TWI673287B - Anti-b7-h3 antibody, antigen-binding fragment thereof and pharmaceutical use thereof - Google Patents

Abstract

The invention relates to an anti-B7-H3 antibody, an antigen-binding fragment thereof, and a medicinal use thereof. Further, the present invention relates to a chimeric antibody, a humanized antibody comprising the CDR region of the anti-B7-H3 antibody, and a pharmaceutical composition comprising a human anti-B7-H3 antibody and an antigen-binding fragment thereof, and as an anticancer drug the use of. In particular, the invention relates to the use of a humanized anti-B7-H3 antibody in the manufacture of a medicament for the treatment of a B7-H3-mediated disease or disorder.

Description

Anti-B7-H3 antibody, its antigen-binding fragment and its medical use

The present invention relates to an anti-B7-H3 antibody having immunoreactivity to a human B7-H3 receptor, and an antigen-binding fragment thereof, a chimeric antibody and a humanized antibody comprising the CDR region of the anti-B7-H3 antibody. ), And a pharmaceutical composition comprising a human anti-B7-H3 antibody and an antigen-binding fragment thereof, and its use as an anticancer drug.

Tumor immunotherapy is a long-term focus in the field of tumor therapy, of which T cell tumor immunotherapy is at the core. Tumor escape is a huge obstacle faced by tumor immunotherapy. Most tumors show antigens that can be recognized by the host's immune system to varying degrees, but in many cases, insufficient activation is caused by ineffective activation of T cells. The immune response, so tumor cells use their own suppressive effect on the immune system to promote the crazy growth of tumors. Tumor immunotherapy is to make full use of and mobilize the cytocidal T cells in the tumor patients to cytokine the tumor.

Studies of the CD28 receptor and its ligands have led to the characterization of related molecules called the B7 superfamily. B7 family members include B7.1 (CD80), B7.2 (CD86), inducible costimulatory ligand (ICOS-L / B7-H2), and programmed death-1 ligand (PD-L1 / B7-H1), programmed death-2 ligand (PD-L2 / B7-DC), B7-H3 and B7-H4, etc., have immunoglobulin V-like domains (IgV) and immunoglobulin C-like structures Domain (IgC) immunoglobulin superfamily , Each encoded by a single exon and predicted to form a back-to-back, non-covalent homodimer on the cell surface.

Recent studies have shown that B7-H3 may inhibit T cell activation through NFAT (nuclear factor for activated T cells), NF-κB (nuclear factor κB) and AP-1 (activating protein 1) factor (Yi.KH Et al., Immunol. Rev. 229: 145-151) and are thought to inhibit Th1, Th2 or Th17 in vivo (Fukushima, A. et al., Immunol. Lett. 113: 52-57; Yi.KH et al., Immunol. Rev. 229: 145-151). Several studies have shown that human malignant tumor cells show a significant increase in B7-H3 protein expression, and this increase in expression is related to increased disease severity, indicating that B7-H3 is used by tumors as an immune escape pathway (Hofmeyer, K. et al. , Proc. Natl. Acad. Sci. 105: 10277-10278).

Human B7-H3 is currently known to be expressed on a variety of cancer cells such as gastric cancer, ovarian cancer, non-small cell lung cancer, and neuroblastoma, and the expression of B7-H3 protein has been detected in tumor cell lines by immunohistochemistry. The expression of B7-H3 mRNA was found in the heart, kidney, testes, lungs, liver, pancreas, prostate, colon and osteoblasts, and the amount of protein was found in human liver, lung, bladder, testes, prostate, breast, B7-H3 manifestations have been found in placenta and lymphoid organs.

At present, many multinational pharmaceutical companies are developing monoclonal antibodies against B7-H3 to improve the patient's own immune system response to tumors, thereby achieving the purpose of cytocidal tumor cells. Related patents such as WO2011109400, WO2008116219, WO2012147713, WO2014160627, WO2016044383 and others. Macrogenics' anti-B7-H3 monoclonal antibody has completed phase I clinical trials and has shown good safety and anti-tumor activity in patients with prostate cancer, bladder cancer and melanoma. It can be used alone or with anti-PD- 1 antibody combination.

The invention provides an anti-B7-H3 antibody with high affinity, high selectivity and high biological activity, which is used for tumor monoclonal antibody immunotherapy and related applications. Medicine, composition and method for treating B7-H3 positive tumor.

The invention provides an anti-B7-H3 antibody or an antigen-binding fragment thereof, comprising: an antibody light chain variable region, the antibody light chain variable region comprising at least one LCDR selected from the following sequence: SEQ ID NO : 6, SEQ ID NO: 7, SEQ ID NO: 8; SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16; and an antibody heavy chain variable region, said antibody heavy chain variable region Contains at least one HCDR selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

The LCDR preferably includes one or more of LCDR1, LCDR2, and LCDR3. The amino acid sequence of the LCDR1 is shown in SEQ ID NO: 6 or SEQ ID NO: 14 in the sequence listing; the amine of the LCDR2 The amino acid sequence is as shown in SEQ ID NO: 7 or SEQ ID NO: 15 in the Sequence Listing; the amino acid sequence of the LCDR3 is shown as SEQ ID NO: 8 or SEQ ID NO: 16 in the Sequence Listing.

The HCDR preferably includes one or more of HCDR1, HCDR2 and HCDR3. The amino acid sequence of the HCDR1 is shown as SEQ ID NO: 3 or SEQ ID NO: 11 in the sequence listing; the amino acid of the HCDR2 The sequence is shown in SEQ ID NO: 4 or SEQ ID NO: 12 in the Sequence Listing; the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 5 or SEQ ID NO: 13 in the Sequence Listing.

In a preferred embodiment of the present invention, an anti-B7-H3 antibody or an antigen-binding fragment thereof as described above, wherein the antibody light chain variable region comprises sequences such as SEQ ID NO: 6, SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown in 7 and SEQ ID NO: 8.

