CN113527487A

CN113527487A - Monoclonal antibody of anti-human B7-H3 and application thereof

Info

Publication number: CN113527487A
Application number: CN202010324347.7A
Authority: CN
Inventors: 张雷; 徐敏; 高明明
Original assignee: Fosun Kaite Biotechnology Co ltd
Current assignee: Fosun Kaite Biotechnology Co ltd
Priority date: 2020-04-22
Filing date: 2020-04-22
Publication date: 2021-10-22
Also published as: WO2021213478A1; CN117024590A; CN117024591A

Abstract

The invention discloses a monoclonal antibody resisting human B7-H3 and application thereof. Specifically, the invention discloses a novel monoclonal antibody, especially a single-chain antibody, targeting B7-H3. The invention also discloses a method for preparing the monoclonal antibody. The monoclonal antibody can be combined with B7-H3 antigen with high specificity, has high affinity and has remarkable anti-tumor activity and the like.

Description

Monoclonal antibody of anti-human B7-H3 and application thereof

Technical Field

The invention relates to biomedical or biopharmaceutical technology, in particular to a monoclonal antibody of human B7-H3 and derivatives thereof, such as single-chain antibodies, bispecific antibodies, antibody-conjugated drugs, CAR-T cells and the like, and corresponding applications thereof.

Background

B7-H3 (also known as CD276) is a type I transmembrane protein belonging to the B7 immunoglobulin superfamily. In humans, mRNA of B7-H3 is widely expressed in various normal tissues, but protein is not expressed or is under expressed in normal tissues. The B7-H3 protein is widely expressed in various cancer tissues, such as lung cancer, prostate cancer, breast cancer, colorectal cancer, kidney cancer, ovarian cancer, liver cancer and the like, and the high expression of the B7-H3 protein is associated with the disease progression and poor prognosis of various cancers. In addition, in the tumor microenvironment, B7-H3 is also overexpressed on tumor-associated vascular endothelium and fibroblasts, but not on normal vascular endothelium as well as on physiological angiogenic endothelial cells. In view of the property that B7-H3 is not expressed or is low expressed in normal tissues and is highly expressed in various cancer tissues, B7-H3 is a tumor-associated antigen with great potential.

Therefore, there is a great need in the art to develop B7-H3 binding antibodies with high affinity and high specificity.

Disclosure of Invention

The invention aims to provide a high-affinity and high-specificity B7-H3 binding antibody.

In a first aspect of the invention, there is provided an anti-B7-H3 antibody having a heavy chain variable region and a light chain variable region,

the heavy chain variable region has 3 complementarity determining regions VH-CDR, 3 VH-CDR selected from the group consisting of:

VH-CDR1 shown in SEQ ID NO.8n +2,

VH-CDR2 shown in SEQ ID NO.8n +3, and

VH-CDR3 shown in SEQ ID NO.8n + 4;

wherein each n is independently 0, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34;

and/or the light chain variable region has 3 complementarity determining regions VL-CDR, 3 VL-CDR selected from the group consisting of:

VL-CDR1 shown in SEQ ID NO.8n +6,

VL-CDR2 as shown in SEQ ID NO.8n +7, and

VL-CDR3 shown in SEQ ID NO.8n + 8;

wherein, any one of the amino acid sequences also comprises a derivative sequence which is optionally added, deleted, modified and/or substituted by at least one amino acid and can retain the binding affinity of B7-H3.

In another preferred embodiment, the heavy chain of the antibody has the amino acid sequence shown in any one of SEQ ID No.8n +1, wherein n is independently 0, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34.

In another preferred embodiment, the light chain of the antibody has the amino acid sequence shown in any one of SEQ ID No.8n +5, wherein n is independently 0, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34.

In another preferred embodiment, n is any integer of 0 to 8.

In another preferred embodiment, n is any integer from 9 to 16.

In another preferred embodiment, the amino acid sequence of any one of the above CDRs comprises a derivative CDR sequence with 1, 2 or 3 amino acids added, deleted, modified and/or substituted, and such that a derivative antibody comprising a VH and VL comprising said derivative CDR sequence retains the affinity for binding to B7-H3.

In another preferred embodiment, the ratio of the affinity of the derivatized antibody to bind to B7-H3, F1, to the affinity of the corresponding non-derivatized antibody to bind to B7-H3, F0 (F1/F0) is from 0.5 to 2, preferably from 0.7 to 1.5, and more preferably from 0.8 to 1.2.

In another preferred embodiment, the number of the amino acids to be added, deleted, modified and/or substituted is 1 to 5 (e.g., 1 to 3, preferably 1 to 2, and more preferably 1).

In another preferred embodiment, said derived sequence which is added, deleted, modified and/or substituted with at least one amino acid and which retains the binding affinity of B7-H3 is an amino acid sequence having a homology or sequence identity of at least 95%.

In another preferred embodiment, the antibody further comprises a heavy chain constant region and/or a light chain constant region.

In another preferred embodiment, said heavy chain constant region is of human origin and/or said light chain constant region is of human origin.

In another preferred embodiment, the heavy chain constant region is a human antibody heavy chain IgG1 constant region and the light chain constant region is a human antibody light chain kappa constant region.

In another preferred embodiment, the heavy chain variable region of the antibody further comprises a framework region of human origin, and/or the light chain variable region of the antibody further comprises a framework region of human origin.

In another preferred embodiment, the antibody is selected from the group consisting of: an antibody of animal origin, a chimeric antibody, a humanized antibody, a fully human antibody, or a combination thereof.

In another preferred embodiment, the antibody is a partially or fully humanized, or fully human monoclonal antibody.

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

In another preferred embodiment, the antibody is a full-length protein, or an antigen-binding fragment of an antibody.

In another preferred embodiment, the antibody is a bispecific antibody, or a multispecific antibody.

In another preferred embodiment, the antibody is in the form of a drug conjugate.

In another preferred embodiment, the antibody has one or more properties selected from the group consisting of:

(a) inhibiting tumor cell migration or metastasis;

(b) inhibiting tumor growth.

