Anti-CD276 nanobody, preparation method therefor and use thereof

Abstract

Provided are an anti-CD276 nanobody, and a preparation method therefor and the use thereof. Specifically, further provided are a corresponding anti-CD276 antibody, a chimeric antigen receptor, a fusion protein, a recombinant protein, an antibody-drug conjugate, and an encoding nucleic acid, an expression vector, a host cell, etc. thereof. The anti-CD276 nanobody is high in affinity, simple in structure and easy to prepare, and the anti-CD276 nanobody-drug conjugate has a significant killing activity, thereby having application prospects in the fields such as tumor treatment and immunodetection.

Description

Anti-CD276 nanoantibody and its preparation method and application Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-CD276 nanobody and a preparation method and application thereof. Background Art

[0002] The CD276 molecule, also known as B7-H3, is a type I transmembrane glycoprotein belonging to the B7 family of immune co-stimulatory and co-inhibitory globulins. Existing research indicates that CD276 has both immune-activating and immunosuppressive effects. CD276 can exert immunosuppressive effects by attenuating the secretion of the type I cytokine IFN-γ. Numerous studies support the negative immunoregulatory effects of CD276 in models of graft-versus-host disease, cardiac transplant rejection, airway inflammation, and autoimmune encephalomyelitis. Furthermore, CD276 has been found to provide immune co-stimulatory signals to T cells in autoimmune diseases both in vivo and in vitro.

[0003] Numerous studies have shown that CD276 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, and breast cancer, but its expression is limited in normal tissues. It has also been reported to be expressed in important components of the tumor microenvironment, such as cancer stem cells, tumor stromal cells, and tumor angiogenesis. Overexpression of CD276 often reduces the infiltration of tumor-associated lymphocytes within the tumor, accelerates disease progression, and is often closely associated with poor prognosis in cancers such as pancreatic ductal carcinoma, prostate cancer, ovarian cancer, lung cancer, and clear cell renal carcinoma. CD276 is a very promising target for tumor therapy.

[0004] However, there is currently a lack of anti-CD276 nanobodies with high affinity, high specificity and high biological activity against CD276. Therefore, there is an urgent need in the art to develop anti-CD276 nanobodies with high affinity, high specificity and high biological activity. Summary of the Invention

[0005] The purpose of the present invention is to provide an anti-CD276 nanobody with high affinity, high specificity and high biological activity.

[0006] In a first aspect of the present invention, an anti-CD276 nanobody is provided, wherein the anti-CD276 nanobody has one or more complementary determining regions (CDRs) selected from the group consisting of:

[0007] (1) CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 7, and CDR3 shown in SEQ ID NO: 8;

[0008] (2) CDR1 shown in SEQ ID NO: 9, CDR2 shown in SEQ ID NO: 10, and CDR3 shown in SEQ ID NO: 11;

[0009] (3) CDR1 shown in SEQ ID NO: 12, CDR2 shown in SEQ ID NO: 13, and CDR3 shown in SEQ ID NO: 14;

[0010] (4) CDR1 shown in SEQ ID NO: 15, CDR2 shown in SEQ ID NO: 16, and CDR3 shown in SEQ ID NO: 17;

[0011] (5) CDR1 shown in SEQ ID NO: 18, CDR2 shown in SEQ ID NO: 19, and CDR3 shown in SEQ ID NO: 20.

[0012] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0013] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-CD276 nanobody is as shown in any one of SEQ ID NOs: 1-5.

[0014] In another preferred example, the VHH chain of the anti-CD276 Nanobody comprises an amino acid sequence having a sequence similarity of at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% to any one of SEQ ID NOs: 1-5.

[0015] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-CD276 Nanobody comprises one or more amino acid substitutions compared to any one of SEQ ID NOs: 1-5, preferably conservative amino acid substitutions.

[0016] 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, more preferably 1) amino acid and can retain the ability to specifically bind to CD276.

[0017] In another preferred embodiment, the anti-CD276 nanobody includes humanized antibodies, animal-derived antibodies, and chimeric antibodies.

[0018] In the second aspect of the present invention, an anti-CD276 multivalent antibody or an antibody against multiple epitopes is provided, wherein the multivalent antibody or the antibody against multiple epitopes comprises at least one antibody element against a CD276 epitope, and the antibody element is the anti-CD276 nanobody described in the first aspect of the present invention.

[0019] In another preferred embodiment, the anti-CD276 multivalent antibody or antibody targeting multiple epitopes includes one or more anti-CD276 nanobodies.

[0020] In another preferred embodiment, the anti-CD276 multivalent antibody includes a monomer, a bivalent body (divalent antibody), a tetravalent body (tetravalent antibody), and / or a multivalent body (multivalent antibody).

[0021] In another preferred embodiment, the anti-CD276 multivalent antibody comprises one or more VHH chains having an amino acid sequence as shown in any one of SEQ ID NOs: 1-5.

[0022] In another preferred embodiment, the anti-CD276 multivalent antibody comprises two VHH chains having the amino acid sequences shown in SEQ ID NO: 1-5.

[0023] In another preferred embodiment, the anti-CD276 nanobody includes humanized antibodies, animal-derived antibodies, and chimeric antibodies.

[0024] In a third aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has:

[0025] (i) the anti-CD276 Nanobody according to the first aspect of the invention, or the anti-CD276 multivalent antibody or antibody against multiple epitopes according to the second aspect of the invention; and

[0026] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0027] In another preferred embodiment, the tag sequence includes a Flag tag, an Fc tag, an HA tag and a His tag.

[0028] In another preferred embodiment, the tag sequence is a His tag.