In a preferred embodiment of the present invention, an anti-B7-H3 antibody or an antigen-binding fragment thereof as described above, wherein the antibody light chain variable region comprises sequences such as SEQ ID NO: 14, SEQ ID NO: LCDR1, LCDR2 and LCDR3 shown in 15 and SEQ ID NO: 16.

In a preferred embodiment of the present invention, an anti-B7-H3 antibody or an antigen-binding fragment thereof as described above, wherein the antibody heavy chain variable region comprises sequences such as SEQ ID NO: 3, SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown by 4 and SEQ ID NO: 5.

In a preferred embodiment of the present invention, an anti-B7-H3 antibody or an antigen-binding fragment thereof as described above, wherein the antibody heavy chain variable region comprises sequences such as SEQ ID NO: 11, SEQ ID NO: HCDR1, HCDR2 and HCDR3 shown in 12 and SEQ ID NO: 13.

In a preferred embodiment of the present invention, an anti-B7-H3 antibody or an antigen-binding fragment thereof as described above, wherein the variable region of the light chain of the antibody comprises sequences such as: SEQ ID NO: 6, SEQ ID NO : LCDR1, LCDR2 and LCDR3 shown in 7 and SEQ ID NO: 8; or LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

In a preferred embodiment of the present invention, an anti-B7-H3 antibody or an antigen-binding fragment thereof as described above, wherein the variable region of the heavy chain of the antibody comprises sequences such as: SEQ ID NO: 3, SEQ ID NO : 4 and HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 5; or HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.

Particularly preferably, the anti-B7-H3 antibody or antigen-binding fragment thereof may be selected from any one of the following:

(1) The antibody light chain variable region containing sequences are as follows: LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8; the antibody heavy chain variable region containing sequences are as follows: : HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

(2) The antibody light chain variable region contains sequences such as: LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; the antibody heavy chain variable region contains sequences such as : LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.

The anti-B7-H3 antibody or the antigen-binding fragment thereof is preferably a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

Preferably, the antibody light chain variable region of the mouse-derived antibody further comprises a light chain FR region of a mouse-derived κ, λ chain or a variant thereof.

Preferably, the mouse-derived antibody further comprises a light-chain constant region of a mouse-derived κ, λ chain, or a variant thereof.

Preferably, the B7-H3 chimeric antibody further comprises a light chain constant region of a human-derived κ, λ chain or a variant thereof.

Preferably, the B7-H3 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof.

In a preferred embodiment of the present invention, the amino acid sequence of the heavy chain variable region of the murine antibody or the chimeric antibody is as shown in SEQ ID NO. 1 in the sequence listing, and the light chain is variable The amino acid sequence of the region is shown in SEQ ID NO. 2 in the sequence listing; or the amino acid sequence of the heavy chain variable region of the murine antibody or the chimeric antibody is shown in SEQ ID NO in the sequence listing. As shown in FIG. 9, the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 10 in the sequence listing.

Preferably, the antibody light chain variable region of the humanized antibody further comprises a light chain FR region of a human-derived κ, λ chain, or a variant thereof. The light chain FR region sequence on the light chain variable region of the humanized antibody is preferably derived from the human germline light chain IGKV1-33 sequence shown in SEQ ID NO: 24; or derived from the sequence such as SEQ ID NO : 26 human germline light chain IGKV1-9 sequence shown.

Preferably, the amino acid sequence of the light chain variable region of the humanized antibody is as shown in SEQ ID NO: 30 or SEQ ID NO: 33 in the sequence listing. More preferably, the humanized antibody further comprises a light chain constant region of a human-derived κ, λ chain, or a variant thereof. Further preferably, the light chain sequence of the humanized antibody is the sequence shown in SEQ ID NO: 18 or SEQ ID NO: 20 or a variant thereof; the variant is preferably located in the variable region of the light chain. The amino acid change of 0-10 is preferably a mutation of amino acid sites at 4 and 9, and the amino acid after the 4th mutation is methionine (M).

Preferably, the heavy chain variable region of the humanized antibody further comprises a heavy chain FR region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably a human chain of IgG1, IgG2 or IgG4 heavy chain FR It is more preferable to use IgG1 which enhances ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation. The heavy chain FR region sequence on the heavy chain variable region of the humanized antibody is preferably derived from the human germline heavy chain IGHV3-23 sequence shown in SEQ ID NO: 23; or derived from the sequence such as SEQ ID NO : 25 human germline heavy chain IGHV1-2 sequence.

Preferably, the amino acid sequence of the heavy chain variable region of the humanized antibody is as shown in SEQ ID NO: 27 or SEQ ID NO: 31 in the sequence listing. More preferably, the humanized antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof. Further preferably, the heavy chain sequence of the humanized antibody is the sequence shown in SEQ ID NO: 17 or SEQ ID NO: 19 or a variant thereof; the variant is preferably provided in the variable region of the heavy chain. 0-10 amino acid changes, preferably mutations in amino acid sites at 9, 13 and 49, the amino acid after the 9th mutation is proline (P), The amino acid after mutation at position 13 is glutamine (Q), and the amino acid after mutation at position 49 is alanine (A).

In a preferred embodiment of the present invention, the humanized antibody is a humanized antibody huA9 or a humanized antibody huA3; the sequence of the heavy chain variable region of the humanized antibody huA9 is as SEQ ID NO: As shown in 31, the sequence of the light chain variable region is shown in SEQ ID NO: 33; the sequence of the heavy chain variable region of the humanized antibody huA3 is shown in SEQ ID NO: 27, and the sequence of the light chain variable region is shown in SEQ ID NO: 30. More preferably, the humanized antibody huA9 comprises a heavy chain antibody sequence of SEQ ID NO: 19 and a light chain antibody sequence of SEQ ID NO: 20; wherein the humanized antibody huA3 comprises a heavy chain antibody sequence of SEQ ID NO : 17, and the light chain antibody sequence SEQ ID NO: 18.

In a preferred embodiment of the present invention, an anti-B7-H3 antibody or an antigen-binding fragment thereof as described above, wherein the antigen-binding fragment is Fab, Fv, sFv, F (ab ') 2, a linear antibody, Single chain antibodies, Nanobodies, domain antibodies and multispecific antibodies.

