was used to detect the binding of the humanized antibody to the antigen on the surface of the recombinant cell CHO-K1-CD276 cell membrane. The human-mouse chimeric antibody constructed with the original mouse antibody VH and VL was used as a control.

[0391] FACS test results Fig. 9 As shown, the humanized antibodies all bound to recombinant CHO-K1-CD276 cells, and the binding force was comparable to that of human-mouse chimeric antibodies. The combined T1H1-T1L1 and T2H1-T2L1 expressed purified antibodies were selected for subsequent affinity testing.

[0392] The SDS-PAGE results of humanized antibodies T1H1-T1L1 and T2H1-T2L1 are as follows Fig.10 shown.

[0393] The sequences of the antibody before and after humanization were compared, and the alignment results of T1H1-T1L1 were as follows: Fig.11 As shown, the alignment results of T2H1-T2L1 are as follows Fig.12 shown.

[0394] Example 8 Humanized Antibody Affinity Determination

[0395] 8.1 Experimental Materials and Reagents Involved in This Example

[0396] S series CM5 chip: GE, lot: 10261136

[0397] Buffer: HBS-EP+10×, GE, lot: BCBV7063. Dilute 10 times with deionized water before use.

[0398] Amino coupling kit: GE, lot: 2067793

[0399] Regeneration reagent: 10mM Glycine 1.5, GE, lot: 10240798

[0400] 8.2 Ligand Coupling Pre-enrichment

[0401] Ligand: CD276 recombinant protein

[0402] Test pH: 10mM Acetate 5.5 / 5.0 / 4.5 / 4.0

[0403] Flow rate: 10 μl / min

[0404] Buffer: HBS-EP+buffer

[0405] The results are shown in Figure 13. 10 mM Acetate 4.0 was selected as the coupling buffer.

[0406] 8.3 Ligand Coupling

[0407] Ligand: CD276 recombinant protein

[0408] Coupling buffer: 10 mM Acetate 4.0

[0409] Target conjugation amount: 15RU

[0410] The results are shown in Figure 14, and the final coupling amount is 8.3RU.

[0411] 8.4 Affinity determination of CD276 Chimeric, CD276T1H1-T1L1, CD276T2H1-T2L1 and CD276 recombinant protein

[0412] Experimental conditions:

[0413] Antibody concentration: 1.5625 / 3.125 / 6.25 / 12.5 / 25 / 50ug / ml

[0414] Flow rate: 30 μl / min

[0415] Combination time: 300s

[0416] Dissociation time: 300s

[0417] Regeneration reagent: Glycine 2.5

[0418] The affinity assay results of CD276 Chimeric are shown in Fig.15 As shown, the affinity determination results of CD276T1H1-T1L1 are as follows Fig.16As shown, the affinity determination results of CD276T2H1-T2L1 are as follows Fig.17 shown.

[0419] The results of affinity testing of each antibody are shown in Table 1.

[0420] Table 1

[0421]

[0422] The affinity test results showed that the affinity of humanized antibodies was basically consistent with that of chimeric antibodies.

[0423] Example 9 CD276 Antibody Membrane Proteome Array

[0424] The Membrane Proteome Array method was used to detect the specificity and targeting of CD276 humanized antibody (CD276T1H1-T1L1) to see if there is any off-target toxicity.

[0425] The Membrane Proteome Array (MPA) is a platform that can analyze the specificity of antibodies or ligands targeting human membrane proteins. MPA can be used to determine the targeting specificity of antibodies. This platform uses flow cytometry to directly detect the binding of antibodies to unfixed cell surface membrane proteins. Therefore, all target proteins have the original native conformation of the protein and appropriate post-translational modifications.

[0426] The humanized antibody CD276T1H1-T1L1 was tested against a library of more than 5,300 human membrane proteins, including 94% of all single-pass, multi-pass, and GPI-anchored transmembrane proteins.

