WO2018161872A1 - Anti-b7-h3 antibody, antigen-binding fragment thereof, and pharmaceutical use thereof - Google Patents

Abstract

Provided are an anti-B7-H3 antibody, an antigen-binding fragment thereof, and a pharmaceutical composition comprising the antibody or the antigen-binding fragment. Also provided is a use of the antibody or the antigen-binding fragment in preparation of a drug for treating a B7-H3 mediated disease or disorder. The antibody can be a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody. The pharmaceutical composition can be used for treating cancers.

Description

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

The present application claims priority from Chinese Patent Application No. CN201710129023.6, filed on March 6, 2017. This application cites the entire text of the above-mentioned Chinese patent application.

Technical field

The present invention relates to an anti-B7-H3 antibody immunoreactive to a human B7-H3 receptor, and an antigen-binding fragment thereof, 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 use thereof as an anticancer drug.

Background technique

Tumor immunotherapy is a long-term hotspot in the field of cancer treatment, and T cell tumor immunotherapy is at the core. Tumor escape is a huge obstacle to tumor immunotherapy. Most tumors express antigens that can be recognized by the host immune system to varying degrees, but in many cases, due to inefficient activation of effector T cells, an inadequate immune response is triggered. Tumor cells use their own inhibition of the immune system to promote the crazy growth of tumors. Tumor immunotherapy is to fully utilize and mobilize the killing T cells in tumor patients to kill the tumor.

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

Recent studies have shown that B7-H3 may inhibit T cell activation by NFAT (nuclear factor for activated T cells), NF-κB (nuclear factor κB) and AP-1 (activator 1) factors (Yi.KH) Etc., Immunol. Rev. 229: 145-151), and is believed 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 associated with increased disease severity, suggesting that B7-H3 is utilized by tumors as an immune evasion pathway (Hofmeyer, K. et al. , Proc. Nat l. Acad. Sci. 105: 10277-10278).

It is known that human B7-H3 is expressed on various 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 by immunohistochemistry in tumor cell lines. Expression of B7-H3 mRNA in heart, kidney, testis, lung, liver, pancreas, prostate, colon and osteoblasts, protein level, in human liver, lung, bladder, testis, prostate, breast, placenta B7-H3 expression has been found in lymphoid organs.

At present, a number of 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 killing tumor cells. Related patents such as WO2011109400, WO2008116219, WO2012147713, WO2014160627, WO2016044383 Wait. Macrogenics' anti-B7-H3 monoclonal antibody has now completed Phase I clinical trials and has shown superior safety and anti-tumor activity in prostate, bladder and melanoma patients, either alone or with anti-PD- 1 antibody combination.

The present invention provides anti-B7-H3 antibodies with high affinity, high selectivity and high biological activity, monoclonal antibody immunotherapy for tumors and related applications. Drugs, compositions, and methods for the treatment of B7-H3 positive tumors.

Summary of the invention

The present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, comprising:

An antibody light chain variable region comprising at least one LCDR selected from the group consisting of 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;

An antibody heavy chain variable region comprising 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, and the amino acid sequence of the LCDR1 is as shown in SEQ ID NO: 6 or SEQ ID NO: 14 in the Sequence Listing; The sequence is as SEQ ID NO: 7 or SEQ ID NO: 15 in the Sequence Listing; the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 8 or SEQ ID NO: 16 in the Sequence Listing.

Preferably, the HCDR comprises one or more of HCDR1, HCDR2 and HCDR3, and the amino acid sequence of the HCDR1 is as shown in SEQ ID NO: 3 or SEQ ID NO: 11 in the Sequence Listing; The 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 invention, an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the antibody light chain variable region comprises the sequence SEQ ID NO: 6, SEQ ID NO :7 and LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 8.

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

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

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

In a preferred embodiment of the invention, the anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the antibody light chain variable region comprises a sequence such as:

LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8;

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 invention, an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein said antibody heavy chain variable region comprises a sequence such as:

HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5;

HCDR1, HCDR2 and HCDR3 set forth 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 of the following:

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

(2) The antibody light chain variable region comprises the sequences of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; the antibody heavy chain variable region comprises 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 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 murine antibody further comprises a light chain FR region of a murine κ, λ chain or variant thereof.

Preferably, the murine antibody further comprises a light chain constant region of a murine kappa, lambda chain or variant thereof.

Preferably, the B7-H3 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or 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 of the Sequence Listing, and the light chain variable region is The amino acid sequence is as shown in SEQ ID NO. 2 of the Sequence Listing; or wherein the amino acid sequence of the murine antibody or the heavy chain variable region of the chimeric antibody is as shown in SEQ ID NO. 9 of the Sequence Listing, light chain The amino acid sequence of the 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 kappa, lambda chain or variant thereof. The light chain FR region sequence on the light chain variable region of the humanized antibody, preferably derived from the human germline light chain IGKV1-33 sequence set forth in SEQ ID NO: 24; or derived from SEQ ID NO : Human germline light chain IGKV1-9 sequence shown in 26.