In another preferred embodiment, the antibody is a single chain antibody (scFV).

In another preferred embodiment, the antibody has a structure represented by formula I or II below:

Z1-L1-Z2-Z3 (I)

Z2-L1-Z1-Z3 (II)

in the formula,

z1 is the heavy chain variable region VH;

z2 is the light chain variable region VL;

l1 is nothing or a peptide linker;

z3 is an antibody constant region (Fc region);

each "-" represents a peptide bond.

In another preferred embodiment, L1 is a flexible peptide linker, preferably (G)₄S)₃。

In another preferred embodiment, the linking fragment (linker) of the scFv is GSTSGSGKPGSGEGS or GKPGSGKPGSGKPGSGKPGS.

In another preferred embodiment, Z3 is an Fc of IgG, preferably IgG 1.

In another preferred embodiment, Z3 is the Fc of a human antibody.

In another preferred embodiment, the antibody has the structure shown in formula III below:

VH-(G₄S)₃-VL-huIgG1Fc (III)

in the formula,

VH is the heavy chain variable region;

VL is a light chain variable region;

(G₄S)₃is a peptide linker;

huIgG1Fc is the constant region of human IgG1 antibody.

In another preferred embodiment, the sequence numbers of VH, VL and the respective CDRs of said antibody are as shown in table a:

SEQ ID NOs of VH, VL and respective CDRs of antibody clones of Table A (SEQ ID NO:)

In another preferred embodiment, the heavy chain variable region of the antibody further comprises a Framework Region (FR), and the light chain variable region of the antibody further comprises a Framework Region (FR).

In another preferred embodiment, the amino acid sequence in the framework region of the VH and/or the framework region of the VL may comprise a derivative sequence optionally having at least one amino acid added, deleted, modified and/or substituted and capable of retaining its B7-H3 binding affinity.

In another preferred embodiment, said heavy chain variable region and said light chain variable region comprise CDRs selected from the group consisting of:

In another preferred embodiment, the antibody is selected from the group consisting of:

in another preferred embodiment, the amino acid sequence of said heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with the amino acid sequence as shown in SEQ ID No.1, 9, 17, 25, 33, 41, 49, 57, 65.

In another preferred embodiment, the amino acid sequence of the light chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology or sequence identity with the amino acid sequence shown in SEQ ID No.5, 13, 21, 29, 37, 45, 53, 61, 69.

In a second aspect of the present invention, there is provided a recombinant protein comprising:

(i) an antibody according to the first aspect of the invention; and

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

In another preferred embodiment, the recombinant protein is a fusion protein.

In another preferred embodiment, the recombinant protein is a membrane protein.

In another preferred embodiment, the recombinant protein is a Chimeric Antigen Receptor (CAR).

In another preferred embodiment, the tag sequence comprises a 6His tag.

In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

In a third aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

(1) the antibody of the first aspect; and

(2) the recombinant protein of the second aspect.

In a fourth aspect of the invention, there is provided a vector comprising the polynucleotide of the third aspect.

In another preferred embodiment, the carrier comprises: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

In a fifth aspect of the invention there is provided a genetically engineered host cell comprising a vector or genome according to the fourth aspect having integrated therein a polynucleotide according to the third aspect.

In another preferred embodiment, the host cell comprises a human cell.

In another preferred embodiment, the host cell comprises an immune cell.

In a sixth aspect of the invention, there is provided an antibody conjugate comprising:

(a) an antibody portion, said antibody being as described in the first aspect, or a combination thereof; and

(b) a coupling moiety coupled to the antibody moiety, the coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

In another preferred embodiment, said antibody moiety is coupled to said coupling moiety by a chemical bond or a linker.

In a seventh aspect of the present invention, there is provided an immune cell expressing or exposed outside the cell membrane the antibody of the first aspect and a fusion protein comprising the antibody.

In another preferred embodiment, the immune cells comprise NK cells and T cells.

In another preferred embodiment, the immune cell is from a human or non-human mammal (e.g., a mouse).

In another preferred embodiment, the fusion protein is a Chimeric Antigen Receptor (CAR).

In an eighth aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) an active ingredient selected from the group consisting of: the antibody of the first aspect, the recombinant protein of the second aspect, the antibody conjugate of the sixth aspect, the immune cell of the seventh aspect, or a combination thereof; and

(ii) a pharmaceutically acceptable carrier.

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

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

In another preferred embodiment, the pharmaceutical composition comprises (i)0.01 to 99.99 wt% of the antibody according to the first aspect, the recombinant protein according to the second aspect, the antibody conjugate according to the sixth aspect, the immune cell according to the seventh aspect, or the combination thereof, and (ii)0.01 to 99.99 wt% of a pharmaceutically acceptable carrier, wherein the percentages are weight percentages of the pharmaceutical composition.

In a ninth aspect of the invention, there is provided the use of an active ingredient selected from the group consisting of: the antibody of the first aspect, the recombinant protein of the second aspect, the antibody conjugate of the sixth aspect, the immune cell of the seventh aspect, or a combination thereof, wherein the active ingredient is used (a) in the preparation of a diagnostic reagent or kit; and/or (B) preparing a medicament for preventing and/or treating diseases related to B7-H3 expression or dysfunction.

In another preferred embodiment, the diagnostic reagent is a test strip or test plate.

In another preferred embodiment, the diseases associated with B7-H3 expression or dysfunction are selected from the group consisting of: cancer, autoimmune disease.

In another preferred embodiment, the cancer is selected from the group consisting of: lung, prostate, breast, colorectal, kidney, ovary and liver cancer.

In another preferred embodiment, the diagnostic reagent or kit is used for:

(1) detecting the B7-H3 protein in the sample; and/or

(2) Detecting endogenous B7-H3 protein in tumor cells; and/or

(3) Detecting the tumor cells expressing the B7-H3 protein.

In another preferred embodiment, the medicament is used for preventing and/or treating diseases related to B7-H3 expression or dysfunction, and the diseases related to B7-H3 expression or dysfunction are selected from the group consisting of: cancer, autoimmune diseases.

