Anti-human B7-H3 antibody and its use

Abstract

Anti-human B7-H3 monoclonal antibodies and their uses are provided. Using the recombinant human B7-H3 extracellular domain as an immunogen, murine anti-human B7-H3 monoclonal antibodies can be produced using hybridoma technology. These murine anti-human B7-H3 antibodies can bind to various domains of the B7-H3 extracellular domain. Human-mouse chimeric antibodies constructed based on murine antibodies can specifically bind to B7-H3 on the cell surface. Humanized antibodies produced by CDR transplantation and CDR mutation retain the ability to specifically bind to the human B7-H3 extracellular domain and cell membrane surface B7-H3 and can mediate intracellular internalization via cell membrane surface B7-H3.

Description

[Technical Field]

[0001] This application was filed with the China National Intellectual Property Administration on December 2, 2020, and claims priority and benefits of China Patent Application No. 202011398727.1. This patent application, in its entirety, constitutes part of this specification by reference.

[0002] This invention belongs to the field of antibody engineering and, more particularly, to anti-human B7-H3 monoclonal antibodies and their uses. In particular, this invention relates to monoclonal antibodies, chimeric antibodies, and humanized antibodies that have high affinity for human B7-H3, cross-react with monkey B7-H3, and do not bind to mouse B7-H3, as well as their uses. [Background technology]

[0003] B7-H3, also known as CD276, is a type I transmembrane protein belonging to the B7-CD28 superfamily. Molecules in the B7 family can provide stimulating signals that enhance and maintain T cell immune responses, and can also generate inhibitory signals that limit and attenuate T cell immune responses. Human B7-H3 protein exists in two forms: membrane-bound and soluble. Membrane-bound B7-H3 protein is mainly distributed on the surface of tumor cells, and is also found in the nucleus, transporters, and exosomes within cells. Soluble B7-H3 is hydrolyzed from the cell membrane by proteases, and high levels of soluble B7-H3 can be detected in the serum of tumor patients, suggesting that B7-H3 can be used as a biomarker (Non-Patent Literature 1). Recent clinical and commercial successes of anti-cancer antibodies have drawn significant interest in antibody-based therapies. Therefore, there is still room for research into B7-H3-based antibody therapies for the treatment of cancer. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] xiec, 2016 [Overview of the Initiative]

[0005] To address the shortcomings of conventional anti-human B7-H3 antibodies, such as off-target effects and adverse reactions caused by insufficient affinity and specificity for B7-H3 proteins on the cell membrane surface, this disclosure provides anti-human B7-H3 monoclonal antibodies and their applications. Mouse anti-human B7-H3 monoclonal antibodies were produced by hybridoma technology using recombinant human B7-H3 extracellular domains as immunogens. Mouse anti-human B7-H3 antibodies could specifically bind to various B7-H3 extracellular domains, and human-mouse chimeric antibodies constructed based on mouse antibodies could specifically bind to B7-H3 on the cell surface. Humanized antibodies produced by CDR grafting and CDR mutation retained the ability to specifically bind to human B7-H3 extracellular domains and B7-H3 on the cell membrane surface, and could also be mediated by B7-H3 on the cell membrane surface and translocate into cells. Anti-human B7-H3 humanized antibody drug conjugates (ADCs) have been shown to specifically kill tumor cells in cytological experiments and nude mouse transplant tumor models. Anti-human B7-H3 antibodies inhibit the extracellular domain of human B7-H3 by 5 × 10⁻¹⁴ units. -10 The antibody exhibits affinity less than M, specifically binding to B7-H3 on the surface of recombinant and tumor cells. It contributes significantly to tumor-killing effects in cell and animal models, and no apparent adverse reactions were observed. Therefore, the anti-human B7-H3 antibody has excellent prospects for clinical antitumor applications.

[0006] Antibodies and methods for producing them According to one embodiment of the present invention, the present disclosure provides an anti-human B7-H3 antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region. According to the embodiment of the present invention, the heavy chain variable region (VH) comprises complementarity-determining regions (CDRs) 1, 2, and 3, wherein CDR1 of VH comprises an amino acid sequence having at least 75% identity to the amino acid sequence of a selected CDR1 of VH, CDR2 of VH comprises an amino acid sequence having at least 75% identity to the amino acid sequence of a selected CDR2 of VH, and CDR3 of VH comprises an amino acid sequence having at least 75% identity to the amino acid sequence of a selected CDR3 of VH, and the light chain variable region (VL) comprises CDR1, CDR2, and CDR3. Here, VL's CDR1 includes an amino acid sequence having at least 75% identity with the selected VL's CDR1, VL's CDR2 includes an amino acid sequence having at least 75% identity with the selected VL's CDR2, and VL's CDR3 includes an amino acid sequence having at least 75% identity with the selected VL's CDR3, where the amino acid sequences of the selected VH's CDR1, CDR2, and CDR3, and the amino acid sequences of the selected VL's CDR1, CDR2, and CDR3, are selected from the group consisting of the following: The amino acid sequences of CDR1, CDR2, and CDR3 of the selected VH are as shown in SEQ ID NOs. 40, 41, and 42, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the selected VL are as shown in SEQ ID NOs. 43, 44, and 45, respectively. The amino acid sequences of CDR1, CDR2, and CDR3 of the selected VH are as shown in SEQ ID NOs. 46, 47, and 48, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the selected VL are as shown in SEQ ID NOs. 49, 50, and 51, respectively, and The amino acid sequences of CDR1, CDR2, and CDR3 of the selected VH are as shown in SEQ ID NOs. 52, 53, and 54, respectively, and the amino acid sequences of CDR1, CDR2, and CDR3 of the selected VL are as shown in SEQ ID NOs. 55, 56, and 57, respectively.