Preferably, the amino acid sequence of the light chain variable region of the humanized antibody is as set forth 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 kappa, lambda chain or variant thereof. Further preferably, the light chain sequence of the humanized antibody is the sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 20 or a variant thereof; said variant preferably having a light chain variable region The amino acid change of 0-10 is preferably a mutation of amino acid positions 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 comprising a human IgG1, IgG2 or IgG4 heavy chain FR In the region, it is more preferable to use IgG1 which enhances the toxicity of antibody-dependent cell-mediated cytotoxicity (antibody-dependent cell-mediated cytotoxicity). The heavy chain FR region sequence on the heavy chain variable region of the humanized antibody, preferably derived from the human germline heavy chain IGHV3-23 sequence set forth in SEQ ID NO: 23; or derived from SEQ ID NO The human germline heavy chain IGHV1-2 sequence shown in :25.

Preferably, the amino acid sequence of the heavy chain variable region of the humanized antibody is as set forth in SEQ ID NO: 27 or SEQ ID NO: 31 of 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 set forth in SEQ ID NO: 17 or SEQ ID NO: 19 or a variant thereof; the variant preferably has a variable region in the heavy chain The amino acid change of 0-10 is preferably a mutation of amino acid positions 9, 13, and 49, the amino acid after the 9th mutation is proline (P), and the amino acid after the 13th mutation is glutamine (Q). The amino acid after the 49th mutation 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 heavy chain variable region sequence of the humanized antibody huA9 is SEQ ID NO: 31 Shown that the light chain variable region sequence is set forth in SEQ ID NO: 33; the heavy chain variable region sequence of the humanized antibody huA3 is set forth in SEQ ID NO:27, and the light chain variable region sequence is SEQ. ID NO: 30 is shown. More preferably, the humanized antibody huA9 comprises the heavy chain antibody sequence SEQ ID NO: 19, and the light chain antibody sequence SEQ ID NO: 20; wherein the humanized antibody huA3 comprises the heavy chain antibody sequence SEQ ID NO :17, and the light chain antibody sequence SEQ ID NO:18.

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

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

The invention further provides an expression vector comprising the DNA sequence as described above.

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

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

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

In a preferred embodiment of the 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 invention also provides a multispecific antibody comprising a light chain variable region and a heavy chain variable region as previously described.

The invention also provides a single chain antibody comprising a light chain variable region and a heavy chain variable region as previously described.

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

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

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

DRAWINGS

FIG 1 is an antibody in vitro ELISA binding experiments, murine antibodies displayed A3 and A9 were purified h-B7H3-Fc-antigen binding activity, EC ₅₀ about 0.03ug / mL.

Figure 2 is a graph showing the in vitro binding activity of chimeric antibodies to CHO cells highly expressing B7-H3. Murine antibodies A3 and A9, chimeric antibodies A3C and A9C all showed significant binding activity to target cells at nanomolar (nM) concentration levels.

Figure 3 is a graph showing the in vitro binding activity of humanized antibodies to MDA-MB-231 cells highly expressing B7-H3. Both huA3 and huA9 have significant binding activity to target cells at nanomolar levels.

Detailed description of the invention

First, the term

In order to more easily understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined 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 code and the one-letter code for amino acids used in the present invention are as described in J. Biol. Chem, 243, p3558 (1968).

The term "antibody" as used in the present invention refers to an immunoglobulin which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds. The immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, respectively. , α chain and ε chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either a kappa chain or a lambda chain by the constant region. Each of the five types of Ig may have a kappa chain or a lambda chain.

In the present invention, the antibody light chain variable region of the present invention may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or a variant thereof.

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

The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C region). The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR). Each of the light chain variable region (VL) and the heavy chain variable region (VH) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The CDR amino acid residues of the VL and VH regions of the antibodies or antigen-binding fragments of the present invention conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3) in number and position, or conform to the Kabat and Chothia numbering rules ( HCDR1).

The term "antigen presenting cell" or "APC" is a cell which displays a foreign antigen complexed with MHC on its surface. T cells recognize this complex using the T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (DC), peripheral 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 MHC-I/MHC-II conjugates.