[0029] In another preferred embodiment, the recombinant protein specifically binds to CD276 protein.

[0030] In a fourth aspect of the present invention, an antibody-drug conjugate is provided, wherein the antibody-drug conjugate comprises:

[0031] (a) the Nanobody as described in the first aspect of the invention, the multivalent antibody or antibody against multiple epitopes as described in the second aspect of the invention, or the recombinant protein as described in the third aspect of the invention; and

[0032] (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.

[0033] In another preferred embodiment, the antibody portion and the coupling portion are coupled via a chemical bond or a linker.

[0034] In the fifth aspect of the present invention, a chimeric antigen receptor (CAR) is provided, wherein the CAR contains an extracellular domain, and the extracellular domain comprises the anti-CD276 nanobody as described in the first aspect of the present invention, or the anti-CD276 multivalent antibody as described in the second aspect of the present invention.

[0035] In another preferred embodiment, the extracellular domain further includes a signal peptide.

[0036] In another preferred embodiment, the extracellular domain also includes other exogenous proteins.

[0037] In another preferred embodiment, the CAR has a structure shown in formula (I): L-Nb-H-TM-C-CD3ζ (I)

[0038] Where,

[0039] The "-" is a connecting peptide or peptide bond;

[0040] L is none or a signal peptide sequence;

[0041] Nb is the antigen-binding domain that specifically binds to CD276;

[0042] H is none or hinge region;

[0043] TM is the transmembrane domain;

[0044] C is the costimulatory signaling domain;

[0045] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type, or mutants / modified forms thereof).

[0046] In another preferred embodiment, the L is selected from the signal peptides of the following histones: CD28, 4-1BB, GM-CSF, CD3, CD8a or a combination thereof.

[0047] In another preferred embodiment, the sequence of Nb is shown in any one of SEQ ID NOs: 1-5.

[0048] In another preferred embodiment, the H is selected from the hinge region of the following histones: CD8, CD28, CD137, IgG, or a combination thereof.

[0049] In another preferred embodiment, the C is selected from the costimulatory domains of the following histones: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or mutants / modified forms thereof, or combinations thereof.

[0050] In the sixth aspect of the present invention, a polynucleotide is provided, which encodes a protein selected from the following group: the nanobody described in the first aspect of the invention, or the multivalent antibody or antibody against multiple epitopes described in the second aspect of the invention, the recombinant protein described in the third aspect of the invention, or the CAR described in the fifth aspect of the invention.

[0051] In the seventh aspect of the present invention, an expression vector is provided, wherein the expression vector contains the polynucleotide described in the sixth aspect of the present invention.

[0052] In another preferred embodiment, the expression vector is selected from the following group: DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or a combination thereof.

[0053] In another preferred embodiment, the expression vector is a lentiviral vector.

[0054] In the eighth aspect of the present invention, a host cell is provided, which contains the expression vector described in the seventh aspect of the present invention, or the polynucleotide described in the sixth aspect of the present invention is integrated into its genome, or expresses the nanoantibody described in the first aspect of the present invention, the multivalent antibody or antibody against multiple epitopes described in the second aspect of the present invention, the recombinant protein described in the third aspect of the present invention, or the CAR described in the fifth aspect of the present invention.

[0055] In another preferred embodiment, the cell is an isolated cell, and / or the cell is a genetically engineered cell.

[0056] In another preferred embodiment, the cells are mammalian cells.

[0057] In another preferred embodiment, the cells are T cells.

[0058] In another preferred embodiment, the host cell is an engineered immune cell.

[0059] In another preferred embodiment, the engineered immune cells are selected from the following group:

[0060] (i) Chimeric antigen receptor αβ T cells (CAR-T cells);

[0061] (ii) chimeric antigen receptor γδ T cells (CAR-T cells);

[0062] (iii) chimeric antigen receptor NKT cells (CAR-NKT cells);

[0063] (iv) chimeric antigen receptor NK cells (CAR-NK cells);

[0064] (v) Chimeric antigen receptor macrophages.

[0065] In another preferred embodiment, the engineered immune cells include autologous or allogeneic αβT cells, γδT cells, NKT cells, NK cells, or a combination thereof.

[0066] In another preferred embodiment, the engineered immune cells are CAR-T cells.

[0067] In the ninth aspect of the present invention, a preparation is provided, which contains the nanobody described in the first aspect of the present invention, the multivalent antibody or the antibody against multiple epitopes described in the second aspect of the present invention, the recombinant protein described in the third aspect of the present invention, the antibody-drug conjugate described in the fourth aspect of the present invention, the CAR described in the fifth aspect of the present invention, or the vector described in the seventh aspect of the present invention, or an immune cell expressing the CAR described in the fifth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0068] In a tenth aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0069] (i) the Nanobody of the first aspect of the invention, the multivalent antibody or the antibody against multiple epitopes of the second aspect of the invention, the recombinant protein of the third aspect of the invention, the antibody-drug conjugate of the fourth aspect of the invention, or the host cell of the eighth aspect of the invention; and

[0070] (ii) a pharmaceutically acceptable carrier.

[0071] In another preferred embodiment, the pharmaceutical composition further contains other drugs for treating diseases.

[0072] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug for preventing and / or treating diseases or conditions associated with CD276.

[0073] In another preferred embodiment, the disease or condition associated with CD276 is selected from the group consisting of leukemia, gastric cancer, breast cancer, non-small cell lung cancer, ovarian cancer, osteosarcoma, neuroblastoma, pancreatic cancer, breast cancer, or a combination thereof.