The present invention further provides a DNA sequence encoding an anti-B7-H3 antibody or an antigen-binding fragment thereof as described above.

The present invention further provides a performance vector containing a DNA sequence as described above.

The invention further provides a host cell transformed with a performance vector as described above.

In a preferred embodiment of the present invention, a host cell as described above, the host cell is a bacterium, preferably Escherichia coli .

In a preferred embodiment of the present invention, a host cell as described above is a yeast, preferably Pichia pastoris .

In a preferred embodiment of the present invention, a host cell as described above is a mammalian cell, preferably a Chinese Hamster Ovary (CHO) cell or a Human Embryonic Kidney (HEK) 293 cell.

The present invention also provides a multispecific antibody containing the light chain variable region and the heavy chain variable region as described above.

The present invention also provides a single-chain antibody comprising the light chain variable region and the heavy chain variable region as described above.

The invention further provides a pharmaceutical composition comprising the anti-B7-H3 antibody or antigen-binding fragment thereof as described above and a pharmaceutically acceptable excipient, diluent or carrier.

The present invention further provides an anti-B7-H3 antibody or an antigen-binding fragment thereof as described above for use in preparing a medicament for treating a B7-H3-mediated disease or condition; wherein the disease is preferably cancer; more preferably B7-H3 cancer; the cancer is most preferably breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder Cancer, glioblastoma, and melanoma.

The present invention further provides a method of treating and preventing a B7-H3-mediated disease or disorder, which method comprises administering to a patient in need thereof a therapeutically effective amount of an anti-B7-H3 antibody or an antigen-binding fragment thereof as described above, or an antibody comprising the same A pharmaceutical composition; wherein the disease is preferably cancer; more preferably, the cancer expresses B7-H3; the cancer is most preferably breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer , Colon, bladder, esophageal, cervical, gallbladder, glioblastoma, and melanoma.

Figure 1 is an in vitro ELISA binding experiment of antibodies, showing that both mouse antibodies A3 and A9 have binding activity to purified h-B7H3-Fc antigen, with an EC50 of about 0.03ug / mL.

FIG. 2 shows the in vitro binding activity experiments of chimeric antibodies and CHO cells with high expression of B7-H3. The mouse-derived antibodies A3 and A9, and the chimeric antibodies A3C and A9C all had significant binding activity to target cells at the nanomolar (nM) concentration level.

Figure 3 shows the in vitro binding activity of humanized antibodies to MDA-MB-231 cells with high expression of B7-H3. Both huA3 and huA9 have significant binding activity to target cells at nanomolar concentration levels.

I. Terminology

To make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

The three-letter and one-letter codes for amino acids used in the present invention are described in J. Biol. Chem, 243, p3558 (1968).

The term "antibody" in the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by an interchain disulfide bond. The composition and arrangement of amino acids in the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. According to this, immunoglobulins can be divided into five categories, or isotypes called immunoglobulins, that is, IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are μ, δ, and γ chains, respectively. Alpha and epsilon chains. The same type of Ig can be divided into different subclasses according to the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chain is divided into a kappa chain or a lambda chain by different constant regions. Each of the five types of Ig may have a κ chain or a λ chain.

In the present invention, the antibody light chain variable region of the present invention may further include a light chain constant region, and the light chain constant region comprises a human or murine κ, λ chain or a variant thereof.

In the present invention, the antibody heavy chain variable region of the present invention may further include a heavy chain constant region, and the heavy chain constant region includes human or murine IgG1,2,3,4 or a variant thereof.

The sequence of about 110 amino acids near the N-terminus of the heavy and light chains of the antibody varies greatly and is a variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). Three hypervariable regions determine the specificity of an antibody, also known as complementarity determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) is composed of three CDR regions and four FR regions. The sequence from amine end to carboxyl end is: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. The CDR amino acid residues of the VL and VH regions of the antibody or antigen-binding fragment according to the present invention conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3) in terms of number and position, or comply with Kabat and Chothia numbering. Rules (HCDR1).

The term "antigen-presenting cell" or "APC" is a cell that displays a foreign antigen complexed with MHC on its surface. T cells use the T cell receptor (TCR) to recognize this complex. Examples of APC include, but are not limited to, dendritic cells (DC), topical blood mononuclear cells (PBMC), monocytes, B lymphoblasts, and monocyte-derived dendritic cells (DC). The term "antigen presentation" refers to the process by which APCs capture antigens and enable them to be recognized by T cells, for example as a component of a MHC-I / MHC-II conjugate.

The term "B7-H3" refers to a member of the human B7 protein family, also known as CD276, which is a type I transmembrane protein with four Ig-like extracellular domains. B7-H3 is one of the immune checkpoint proteins expressed on the surface of antigen-presenting cells or cancer cells, and it can inhibit the functional activation of T cells. The term "B7-H3" includes any variant or isoform of B7-H3 that is naturally expressed by the cell. The antibodies of the invention are cross-reactive with B7-H3 obtained from non-human species. Alternatively, the antibody may be specific for human B7-H3 and may not show cross-reactivity with other species. B7-H3 or any of its Any variants or isoforms can be isolated from cells or tissues in which they are naturally expressed, or produced by recombinant techniques using techniques commonly used in the art and those described herein. Preferably, the anti-B7-H3 antibody targets human B7-H3 with a normal glycosylation pattern.

The term "recombinant human antibody" includes human antibodies prepared, expressed, created, or isolated by recombinant methods. The techniques and methods involved are well known in the art, such as (1) transgenes, transchromosomes from human immunoglobulin genes Antibodies isolated from animals (e.g., mice) or fusion tumors prepared therefrom; (2) antibodies isolated from host cells transformed to express antibodies, such as transfected tumors; (3) isolated from recombinant human antibody libraries Antibodies; and (4) antibodies prepared, expressed, created, or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include rearrangements and mutations that subsequently occur, such as during antibody maturation.