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 immunological checkpoint proteins expressed on the surface of antigen-presenting cells or cancer cells, and has an inhibitory effect on the functional activation of T cells. The term "B7-H3" includes any variant or isoform of B7-H3 naturally expressed by a cell. The antibodies of the invention can be cross-reactive with B7-H3 from non-human species. Alternatively, the antibody may also be specific for human B7-H3 and may not exhibit cross-reactivity with other species. B7-H3, or any variant or isoform thereof, can be isolated from cells or tissues in which they are naturally expressed, or produced by recombinant techniques using techniques common in the art and 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 which are prepared, expressed, created or isolated by recombinant methods, and the techniques and methods involved are well known in the art, such as (1) transgenes from human immunoglobulin genes, transgenic chromosomes. An antibody isolated from an animal (eg, a mouse) or a hybridoma prepared therefrom; (2) an antibody isolated from a host cell transformed with an antibody, such as a transfectoma; (3) isolated from a recombinant combinatorial human antibody library And (4) an antibody prepared, expressed, created or isolated by splicing a human immunoglobulin gene sequence to other DNA sequences. Such recombinant human antibodies comprise variable regions and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as occur during antibody maturation.

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

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

The term "humanized antibody", also referred to as a CDR-grafted antibody, refers to an antibody produced by grafting a CDR sequence of a mouse into a human antibody variable region framework. It is possible to overcome the strong immune response induced by chimeric antibodies by carrying a large amount of mouse protein components. To avoid a decrease in activity while reducing immunogenicity, the human antibody variable region can be subjected to minimal reverse mutation to maintain activity.

The term "chimeric antibody" is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody. To establish a chimeric antibody, a hybridoma that secretes a murine-specific monoclonal antibody is selected, and then the variable region gene is cloned from the mouse hybridoma cell, and the constant region gene of the human antibody is cloned as needed to change the mouse. The region gene and the human constant region gene are ligated into a chimeric gene and inserted into a human vector, and 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 region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising a human IgG2 or IgG4 heavy chain constant region, or without ADCC (antibody-dependent) after amino acid mutation Cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG1.

The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and an antibody analog, which typically includes at least a portion of an antigen binding or variable region (eg, one or more CDRs) of a parental antibody. The antibody fragment retains at least some of the binding specificity of the parent antibody. Generally, when the activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parental 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 to 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 comprising the CH1 and CH2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic action of the CH3 domain.

A "Fab' fragment" contains a light chain and a portion of a heavy chain comprising a VH domain and a CH1 domain and a region between the CH1 and CH2 domains, thereby being between the two heavy chains of the two Fab' fragments An interchain disulfide bond is formed to form a F(ab')2 molecule.

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

The "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 to encompass antibodies having 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; having two or more Antibodies of the VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region Different targets or different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, covalently or non-covalently linked Along with antibody fragments and the like.

The term "single-chain antibody" is a single-stranded recombinant protein joined by a heavy chain variable region (VH) and a light chain variable region (VL) of a antibody via a ligation peptide, which is the smallest with complete antigen binding sites. Antibody fragment.

The term "domain antibody fragment" is an immunologically functional immunoglobulin fragment containing only a heavy chain variable region or a light chain variable region chain. In some cases, two or more VH regions are covalently joined 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 means that it can interact with human B7-H3. The term "antigen binding site" as used in the present invention refers to a three-dimensional spatial site that is discrete on an antigen and is recognized by an antibody or antigen-binding fragment of the present invention.

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

As used herein, the terms "specifically bind" and "selectively bind" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, when recombinant human B7-H3 is used as an analyte and an antibody is used as a ligand, the antibody has an equilibrium solution of less than about 10 ^-7 M or even less when measured by surface plasmon resonance (SPR) techniques in an instrument. The isolating constant (K _D ) binds to a predetermined antigen, and its affinity for binding to a predetermined antigen is at least twice its affinity for binding to a non-specific antigen other than the predetermined antigen or a closely related antigen (such as BSA, etc.). The term "antibody recognizing an antigen" can be used interchangeably herein with the term "specifically bound antibody".

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

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

The term "inhibiting growth" (eg, involving cells) is intended to include any measurable reduction in cell growth.

The terms "inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to the stimulation (ie, passive or adaptive) of an immune response to a particular antigen. The term "inducing" for inducing CDC or ADCC refers to stimulating a specific direct cell killing mechanism.

The "ADCC" described in the present invention, that is, antibody-dependent cell-mediated cytotoxicity, is an antibody-dependent cell-mediated cytotoxicity, which means that a cell expressing an Fc receptor directly kills an antibody by Fc segment of the recognition antibody. Target cells. The ADCC effector function of the antibody can be reduced or eliminated by modification of the Fc fragment on IgG. Said modification refers to mutations in the heavy chain constant region 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 the Cold Spring Harbor Antibody Technical Guide, Chapters 5-8 and 15. For example, a mouse can be immunized with human B7-H3 or a fragment thereof, and the obtained antibody can be renatured, purified, and subjected to amino acid sequencing by a conventional method. The antigen-binding fragment can also be prepared by a conventional method. The antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions in a non-human CDR region. Human FR germline sequences are available on the website of ImMunoGeneTics (IMGT) http://imgt.cines.fr or from the Journal of Immunoglobulins, 2001 ISBN 014441351.