[0074] In the eleventh aspect of the present invention, a kit is provided, wherein the kit contains the polynucleotide described in the sixth aspect of the present invention, the CAR described in the fifth aspect of the present invention, or the vector described in the seventh aspect of the present invention.

[0075] In another preferred embodiment, the kit is used to prepare immune cells expressing the CAR described in the fifth aspect of the present invention.

[0076] In the twelfth aspect of the present invention, there is provided the use of the nanobody described in the first aspect of the present invention, the multivalent antibody or the antibody against multiple epitopes described in the second aspect of the present invention, the recombinant protein described in the third aspect of the present invention, the antibody-drug conjugate described in the fourth aspect of the present invention, or the immune cell expressing the CAR described in the fifth aspect of the present invention for the preparation of a medicament for preventing and / or treating cancers or diseases associated with CD276.

[0077] In another preferred embodiment, the CD276-related cancer or disease is selected from the group consisting of leukemia, gastric cancer, breast cancer, non-small cell lung cancer, ovarian cancer, osteosarcoma, neuroblastoma, pancreatic cancer, breast cancer, or a combination thereof.

[0078] In the thirteenth aspect of the present invention, a method for treating a disease is provided, which comprises administering to a subject in need of treatment: the nanoantibody described in the first aspect of the invention, the multivalent antibody or antibody against multiple epitopes described in the second aspect of the invention, the recombinant protein described in the third aspect of the invention, the antibody-drug conjugate described in the fourth aspect of the invention, or an immune cell expressing the CAR described in the fifth aspect of the invention.

[0079] In another preferred embodiment, the disease is a tumor that is positive for CD276 expression.

[0080] In another preferred embodiment, the subject includes mammals, such as humans.

[0081] In another preferred embodiment, the tumor is selected from the group consisting of leukemia, gastric cancer, breast cancer, non-small cell lung cancer, ovarian cancer, osteosarcoma, neuroblastoma, pancreatic cancer, breast cancer, or a combination thereof.

[0082] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] FIG1 shows the agarose gel electrophoresis results of the VHH fragment PCR products.

[0084] Figure 2 shows the diversity analysis results of the phage display library.

[0085] Figure 3 shows the results of phage ELISA detection of single clones of the enriched product.

[0086] FIG4 shows the flow cytometry results of the binding of candidate antibodies to target proteins.

[0087] FIG5 shows the results of endocytic activity assay of candidate antibodies.

[0088] FIG6 shows the killing activity test results of the candidate antibody ADC.

[0089] FIG7 shows the antibody affinity test results. DETAILED DESCRIPTION

[0090] After extensive and in-depth research, the present inventors unexpectedly obtained, through extensive screening, for the first time, anti-CD276 nanoantibodies with high affinity and high specificity. Specifically, the present invention used CD276 recombinant protein to immunize camels, thereby obtaining a high-quality immune library. Phage display technology was used to screen the immune library, resulting in a class of specific nanoantibodies against CD276. Based on the nanoantibodies, the present invention also provides antibody-drug conjugates with significant anti-CD276 effects. The anti-CD276 nanoantibodies of the present invention have high affinity, simple structure, and are easy to prepare, with broad application prospects in fields such as tumor treatment and immunoassays. This is the basis for the completion of the present invention.

[0091] the term

[0092] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0093] The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0094] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0095] Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

[0096] Antibody

[0097] As used herein, the term "antibody" refers to immunoglobulins, which are tetrapeptide chains composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.

[0098] As used herein, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning and refer to the cloning of the variable region of an antibody heavy chain to construct a single-domain antibody (VHH) consisting of only a single heavy chain variable region. This is the smallest antigen-binding fragment with complete function. Typically, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only a single heavy chain variable region.

[0099] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD276). It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment of an antibody" include:

[0100] (i) Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains;

[0101] (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region;

[0102] (iii) an Fd fragment consisting of the VH and CH1 domains;

[0103] (iv) An Fv fragment consisting of the VH and VL domains of a single arm of an antibody.

[0104] Fv antibodies contain the variable regions of the antibody heavy chain and light chain, but no constant region, and are the smallest antibody fragments with all antigen-binding sites. Generally, Fv antibodies also include a polypeptide linker between the VH and VL domains and are able to form the structure required for antigen binding.

[0105] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication, No. 91-3242).

[0106] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on a CD276 molecule). An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation.

[0107] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. -7 M, for example, less than approximately 10 -8 M, 10 -9 M or 10 -10 Binds with an affinity (KD) of M or less.

[0108] 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.

[0109] 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.

[0110] In the present invention, the antibodies of the present invention also include conservative variants thereof, which refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties, compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A below.

[0111] Table A

[0112] Anti-CD276 nanobody

[0113] As used herein, the term "CD276" generally refers to natural or recombinant human CD276, as well as non-human homologs of human CD276.

[0114] As used herein, the terms "Nanoantibodies of the present invention", "single domain antibodies of the present invention", and "anti-CD276 Nanoantibodies of the present invention" are used interchangeably and all refer to the highly specific and high affinity Nanoantibodies of the present invention against CD276.

[0115] Compared with traditional monoclonal antibodies, the nanoantibodies of the present invention have small molecular weight, strong penetration, high sensitivity, strong specificity and good stability; once they enter the body, they can efficiently penetrate cells to quickly capture antigens to achieve therapeutic purposes.

[0116] In the present invention, the anti-CD276 nanobody includes monomers, bivalents (divalent antibodies), tetravalents (tetravalent antibodies), and / or multivalents (multivalent antibodies).