[0427] The results are as follows Fig.18 As shown, the humanized CD276 antibody (CD276T1H1-T1L1) only specifically binds to the CD276 protein, and does not specifically bind to other membrane proteins, indicating that the CD276 humanized antibody of the present invention will only specifically recognize the CD276 protein, will not bind to other proteins, and will not produce off-target toxicity, further proving the specificity and safety of the humanized CD276 antibody of the present invention.

[0428] Example 10 Antigen recognition epitope of CD276 humanized antibody (CD276T1H1-T1L1)

[0429] 10.1 Preparation of CD276 antigen fragment expression vector

[0430] The amino acid sequence information of the extracellular segment of the target antigen CD276 was analyzed, and truncated fragments of the antigen were designed. Each fragment was truncated by 10 amino acids, and the truncations were performed sequentially. Primers were designed, PCR was performed, and subcloned into the target vector Lenti-CMV-puro (the signal peptide used the IL2 signal peptide, MYRMQLLSCIALSLALVTNS (SEQ ID NO.: 31), and the EcoRI restriction site was used).

[0431] The information of the truncated amino acid sequence of CD276 is shown in Table 2.

[0432] Table 2

[0433]

[0434]

[0435] 10.2 Expression and flow cytometry of different truncated forms of CD276

[0436] 1. Take out the LVTransm transfection reagent and antibody expression vector from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down. Take out the PBS buffer and warm it to room temperature. Take 0.5mL PBS to one well of a 6-well plate, add 6μg of CD276 different truncated expression vectors respectively, pipette up and down to mix thoroughly, add 18μL LVTransm, and immediately pipette up and down to mix thoroughly, and let stand at room temperature for 10 minutes.

[0437] 2. Add the above DNA / LVTransm complex to 1.5mL 293F cells and shake gently to mix thoroughly. Place the cells in a 37°C, 5% CO2 incubator at 130RPM for 6-8 hours, then add 1.5mL fresh FreeStyle TM 293 Expression Medium culture medium, put the cells back into the incubator and continue culturing.

[0438] 3. After continuous culture for 48 hours, centrifuge and collect cells separately for flow cytometry detection.

[0439] 10.3 Flow cytometry detection of binding of CD276 antibody to different truncated antigens

[0440] 1. Collect the cells transfected in step 10.2 and divide each cell type into 2 portions, with the number of cells in each portion being 5×10 5 cells;

[0441] 2. Incubate with CD276 antibody (1ug / well), mix thoroughly, and incubate at room temperature for 1 hour;

[0442] 3. Centrifuge at 800xg for 5 minutes at room temperature, remove the supernatant containing antibodies, and wash the cells 3 times with PBS;

[0443] 4. Add 1uL PE-labeled Anti-human IgG, mix thoroughly, and incubate at room temperature in the dark for 30 minutes;

[0444] 5. Centrifuge at 800xg for 5 minutes at room temperature, remove the supernatant containing the secondary antibody, and wash the cells three times with PBS;

[0445] 6. Resuspend the cells in 500uL PBS and perform flow cytometry analysis.

[0446] The flow cytometry results are as follows Fig.19 As shown, the antigen epitope of CD276 is in the first 10 amino acids of the extracellular segment. Through signal peptide prediction analysis, the cleavage position of the IL2 signal peptide will cut two more amino acids, so the antigen epitope should be between 3-12 amino acids.

[0447] In order to ensure the structural integrity of the antigen, the antigen epitope was further detected, and each amino acid was mutated to Gly with the smallest side chain through a point mutation scheme. Subsequent experiments designed point mutations of 3-12 amino acids of the antigen, each site mutated to Gly, and after gene synthesis, subcloned into Lenti-CMV-puro to construct an antigen mutation vector.

[0448] The flow cytometry results of CD276 antibody binding to antigens with different mutation sites are shown in Fig. 20 The results showed that the antigen epitope of CD276 antibody was mainly located at amino acids 31-40, with the main sites being 32Q and 40A.