The engineered antibodies or antigen-binding fragments of the invention can be prepared and purified by conventional methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the FC region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones were expanded in serum-free medium in a bioreactor to produce antibodies. The culture medium from which the antibody is secreted can be purified and collected by a conventional technique. The antibody can be concentrated by filtration in a conventional manner. Soluble mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange. The resulting product needs to be frozen immediately, such as -70 ° C, or lyophilized.

The antibody of the present invention refers to a monoclonal antibody. The monoclonal antibody (mAb) of the present invention refers to an antibody obtained from a single clonal cell strain, and the cell strain is not limited to a eukaryotic, prokaryotic or phage clonal cell strain. Monoclonal antibodies or antigen-binding fragments can be obtained recombinantly using, for example, hybridoma technology, recombinant techniques, phage display technology, synthetic techniques (e.g., CDR-grafting), or other prior art techniques.

"Administration" and "treatment" when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic agent, diagnostic agent or composition and animal, human, subject Contact of the test subject, cell, tissue, organ or biological fluid. "Administration" and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells. "Administering" and "treating" also means treating, for example, cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell. "Treatment", when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.

"Treatment" means administering to a patient a therapeutic agent for internal or external use, such as a composition comprising any of the binding compounds of the present invention, the patient having one or more symptoms of the disease, and the therapeutic agent is known to have Therapeutic effect. Generally, a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases, whether by inducing such symptoms to degenerate or inhibiting the progression of such symptoms to any degree of clinical right measurement. The amount of therapeutic agent (also referred to as "therapeutically effective amount") effective to alleviate 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 a desired effect in the patient. Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition. Embodiments of the invention (e.g., methods of treatment or preparations) may be ineffective in alleviating the symptoms of a target disease in each patient, but according to any statistical test methods known in the art such as Student's t test, chi-square test, according to Mann It was determined by Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test that it should alleviate the target disease symptoms in a statistically significant number of patients.

The term "consisting essentially of" or variations thereof, as used throughout the specification and claims, includes all such elements or groups of elements, and optionally includes other elements that are similar or different in nature to the elements, the other The element does not significantly alter the essential or novel properties of a given dosage regimen, method or composition. As a non-limiting example, a binding compound consisting essentially of the amino acid sequence recited may also include one or more amino acids that do not significantly affect the properties of the binding compound.

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

An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate the diagnosis. An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the route and dosage of the method of administration, and the severity of the side effects. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

"Exogenous" refers to a substance that is produced outside of a living organism, cell, or human body depending on the background. "Endogenous" refers to a substance produced in a cell, organism or human body depending on the background.

"Homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in both comparison sequences are 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 molecule is homologous at that position . The percent homology between the two sequences is a function of the number of matches or homology positions shared by the two sequences divided by the number of positions compared × 100%. For example, in the optimal alignment of sequences, if there are 6 matches or homologs in 10 positions in the two sequences, then the two sequences are 60% homologous. In general, comparisons are made when the maximum sequence of homology is obtained by aligning the two sequences.

The expression "cell", "cell line" and "cell culture" as used herein are used interchangeably and all such names include their progeny. Thus, the words "transformants" and "transformed cells" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included. In the case of a different name, it is clearly visible from the context.

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

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

detailed description

The invention is further described in the following examples, but these examples are not intended to limit the scope of the invention. The experimental methods in the examples of the present invention which do not specify the specific conditions are usually in accordance with conventional conditions, such as the cold spring harbor antibody technology experiment manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer. Reagents without specific source are routine reagents purchased from the market.

Example 1 Sequence of immunizing antigen, screening antigen and preparation thereof

The human B7-H3 (h-B7H3-his) sequence encoding the His tag, and the human B7-H3 (h-B7H3-Fc) sequence encoding the huFc tag were synthesized by Integrated DNA Technology (IDT) (recombinant B7-H3 above) The proteins were all designed into the pTT5 vector (Biovector) by the design of the template sequence of the present invention. The recombinant B7-H3 protein was purified by Example 2 after expression in 293T cells. 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 in SEQ ID NO. 21 and 22, respectively, in the Sequence Listing.

Example 2 Preparation of B7-H3 recombinant protein

1. Purification steps of His-tagged B7H3 recombinant protein:

The supernatant sample of the HEK293 cell sheet (purchased from the American type culture collection, ATCC) was 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. The nickel column was equilibrated with PBS solution containing 5 mM imidazole and rinsed 2-5 column volumes. The displaced supernatant sample was applied to the column. The column was washed with PBS containing 5 mM imidazole until the A280 reading dropped to baseline. The column was washed with PBS + 10 mM imidazole, the non-specifically bound heteroprotein was removed, and the effluent was collected. The protein of interest was eluted with PBS containing 300 mM imidazole, and the eluted peak was collected.