In another preferred embodiment, the antibody is in the form of A Drug Conjugate (ADC).

In another preferred embodiment, the diagnostic reagent or the kit is used for diagnosing B7-H3 related diseases.

In another preferred embodiment, the diagnostic reagent or kit is used for detecting the B7-H3 protein in a sample.

In a tenth aspect of the invention, there is provided a method for in vitro detection (including diagnostic or non-diagnostic) of B7-H3 protein in a sample, said method comprising the steps of:

(1) contacting said sample in vitro with an antibody according to the first aspect of the invention;

(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex indicates the presence of B7-H3 protein in the sample.

In an eleventh aspect of the present invention, there is provided a detection panel comprising: a substrate (support plate) and a test strip comprising an antibody according to the first aspect of the invention.

In a twelfth aspect of the present invention, there is provided a kit comprising:

(1) a first container comprising an antibody according to the first aspect of the invention; and/or

(2) A second container comprising a secondary antibody directed against the antibody of the first aspect of the invention;

or,

the kit contains the detection plate.

In a thirteenth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:

(a) culturing a host cell according to the fifth aspect of the invention under conditions suitable for expression;

(b) isolating a recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the first aspect of the invention or a recombinant protein according to the second aspect of the invention.

In a fourteenth aspect of the present invention, there is provided a pharmaceutical combination comprising:

(i) a first active ingredient selected from the group consisting of: the antibody of the first aspect, the recombinant protein of the second aspect, the antibody conjugate of the sixth aspect, the immune cell of the seventh aspect, or a combination thereof;

(ii) a second active ingredient comprising a second antibody, or a chemotherapeutic agent.

In another preferred embodiment, the second antibody is selected from the group consisting of: CTLA4 antibody, PD-1 antibody, PD-L1 antibody, 4-1BB antibody.

In another preferred embodiment, the chemotherapeutic agent is selected from the group consisting of: docetaxel, carboplatin, or a combination thereof.

In a fifteenth aspect of the present invention, there is provided a method of treating a disease associated with B7-H3 expression or dysfunction, comprising the steps of: administering to a subject in need thereof an effective amount of an antibody according to the first aspect, a recombinant protein according to the second aspect, an antibody conjugate according to the sixth aspect, an immune cell according to the seventh aspect, or a combination thereof.

In another preferred embodiment, the disease associated with B7-H3 expression or dysfunction is selected from the group consisting of: cancer, autoimmune disease.

In another preferred embodiment, the cancer is selected from the group consisting of: lung cancer, prostate cancer, breast cancer, colorectal cancer, kidney cancer, ovarian cancer, liver cancer and the like.

In another preferred example, the method further comprises: administering to the subject a safe and effective amount of a second antibody before, during and/or after administration of the first active ingredient.

In another preferred embodiment, the second antibody is selected from the group consisting of: PD-1 antibody, CTLA4 antibody, PD-L1 antibody, 4-1BB antibody.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the results of screening human 4Ig-B7-H3 overexpressed embryonic kidney cell HEK 293T cell line (designated B cell).

FIG. 2 shows the results of a screen of mouse 2Ig-B7-H3 over-expressed embryonic kidney cell HEK 293T cell line (designated C cell).

FIG. 3 shows SDS-PAGE of recombinant proteins (extracellular region of B7-H3 protein), wherein lanes B7-H3ECD (29-465) -6His (R) represent reducing samples of recombinantly expressed human B7-H3 extracellular region protein (with a 6His tag); lanes B7-H3ECD (29-465) -6His (NR) represent non-reducing samples of recombinantly expressed human B7-H3 extracellular domain protein (with a 6His tag).

FIG. 4 shows the SEC-HPLC profile of the recombinant protein (extracellular region of the B7-H3 protein).

FIG. 5 shows an alignment of the amino acid sequences of CDRs 1 of a portion of the scFv single chain antibody VH.

FIG. 6 shows an alignment of the amino acid sequences of CDRs 2 of a portion of the scFv single chain antibody VH.

FIG. 7 shows an alignment of the amino acid sequences of CDRs 3 of a portion of the scFv single chain antibody VH.

FIG. 8 shows an alignment of the amino acid sequences of CDR1 of a partial scFv single chain antibody VL.

FIG. 9 shows an alignment of the CDR2 amino acid sequences of a partial scFv single chain antibody VL.

FIG. 10 shows an alignment of the amino acid sequences of CDR3 of a partial scFv single chain antibody VL.

FIG. 11 shows the affinity curves and IC50 values of the partial scFv single-chain antibody and human 4Ig-B7-H3ECD protein detected by enzyme-linked immunosorbent assay (ELISA). In fig. 11, a to I show the detection results of different antibodies, respectively.

FIG. 12 shows binding of a portion of scFv single chain antibodies to A, B, C cells as detected by flow cytometry. Wherein, the combination condition of the following single-chain antibodies is included from top to bottom.

(A)96-3,96-14,96-2,96-8,90-26,90-16,81A1,81A15 single chain antibody;

(B)81a55,81a9,81a58,81a68,81a53,81a40,81a14,81a5 single chain antibodies;

(C)81a37,81a2,81a57,81a30,81a21,81a18,81a31,81a19 single chain antibodies;

(D)81a6,81A8,81a71,81a56,81a66,81a70 single chain antibodies;

(E)81A22,81A4,81A25,81A24,81A34 single chain antibody.

Detailed Description

The present inventors have conducted extensive and intensive studies, and as a result, have surprisingly obtained a group of antibodies B7-H3 having a novel amino acid sequence by screening a large number of cell lines stably expressing B7-H3 protein and human B7-H3 protein as immunogens. The B7-H3 antibody is a B7-H3 antibody with excellent biological characteristics, has high affinity and specificity to human B7-H3 protein, and can be applied to treatment of tumors and other related diseases. The present invention has been completed based on this finding.