[0449] The antigen epitope is as follows (wherein the sequence marked in italics is the signal peptide, and the sequence marked in bold underline is the antigen recognition epitope of the CD276 antibody):

[0450]

[0451] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Boshengji Pharmaceutical Technology (Suzhou) Co., Ltd. <120> Monoclonal antibody targeting human CD276 and its application <130> P2020-0619 <160> 33 <170> SIPOSequenceListing 1.0 <210> 1 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 1 Gly Phe Thr Phe Ser Thr Tyr 1 5 <210> 2 <211> 5 <212> PRT <213> Mouse (Mus musculus) <400> 2 Leu Ser Gly Gly Thr 1 5 <210> 3 <211> 12 <212> PRT <213> Mouse (Mus musculus) <400> 3 Arg Tyr Gly His Asp His Tyr Tyr Ala Met Asp Tyr 1 5 10 <210> 4 <211> 12 <212> PRT <213> Mouse (Mus musculus) <400> 4 Ala Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His 1 5 10 <210> 5 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 5 Ser Thr Ser Asn Leu Ala Ser 1 5 <210> 6 <211> 8 <212> PRT <213> Mouse (Mus musculus) <400> 6 Gln Tyr His Arg Ser Pro Trp Thr 1 5 <210> 7 <211> 12 <212> PRT <213> Mouse (Mus musculus) <400> 7 Arg Tyr Gly His Asp Arg Tyr Tyr Ala Met Asp Tyr 1 5 10 <210> 8 <211> 120 <212> PRT <213> Mouse (Mus musculus) <400> 8 Glu Val Thr Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile Leu Ser Gly Gly Thr Ser Phe Tyr Ala Asp Asn Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Arg Asn Ile Leu Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Val 85 90 95 Arg Arg Tyr Gly His Asp Arg Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 9 <211> 120 <212> PRT <213> Mouse (Mus musculus) <400> 9 Glu Val Thr Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile Leu Ser Gly Gly Thr Ser Phe Tyr Ala Asp Asn Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Arg Asn Ile Leu Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Val 85 90 95 Arg Arg Tyr Gly His Asp His Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 10 <211> 108 <212> PRT <213> Mouse (Mus musculus) <400> 10 Gln Ile Val Leu Thr Gln Ser Pro Ala Asn Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Ala Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Gly Arg Phe Asn 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro 85 90 95 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 11 <211> 324 <212> DNA <213> Artificial Sequence <400> 11 gagatcgtc tgacacagtc tcccgccaca ctgtcactgt ctccaggcga aagagccaca ctgagctgcg ctgcctctag ctctgtgtcc agcagctacc tgcactggta tcagcagaag cctggactgg cccctcggct gctgatctac cake atctggccag cggcatcccc 180 gatagattt ccggctctgg ctccggcacc gacttcaccc tgacaatcag cagactgga 300. cccgaggact tcgccgtgta ctactgccag cagtaccaca gaagcccctg gacatttggc ggaggcacca aggtggaat caag <210> 12 <211> 324 <212> DNA <213> Artificial Sequence <400> 12 gagatcgtc tgacacagtc tcccgccaca ctgtcactgt ctccaggcga aagagccaca ctgagctgcg ctgcctctag ctctgtgtcc agcagctacc tgcactggta tcagcagaag cctggcagcg ctcccagact gctgatctac agcacaagca atctggccag cggcatcccc 180 gatagattt ccggctctgg ctccggcacc gacttcaccc tgacaatcag cagactgga 300. cccgaggact tcgccgtgta ctactgccag cagtaccaca gaagcccctg gacatttggc ggaggcacca aggtggaat caag <210> 13 <211> 324 <212> DNA <213> Artificial Sequence <400> 13 gagatcgtc tgacacagtc tcccgccaca ctgtcactgt ctccaggcga aagagccaca ctgagctgcg ctgcctctag ctctgtgtcc agcagctacc tgcactggta tcagcagaag cctggcagcg ctcccagact gctgatctac agcacaagca atctggccag cggcatcccc 180 gatagattt ccggctctgg ctccggcacc gacttcaccc tgacaatctc tagactgga gccgaggact tcgccgtgta ctactgccag cagtaccaca gaagcccctg gacatttggc ggaggcacca aggtggaat caag <210> 14 <211> 324 <212> DNA <213> Artificial Sequence <400> 14 gacatccaga tgacacagag ccccagcaca ctgtctgcca gcgtgggaga cagagtgacc attackgtg ccgccagcag cagcgtgtcc agctcttacc tgcactggta tcagcagaag cccggcaagg cccctaagct gctgatctac agcaccagca atctggccag cggcgtgcca 180 agcagatttt ctggctctgg cagcggcacc gagttcaccc tgaccatctc tagcctgcag 240 cctgacgact tcgccaccta ctactgccag cagtaccaca gaagcccctg gacatttggc 