The collected eluate was further purified by ion exchange (SP column). Configure solution A: 0.01 M PB, pH 8.0. Configure liquid B: liquid A + 1 M NaCl. First, the imidazole in PBS solution was eluted to the A solution, and the SP column was equilibrated with the A solution. The concentration of the B solution was 0-100%, and 10 column volumes were eluted to collect the elution peaks. The obtained protein was electrophoresed, peptide map, and LC-MS was identified and used.

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

The supernatant sample expressed by HEK293 cells was centrifuged at high speed to remove impurities, and the buffer was exchanged for PBS. The Protein A affinity column was equilibrated with 10 mM phosphate buffer and rinsed 2-5 column volumes. The displaced supernatant sample was applied to the column. Rinse the column with 25 column volumes of buffer until the A280 reading drops to baseline. The target protein was eluted with 0.8% acetate buffer at pH 3.5, and the elution peak was collected. Immediately after the addition, the mixture was neutralized with 1 M Tris-Cl pH 8.0 buffer, and then the solution was replaced with Millipore's Amico-15 filter column. . The obtained protein was electrophoresed, peptide map, and LC-MS was identified and used.

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

The full-length sequence encoding the human or cynomolgus B7-H3 protein (huB7H3 or cyB7H3) was synthesized by Integrated DNA Technology (IDT) (the above B7-H3 recombinant proteins were designed by the template sequence of the present invention) and cloned into pcDNA3.1/, respectively. Puro (Invitrogen #V79020). CHO-S (ATCC) cells were cultured to 0.5 x 10 ⁶ /ml in CD-CHO medium (Life Technologies, #10743029). 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 Opti-MEM medium (Life Technologies, #31985088), incubated for 20 minutes at room temperature, and then added to CHO cell culture medium. And put it into a carbon dioxide incubator for cultivation. After 24 hours, the new medium was replaced and 10 μg/ml of puromycin was added. After that, the new culture solution was changed every 2-3 days, and after 10-12 days of screening, a stable CHO-S cell pool was obtained.

Example 3 Preparation of antibodies

CRL-8287 ^TM)进行融合得到杂交瘤细胞。 Anti-human B7H3 monoclonal antibodies are produced by immunizing mice. The experiment used Swiss Webster white mice, female, 6 weeks old (Charles River). Feeding environment: SPF level. After the mice were purchased, the laboratory environment was kept for 1 week, 12/12 hours light/dark cycle adjustment, temperature 20-25 ° C; humidity 40-60%. The immunizing antigen is the Fc-tagged human B7H3 recombinant protein (huB7H3-Fc). Titermax (sigma Lot Num: T2684) was used as an adjuvant. The ratio of antigen to adjuvant (titermax) was 1:1, and the antigen was emulsified and inoculated for 0, 21, 35, 49, and 63 days. Day 0 intraperitoneal (IP) injection of 15 μg + footpad 25 / emulsified antigen. 21, 35, 49, 63 days of intraperitoneal (IP) injection of 15 μg + footpad 15 / emulsified antigen, boosted 3 days before spleen cell fusion, intraperitoneal (IP) injection 15μg + claw pad (footpad 15% of the antigen solution prepared in physiological saline. Blood tests were performed on the 42nd, 56th, and 70th day, and the serum of the mice was detected by ELISA and FACS to determine the antibody titer in the serum of the mice. After the fifth immunization, splenocytes were fused to mice with high antibody titers in serum and titers to the platform, and spleen lymphocytes and myeloma cells Sp2/0 cells (ATCC) were optimized using an electrofusion procedure.

CRL-8287 ^(TM ) was fused to obtain hybridoma cells.

After the conjugated hybridoma cells were cultured for 7-14 days, the supernatant of the culture medium was taken, and the hybridoma supernatant was subjected to antibody screening using the B7-H3 recombinant protein huB7H3-Fc. The positive antibody strain obtained was further stably expressed B7. -H3 CHO-S cells, comparing blank CHO-S cells to exclude non-specific binding antibody hybridoma strains, and screening by flow sorting method, thereby selecting two hybridomas that bind to recombinant protein and also bind to cell-expressing antigen . Hybridoma cells in logarithmic growth phase were collected, RNA was extracted with Trizol (Invitrogen, 15596-018) and reverse transcription ^{(PrimeScript TM Reverse Transcriptase, Takara #} 2680A). The cDNA obtained by reverse transcription was subjected to PCR amplification using a mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503), and then sequenced, and finally the sequences of two murine mouse antibodies A3 and A9 were obtained.