The term "murine antibody" in the present invention is a monoclonal antibody to human B7-H3 prepared according to the knowledge and skills in the art. The test subject is injected with the B7-H3 antigen during preparation, and then a fusion tumor expressing an antibody having a desired sequence or functional characteristics is isolated. In a preferred embodiment of the present invention, the mouse-derived B7-H3 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a mouse-derived kappa, lambda chain or a variant thereof, or further comprise a mouse-derived IgG1 , IgG2, IgG3 or IgG4 or variants of the heavy chain constant region.

The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term "human antibody" does not include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, has been grafted onto a human backbone sequence (ie, a "humanized antibody").

The term "humanized antibody", also known as a CDR-grafted antibody, refers to an antibody produced by transplanting a mouse CDR sequence into a human antibody variable region framework. It can overcome the strong immune response induced by the chimeric antibody because it carries a large amount of mouse protein components. In order to avoid the decrease in activity while the decrease in immunogenicity, the human antibody variable region may be subjected to a minimum of reverse mutations to maintain the activity.

The term "chimeric antibody" is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, and can alleviate the immune response response induced by the murine antibody. To establish a chimeric antibody, select a fusion tumor that secretes a mouse-specific monoclonal antibody, then copy the variable region gene from the mouse fusion tumor cell, and then copy the constant region gene of the human antibody according to the needs, and the mouse variable region The gene is linked to the human constant region gene to form a chimeric gene and inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system. The constant region of a human antibody may be selected from the heavy chain constant regions of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably including human IgG2 or IgG4 heavy chain constant regions, or without ADCC (antibody after amino acid mutation) -dependent cell-mediated cytotoxicity) Toxic IgG1.

The term "antigen-binding fragment" refers to an antigen-binding fragment and an antibody analog of an antibody, which typically includes at least a portion of a parental antibody's antigen-binding region or variable region (eg, one or more CDRs). The antibody fragment retains at least some of the binding specificity of the parent antibody. Generally, when activity is expressed on a mole basis, antibody fragments retain at least 10% of the maternal binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parent antibody for the target. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab ' , F (ab ' ) 2, Fv fragments, linear antibodies, single chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson (2005) Nat. Biotechnol. 23: 1126-1136.

A "Fab fragment" consists of a light chain and a heavy chain CH1 and a variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

The "Fc" region contains two heavy chain fragments containing the CH1 and CH2 domains of the antibody. Two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domain.

A "Fab ' fragment" contains a light chain and a portion of a heavy chain containing a region between the VH and CH1 domains and between the CH1 and CH2 domains, so that it can be placed between the two heavy chains of two Fab ' fragments Inter-chain disulfide bonds are formed to form F (ab ' ) 2 molecules.

The "F (ab ' ) 2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F (ab ' ) 2 fragment consists of two Fab ' fragments held together by a disulfide bond between the two heavy chains.

An "Fv region" contains variable regions from both heavy and light chains, but lacks a constant region.

The term "multispecific antibody" is used in its broadest sense and encompasses antibodies with multi-epitope specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has multi-epitope specificity; has two or more Antibodies for VL and VH regions, each VH-VL unit binds to a different target or different epitopes of the same target; antibodies with two or more single variable regions, each single variable region and Different targets or different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, covalently or non-covalently linked together Antibody fragments and so on.

The term "single-chain antibody" is a single-chain recombinant protein composed of an antibody's heavy chain variable region (VH) and light chain variable region (VL) connected by a linker peptide. It is the smallest protein with a complete antigen-binding site. Antibody fragments.

The term "domain antibody fragment" is an immunologically functional immunoglobulin fragment containing only the heavy or light chain variable region chains. In some cases, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment. The two VH regions of a bivalent domain antibody fragment can target the same or different antigens.

The term "binding to B7-H3" in the present invention refers to the ability to interact with human B7-H3. The term "antigen-binding site" in the present invention refers to a three-dimensional spatial site on the antigen that is discontinuous and recognized by the antibody or antigen-binding fragment of the present invention.

The term "epitope" refers to a site on an antigen that specifically binds an immunoglobulin or antibody. Epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by the tertiary folding of a protein. An epitope formed by an adjacent amino acid is typically maintained after exposure to a denaturing solvent, while an epitope formed by tertiary folding is usually lost after treatment with a denaturing solvent. Epitope usually includes at least 3-15 amino acids in a unique steric conformation. Methods to determine what epitopes are bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of an epitope include techniques in the art and techniques described herein, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.

The terms "specific binding" and "selective binding" used in the present invention refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, when recombinant human B7-H3 is used as the analyte and the antibody is used as the ligand, as measured by surface plasmon resonance (SPR) technology in the instrument, the antibody has an equilibrium dissociation constant below about 10-7M or even less (KD) binds to a predetermined antigen, and has an affinity for the predetermined antigen that is at least twice that of a non-specific antigen (such as BSA, etc.) for binding to the predetermined antigen or a closely related antigen. The term "antibody that recognizes an antigen" is used interchangeably herein with the term "antibody that specifically binds".

The term "cross-reactivity" refers to the ability of an antibody of the invention to bind to B7-H3 from different species. For example, an antibody of the invention that binds human B7-H3 can also bind to another species B7-H3. Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays, such as SPR and ELISA, or binding or functional interaction with cells that physiologically express B7-H3. Methods to determine cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.

The terms "inhibit" or "block" are used interchangeably and cover both partial and complete inhibition / blocking. The inhibition / blocking of the ligand preferably reduces or changes the normal level or type of activity that occurs when ligand binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in ligand binding affinity when contacted with an anti-B7-H3 antibody, compared to a ligand that has not been contacted with an anti-B7-H3 antibody.

The term "inhibiting growth" (e.g., involving a cell) is intended to include any measurable reduction in cell growth.

The terms "induced immune response" and "enhancing immune response" are used interchangeably and refer to the stimulation (i.e., passive or adaptive) of a particular antigen by an immune response. The term "induction" with respect to the induction of CDC or ADCC refers to the stimulation of specific direct cell killing mechanisms.