[0117] In a preferred embodiment of the present invention, the amino acid sequence of the VHH chain of the anti-CD276 nanobody is selected from one or more of SEQ ID NOs: 1 to 5. The preferred nanobody names, VHH sequence numbers and corresponding CDR region sequence numbers of the present invention are shown in Table 1:

[0118] Table 1

[0119] Note: Each numerical value in the table represents a sequence number, i.e., "1" represents "SEQ ID NO: 1".

[0120] Antibody-drug conjugates (ADCs)

[0121] The present invention also provides an antibody-drug conjugate (ADC) based on the Nanobody of the present invention.

[0122] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0123] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent may include 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 may include any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (where the coupling site is an acylhydrazone).

[0124] 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 reagents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0125] Antibodies can be coupled to drugs to form antibody-drug conjugates (ADCs). Typically, ADCs contain a linker positioned between the drug and the antibody. The linker can be a degradable or non-degradable linker. Degradable linkers typically readily degrade in the intracellular environment, for example, at the target site, where the linker degrades, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including linkers containing peptidyl groups that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidases. 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 (e.g., linkers that hydrolyze at a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug when the antibody is hydrolyzed by proteases.

[0126] 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 via the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethylketones (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, where the counter ion is acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. Linkers may include, for example, maleimides attached to the antibody via thiosuccinimide.

[0127] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, a linker connects the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group capable of forming a bond with the linker. In cases where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0128] 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, and the like. In the present invention, drug-linkers can be used to form ADCs in a single step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step process. For example, a cysteine ​​residue reacts with a reactive portion of a linker in a first step, and in a subsequent step, the functional group on the linker reacts with the drug to form an ADC.

[0129] Typically, the functional group on the linker is selected to facilitate specific reaction with an appropriate reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. 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 linking 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 between the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0130] The present invention also provides a method for preparing an ADC, which may further comprise: combining an antibody and a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0131] In certain embodiments, the methods of the present invention comprise conjugating an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the present invention further comprise conjugating the antibody-linker conjugate to a drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody via the linker.

[0132] In some embodiments, the antibody drug conjugate ADC has the following molecular formula:

[0133] in:

[0134] Ab is the Nanobody of the present invention,

[0135] LU is the connector;

[0136] D is for medicine;

[0137] And the subscript p is a value selected from 1 to 8.

[0138] Pharmaceutical composition

[0139] 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 properties 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.

[0140] The antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used in chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

[0141] The pharmaceutical composition of the present invention can be used to directly 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.

[0142] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) 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, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. 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 μg / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0143] 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 generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health condition, which are all within the skill of a skilled physician.

[0144] The main advantages of the present invention include:

[0145] (1) The anti-CD276 nanobody of the present invention has high specificity and affinity.

[0146] (2) The anti-CD276 nanoantibody of the present invention has a small molecular weight, strong penetrating power and high sensitivity.

[0147] (3) The anti-CD276 nanobody of the present invention can be used to prepare antibody-drug conjugates with significant killing effects.

[0148] (3) The anti-CD276 nanobody of the present invention has a simple structure and is easy to prepare, and has application prospects in the fields of tumor treatment and immune detection.

[0149] 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 invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally 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 as recommended by the manufacturer. Unless otherwise indicated, percentages and fractions are by weight.

[0150] Example 1: Screening of single domain antibodies against CD276

[0151] 1.1 Alpaca Immunity

[0152] Alpaca were immunized with CD276 recombinant protein (Q5ZPR3, Leu29-Ala466) at an interval of 21 days. Ten days after the last immunization, 5 mL of peripheral blood was collected and the serum was separated and tested for the immune effect using ELISA.

[0153] 1.2 Detection of immune titer

[0154] Collect 5 mL of peripheral blood and 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; place the centrifuge tube containing the blood sample in a centrifuge and centrifuge at 5000 rpm for 20 minutes; separate the upper serum and transfer the serum to a new sterile centrifuge tube to collect the immune serum. Dilute the target recombinant protein with sterile CBS (carbonate buffer) to a final concentration of 1 μg / mL. Take a new 96-well ELISA plate and add 100 μL / well to coat at 4°C overnight. Remove the antigen coating solution and wash 5 times with PBST (containing 0.05% Tween 20). Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours; after removing the blocking buffer, wash the plate 5 times with PBST; add 100 μL of serially diluted serum (100 μL / well) and incubate at room temperature for 1 hour. The control well is PBS; remove the liquid in the well and wash 5 times with PBST; add 100 μL of HRP anti-Lama IgG (H+L) antibody (1:50000 dilution) and incubate at room temperature for 1 hour; after removing the liquid in the well, wash the plate 5 times with PBST; add 100 μL / well TMB colorimetric solution; incubate at room temperature in the dark for 10-15 minutes; add 50 μL / well stop solution; read the OD in the well using a microplate reader. 450 value.

[0155] The results are shown in Table 2. According to the immune titer ELISA test results, the CD276 immune titer was high, so pulse immunization was performed, and 100 mL of peripheral blood was collected 10 days later for the construction of the phage display library.

[0156] Table 2 Immune titer test results

[0157] 1.3 PBMC isolation and VHH antibody fragment cloning

[0158] 1. Collect 100 mL of peripheral blood and separate PBMCs using lymphocyte separation medium.