The heavy and light chain variable region sequences of murine mAb A3 are as follows:

Table 1 CDRs contained in the heavy and light chain variable regions of murine mAb A3

name sequence Numbering HCDR1 RYGMS SEQ ID NO: 3 HCDR2 ISSGGGSIYYPDTVKG SEQ ID NO: 4 HCDR3 TRHYLLFEMDY SEQ ID NO: 5

LCDR1 KASQNVNTAVA SEQ ID NO: 6 LCDR2 SASNRYT SEQ ID NO:7 LCDR3 QQYSSSLT SEQ ID NO:8

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

Table 2 CDRs contained in the heavy and light chain variable regions of murine mAb A9

name sequence Numbering HCDR1 DYAMH SEQ ID NO: 11 HCDR2 VISTYYGNTNYNQKFKG SEQ ID NO: 12 HCDR3 PVTTMVPRGGYYFDY SEQ ID NO: 13 LCDR1 RASKSINKYLA SEQ ID NO: 14 LCDR2 SGSTLQS SEQ ID NO: 15 LCDR3 QQHNEYPLT SEQ ID NO: 16

The heavy and light chain variable regions of each mouse antibody were cloned 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 anti-resistant The chimeric antibodies A3C and A9C of B7-H3 were purified, identified, and assayed for activity as described in Example 2 (purification with Fc-tag protein).

Example 4 Determination of in vitro binding activity of antibodies

The neutralizing avidin for binding to biotin was diluted to 1 μg/ml with PBS buffer, added to a 96-well plate in a volume of 100 μl/well, and left at 4 ° C for 16 h to 20 h. After washing the plate once with PBST (pH 7.4 PBS containing 0.05% Tween-20) buffer, 120 μl/well of PBST/1% milk was added, and the mixture was incubated for 1 hour at room temperature for blocking. After washing the plate once with PBST buffer, 1 μg/ml of biotin-labeled h-B7H3-Fc diluted with PBST/1% milk was added and incubated for 1 h at room temperature. After washing the plate three times with PBST buffer, the B7-H3 antibody to be tested diluted with PBST/1% milk was added to an appropriate concentration, and incubated at room temperature for 1.5 h. The reaction system was removed, and the plate was washed three times with PBST, and then a horseradish peroxidase (HRP)-labeled anti-mouse antibody secondary antibody (The Jackson Laboratory) diluted with PBST/1% milk was added at 100 μl/well. Incubate for 1 h at room temperature. After washing the plate 3 times with PBST, 100 μl/well of TMB was added and incubated for 5-10 min at room temperature. The reaction was stopped by the addition of 100 μl/well of 1 MH ₂ SO ₄ , and the absorbance was read at 450 nm, and the ELISA binding EC ₅₀ value was calculated. As a result, as shown in Fig. 1, the EC _{50 of the} antibodies A3 and A9 were both about 0.03 μg/mL, and the murine antibodies A3 and A9 all had binding activity to the purified h-B7H3-Fc antigen.

CHO-S cells highly expressing huB7-H3 were centrifuged at 1000 rpm for 5 minutes, and the precipitate was collected and suspended in 10-15 ml of pre-cooled flow buffer, and the cells were counted. The cells were collected by centrifugation at 1000 rpm for 5 minutes in a 50 ml centrifuge tube, the supernatant was discarded, and the pellet was resuspended in a pre-cooled blocking buffer at a density of 0.5-1.0 x 10 ⁷ cells/ml. After incubating for 30 minutes at 4 ° C, it was resuspended to 100 μl per well and added to a 96-well plate. After centrifugation of the 96-well plate at 1500 rpm for 5 minutes, the supernatant was discarded. 100 μl of the antibody to be tested was added to each well at a concentration gradient of 0.01 nM to 670 nM, the cells were resuspended, and incubated at 4 ° C for 60 minutes in the dark. The supernatant was discarded by centrifugation, and 100 μl of a 1:400 diluted FITC-labeled secondary antibody (BD Biosciences) was added. The cells were resuspended and incubated for 60 minutes at 4 ° C in the dark. The cells were washed twice with flow buffer and resuspended in 1% paraformaldehyde for flow cytometry. As shown in Fig. 2, A3, A9 and their corresponding chimeric antibodies all showed significant binding to huB7H3 high expressing cells at nanomolar (nM) concentration level, and their binding was stronger than that of Macrogenics' reference antibody BRCA84D.