The “ADCC” in the present invention, that is, antibody-dependent cell-mediated cytotoxicity, refers to the cell that expresses Fc receptors is directly cytokilled by the antibody package by recognizing the Fc segment of the antibody. Covered target cells. By modifying the Fc segment on IgG, the ADCC function of reducing or eliminating antibodies can be enhanced or reduced. The modification refers to mutation in the constant region of the heavy chain of the antibody.

Methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as Cold Spring Harbor's antibody experimental technical guide, Chapters 5-8 and 15. For example, mice can be immunized with human B7-H3 or fragments thereof, and the resulting antibodies can be coated, purified, and can be subjected to amino acid sequencing by conventional methods. Antigen-binding fragments can also be prepared by conventional methods. The antibody or antigen-binding fragment of the invention is genetically engineered to add one or more human FRs to a non-human CDR region. Area. Human FR germline sequences can be obtained from the website of ImMunoGeneTics (IMGT) http://imgt.cines.fr, or from the Journal of Immunoglobulins, 2001ISBN012441351.

The engineered antibodies or antigen-binding fragments of the present invention can be prepared and purified by conventional methods. The cDNA sequence of the corresponding antibody can be copied and recombined into the GS expression vector. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems cause glycosylation of antibodies, especially the highly conserved N-terminus of the FC region. Stable replication is obtained by antibodies that exhibit specific binding to human-derived antigens. Positive replicates were expanded in serum-free medium in the bioreactor to produce antibodies. The culture medium in which the antibody is secreted can be purified and collected by conventional techniques. The antibody can be concentrated by filtration using a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product needs to be immediately frozen, such as -70 ° C, or lyophilized.

The antibody of the present invention refers to a monoclonal antibody. The monoclonal antibody (mAb) according to the present invention refers to an antibody obtained from a single replicating cell strain, and the cell strain is not limited to a eukaryotic, prokaryotic, or phage replicating cell strain. Monoclonal antibodies or antigen-binding fragments can be recombined using techniques such as fusion tumor technology, recombinant technology, phage display technology, synthetic technology (such as CDR-grafting), or other existing technologies.

"Administration" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions related to animals, humans, and recipients. Contact with a subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" may refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. Treatment of a cell includes contact of a reagent with a cell, and contact of a reagent with a fluid, wherein the fluid is in contact with the cell. "Administering" and "treating" also mean in vitro and ex vivo treatment of cells, such as cells, by an agent, diagnostic, binding composition, or by another cell. "Treatment" when applied to human, veterinary or research subjects refers to therapeutic treatment, preventive or preventative measures, research and diagnostic applications.

"Treatment" means the administration to a patient of an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the invention, said patient having one or more symptoms of a disease, and said therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, a therapeutic agent is administered in a treated patient or population in an amount effective to alleviate the symptoms of one or more diseases, whether by inducing the deterioration of such symptoms or inhibiting the development of such symptoms to any clinically right degree. The amount of therapeutic agent (also known as a "therapeutically effective amount") that is effective in alleviating the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired effect in the patient. Evaluate whether the symptoms of the disease have been alleviated by any clinical test method that a doctor or other health care professional usually uses to assess the severity or progression of the symptoms. Embodiments of the present invention (e.g., treatment methods or articles of manufacture) may not be effective in alleviating symptoms of the target disease that each patient has, but according to any statistical test method known in the art such as Student's t-test, chi-square test, basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce symptoms of the target disease in a statistically significant number of patients.

The term "consisting essentially of" or its variants used throughout the specification and the scope of the patent application is meant to include all of the elements or groups of elements, and optionally other elements of similar or different nature to the elements The other elements do not significantly change the basic or novel properties of a given dosing regimen, method or composition. As a non-limiting example, a binding compound consisting essentially of the mentioned amino acid sequence may also include one or more amino acids, which does not significantly affect the properties of the binding compound.

The term “naturally occurring” applied to an object according to the present invention refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism (including a virus) that can be isolated from natural sources and has not been intentionally modified in the laboratory is naturally occurring.

An "effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's general health, the route and dosage of administration, and the severity of the side effects. An effective amount may be the maximum dose or dosage regimen to avoid significant side effects or toxic effects.

"Exogenous" refers to substances that are produced outside the body, cell, or human depending on the context. "Endogenous" refers to a substance that is produced in a cell, organism, or human body by context.

"Homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are identical at that position Source. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of compared positions x 100%. For example, when the sequences are optimally aligned, if there are 6 matches or homology at 10 positions in the two sequences, then the two sequences are 60% homologous. In general, comparisons are made when the two sequences are aligned for the greatest percentage of homology.

As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such names include their progeny. Thus, the words "transformants" and "transformed cells" include primary test cells and cultures derived from them, regardless of the number of metastases. It should also be understood that due to intentional or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Included are mutant offspring that have the same functional or biological activity as those originally screened in the transformed cells. Where different names are meant, they are clearly visible from the context.

"Optional" or "optionally" means that the event or environment described later can, but need not, occur, and the description includes situations where the event or environment occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that an antibody heavy chain variable region of a particular sequence may, but need not, be present.

"Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or a physiological / pharmaceutically acceptable salt or prodrug thereof with other chemical components, as well as other components such as physiological / pharmaceutically acceptable carriers and excipient. The purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and then exerts the biological activity.

The following examples are used to further describe the present invention, but these examples do not limit the scope of the present invention. The experimental methods without specific conditions in the examples of the present invention generally follow conventional conditions, such as the manual of antibody technology experiments and molecular reproduction manuals of Cold Spring Harbor; or according to the conditions recommended by the raw material or commodity manufacturers. The reagents without specific sources are conventional reagents purchased on the market.

Example 1 Sequence and preparation of immune antigen and screening antigen

The sequence encoding the human B7-H3 (h-B7H3-his) with His tag and the sequence encoding the human B7-H3 (h-B7H3-Fc) with huFc tag were synthesized by Integrated DNA Technology (IDT) company (recombination of B7-H3 above) The proteins are template sequences designed according to the present invention) and copied to the pTT5 vector (Biovector). After the recombinant B7-H3 protein was expressed on 293T cells, it was purified in Example 2. The purified protein can be used in the experiments of the following examples.