300 cagggcacca agctggaaat caag 324 <210> 15 <211> 324 <212> DNA <213> Artificial Sequence <400> 15 gacatccaga tgacacagag ccccagcaca ctgtctgcca gcgtgggaga cagagtgacc 60 attacatgtg ccgccagcag cagcgtgtcc agctcttacc tgcactggta tcagcagaag 120 cccggcaagg cccctaagct gctgatctac agcaccagca atctggccag cggcgtgcca 180 agcagatttt ctggctctgg cagcggcacc gagttcaccc tgaccatctc tagcctgcag 240 cctgatgatg ccgccaccta ctactgccag cagtaccaca gaagcccctg gacatttggc 300 cagggcacca agctggaaat caag 324 <210> 16 <211> 360 <212> DNA <213> Artificial Sequence <400> 16 gaggtgcagc tggttgaatc tggcggcgga ctgattcagc ctggcggatc tctgagactg 60 agctgtgccg ccagcggctt cacctttagc acctacgtga tgagctgggt ccgacaggcc 120 cctggcaaag gacttgaatg ggtgtccagc atcctgagcg gcggcacaag cttctacgcc 180 gacaatgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240 cagatgaaca gcctgagagc cgaggacacc gccgtgtact actgcgccag aagatacggc 300 cacgaccact actacgccat ggactattgg ggccagggca ccctggttac cgttagctct 360 <210> 17 <211> 360 <212> DNA <213> Artificial Sequence <400> 17 gaggtgcagc tggttgaatc tggcggcgga ctgattcagc ctggcggatc tctgagactg 60 agctgtgccg ccagcggctt cacctttagc acctacgtga tgagctgggt ccgacagaca 120 cctggcaaag gcctggaatg ggtgtccagc attctgagcg gcggcaccag cttctacgcc 180 gacaatgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240 cagatgaaca gcctgagagc cgaggacacc gccgtgtact actgcgccag aagatacggc 300 cacgaccact actacgccat ggactattgg ggccagggca ccctggttac cgttagctct 360 <210> 18 <211> 360 <212> DNA <213> Artificial Sequence <400> 18 gaggtgcagc tggttgaatc tggcggcgga ctgattcagc ctggcggatc tctgagactg 60 agctgtgccg ccagcggctt cacctttagc acctacgtga tgagctgggt ccgacagaca 120 cctggcaaag gcctggaatg ggtgtccagc attctgagcg gcggcaccag cttctacgcc 180 gacaatgtga agggcagatt caccatcagc cgggacaaca gcaagaacac cctgtacctg 240 cagatgaaca gcctgagagc cgaggacacc gccatgtact actgcgccag aagatacggc 300 cacgaccact actacgccat ggattattgg ggccagggca ccctggtcac cgtttcttct 360 <210> 19 <211> 360 <212> DNA <213> Artificial Sequence <400> 19 gaggtgcagc tggttgaatc tggtggcggc gttgtcagac ctggcggatc tctgagactg 60 agctgtgccg ccagcggctt cacctttagc acctacgtga tgagctgggt ccgacaggcc 120 cctggcaaag gacttgaatg ggtgtccagc atcctgagcg gcggcacaag cttctacgcc 180 gacaatgtga agggcagatt caccatcagc cgggacaacg ccaagaacag cctgtacctg 240 cagatgaact ccctgagagc cgaggacaca gccctgtatc actgcgccag aagatacggc 300 cacgaccact actacgccat ggactattgg ggccagggca ccaccgtgac agtctcttct 360 <210> 20 <211> 360 <212> DNA <213> Artificial Sequence <400> 20 gaggtgcagc tggttgaatc tggtggcggc gttgtcagac ctggcggatc tctgagactg 60 agctgtgccg ccagcggctt cacctttagc acctacgtga tgagctgggt ccgacagaca 120 cctggcaaag gcctggaatg ggtgtccagc attctgagcg gcggcaccag cttctacgcc 180 gacaatgtga agggcagatt caccatcagc cgggacaacg ccaagaacag cctgtacctg 240 cagatgaact ccctgagagc cgaggacaca gccctgtatc actgcgccag aagatacggc 300 cacgaccact actacgccat ggactattgg ggccagggca ccaccgtgac agtctcttct 360 <210> 21 <211> 108 <212> PRT <213> Artificial Sequence <400> 21 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ala Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Leu Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Arg Ser Pro 85 90 95 Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 22 <211> 108 <212> PRT <213> Artificial Sequence <400> 22 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ala Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Arg Ser Pro 85 90 95 Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 23 <211> 108 <212> PRT <213> Artificial Sequence <400> 23 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ala Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Ala Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr His Arg Ser Pro 85 90 95 Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 24 <211> 108 <212> PRT <213> Artificial Sequence <400> 24 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ala Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Arg Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 