In the same manner, CHO-S cells with high expression of cyB7-H3 were used to detect the binding of each antibody to cynomolgus B7-H3 at a single concentration of 10 nM. The results are shown in Table 3 below. A3C has obvious binding to monkey antigen. Signal, while A9C and the reference antibody BRCA84D have no binding.

table 3

Antibody name A3C A9C BRCA84D Negative control Signal strength (MFI) 54.5 4.59 5.25 3.98

Example 5 In vitro binding affinity and kinetic experiments

This experiment was measured by surface plasmon resonance (SPR) method. The anti-mouse IgG polyclonal antibody was covalently linked to a CM5 (GE) chip using a standard amino coupling method using a kit supplied by Biacore, and then the purified mouse antibody to be tested of the present invention was captured to the stationary phase using the antibody. . The h-B7H3-Fc or h-B7H3-His protein (Example 1) diluted in the same buffer at a concentration of 12.5-800 nM was injected at each cycle before and after the injection, and the regeneration reagent was regenerated with the reagent in the kit. . Antigen-antibody binding kinetics were followed for 3 minutes and dissociation kinetics were followed for 10 minutes. The data were analyzed using the 1:1 (Langmuir) binding model using GE's BIAevaluation software, and the k _a (k _on ), kd (k _off ), and K _D values determined by this method are shown in Table 4 below.

Table 4

Example 6 Mouse antibody humanization experiment

Humanization of murine anti-human B7-H3 monoclonal antibodies was performed as disclosed in many literatures in the art. Briefly, the human constant domain is used in place of the parental (murine antibody) constant domain, and the human antibody sequence is selected based on the homology of the murine antibody and the human antibody. The present invention humanizes the candidate molecules A3 and A9.

Based on the typical structure of the obtained murine antibody VH/VL CDR, the heavy and light chain variable region sequences were compared with the human antibody germline database to obtain a human germline template with high homology. Wherein 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, and 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 preferably human germline light chain template IGkV1-33 (SEQ ID NO: 24):

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

A9 is preferably a human germline light chain template IGkV1-9 (SEQ ID NO: 26):

The CDR regions of murine antibodies A3 and A9 are grafted onto the selected corresponding humanized template, the humanized variable region is replaced, and recombined with the IgG constant region (preferably the heavy chain is IgG1 and the light chain is κ). Then, based on the three-dimensional structure of the murine antibody, the residues which have an important interaction with the CDRs and the residues of the CDRs, and the residues which have an important influence on the conformation of VL and VH are subjected to back mutation and the CDR regions are The chemically labile amino acid residues were optimized, and antibodies assembled from the following humanized light and heavy chain variable region sequences were designed and tested.

The final humanized huA3 (using the H1 heavy chain and the L2 light chain) and the huA9 antibody molecule (using the H1 heavy chain and the L1 light chain) were selected by expression test and back mutation number comparison, the respective sequences of which are SEQ ID NO: 17-20.

The last three nucleotides "TGA" of the following gene sequences SEQ ID NOs: 35-38 are stop codons and do not encode any amino acids.

huA3 antibody heavy chain sequence:

huA3 antibody heavy chain sequence encoding gene sequence:

huA3 antibody light chain sequence:

huA3 antibody light chain sequence encoding gene sequence:

huA9 antibody heavy chain sequence:

huA9 antibody heavy chain sequence encoding gene sequence:

huA9 antibody light chain sequence:

The huA9 antibody light chain sequence encodes a gene sequence (wherein the first 60 nucleotides of the following sequence are not involved in the coding of the final huA9 antibody light chain):

A cDNA fragment was synthesized based on the gene sequences of the above humanized antibody light and heavy chains, and inserted into a pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). The expression vector and the 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 CO ₂ incubator for incubation 4- 5 days. After the expressed antibody was recovered by centrifugation, antibody purification was carried out by the method of Example 2 (purification with Fc-tag protein) to obtain a humanized antibody protein of the present invention.

Example 7 Humanized Antibody Activity Assay

The following experimental assays were performed on huA3 and huA9 and other derivatized humanized antibodies in vitro:

1. Cell binding assay (method procedure is the same as in Example 4). The results are shown in Figure 3. Humanized antibodies huA3 and huA9 are positively bound to MDA-MB-231 cells with high expression of B7-H3, and binding ability and incorporation The antibody A3C is equivalent.

2. Affinity kinetics experiment (the method steps are the same as in Example 5). The results are shown in the following table. The final preferred humanized antibodies huA3 and huA9 have a KD of less than 1 nM for the human B7-H3 antigen protein, showing a strong Affinity.

table 5

While the invention has been described with respect to the preferred embodiments of the embodiments of the embodiments of the invention modify. Accordingly, the scope of the invention is defined by the appended claims.