[0159] 2. Extract RNA using PrimeScript TM Perform reverse transcription using the II 1st Strand cDNA Synthesis Kit to prepare cDNA. Prepare the reaction mixture Mix 1 shown in Table 3 in 200 μL PCR:

[0160] Table 3 Reaction mixture Mix1 configuration system

[0161] After incubation at 65°C for 5 minutes, cool rapidly on ice. Prepare the reaction solution shown in Table 4 in the above PCR tube:

[0162] Table 4 PCR reaction solution

[0163] After mixing by pipetting, the mixture was divided into 80 μL / tubes, placed in a PCR instrument at 42°C for 1 hour, and heat-inactivated at 70°C for 15 minutes. Finally, the cDNA sample was placed on ice or stored at -20°C for a long time.

[0164] 3. Amplification of VHH fragments:

[0165] (1) Prepare the first round of PCR reaction system as shown in Table 5 (50 μL / tube):

[0166] Table 5 First round PCR reaction system

[0167] After configuring the PCR reaction system, set up the PCR instrument according to the program shown in Table 6:

[0168] Table 6 PCR program settings

[0169] (2) Agarose gel electrophoresis analysis of PCR products: PCR products were analyzed by electrophoresis using 1% agarose gel to separate fragments with a molecular weight of approximately 750 bp. PCR products were recovered using a gel recovery kit and their concentrations were determined using NanoDrop.

[0170] (3) Prepare the second round of PCR reaction system as shown in Table 7 (50 μL / tube):

[0171] Table 7 Second round PCR reaction system

[0172] After configuring the PCR reaction system, set up the PCR instrument according to the program shown in Table 8:

[0173] Table 8 Second round PCR program settings

[0174] (4) Agarose gel electrophoresis analysis of the second-round PCR products: PCR products were analyzed by electrophoresis using 1% agarose gel to isolate VHH fragments with a molecular weight of approximately 400 bp. The VHH PCR products were recovered using a gel recovery kit and the concentration was determined using NanoDrop.

[0175] Agarose gel electrophoresis of PCR products is shown in Figure 1. In the first round of PCR, PCR bands of approximately 1000 bp and 750 bp were obtained, respectively. The 750 bp fragment was recovered and used as a template for the second round of PCR. A band of approximately 400 bp was obtained in the second round of PCR, representing the VHH fragment. The pDisplay vector and the VHH fragment obtained above were digested with SfiI, respectively, and recovered from the gel. The digested pDisplay vector and VHH fragment were ligated using T4 ligase, incubated overnight at 16°C, and then transformed into E. coli.

[0176] 1.4 Construction of phage display library

[0177] 1. Construction of single domain antibody phage display vector

[0178] The pDisplay vector and the VHH PCR products obtained above were digested with SfiI, respectively. The digestion systems are shown in Tables 9 and 10:

[0179] Table 9 Enzyme Digestion System

[0180] The enzyme digestion system shown in Table 9 was divided into 100 μL / tube and placed in a PCR instrument at 50°C for overnight digestion;

[0181] Table 10 Enzyme digestion system

[0182] Aliquot 100 μL of the digestion system listed in Table 10 into 100 μL tubes and digest overnight at 50°C in a PCR instrument. Separate the pDisplay vector fragments using a 1% agarose gel and excise a 5000 bp fragment. Purify the PCR digestion product using a DNA fragment recovery kit and determine its concentration using a NanoDrop. Ligate the digested pDisplay vector and VHH fragment using T4 ligase at 16°C overnight.

[0183] 2. Electrotransformation of phage ligation products into E. coli

[0184] Prepare the electroporation cuvettes, ligation product, and competent medium for electroporation and pre-chill them on ice. Add the pre-chilled library construction ligation product to the competent medium for electroporation and incubate on ice for 1 minute. Add 300 μL of the DNA / competent medium mixture to each cuvette and place the cuvette on ice. Electroporate at 2500V for 5 ms. Immediately resuspend the cells in S℃ medium equilibrated to room temperature and incubate at 37℃ with shaking for 1 hour. Use 15 mL of the bacterial solution directly for phage rescue. Add an equal volume of 50% glycerol to the remaining 5 mL of electroporation product, mix thoroughly, and store at -80℃. Separately, dilute 20 μL of the bacterial solution with 980 μL of 2YT medium. Take 100 μL of this dilution and dilute it again with 900 μL of 2YT medium. Spread 50 μL evenly on an LB plate containing ampicillin and incubate overnight at 37℃. The next day, remove the plates and count the number of colonies generated from each ligation to calculate the library capacity. At the same time, pick 20 single clones from the plate and transfer them to 2YT medium containing ampicillin. Incubate at 37°C with shaking for approximately 6-8 hours. Then send the culture solution for sequencing (sequencing universal primer M13R). Randomly select 20 single clones for sequencing to analyze the diversity of the constructed phage display library.

[0185] The results are shown in Figure 2. According to the sequencing results, the empty rate and antibody duplication rate of the phage display library are no more than 5%, and the library capacity of the E. coli library is: 1×10 9 .