25 <211> 108 <212> PRT <213> Artificial Sequence <400> 25 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ala Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr His Arg Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 26 <211> 120 <212> PRT <213> Artificial Sequence <400> 26 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Leu Ser Gly Gly Thr Ser Phe Tyr Ala Asp Asn Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Arg Tyr Gly His Asp His Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 27 <211> 120 <212> PRT <213> Artificial Sequence <400> 27 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Leu Ser Gly Gly Thr Ser Phe Tyr Ala Asp Asn Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Arg Tyr Gly His Asp His Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 28 <211> 120 <212> PRT <213> Artificial Sequence <400> 28 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Leu Ser Gly Gly Thr Ser Phe Tyr Ala Asp Asn Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Arg Arg Tyr Gly His Asp His Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 29 <211> 120 <212> PRT <213> Artificial Sequence <400> 29 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Leu Ser Gly Gly Thr Ser Phe Tyr Ala Asp Asn Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr His Cys Ala 85 90 95 Arg Arg Tyr Gly His Asp His Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 30 <211> 120 <212> PRT <213> Artificial Sequence <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Leu Ser Gly Gly Thr Ser Phe Tyr Ala Asp Asn Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr His Cys Ala 85 90 95 Arg Arg Tyr Gly His Asp His Tyr Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 31 <211> 20 <212> PRT <213> Artificial Sequence <400> 31 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser 20 <210> 32 <211> 288 <212> PRT <213> Artificial Sequence <400> 32 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 Arg 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 Val 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 Gly Ser Val Thr 195 200 205 Ile Thr Gly Gln Pro Met Thr Phe Pro Pro Glu Ala Leu Trp Val Thr 210 215 220 Val Gly Leu Ser Val Cys Leu Ile Ala Leu Leu Val Ala Leu Ala Phe 225 230 235 240 Val Cys Trp Arg Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly 245 250 255 Ala Glu Asp Gln Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu Gln 260 265 270 Pro Leu Lys His Ser Asp Ser Lys Glu Asp Asp Gly Gln Glu Ile Ala 275 280 285 <210> 33 <211> 316 <212> PRT <213> Artificial Sequence <400> 33 Met Leu Arg Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala 1 5 10 15 Ala Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln 20 25 30 Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu 35 40 45 Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn 50 55 60 Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Ala 65 70 75 80 Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe 85 90 95 Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val 100 105 110 Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp 115 120 125 Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys 130 135 140 Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr 145 150 155 160 Val Thr Ile Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val 165 170 175 Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr 180 185 190 Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Val Leu 195 200 205 Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn 210 215 220 Pro Val Leu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly Gln 225 230 235 240 Pro Met Thr Phe Pro Pro Glu Ala Leu Trp Val Thr Val Gly Leu Ser 245 250 255 Val Cys Leu Ile Ala Leu Leu Val Ala Leu Ala Phe Val Cys Trp Arg 260 265 270 Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln 275 280 285 Asp Gly Glu Gly Glu Gly Ser Lys Thr Ala Leu Gln Pro Leu Lys His 290 295 300 Ser Asp Ser Lys Glu Asp Asp Gly Gln Glu Ile Ala 305 310 315