Claims (20)

An anti-B7-H3 antibody or antigen-binding fragment thereof comprising: An antibody light chain variable region comprising at least one LCDR selected from the group consisting of 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; An antibody heavy chain variable region comprising 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 anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 1, wherein the LCDR comprises one or more of LCDR1, LCDR2 and LCDR3, and the amino acid sequence of the LCDR1 is SEQ ID NO: 6 or SEQ ID NO: 14; the amino acid sequence of the LCDR2 is SEQ ID NO: 7 or SEQ ID NO: 15 in the Sequence Listing; the amino acid sequence of the LCDR3 is SEQ ID NO: 8 or SEQ ID in the Sequence Listing. NO: 16; And/or, the HCDR comprises one or more of HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 being represented by SEQ ID NO: 3 or SEQ ID NO: 11 in the Sequence Listing; 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. The anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 2, wherein said antibody light chain variable region comprises a sequence as set forth in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. LCDR1, LCDR2 and LCDR3, or LCDR1, LCDR2 and LCDR3 having sequences as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; And/or the antibody heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 sequences as set forth in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, or comprises the sequences respectively as SEQ ID NO: 11, HCDR1, HCDR2 and HCDR3 represented by SEQ ID NO: 12 and SEQ ID NO: 13. The anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 3, wherein said antibody light chain variable region comprises, for example, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. LCDR1, LCDR2 and LCDR3 are shown; and the antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively; Alternatively, wherein the antibody light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; the antibody heavy chain variable region comprises For example, 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 antigen-binding fragment thereof according to any one of claims 1 to 4, 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 5, 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. The amino acid sequence of the light chain variable region is set forth in SEQ ID NO. 2 of the Sequence Listing; or wherein the amino acid sequence of the murine antibody or the heavy chain variable region of the chimeric antibody is SEQ ID NO in the Sequence Listing As shown in .9, the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 10 in the Sequence Listing. The anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 5, wherein the light chain FR region sequence on the variable region of the humanized antibody light chain is derived from the human as set forth in SEQ ID NO: The germline light chain IGKV1-33 sequence; or the human germline light chain IGKV1-9 sequence as set forth in SEQ ID NO:26. The anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 5, wherein the humanized antibody light chain sequence is the sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 20 or a variant thereof; Preferably, the variant has a 0-10 amino acid change in the light chain variable region, preferably a mutation in the 4 and 9 amino acid positions. The anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 5, 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 Preferably, it comprises a human IgG1, IgG2 or IgG4 heavy chain FR region, more preferably IgG1 which enhances ADCC toxicity after amino acid mutation. The anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 5, wherein the heavy chain FR region sequence on the heavy chain variable region of the humanized antibody is derived from a human as set forth in SEQ ID NO: The germline heavy chain IGHV3-23 sequence; or the human germline heavy chain IGHV1-2 sequence as set forth in SEQ ID NO:25. The anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 5, wherein the humanized antibody heavy chain sequence is the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 19 or a variant thereof; Preferably, the variant has a 0-10 amino acid change in the heavy chain variable region, preferably a mutation at the amino acid positions 9, 13, and 49. The anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 5, wherein the humanized antibody is a humanized antibody huA9 or a humanized antibody huA3; The heavy chain variable region sequence of the humanized antibody huA9 is set forth in SEQ ID NO: 31, and the light chain variable region sequence is set forth in SEQ ID NO: 33; The heavy chain variable region sequence of the humanized antibody huA3 is set forth in SEQ ID NO:27, and the light chain variable region sequence is set forth in SEQ ID NO:30. The anti-B7-H3 antibody or antigen-binding fragment thereof according to claim 12, wherein 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; The humanized antibody huA3 comprises the heavy chain antibody sequence SEQ ID NO: 17, and the light chain antibody sequence SEQ ID NO: 18. A DNA sequence encoding an anti-B7-H3 antibody or antigen-binding fragment of any of claims 1-13. An expression vector comprising the DNA sequence of claim 14. A host cell comprising the expression vector of claim 15. The host cell according to claim 16, wherein the host cell is a bacterial, yeast or mammalian cell; The bacterium is preferably Escherichia coli; The yeast is preferably Pichia pastoris; The mammalian cells are preferably Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells. A pharmaceutical composition comprising the anti-B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 and a pharmaceutically acceptable excipient, diluent or carrier. Use of an anti-B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 for the preparation of a medicament for the treatment or prevention of a B7-H3 mediated disease or condition; wherein said disease is preferred More preferably, it is a cancer expressing B7-H3; 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. A method of treating and preventing a B7-H3 mediated disease or condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of the anti-B7-H3 antibody of any of claims 1-13 or antigen binding thereof A fragment, or a pharmaceutical composition according to claim 18, wherein said disease is preferably cancer; more preferably a cancer expressing B7-H3; said cancer being most preferably breast cancer, ovarian cancer, prostate cancer, pancreas Cancer, kidney cancer, lung cancer, liver cancer, stomach cancer, colon cancer, bladder cancer, esophageal cancer, gallbladder cancer, cervical cancer, glioblastoma and melanoma.

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