[0186] 1.5 Phage library preparation and phage precipitation

[0187] The electroporated product was diluted with 2YT to adjust the OD 600 The final concentration of ampicillin was 100 μg / mL, and the cells were cultured in a constant temperature shaker at 37°C and 225 rpm until the OD 600 Stop when the OD value is 0.5; add M13KO7, shake well, let it stand at 37℃ for 30 minutes, and then incubate at 37℃, 225rpm for 1 hour. M13KO7 volume = 10 × volume × OD 600 ×5×10 8 / M13KO7 titer. Centrifuge the bacterial solution at 6000 rpm for 10 minutes, resuspend in 2YT-AK medium, and culture overnight at 25°C and 200 rpm. Centrifuge the bacterial solution at 10000 rpm for 15 minutes. Discard the precipitate and transfer the supernatant to a new centrifuge tube. Add PEG / NaCl (1 / 5 the volume of the bacterial solution) to the tube, mix thoroughly, and incubate at 4°C for 2 hours. The supernatant of the precipitated phage was centrifuged at 10,000 rpm and 4°C for 30 min. The supernatant was discarded and the precipitate (phage) in each 50 mL centrifuge tube was resuspended with 1 mL of sterile PBS. The resuspended phage was transferred to a 1.5 mL EP tube and placed in a centrifuge at 12,000 g and 4°C for 5 min. The supernatant was transferred to a new 1.5 mL EP tube, 250 μL of PEG / NaCl was added to each tube, mixed and allowed to stand at 4°C for 10 min, centrifuged at 12,000 g for 10 min, the supernatant was discarded, 1 mL of PBS was added to resuspend, centrifuged at 12,000 g for 5 min, the precipitate was discarded, the supernatant was transferred to a new 1.5 mL EP tube, centrifuged at 12,000 g for 5 min, and the supernatant was transferred to a new 1.5 mL EP tube to obtain the original phage library. 10 μL of the precipitate was added to 90 μL of 2YT medium and recorded as 10-1. It was then diluted 10 times to 10 -9 , take 10 -7 , 10 -8 , 10 -9 Three gradient 20 μL diluted samples were added to 200 μL of pre-prepared OD 600 ER2738 at 0.5, mix thoroughly, place in a 37°C water bath, and let stand for 10 minutes. Apply 100 μL of the solution to one LB-AMP plate. Incubate at 37°C overnight. The next day, count the plaques to determine the titer. Titer calculation: Select plates with 30-300 plaques, average the two plates, and multiply the number of plaques by the dilution factor, then multiply by 100 to obtain the titer.

[0188] 1.6 Panning of phage display libraries

[0189] Recombinant protein is used for panning, and then the phage display library is incubated with the recombinant protein. The recombinant phage bound to the target antigen is eluted using TEA and amplified. After 3-4 rounds of panning, single clones are selected for sequencing.

[0190] Take about 6x10 11150 μL of Pfu Phage library was diluted to about 1 mL with 1% PBSA and added to the ELISA plate coated with the target protein. The plate was incubated at 4°C for 1 hour. The bound phage was eluted with 600 μL 1xTEA for 10 minutes. The eluted product was transferred to a pre-sealed EP tube and neutralized by adding 300 μL Tris-HCl. 10 μL of the output product was added to 90 μL 2YT medium, recorded as 100, and then diluted 10-fold to 10 -2 , take 101, 100, 10 -1 , 10 -2 Four gradient 20 μL diluted samples were added to 200 μL of pre-prepared OD 600 Add 0.5% ER2738 (101 is to directly add 20 μL of undiluted product to ER2738), mix thoroughly, place in a 37°C water bath, and let it stand for 10 minutes. Apply 100 μL of solution to one LB-AMP solid plate and incubate at 37°C overnight. The next day, count the spots to determine the titration. Titration calculation: Select plates with 30-300 spots, average the two plates, and multiply the number of spots by the dilution factor, then by the elution volume.

[0191] The target antigen recombinant protein was co-incubated with the single-domain antibody phage display library to enrich phage that specifically bind to the target antigen. Four enrichment rounds were performed. The input and output data for each round were counted, and the enrichment factor for each round was calculated. The results are shown in Table 11.

[0192] Table 11 Enrichment results for each round

[0193] 1.7 Phage ELISA

[0194] Select the third round of enrichment product monoclonal clones for phage ELISA detection. The experimental method is as follows:

[0195] Aliquot 2YT-Amp medium into 96-well deep-well plates, 500 μL per well, pick single clones from the third round output plate, and culture at 37°C, 225 rpm until OD 600 Until 0.5; among them, E12 and F12 wells were not selected for clones, and only culture medium was placed as blank control; G12 and H12 wells were not selected for clones, and the binding of antigen and positive antibody was detected as positive control; at the same time, antigen was coated on ELISA plate with CBS, concentration of 1μg / mL, 100μL / well, 37℃, and coated for 2h; another 96-well deep-well plate was taken, 2YT-A culture medium was dispensed, 500μL per well, and OD was aspirated in sequence with a dispenser. 60010 μL of the bacterial solution with an OD600 of 0.5 was added to a newly aliquoted 96-well plate and incubated at 37°C, 225 rpm overnight. This was the bacterial solution for sample sequencing. M13KO7 was added to the bacterial solution with an OD600 of 0.5, mixed well, and incubated at 37°C for 15 minutes. The volume of M13KO7 = 10 × volume × OD 600 ×5×10 8 / M13KO7 titer: Place the infected bacterial solution on a shaker and culture at 37°C, 225 rpm for 45 min; place the bacterial solution in a centrifuge and centrifuge at 4000 rpm for 10 min, discard the supernatant, resuspend in 2YT-AK medium, 800 μL per well, return to the shaker, and culture overnight at 30°C, 210 rpm. At the same time, the ELISA plate was shaken off the antigen, washed three times with PBST solution, and then blocked with 3% MPBS at 250μL / well and incubated at 4°C overnight; and an additional blank plate was blocked as BLANK; the next day, the 96-well deep-well plate was placed in a centrifuge and centrifuged at 4000rpm for 10 minutes; the milk in the ELISA plate was discarded and washed 4 times with 200μL PBST; 50μL PBST was added to each well first, and then 50μL of the phage supernatant after centrifugation was added one by one, and incubated at 4°C for 1h; the supernatant was discarded and washed 5 times with PBST; HRP-Anti M13 secondary antibody was diluted with PBST, 100μL per well, incubated at 4°C for 45min, the secondary antibody was washed away, washed 5 times with PBST, TMB color was developed at room temperature for 10min, hydrochloric acid was terminated, and the reading was performed. Clones with an S / N ratio greater than 6 were selected and sent for testing with the preserved bacterial solution; the sequencing results were analyzed, and different antibody sequences were selected to construct eukaryotic expression vectors.