The amino acid sequences of h-B7H3-Fc and h-B7H3-his are shown as SEQ ID NOs. 21 and 22 in the sequence listing, respectively.

Example 2 Preparation of B7-H3 Recombinant Protein

1. Purification steps of B7H3 recombinant protein with His tag:

The supernatant samples of HEK293 cells (purchased from American Type Culture Collection, ATCC) were centrifuged at high speed to remove impurities, and the buffer was replaced with PBS, and imidazole was added to a final concentration of 5 mM. Equilibrate the nickel column with 5 mM imidazole in PBS and rinse 2-5 times the column volume. The replaced supernatant sample was applied to the column. The column was rinsed with 5 mM imidazole in PBS until the A280 reading dropped to baseline. After washing the column with PBS + 10mM imidazole, remove Non-specifically bound heteroproteins, and the effluent was collected. The target protein was eluted with 300 mM imidazole in PBS, and the elution peak was collected.

The collected eluate was further purified by ion exchange (SP column). Solution A: 0.01M PB, pH 8.0. Configure liquid B: liquid A + 1M NaCl. Firstly, the target protein eluting with the PBS solution of imidazole was replaced with liquid A, and the SP column was equilibrated with liquid A, and the concentration of liquid B was gradientd from 0 to 100%, and the column volume was eluted at 10 times. The obtained proteins were identified by electrophoresis, peptide mapping, and LC-MS.

2. Purification steps of Fc-tagged B7H3 recombinant protein (h-B7H3-Fc):

The supernatant sample of HEK293 cells was centrifuged at high speed to remove impurities, and the buffer was replaced with PBS. The Protein A affinity column was equilibrated with 10 mM phosphate buffer, and the column volume was washed 2-5 times. The replaced supernatant sample was applied to the column. Flush the column with 25 volumes of column buffer until the A280 reading drops to baseline. The target protein was eluted with 0.8% acetic acid buffer at pH 3.5, and the eluted peaks were collected. Immediately after aliquoting, 1M Tris-Cl pH8.0 buffer was added to neutralize, and then the solution was replaced with Millipore's Amico-15 filter column to PBS. . The obtained proteins were identified by electrophoresis, peptide mapping, and LC-MS.

3. Preparation of stable CHO cell line expressing human or monkey B7-H3 antigen:

The full-length sequence encoding the human or cynomolgus monkey ( Macaca fascicularis ) B7-H3 protein (huB7H3 or cyB7H3) was synthesized by Integrated DNA Technology (IDT) company (the above B7-H3 recombinant proteins were all designed by the template sequence of the present invention) and were copied to pcDNA3 .1 / puro (Invitrogen # V79020). CHO-S (ATCC) cells were cultured in CD-CHO medium (Life Technologies, # 10743029) to 0.5 x 106 / ml. 10 μg of the vector encoding the huB7H3 or cyB7H3 gene was mixed with 50 ul of LF-LTX (Life Technologies, # A12621) in 1 ml of Opti-MEM medium (Life Technologies, # 31985088). After incubating at room temperature for 20 minutes, it was added to the CHO cell culture solution. And put in a carbon dioxide incubator. After 24 hours, the medium was replaced with new medium and 10 μg / ml puromycin was added. After that, a new culture medium was replaced every 2-3 days, and a stable CHO-S cell pool was obtained after screening for 10-12 days.

Example 3 Preparation of antibodies

Anti-human B7H3 single-replicated antibodies were produced by immunizing mice. Swiss Webster white mice, female, 6 weeks old (Charles River) were used in the experiments. Rearing environment: SPF level. After the mice were purchased, they were reared in a laboratory environment for 1 week, with a 12/12 hour light / dark cycle adjustment, a temperature of 20-25 ° C, and a humidity of 40-60%. The immune antigen was an Fc-tagged human B7H3 recombinant protein (huB7H3-Fc). Titermax (sigma Lot Num: T2684) was used as an adjuvant. The ratio of the antigen to the adjuvant (titermax) is 1: 1. The antigen is emulsified and vaccinated for the first 0, 21, 35, 49, 63 days. On day 0, 15 μg + 25 / foot of emulsified antigen were injected intraperitoneally (IP). 21, 35, 49, 63 days Intraperitoneal (IP) injection of 15 μg + footpad (post-emulsified antigen), 3 days before spleen cell fusion to strengthen immunity, intraperitoneal (IP) injection of 15 μg + footpad (footpad) ) 15 / antigen solution in normal saline. Blood tests were performed on days 42, 56, and 70, and mouse sera were detected by ELISA and FACS methods to determine antibody titers in mouse sera. After the 5th immunization, mice with high antibody titers and plateaus in the serum were selected for splenocyte fusion, and spleen lymphocytes were splenocytes and myeloma cells Sp2 / 0 cells (ATCC® CRL) using an optimized electrofusion procedure. -8287 ^(TM )) to obtain fusion tumor cells.

After fused fusion tumor cells are cultured for 7-14 days, the supernatant of the medium is taken, and the B7-H3 recombinant protein huB7H3-Fc is used for antibody screening of the fusion tumor supernatant by ELISA. -H3 CHO-S cells, compared with blank CHO-S cells to exclude non-specific binding antibody fusion tumor strains, and screened by flow sorting method to select two fusion tumors that bind recombinant proteins and also bind cells to express antigens . Fusion tumor cells in logarithmic growth phase were collected, RNA was extracted with Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript ^™ Reverse Transcriptase, Takara # 2680A). The reverse-transcribed cDNA was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and sequenced, and the sequences of two mouse antibodies A3 and A9 were finally obtained.

The sequence of the variable region of the heavy and light chains of murine mAb A3 is as follows:

A3 HCVR

SEQ ID NO: 1

A3 LCVR

SEQ ID NO: 2

The heavy and light chain variable region sequences of A9 are as follows:

A9 HCVR

SEQ ID NO: 9

A9 LCVR

SEQ ID NO: 10

The heavy and light chain variable regions of each mouse antibody were copied into pTT vector plasmid (Biovector) containing human IgG1 heavy chain constant region and kappa light chain constant region, respectively, and then transiently transfected into HEK293 cells to obtain antibody The chimeric antibodies A3C and A9C of B7-H3 were purified and identified according to the method described in Example 2 (purification of Fc-tagged proteins), and the activity was detected as described below.