Claims

1. An antibody targeting CD276, comprising a heavy chain variable region and a light chain variable region, characterized in that: The heavy chain variable region includes the following three complementarity determining regions CDR: CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3 or 7, The light chain variable region includes the following three complementarity determining regions CDR: CDR1' shown in SEQ ID NO:4, CDR2' shown in SEQ ID NO:5, and CDR3' shown in SEQ ID NO:

6.

2. The antibody according to claim 1, characterized in that The antibody is selected from: an animal-derived antibody, a chimeric antibody, a humanized antibody or a combination thereof.

3. The antibody according to claim 1, characterized in that The heavy chain variable region sequence of the antibody is shown in any one of SEQ ID NOs: 8, 9, 26-30; and The light chain variable region sequence of the antibody is shown in any one of SEQ ID NOs: 10, 21-25.

4. The antibody according to claim 1, characterized in that The heavy chain variable region sequence of the antibody is shown in SEQ ID NO: 8 or 9; and the light chain variable region sequence of the antibody is shown in SEQ ID NO:

10.

5. The antibody according to claim 1, characterized in that The heavy chain variable region sequence of the antibody is shown in SEQ ID NO: 26; and the light chain variable region sequence of the antibody is shown in SEQ ID NO:

21.

6. The antibody according to claim 1, characterized in that The heavy chain variable region sequence of the antibody is shown in SEQ ID NO: 29; and the light chain variable region sequence of the antibody is shown in SEQ ID NO:

24.

7. The antibody according to claim 1, characterized in that The antibody is a partially or fully humanized monoclonal antibody.

8. Use of the antibody according to claim 1 in preparing a detection reagent or a kit for detecting CD276 protein.

9. An antibody-drug conjugate, comprising: (a) an antibody portion, wherein the antibody portion is the antibody of claim 1; and (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

10. A pharmaceutical composition, comprising: (i) an active ingredient, wherein the active ingredient is selected from the group consisting of the antibody according to claim 1, the antibody-drug conjugate according to claim 9, or a combination thereof; and (ii) a pharmaceutically acceptable carrier.

11. A polynucleotide encoding the antibody according to claim 1.

12. A vector comprising the polynucleotide according to claim 11.

13. An in vitro non-diagnostic method for detecting CD276 protein in a sample, the method comprising the steps of: (1) contacting the sample with the antibody according to claim 1 or the antibody-drug conjugate according to claim 9 in vitro; (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CD276 protein in the sample.

14. A detection board, comprising: A substrate and a test strip, wherein the test strip contains the antibody according to claim 1 or the antibody-drug conjugate according to claim 9.

15. A kit, comprising: (1) a first container, wherein the first container contains the antibody according to claim 1; and (2) a second container, wherein the second container contains a secondary antibody against the antibody of claim 1; Alternatively, the kit contains the detection plate as described in claim 14.

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