Term(s) for

In the present invention, "VH-CDR 1" and "CDR-H1" are used interchangeably and refer to CDR1 of the heavy chain variable region; "VH-CDR 2" and "CDR-H2" are used interchangeably and refer to CDR2 of the heavy chain variable region; "VH-CDR 3" and "CDR-H3" are used interchangeably and refer to CDR3 of the heavy chain variable region. "VL-CDR 1" and "CDR-L1" are used interchangeably and refer to CDR1 of the light chain variable region; "VL-CDR 2" and "CDR-L2" are used interchangeably and refer to CDR2 of the light chain variable region; "VL-CDR 3" and "CDR-L3" are used interchangeably and refer to CDR3 of the light chain variable region.

The term "about" can refer to a value or composition that is within an acceptable error range for the 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 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which may 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.

B7-H3

B7-H3 (also known as CD276) is a type I transmembrane protein belonging to the B7 immunoglobulin superfamily, and B7-H3 shares 20-27% homology with other family members. In mice, the B7-H3 gene is located on mouse chromosome 9, and the extracellular structure of the protein consists of one IgV and one IgC (VC, 2 Ig-B7-H3); in humans, the B7-H3 gene is located on human chromosome 15, and human B7-H3 protein exists in two forms, one is composed of one IgV and IgC (VC, 2Ig-B7-H3), the other is in a form in which two pairs of VCCs are connected in series (VCVC, 4Ig-B7-H3), and the latter is the most widely expressed form in humans.

In humans, mRNA of B7-H3 is widely expressed in various normal tissues, but the expression of protein is strictly limited and is not expressed or is underexpressed in normal tissues. The B7-H3 protein is widely expressed in various cancer tissues, such as lung cancer, prostate cancer, breast cancer, colorectal cancer, kidney cancer, ovarian cancer, liver cancer and the like, and the high expression of the B7-H3 protein is associated with the disease progression and poor prognosis of various cancers. In addition, in the tumor microenvironment, B7-H3 is also overexpressed on tumor-associated vascular endothelium and fibroblasts, but not on normal vascular endothelium as well as on physiological angiogenic endothelial cells. In addition, it was found that B7-H3 exerts a co-inhibitory effect in immune regulation, which can inhibit T cell division by regulating NFAT, nfkb and AP-1 signaling pathways, and that in B7-H3 deficient mice Th cells differentiate more into Th1 cells than Th2 cells, resulting in more severe airway inflammation.

Antibodies

As used herein, the term "antibody" or "immunoglobulin" is an heterotetrameric glycan protein of about 150000 daltons with the same structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable region (VH) followed by a plurality of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Particular amino acid residues form the interface between the variable regions of the light and heavy chains.

Single chain antibodies (scFv) are antibodies composed of the variable regions of the heavy and light chains of an antibody_HAnd V_L) Composed, and having a size of about 30 KDa. Typically at V_HAnd V_LThe two are connected by a 15-20 amino acid variable short peptide linker (linker), the linker short peptide usually consists of a hydrophobic sequence, and the most common design is that consisting of glycine (G) and serine (S) (G)₄S)₃A linker. Compared with full-length antibodies, the single-chain antibody has smaller molecular weight and can be better and more quickly immersed into tumor tissues or other tissues while retaining the binding specificity and affinity, and meanwhile, the single-chain antibody has shorter half-life in vivo and can be more quickly discharged out of the body from blood through kidneys, so that the exposure time of the drug or radionuclide-linked single-chain antibody in normal tissues can be reduced. Moreover, single chain antibodies can be used to construct the affinity elements of CAR-T cells, have been widely used, and demonstrate their efficacy in clinical applications.

As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding ability and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, which are in a substantially β -sheet configuration, connected by three CDRs that form a connecting loop, and in some cases may form part of a β -sheet structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of the antibody with the CDRs of the other chain (see Kabat et al, NIHPubl. NoNO.91-3242, Vol I, 647-669 (1991)). The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.

As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokines), radionuclides, enzymes, and other diagnostic or therapeutic molecules are conjugated to the antibodies or fragments thereof of the present invention to form conjugates. The invention also comprises a cell surface marker or antigen combined with the anti-B7-H3 protein antibody or the fragment thereof.

As used herein, the terms "heavy chain variable region" and "V_H"may be used interchangeably.

As used herein, the term "variable region" is used interchangeably with "Complementary Determining Region (CDR)".

In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR 3.

In a preferred embodiment of the invention, the heavy chain of the antibody comprises the above-described heavy chain variable region and heavy chain constant region.

In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" are used interchangeably and refer to a polypeptide that specifically binds to B7-H3 protein, e.g., a protein or polypeptide having a heavy chain variable region. They may or may not contain the initial methionine.

The invention also provides other proteins or fusion expression products having an antibody of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain comprising a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of an antibody of the invention.

The antigen binding properties of an antibody can be described by 3 specific regions, called variable regions (CDRs), located in the heavy chain variable region, which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, and the β -sheets formed by the FRs between them are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of antibodies of the same type.

The variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding to antigen. Thus, the invention includes those molecules having an antibody heavy chain variable region with CDRs whose homology to the CDRs identified herein is greater than 90% (preferably greater than 95%, most preferably greater than 98%).

The invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

The antibody of the present invention refers to a polypeptide having the binding activity of B7-H3 protein, including the CDR regions described above. The term also includes variants of the polypeptides comprising the CDR regions described above that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more amino acids, and addition of one or several amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera to an antibody of the invention.

In the present invention, the antibody may be monospecific, bispecific, trispecific, or multispecific.

The invention also provides other polypeptides, such as fusion proteins comprising single domain antibodies or fragments thereof. In addition to almost full-length polypeptides, the invention also encompasses fragments of the single domain antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids of the antibody of the invention, preferably at least about 70 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

In the present invention, "conservative variant of the antibody of the present invention" means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are substituted by amino acids having similar or similar properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

TABLE 1

In the present invention, the number of amino acids to be added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total number of amino acids in the original amino acid sequence.