[0196] The results are shown in Figure 3. Plate 1 shows the OD450 value of monoclonal detection in a 96-well plate coated with the target antigen protein; Plate 2 shows the OD450 value of monoclonal detection in a 96-well plate coated with the BSA control antigen protein; the S / N value in Plate 3 is the value shown in Plate 1 divided by the value in the corresponding well of Plate 2. Clones with an S / N value > 4 were selected for testing.

[0197] The sequence information of the candidate antibodies is shown below (the underlined part is the CDR region).

[0198] Table 12 Antibody CDR sequences

[0199] Note: Each numerical value in the table represents a sequence number, i.e., "1" represents "SEQ ID NO: 1".

[0200] Example 2: Evaluation and identification of single domain antibodies against CD276

[0201] 2.1 Preparation of CD276 candidate nanoantibodies

[0202] Remove the LVTransm transfection reagent and antibody expression vector from the refrigerator. Thaw at room temperature and mix thoroughly by pipetting up and down. Remove the PBS buffer and warm to room temperature. Add 2 mL of PBS to each well of a 6-well plate and add 50 μg of the antibody eukaryotic expression vector. Mix thoroughly by pipetting up and down. Then add 150 μL of LVTransm and immediately mix by pipetting up and down. Let stand at room temperature for 10 minutes. Add the DNA / LVTransm complex to 50 mL of 293F cells and gently shake to mix thoroughly. Continue incubating the cells at 37°C, 5% CO2, and 130 RPM. After 5-7 days of continuous culture, collect the supernatant by centrifugation, filter through a 0.45 μm filter, transfer the filtrate to a sterile centrifuge tube, and purify the antibody using a Protein A affinity column.

[0203] 2.2 Flow cytometry detection of the binding of candidate antibodies to target proteins

[0204] CHO and CHO-CD276 cell lines were revived from liquid nitrogen and adjusted to the logarithmic growth phase using complete culture medium. The cells were divided into several aliquots, with the number of cells in each aliquot being 3×10 5 cells. Incubate the target cells with the expressed antibodies separately, mix thoroughly, and incubate at room temperature for 1 hour. Centrifuge at 800xg for 5 minutes at room temperature, remove the supernatant containing the antibody, and wash the cells three times with PBS. Add Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:1000 dilution), mix thoroughly, and incubate at room temperature in the dark for 30 minutes. Centrifuge at 800xg for 5 minutes at room temperature, remove the supernatant containing the secondary antibody, and wash the cells three times with PBS. Resuspend the cells in 500μL PBS for flow cytometry analysis.

[0205] The results are shown in Figure 4. According to the flow cytometry results, the five candidate antibodies 1-B1, 8-E5, 1-E3, 1-A2, and 4-B11 can effectively bind to CD276; among them, 1-B1 and 1-A2 are cross-binding antibodies to human and hamster CD276.

[0206] 2.3 Candidate Antibody Internalization Experiment

[0207] SKOV3 cells in the logarithmic growth phase were taken and 5×10 5The cells were divided into groups; 10ug / ml purified antibody was added, 200ul / well, and incubated at 4℃ for 30min; centrifuged at 4℃, 800xg for 5min, the supernatant containing the antibody was removed, and the cells were washed three times with pre-cold PBS; the above cells were divided into two equal parts, incubated at 4℃ and 37℃ for 240min respectively. After the incubation, ice-bathed PBS was immediately added to terminate the endocytosis experiment, centrifuged at 4℃, 800xg for 5min, and resuspended with 100ul pre-cold PBS; Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:1000 dilution) was immediately added, and incubated at 4℃ for 30min; centrifuged at 4℃, 800xg for 5min, the supernatant containing the antibody was removed, and the cells were washed three times with pre-cold PBS; the cells were resuspended with 200uL pre-cold PBS and immediately subjected to flow cytometry analysis.

[0208] The results are shown in Figure 5. According to the results of flow cytometry endocytosis assay, all five candidate antibodies had endocytosis activity in SKOV3 cells, among which 8-E5, 1-E3 and 1-B1 antibodies had high endocytosis activity.

[0209] 2.4 Candidate Antibody ADC Killing Experiment

[0210] Absorb 3.0×10 5 Pipette logarithmically growing target SKOV3 cells into a 15mL centrifuge tube, mix thoroughly, and centrifuge at 500g × 5 minutes (based on the cell count for one 96-well plate). Remove the supernatant, resuspend the cells in 10mL of complete DMEM medium, mix thoroughly, and pipette 100μL of the cell suspension into a 96-well plate. Add 200μL of sterile water to unused wells. After inoculation, transfer the plate to a 37°C, 5% CO2 incubator and incubate for 24 hours. After overnight incubation, remove the 96-well plate and add 100μL of serially diluted ADC antibodies to the corresponding wells, with final concentrations of 10, 2, 0.4, 0.08, 0.016, 0.0032, 0.00064, 0.000128, 0.0000256, and 0μg / mL. Transfer the co-cultured 96-well plate to a cell culture incubator at 37°C, 5% CO2 and continue incubation for 96 hours. After the incubation period, the 96-well plate was removed and 20 μL of CCK-8 detection reagent was added. Avoid bubbles when adding the sample and protect from light. The plate was placed at room temperature for 1, 2, 3, and 4 h. The absorbance at OD450 nm was read using a Tecan M1000 multifunctional microplate reader.