Example 4 Determination of in vitro binding activity of antibodies

The neutral avidin used for binding with biotin was diluted to 1 μg / ml with PBS buffer, added to a 96-well plate at a volume of 100 μl / well, and left at 4 ° C. for 16 h-20 h. After washing the plate once with PBST (pH 7.4 PBS containing 0.05% Tween-20) buffer, 120 μl / well PBST / 1% milk was added, and the plate was blocked by incubation at room temperature for 1 hour. After washing the plate with PBST buffer once, 1 μg / ml of biotin-labeled h-B7H3-Fc diluted with PBST / 1% milk was added and incubated at room temperature for 1 h. After washing the plate 3 times with PBST buffer, add the test B7-H3 antibody diluted with PBST / 1% milk to the appropriate concentration, and incubate at room temperature for 1.5h. Remove the reaction system and wash the plate 3 times with PBST, then add 100 μl / well to dilute horseradish with PBST / 1% milk. Oxidase (Horseradish Peroxidase, HRP) labeled anti-mouse antibody secondary antibody (The Jackson Laboratory), incubated at room temperature for 1 h. After washing the plate 3 times with PBST, 100 μl / well of TMB was added and incubated for 5-10 min at room temperature. Add 100 μl / well 1M H2SO4 to stop the reaction, read the absorption value at 450nm, and calculate the ELISA binding EC50 value. The results are shown in Figure 1. The EC50s of antibodies A3 and A9 were about 0.03 μg / mL, and the mouse antibodies A3 and A9 had binding activity to the purified h-B7H3-Fc antigen.

CHO-S cells with high performance huB7-H3 were centrifuged at 1000 rpm for 5 minutes, the pellet was collected and suspended in 10-15 ml of pre-chilled flow buffer, and the cells were counted. The cells were collected by centrifugation in a 50 ml centrifuge tube at 1000 rpm for 5 minutes, the supernatant was discarded, and the pellet was resuspended in a pre-cooled blocking buffer with a density of 0.5-1.0 × 107 cells / ml. After incubation at 4 ° C for 30 minutes, resuspend in 100 μl per well and add to a 96-well plate. The 96-well plate was centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. Add 100 μl of the test antibody to each well with a concentration gradient of 0.01 nM to 670 nM. Resuspend the cells and incubate at 4 ° C in the dark for 60 minutes. The supernatant was discarded by centrifugation, and 100 μl of a 1400 diluted FITC-labeled secondary antibody (BD Biosciences) was added. The cells were resuspended and incubated at 4 ° C in the dark for 60 minutes. Cells were washed twice with flow buffer and resuspended in 1% paraformaldehyde to fix the cells for flow cytometry. The test results are shown in Figure 2. A3, A9 and their corresponding chimeric antibodies all bind significantly to huB7H3 high-performance cells at nanomolar (nM) concentration levels, and their binding is stronger than that of the reference antibody BRCA84D from Macrogenics.

The same method was used to detect the binding of each antibody to cynomolgus monkey B7-H3 at a single spot concentration of 10nM using CHO-S cells with high expression of cyB7-H3. The results are shown in Table 3 below. A3C has significant binding to monkey antigens. Signal, while A9C and reference antibody BRCA84D did not bind.

Example 5 In vitro binding affinity and kinetic experiments

This experiment uses the surface plasmon resonance (SPR) method. Using a kit provided by Biacore, the anti-mouse IgG multi-replicating antibody was covalently linked to the CM5 (GE) chip using a standard amine coupling method, and then the purified mouse-derived antibody to be tested according to the present invention was captured and immobilized using this antibody. phase. The h-B7H3-Fc or h-B7H3-His protein (Example 1) was diluted in the same buffer with a 12.5-800 nM concentration gradient (Example 1). . The antigen-antibody binding kinetics were followed for 3 minutes and the dissociation kinetics were followed for 10 minutes. GE's BIAevaluation software was used to analyze the data with 1: 1 (Langmuir) combined model analysis. The ka (kon), kd (koff), and KD values determined by this method are shown in Table 4 below.

Example 6 Humanization of mouse antibodies

Humanization of murine anti-human B7-H3 single-copy antibodies was performed as disclosed in many literatures in the art. In short, the human constant domain is used instead of the parent (murine antibody) constant domain, and the human antibody sequence is selected based on the homology of the mouse antibody and the human antibody. The present invention humanizes the candidate molecules A3 and A9 .

Based on the obtained typical structure of the mouse-derived antibody VH / VL CDR, the heavy and light chain variable region sequences were compared with human-derived antibody germline databases to obtain human germline templates with high homology. The human germline light chain framework region is derived from a human kappa light chain gene, and the human germline heavy chain framework region is derived from a human heavy chain. The antibody of the present invention is preferably a human germline antibody template shown below.

A3 is preferably a human germline heavy chain template IGHV3-23 (SEQ ID NO: 23):

A3 preferred human germline light chain template IGkV1-33 (SEQ ID NO: 24): DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLP

A9 is preferably a human germline heavy chain template IGHV1-2 (SEQ ID NO: 25):

A9 preferred human germline light chain template IGkV1-9 (SEQ ID NO: 26): DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKKKKLQLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYP

The CDR regions of the murine antibodies A3 and A9 were transplanted onto the selected corresponding humanized template, the humanized variable region was replaced, and then recombined with the IgG constant region (preferably the heavy chain was IgG1 and the light chain was kappa ). Then, based on the three-dimensional structure of the mouse-derived antibody, back-mutate the embedded residues, residues that directly interact with the CDR region, and residues that have an important effect on the conformation of VL and VH, and CDR The chemically labile amino acid residues in the region were optimized. Antibodies composed of the following humanized light and heavy chain variable region sequences were designed and tested.

huA3H1 (SEQ ID NO: 27):

huA3H2 (SEQ ID NO: 28):

huA3L1 (SEQ ID NO: 29):

huA3L2 (SEQ ID NO: 30):

huA9H1 (SEQ ID NO: 31):

huA9H2 (SEQ ID NO: 32):

huA9L1 (SEQ ID NO: 33):

huA9L2 (SEQ ID NO: 34):

After performance testing and comparison of the number of back mutations, the final humanized huA3 (using H1 heavy chain and L2 light chain) and huA9 antibody molecule (using H1 heavy chain and L1 light chain) were selected, and their respective sequences are as SEQ ID NO: 17-20.