In the present invention, more preferably, the number of the amino acids to be added, deleted, modified and/or substituted may be 1 to 7, more preferably 1 to 5, still more preferably 1 to 3, still more preferably 1 to 2.

Nucleic acids

The invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. Also, the polynucleotides that hybridize to the mature polypeptide encode polypeptides having the same biological functions and activities as the mature polypeptide.

Preparation method

The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique, such as PCR amplification, recombination or artificial synthesis. Alternatively, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.

Alternatively, the coding sequence for the antibody and an expression tag (e.g., 6His) can be fused together to form a fusion protein.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a genetically engineered vector, transferring it into a cell, and isolating the relevant sequence from the expanded host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules in an isolated form.

At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

Vectors and host cells

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

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

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, such as E.coli, competent cells capable of DNA uptake can be harvested after the exponential growth phase and treated by the CaCl2 method using procedures well known in the art. Another method is to use MgCl₂. If desired, transformation may also be by electroporationThe method is carried out. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis by Munson et al, anal. biochem.,107:220 (1980).

Antibody-drug conjugates (ADC)

The invention also provides an antibody-conjugated drug (ADC) based on the antibody of the invention.

Typically, the antibody-conjugated drug comprises the antibody, and an effector molecule to which the antibody is conjugated, and preferably chemically conjugated. Wherein 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.

The antibody of the invention may be conjugated to the effector molecule by a coupling agent. Examples of the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. The non-selective coupling agent is a compound which enables effector molecules and antibodies to form covalent bonds, such as glutaraldehyde and the like. The coupling agent using carboxyl can be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (coupling site is acylhydrazone).

Certain residues on the antibody (e.g., Cys or Lys, etc.) are used to attach to a variety of functional groups, including imaging agents (e.g., chromophores and fluorophores), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizing agents (e.g., ethylene glycol polymers) and therapeutic agents. The antibody may be conjugated to a functional agent to form an antibody-functional agent conjugate. Functional agents (e.g., drugs, detection reagents, stabilizers) are coupled (covalently linked) to the antibody. The functional agent may be attached to the antibody directly, or indirectly through a linker.

Antibodies may be conjugated to drugs to form Antibody Drug Conjugates (ADCs). Typically, the ADC comprises a linker between the drug and the antibody. The linker may be degradable or non-degradable. Degradable linkers are typically susceptible to degradation in the intracellular environment, e.g., the linker degrades at the site of interest, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl-containing linkers 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 glucuronidase. The peptidyl linker may comprise, for example, a dipeptide 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 under conditions in which the antibody is hydrolyzed by a protease.

Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and attachment is achieved by the reactive group. Thiol-specific reactive groups are preferred and include: for example maleimide compounds, haloamides (for example iodine, bromine or chlorine); halogenated esters (e.g., iodo, bromo, or chloro); halomethyl ketones (e.g., iodo, bromo, or chloro), benzyl halides (e.g., iodo, bromo, or chloro); vinyl sulfone, pyridyl disulfide; mercury derivatives such as 3, 6-bis- (mercuric methyl) dioxane, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiolsulfonate. The linker may comprise, for example, a maleimide linked to the antibody via a thiosuccinimide.

The drug may be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, the linker links the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or keto group that may form a bond with the linker. In the case of a drug directly attached to a linker, the drug has a reactive group prior to attachment to the antibody.

Useful classes of drugs 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. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs including, for example, auristatins (auristatins), camptothecins (camptothecins), duocarmycins/duocarmycins (duocarmycins), etoposides (etoposides), maytansinoids (maytansinoids) and maytansinoids (e.g., DM1 and DM4), taxanes (taxanes), benzodiazepines (benzodiazepines), or benzodiazepine-containing drugs (e.g., pyrrolo [1,4] benzodiazepines (PBDs), indoloprazoids (indobenzodiazepines) and benzodiazepines (oxyphenodiazepines) and vincristine alkaloids (vincaleoxinoids).

In the present invention, a drug-linker can be used to form an ADC in a single step. In other embodiments, bifunctional linker compounds may be used to form ADCs in a two-step or multi-step process. For example, a cysteine residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, a functional group on the linker is reacted with a drug, thereby forming an ADC.

Generally, the functional group on the linker is selected to facilitate specific reaction with a suitable reactive group on the drug moiety. As a non-limiting example, azide-based moieties may be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker by 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, second edition (Elsevier), are well known to those skilled in the art. It will be appreciated by those skilled in the art that for selective reaction of a drug moiety and a linker, each member of a complementary pair may be used for both the linker and the drug when the reactive functional group of the complementary pair is selected.

The present invention also provides a method of preparing an ADC, which may further comprise: the antibody is conjugated to a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

In certain embodiments, the methods of the invention comprise: the antibody is conjugated to the bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the method of the present invention further comprises: the antibody linker conjugate is bound to the drug moiety under conditions sufficient to covalently link the drug moiety to the antibody through the linker.

In some embodiments, the antibody drug conjugate ADC has the formula:

wherein:

ab is an antibody, and Ab is an antibody,

LU is a joint;

d is a drug;

and subscript p is a value selected from 1 to 8.

Applications of

The invention also provides the use of the antibodies, antibody conjugates ADC, recombinant proteins, and/or immune cells of the invention, for example for the preparation of a diagnostic formulation or for the preparation of a medicament.

Preferably, the medicament is a medicament for preventing and/or treating diseases related to B7-H3 expression or dysfunction.

In the present invention, the diseases associated with B7-H3 expression or dysfunction are diseases associated with B7-H3 expression or dysfunction, which are conventional in the art. Preferably, the disease associated with B7-H3 expression or dysfunction is cancer.

In the present invention, the cancer is a cancer that is conventional in the art, preferably lung cancer, prostate cancer, breast cancer, colorectal cancer, kidney cancer, ovarian cancer and liver cancer.

Uses of the antibodies, ADCs, recombinant proteins, and/or immune cells of the invention include (but are not limited to):

(i) diagnosing, preventing and/or treating tumorigenesis, growth and/or metastasis, in particular tumors with a high expression of B7-H3. Such tumors include (but are not limited to): lung, prostate, breast, colorectal, kidney, ovary and liver cancer.