[0211] The results are shown in Figure 6 and Table 13. All the conjugates of the candidate antibodies and drugs had killing activity, among which 4-B11 and 8-E5 had particularly significant killing activity.

[0212] Table 13 IC50 values ​​of candidate antibody ADCs

[0213] 2.5 Antibody affinity detection

[0214] Affinity testing was performed for 8-E5, 1-E3, and 1-B1. The candidate antibodies were immobilized using an AHC sensor for 40 seconds. The buffer was PBST (PBS + 0.02% tween 20), and the CD276-His protein was diluted to 200, 100, 50, 25, 12.5, and 0 nM. Affinity testing was performed with a 60-second equilibrium, 180-second association, and 180-second dissociation cycle at 25°C.

[0215] The results are shown in Figure 7 and Table 14. The antibodies 8-E5, 1-E3 and 1-B1 of the present invention all have high affinity.

[0216] Table 14 Antibody affinity test results

[0217] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An anti-CD276 nanobody, characterized in that, The anti-CD276 nanobody has one or more complementarity-determining regions CDRs selected from the group consisting of: (1) CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:7, and CDR3 shown in SEQ ID NO:8; (2) CDR1 shown in SEQ ID NO:9, CDR2 shown in SEQ ID NO:10, and CDR3 shown in SEQ ID NO:11; (3) CDR1 shown in SEQ ID NO:12, CDR2 shown in SEQ ID NO:13, and CDR3 shown in SEQ ID NO:14; (4) CDR1 shown in SEQ ID NO:15, CDR2 shown in SEQ ID NO:16, and CDR3 shown in SEQ ID NO:17; (5) CDR1 shown in SEQ ID NO:18, CDR2 shown in SEQ ID NO:19, and CDR3 shown in SEQ ID NO:

20.

2. The nanobody against CD276 according to claim 1, wherein The CDR1, CDR2, and CDR3 are separated by the framework regions FR1, FR2, FR3, and FR4 of the VHH chain.

3. The nanobody against CD276 according to claim 1, wherein The amino acid sequence of the VHH chain of the anti-CD276 nanobody is as shown in any one of SEQ ID NOs: 1-5.

4. The nanobody against CD276 according to claim 1, wherein The anti-CD276 nanobody described above includes humanized antibodies, animal-derived antibodies, and chimeric antibodies.

5. A multivalent anti-CD276 antibody or an antibody directed against multiple epitopes, characterized in that, The multivalent antibody or the antibody against multiple epitopes includes at least one antibody element against the CD276 epitope, and the antibody element is the anti-CD276 nanobody as claimed in claim 1.

6. A recombinant protein, characterized in that, The recombinant protein has: (i) The anti-CD276 nanobody as claimed in claim 1, or the multivalent antibody against CD276 or the antibody against multiple epitopes as claimed in claim 5; and (ii) Optionally, a tag sequence for assisting expression and / or purification.

7. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate contains: (a) The nanobody as claimed in claim 1, the multivalent antibody as claimed in claim 5, or the antibody against multiple epitopes, the recombinant protein as claimed in claim 6; and (b) A conjugate part conjugated to the antibody part, and the conjugate part is selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

8. A chimeric antigen receptor (CAR), characterized in that, The CAR contains an extracellular domain, and the extracellular domain contains the anti-CD276 nanobody as claimed in claim 1, or the anti-CD276 multivalent antibody as claimed in claim 5.

9. A polynucleotide, characterized in that, The polynucleotide encodes a protein selected from the group consisting of: the nanobody as claimed in claim 1, or the multivalent antibody or the antibody against multiple epitopes as claimed in claim 5, the recombinant protein as claimed in claim 6, or the CAR as claimed in claim 8.

10. An expression vector, characterized in that, The expression vector contains the polynucleotide as claimed in claim 9.

11. A host cell, characterized in that, The host cell contains the expression vector as claimed in claim 10, or the polynucleotide as claimed in claim 9 is integrated into its genome, or expresses the nanobody as claimed in claim 1, the multivalent antibody as claimed in claim 5 or the antibody against multiple epitopes, the recombinant protein as claimed in claim 6, or the CAR as claimed in claim 8.

12. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (i) the nanobody as claimed in claim 1, the multivalent antibody as claimed in claim 5 or the antibody against multiple epitopes, the recombinant protein as claimed in claim 6, the antibody-drug conjugate as claimed in claim 7, or the host cell as claimed in claim 11; and (ii) a pharmaceutically acceptable carrier.

13. Use of the nanobody as claimed in claim 1, the multivalent antibody as claimed in claim 5 or the antibody against multiple epitopes, the recombinant protein as claimed in claim 6, the antibody-drug conjugate as claimed in claim 7, or the immune cell expressing the CAR as claimed in claim 8 for the preparation of a medicament for preventing and / or treating CD276-related cancer or disease.

14. The use according to claim 13, characterized in that, The cancer or disease is selected from the group consisting of leukemia, gastric cancer, breast cancer, non-small cell lung cancer, ovarian cancer, osteosarcoma, neuroblastoma, pancreatic cancer, breast cancer, or a combination thereof.

15. A method for treating a disease, characterized in that, The method comprises administering to a subject in need of treatment: the nanobody as claimed in claim 1, the multivalent antibody as claimed in claim 5 or the antibody against multiple epitopes, the recombinant protein as claimed in claim 6, the antibody-drug conjugate as claimed in claim 7, or the immune cell expressing the CAR as claimed in claim 8.

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