The last three nucleotides "TGA" of the following gene sequence SEQ ID NO: 35 ~ 38 are stop codons and do not encode any amino acid.

huA3 antibody heavy chain sequence:

SEQ ID NO: 17

huA3 antibody heavy chain sequence encoding gene sequence:

SEQ ID NO: 35

huA3 antibody light chain sequence:

SEQ ID NO: 18

huA3 antibody light chain sequence encoding gene sequence:

SEQ ID NO: 36

huA9 antibody heavy chain sequence:

SEQ ID NO: 19

huA9 antibody heavy chain sequence encoding gene sequence:

SEQ ID NO: 37

huA9 antibody light chain sequence:

SEQ ID NO: 20

huA9 antibody light chain sequence encoding gene sequence (wherein the first 60 nucleotides of the following sequence do not participate in the encoding of the final huA9 antibody light chain):

SEQ ID NO: 38

A cDNA fragment was synthesized based on the gene sequences of the light and heavy chains of each humanized antibody, and inserted into a pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). The expression vector and transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1: 2 and placed in a CO2 incubator for 4-5 days. . After the expressed antibody was recovered by centrifugation, the antibody was purified according to the method of Example 2 (purification of Fc-tagged protein) to obtain the humanized antibody protein of the present invention.

Example 7 Determination of humanized antibody activity

The following experimental determinations of huA3 and huA9 and other derived humanized antibodies were performed in vitro:

1. Cell binding experiment (method steps are the same as in Example 4). The results are shown in Figure 3. Both humanized antibodies huA3 and huA9 positively bind to MDA-MB-231 cells with high expression of B7-H3. It is equivalent to A3C.

2. Affinity kinetics experiment (method steps are the same as in Example 5). The results are shown in the following table. The final preferred humanized antibodies hnA3 and huA9 have a KD of human B7-H3 antigen protein below 1 nM, showing a strong Affinity.

Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely examples, and various changes or modifications can be made to these embodiments without departing from the principle and essence of the present invention. . Therefore, the protection scope of the present invention is defined by the scope of the attached application patent.

Claims (20)

An anti-B7-H3 antibody or an antigen-binding fragment thereof comprises: an antibody light chain variable region, wherein the antibody light chain variable region comprises LCDR1, LCDR2, LCDR3 as shown in the following sequence: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and an antibody heavy chain variable region, said antibody heavy chain variable region comprising HCDR1, HCDR2, HCDR3 as described in the following sequence: SEQ ID NO: 3, SEQ ID NO : 4, SEQ ID NO: 5. The anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody. The anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 2, wherein the amino acid sequence of the heavy chain variable region of the murine antibody or the chimeric antibody is as shown in SEQ ID NO. 1 in the sequence listing As shown, the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 2 in the sequence listing. The anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 2, wherein the light chain FR region sequence on the light chain variable region of the humanized antibody is derived from a human as shown in SEQ ID NO: 24 Germline light chain IGKV1-33 sequence; and / or, the sequence of the heavy chain FR region on the variable region of the heavy chain of the humanized antibody, derived from the human germline heavy chain IGHV3- as shown in SEQ ID NO: 23 23 sequences. The anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 2, wherein the humanized antibody light chain sequence is the sequence shown in SEQ ID NO: 18; and / or, the humanized antibody The weight chain sequence is the sequence shown in SEQ ID NO: 17. The anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 2, wherein the humanized antibody heavy chain variable region further comprises a heavy chain FR region of human IgG1, IgG2, IgG3, or IgG4 or a variant thereof . The anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 6, wherein the heavy chain variable region of the humanized antibody comprises a heavy chain FR region of IgG1 that enhances ADCC toxicity after amino acid mutation. The anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 2, wherein the humanized antibody is a humanized antibody huA3; the sequence of the heavy chain variable region of the humanized antibody huA3 is as SEQ ID NO: 27 or SEQ ID NO: 28; and / or, the light chain variable region sequence is shown in SEQ ID NO: 29 or SEQ ID NO: 30. The anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 8, the heavy chain variable region sequence of the humanized antibody huA3 is shown in SEQ ID NO: 27, and the light chain variable region sequence is shown in SEQ. ID NO: 30. A DNA sequence encoding the anti-B7-H3 antibody or antigen-binding fragment according to any one of claims 1-9. The DNA sequence encoding the anti-B7-H3 antibody or antigen-binding fragment according to claim 10, the antibody heavy chain coding sequence is shown in SEQ ID NO: 35, and / or, the antibody light chain coding sequence is shown in SEQ ID NO: 36 As shown. A expression vector comprising the DNA sequence according to claim 11. A host cell comprising the expression vector according to claim 12. The host cell according to claim 13, wherein the host cell is a bacterial, yeast, or mammalian cell. The host cell according to claim 14, wherein the host cell is E. coli, Pichia, Chinese hamster ovary cell or human embryonic kidney 293 cell. A pharmaceutical composition comprising the anti-B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1-9 and a pharmaceutically acceptable excipient, diluent, or carrier. An anti-B7-H3 antibody or an antigen-binding fragment thereof according to any one of claims 1-9, for use in the manufacture of a medicament for the treatment or prevention of a B7-H3-mediated disease or disorder. Use according to claim 17, wherein said disease is cancer. The use according to claim 18, wherein the cancer is a cancer expressing B7-H3. Use according to claim 18 or 19, wherein the cancer is breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer , Gallbladder cancer, glioblastoma, and melanoma.