Detection use and kit

The antibodies of the invention or ADCs thereof may be used in detection applications, for example for the detection of samples, to provide diagnostic information.

In the present invention, the specimen (sample) used includes cells, tissue samples and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to the person skilled in the art. Thus biopsies as used in the present invention may comprise e.g. resection samples of tumours, tissue samples prepared by endoscopic methods or needle biopsy of organs.

Samples for use in the present invention include fixed or preserved cell or tissue samples.

The invention also provides a kit containing the antibody (or fragment thereof) of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.

Pharmaceutical composition

The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above antibody or active fragment thereof or fusion protein thereof or ADC thereof or corresponding immune cell, and a pharmaceutically acceptable carrier. Generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated.

The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration. Typically, the route of administration of the pharmaceutical composition of the present invention is preferably injection administration or oral administration. The injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid or liquid form, and may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule, a granule, an injection or an infusion, etc.

The antibody of the present invention may also be used for cell therapy by intracellular expression of a nucleotide sequence, for example, for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.

The pharmaceutical composition is used for preventing and/or treating diseases related to B7-H3 expression or dysfunction.

The pharmaceutical composition can be directly used for combining B7-H3 protein molecules, so that the pharmaceutical composition can be used for preventing and treating diseases such as tumors and the like.

The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or conjugate thereof) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

In the present invention, preferably, the pharmaceutical composition of the present invention further comprises one or more pharmaceutically acceptable carriers. The medicinal carrier is a conventional medicinal carrier in the field, and can be any suitable physiologically or pharmaceutically acceptable medicinal auxiliary material. The pharmaceutical adjuvant is conventional in the field, and preferably comprises pharmaceutically acceptable excipient, filler or diluent and the like. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the protein and 0.01-99.99% of a pharmaceutical carrier, wherein the percentage is the mass percentage of the pharmaceutical composition.

In the present invention, preferably, the pharmaceutical composition is administered in an effective amount, which is an amount that alleviates or delays the progression of the disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be based in part on the consideration of the condition to be treated and the result sought. One skilled in the art can determine an effective amount by using such factors as an individual basis and using no more than routine experimentation.

In the case of pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 20 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The invention provides application of the pharmaceutical composition in preparing a medicament for preventing and/or treating diseases related to B7-H3 expression or dysfunction. Preferably, the diseases related to B7-H3 expression or dysfunction are cancer and autoimmune diseases.

Methods, compositions for detecting B7-H3 protein in a sample

The invention also provides a method for detecting B7-H3 protein (such as detecting over-expressing B7-H3 cells) in a sample, which comprises the following steps: and (3) contacting the antibody with a sample to be detected in vitro, and detecting whether the antibody is combined with the sample to be detected to form an antigen-antibody compound.

The expression "overexpression" is conventional in the art and refers to the overexpression of the B7-H3 protein in the RNA or protein of the sample to be tested (due to increased transcription, post-transcriptional processing, translation, post-translational processing and altered protein degradation), as well as to the local overexpression and increased functional activity (e.g.in the case of increased enzymatic hydrolysis of the substrate) due to altered protein transport patterns (increased nuclear localization).

In the present invention, the above-mentioned detection method for detecting whether or not the antigen-antibody complex is formed by binding is a detection method which is conventional in the art, and is preferably a flow cytometry (FACS) detection method.

The invention provides a composition for detecting B7-H3 protein in a sample, which comprises the antibody, the recombinant protein, the antibody conjugate, the immune cell or the combination thereof as an active ingredient. Preferably, it further comprises a compound consisting of a functional fragment of the above antibody as an active ingredient.

The main advantages of the invention include:

(a) the antibodies of the invention have high affinity for human B7-H3.

(b) The antibody of the invention has high specificity to human B7-H3.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Unless otherwise specified, materials and reagents used in examples of the present invention are commercially available products.

EXAMPLE 1 construction of human 4Ig-B7-H3 overexpressing embryonic Kidney cell HEK 293T cell line (designated B cell)

Recovering HEK 293T cell strain with low generation number, and culturing at 37 deg.C and 5% CO₂Saturated steam, the medium was dmem (Gibco) containing 10% fbs (Gibco), to which 1% penicillin-streptomycin (Gibco) was added. After 2-3 passages of culture, the cells are inoculated into a 6-well plate, and transfection is carried out when the confluency of monolayer cells is 70-90%. Mu.g plasmid per well (expressing B7-H3 and eGFP fluorescent protein) and the Transfection Reagent used was X-tremeGENE HP DNA Transfection Reagent (Roche). After the transfected cells are continuously cultured for 2-3 generations, single cell suspension is prepared, the cells are inoculated into a 96-well plate according to the density of 0.8 cell per well, monoclonals are selected by observing the GFP fluorescence condition of the cells, and the monoclonals are gradually expanded and cultured. Detecting GFP single peak condition by flow cytometry in the process of expanding culture, eliminating multi-peak clone, and finally introducingThe GFP unimodal clone obtained was further verified by flow-through B7-H3 to obtain the desired positive clone.

The nucleotide gene sequence for expressing the 4Ig-B7-H3 protein is as follows:

ATGCTGCGTCGGCGGGGCAGCCCTGGCATGGGTGTGCATGTGGGTGCAGCCCTGGGAGCACTGTGGTTCTGCCTCACAGGAGCCCTGGAGGTCCAGGTCCCTGAAGACCCAGTGGTGGCACTGGTGGGCACCGATGCCACCCTGTGCTGCTCCTTCTCCCCTGAGCCTGGCTTCAGCCTGGCACAGCTCAACCTCATCTGGCAGCTGACAGATACCAAACAGCTGGTGCACAGCTTTGCTGAGGGCCAGGACCAGGGCAGCGCCTATGCCAACCGCACGGCCCTCTTCCCGGACCTGCTGGCACAGGGCAACGCATCCCTGAGGCTGCAGCGCGTGCGTGTGGCGGACGAGGGCAGCTTCACCTGCTTCGTGAGCATCCGGGATTTCGGCAGCGCTGCCGTCAGCCTGCAGGTGGCCGCTCCCTACTCGAAGCCCAGCATGACCCTGGAGCCCAACAAGGACCTGCGGCCAGGGGACACGGTGACCATCACGTGCTCCAGCTACCAGGGCTACCCTGAGGCTGAGGTGTTCTGGCAGGATGGGCAGGGTGTGCCCCTGACTGGCAACGTGACCACGTCGCAGATGGCCAACGAGCAGGGCTTGTTTGATGTGCACAGCATCCTGCGGGTGGTGCTGGGTGCAAATGGCACCTACAGCTGCCTGGTGCGCAACCCCGTGCTGCAGCAGGATGCGCACAGCTCTGTCACCATCACACCCCAGAGAAGCCCCACAGGAGCCGTGGAGGTCCAGGTCCCTGAGGACCCGGTGGTGGCCCTAGTGGGCACCGATGCCACCCTGCGCTGCTCCTTCTCCCCCGAGCCTGGCTTCAGCCTGGCACAGCTCAACCTCATCTGGCAGCTGACAGACACCAAACAGCTGGTGCACAGTTTCACCGAAGGCCGGGACCAGGGCAGCGCCTATGCCAACCGCACGGCCCTCTTCCCGGACCTGCTGGCACAAGGCAATGCATCCCTGAGGCTGCAGCGCGTGCGTGTGGCGGACGAGGGCAGCTTCACCTGCTTCGTGAGCATCCGGGATTTCGGCAGCGCTGCCGTCAGCCTGCAGGTGGCCGCTCCCTACTCGAAGCCCAGCATGACCCTGGAGCCCAACAAGGACCTGCGGCCAGGGGACACGGTGACCATCACGTGCTCCAGCTACCGGGGCTACCCTGAGGCTGAGGTGTTCTGGCAGGATGGGCAGGGTGTGCCCCTGACTGGCAACGTGACCACGTCGCAGATGGCCAACGAGCAGGGCTTGTTTGATGTGCACAGCGTCCTGCGGGTGGTGCTGGGTGCGAATGGCACCTACAGCTGCCTGGTGCGCAACCCCGTGCTGCAGCAGGATGCGCACGGCTCTGTCACCATCACAGGGCAGCCTATGACATTCCCCCCAGAGGCCCTGTGGGTGACCGTGGGGCTGTCTGTCTGTCTCATTGCACTGCTGGTGGCCCTGGCTTTCGTGTGCTGGAGAAAGATCAAACAGAGCTGTGAGGAGGAGAATGCAGGAGCTGAGGACCAGGATGGGGAGGGAGAAGGCTCCAAGACAGCCCTGCAGCCTCTGAAACACTCTGACAGCAAAGAAGATGATGGACAAGAAATAGCCTGA(SEQ ID No:281)

the experimental results are as follows:

the results are shown in FIG. 1, and based on the flow analysis results, the 3E11 clone was finally selected as a B cell for post-screening.

EXAMPLE 2 construction of mouse 2Ig-B7-H3 overexpressing embryonic Kidney cell HEK 293T cell line (designated C cell)

Example 1 was repeated except that the coding sequence of human 4Ig-B7-H3 was replaced with the coding sequence of mouse 2 Ig-B7-H3.

The nucleotide gene sequence for expressing mouse 2Ig-B7-H3 protein is as follows:

ATGCTTCGAGGATGGGGTGGCCCCAGTGTGGGTGTGTGTGTGCGCACAGCACTGGGGGTGCTGTGCCTCTGCCTCACAGGAGCTGTGGAAGTCCAGGTCTCTGAAGACCCCGTGGTGGCCCTGGTGGACACGGATGCCACCCTACGCTGCTCCTTTTCCCCAGAGCCTGGCTTCAGTCTGGCACAGCTCAACCTCATCTGGCAGCTGACAGACACCAAACAGCTGGTGCACAGCTTCACGGAGGGCCGGGACCAAGGCAGTGCCTACTCCAACCGCACAGCGCTCTTCCCTGACCTGTTGGTGCAAGGCAATGCGTCCTTGAGGCTGCAGCGCGTCCGAGTAACCGACGAGGGCAGCTACACCTGCTTTGTGAGCATCCAGGACTTTGACAGCGCTGCTGTTAGCCTGCAGGTGGCCGCCCCCTACTCGAAGCCCAGCATGACCCTGGAGCCCAACAAGGACCTACGTCCAGGGAACATGGTGACCATCACGTGCTCTAGCTACCAGGGCTATCCGGAGGCCGAGGTGTTCTGGAAGGATGGACAGGGAGTGCCCTTGACTGGCAATGTGACCACATCCCAGATGGCCAACGAGCGGGGCTTGTTCGATGTTCACAGCGTGCTGAGGGTGGTGCTGGGTGCTAACGGCACCTACAGCTGCCTGGTACGCAACCCGGTGTTGCAGCAAGATGCTCACGGCTCAGTCACCATCACAGGGCAGCCCCTGACATTCCCCCCTGAGGCTCTGTGGGTAACCGTGGGGCTCTCTGTCTGTCTTGTGGTACTACTGGTGGCCCTGGCTTTCGTGTGCTGGAGAAAGATCAAGCAGAGCTGCGAGGAGGAGAATGCAGGTGCCGAGGACCAGGATGGAGATGGAGAAGGATCCAAGACAGCTCTACGGCCTCTGAAACCCTCTGAAAACAAAGAAGATGACGGACAAGAAATTGCTTGA(SEQ ID No:282)

the results are shown in fig. 2, and based on the flow analysis results, 2B3 clone was finally selected as C cells for subsequent screening.

The construction method of A cell is the same as that of B, C cell, except that the plasmid used only expresses eGFP fluorescent protein, and no other target gene is used as the control of the screening process.

Example 3 extraction of B7-H3ECD protein

The eukaryotic expression system HEK 293-6E (available from Thermo Fisher Scientific) is adopted to express the extracellular region (ECD, amino acids 29-465) recombinant protein of human 4Ig-B7-H3, and the sequence is as follows:

LEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPE(SEQ ID No:283)

the recombinant protein (extracellular region of B7-H3 protein) was prepared by affinity purification using a nickel column.

The SDS-PAGE and SEC-HPLC results are shown in FIG. 3 and 4, respectively, indicating that the purity of the recombinant protein is > 95%.

Example 4 screening of clones capable of binding to cells overexpressing 4Ig-B7-H3

Screening the whole human natural antibody library by using a phage display technology to obtain different Fab libraries.

Method one, screening by ELISA and flow cytometry, and obtaining clone capable of combining B cell and/or C cell. And removing repeated clones through sequencing comparison to obtain specific clones capable of combining B cells and/or C cells.

And secondly, screening a fully human natural antibody library through the B7-H3ECD protein, screening by adopting a method of immune tube solid-phase immunoaffinity and magnetic bead liquid-phase immunoaffinity, and removing repeated cloning through sequencing comparison to obtain specific clone capable of combining B cells and/or C cells.

Performing recheck verification on specific clones obtained by screening the cells by ELISA and flow cytometry, and obtaining 29 clones which have unique sequences and can be combined with B cells and/or C cells after removing clones with repeated sequences after combining sequencing comparison; screening of the fully human natural antibody repertoire by the B7-H3ECD protein resulted in 9 clones with unique sequences that were able to bind to B cells and/or C cells.

Thus, by the above two different technical routes, after excluding 3 clones with sequence redundancy, a total of 35 positive antibody clones with sequence uniqueness capable of binding to B cells were obtained; and wherein 30 of the antibody clones are capable of binding to both B cells and C cells.

Example 5 construction of Single chain antibodies and ordering of affinity Properties

5.1 construction of Single chain antibodies

Passing (G) the heavy chain variable region (VH) and the light chain variable region (VL) of the antibody obtained above₄S)₃The connecting fragments are connected together to construct a single chain antibody (scFv), and the scFv is fused with a human IgG1-Fc fragment to construct a structural form of VH- (G)₄S)₃-VL-huIgG1 Fc. These 35 single-chain antibodies were recombinantly expressed using 293T cells and purified.

5.2 affinity assay

Based on the A, B and C cells and the B7-H3ECD protein, the 35 single-chain antibodies are rechecked by adopting flow cytometry and enzyme-linked immunosorbent assay (ELISA) experiments, and the affinity and the specificity of the single-chain antibodies for combining with B7-H3 molecules are classified and ranked.

5.3 results

The structures of the 35 constructed single-chain antibodies are shown in Table 1.

TABLE 1

The corresponding amino acid sequences are shown in Table 2

TABLE 2 amino acid sequences of VH and VL

The corresponding DNA sequences are shown in Table 3.

TABLE 3 nucleotide sequences of VH and VL

The CDRs of VH and VL of the partial antibody are listed in Table 4, in which the sequence alignment of VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, VL-CDR3 of the partial antibody clone is shown in FIGS. 5-10.

Table 4 CDR amino acid sequences of partial antibodies

The nucleotide sequences of the CDRs of VH and VL of the partial antibodies are shown in table 5.

TABLE 5 nucleotide sequences of the CDRs of partial antibodies

The affinity of the partial antibodies was measured as shown in table 6, fig. 11 and fig. 12.

TABLE 6 affinity of partial scFv Single chain antibodies

As shown in FIG. 11, enzyme-linked immunosorbent assay (ELISA) showed that the scFv single-chain antibody of the present invention and the human 4Ig-B7-H3ECD protein have excellent affinity and EC50 value.

As shown in FIG. 12, flow cytometry revealed that the scFv single-chain antibody of the present invention specifically binds to human B7-H3ECD protein and specifically recognizes murine B7-H3 (the sequence and structure of which are highly conserved with human B7-H3).

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. An anti-B7-H3 antibody, wherein the antibody has a heavy chain variable region and a light chain variable region,

VH-CDR1 shown in SEQ ID NO.8n +2,

VH-CDR2 shown in SEQ ID NO.8n +3, and

VH-CDR3 shown in SEQ ID NO.8n + 4;

VL-CDR1 shown in SEQ ID NO.8n +6,

VL-CDR2 as shown in SEQ ID NO.8n +7, and

VL-CDR3 shown in SEQ ID NO.8n + 8;

2. The antibody of claim 1, wherein the heavy chain of said antibody has the amino acid sequence set forth in any one of SEQ ID No.8n +1, wherein n is independently 0, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34.

3. The antibody of claim 1, wherein the light chain of said antibody has the amino acid sequence set forth in any one of SEQ ID nos. 8n +5, wherein n is independently 0, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34.

4. The antibody of claim 1, wherein said antibody is a single chain antibody (scFV).

5. The antibody of claim 1, wherein said antibody has the structure of formula I or II:

Z1-L1-Z2-Z3 (I)

Z2-L1-Z1-Z3 (II)

in the formula,

z1 is the heavy chain variable region VH;

z2 is the light chain variable region VL;

l1 is nothing or a peptide linker;

z3 is an antibody constant region (Fc region);

each "-" represents a peptide bond.

6. The antibody of claim 1, wherein said antibody has the structure of formula III:

VH-(G₄S)₃-VL-huIgG1Fc (III)

in the formula,

VH is the heavy chain variable region;

VL is a light chain variable region;

(G₄S)₃is a peptide linker;

huIgG1Fc is the constant region of human IgG1 antibody.

7. A recombinant protein, said recombinant protein comprising:

(i) the antibody of any one of claims 1-6; and

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

8. A polynucleotide encoding a polypeptide selected from the group consisting of:

(1) the antibody of any one of claims 1-6; and

(2) the recombinant protein of claim 7.

9. A vector comprising the polynucleotide of claim 8.

10. A genetically engineered host cell comprising the vector or genome of claim 9 having the polynucleotide of claim 8 integrated therein.