[0007] The anti-human B7-H3 antibody or fragment thereof according to embodiments of the present invention can specifically bind to the extracellular domain of human B7-H3 and to sites on the cell membrane surface of B7-H3, exhibiting strong affinity, for example, in some embodiments, 5 × 10⁻¹⁶ -10 In addition to having an affinity of less than M, it possesses good tumor inhibitory effects and low toxicity, giving it excellent prospects for clinical use.

[0008] Furthermore, the antibody or antigen-binding fragment according to the above embodiments of the present invention may have the following additional technical features:

[0009] According to embodiments of the present invention, an amino acid sequence having at least 75% identity with a selected VL CDR3 containing the amino acid sequence shown in Sequence ID No. 51 is QQWSX1X2PLT (where X1 is S or A, and X2 is N, A, S, or Q).

[0010] According to embodiments of the present invention, VH comprises an amino acid sequence having at least 75% identity with SEQ ID NO: 1, and VL comprises an amino acid sequence having at least 75% identity with SEQ ID NO: 2.

[0011] According to embodiments of the present invention, VH comprises an amino acid sequence having at least 75% identity with SEQ ID NO: 3, and VL comprises an amino acid sequence having at least 75% identity with SEQ ID NO: 4.

[0012] According to an embodiment of the present invention, VH contains an amino acid sequence having at least 75% identity to SEQ ID NO: 5, and VL contains an amino acid sequence having at least 75% identity to SEQ ID NO: 6.

[0013] Specifically, the at least 75% identity can be at least 82%, preferably at least 85%, more preferably at least 90%, still more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identity, or any percentage of identity of 75% or more. More specifically, 0, 1, 2, or more amino acids may be inserted, deleted, or substituted.

[0014] It is further explained that the sequence of the heavy chain variable region of an antibody or antigen-binding fragment can be obtained by inserting one or more amino acids into the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, deleting one or more amino acids from these amino acid sequences, mutating one or more amino acids in these amino acid sequences, or modifying one or more amino acids in these amino acid sequences.

[0015] According to an embodiment of the present invention, VH contains the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 17, and VL contains the amino acid sequence shown in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0016] According to an embodiment of the present invention, VH contains the amino acid sequence shown in SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23, and VL contains the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.

[0017] According to an embodiment of the present invention, VH contains the amino acid sequence shown in SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37, and VL contains the amino acid sequence shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33.

[0018] According to an embodiment of the present invention, the anti-human B7-H3 antibody or a fragment thereof can specifically bind to the human B7-H3 extracellular domain and has an affinity constant (KD) of less than 5×10 -10 M for the human B7-H3 extracellular domain.

[0019] According to an embodiment of the present invention, the anti-human B7-H3 antibody or a fragment thereof has a cross-reaction with cynomolgus B7-H3 and no binding activity to mouse B7-H3.

[0020] According to an embodiment of the present invention, the anti-human B7-H3 antibody or a fragment thereof has a weak cross-reaction with cynomolgus B7-H3, and its binding activity to cynomolgus B7-H3 is lower than its binding activity to human B7-H3.

[0021] According to an embodiment of the present invention, the anti-human B7-H3 antibody or a fragment thereof can specifically bind to B7-H3 on the cell surface and can be internalized into the cell mediated by B7-H3.

[0022] Furthermore, according to another aspect of the present invention, the present disclosure provides an anti-human B7-H3 antibody or a fragment thereof. According to an embodiment of the present invention, the anti-human B7-H3 antibody or a fragment thereof can specifically bind to the human B7-H3 extracellular domain and has an affinity constant (KD) of less than 5×10 -10 M for the human B7-H3 extracellular domain.

[0023] According to embodiments of the present invention, an anti-human B7-H3 antibody or a fragment thereof has cross-reactivity with monkey B7-H3 and does not have binding activity to mouse B7-H3.

[0024] According to embodiments of the present invention, an anti-human B7-H3 antibody or a fragment thereof has a weak cross-reactivity with monkey B7-H3, and its binding activity to monkey B7-H3 is lower than its binding activity to human B7-H3.

[0025] According to embodiments of the present invention, an anti-human B7-H3 antibody or a fragment thereof can specifically bind to B7-H3 on the cell surface and can be interposed into the cell by B7-H3.

[0026] According to embodiments of the present invention, examples of antibodies include mouse antibodies, rabbit antibodies, human antibodies, and chimeric antibodies.

[0027] According to embodiments of the present invention, an anti-human B7-H3 antibody or a fragment thereof has a heavy chain CDR of a heavy chain variable region indicated by any one amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, and / or a light chain CDR of a light chain variable region indicated by any one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6.

[0028] According to embodiments of the present invention, an anti-human B7-H3 antibody or a fragment thereof has a heavy chain variable region represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, and / or a light chain variable region represented by any one amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6.

[0029] According to embodiments of the present invention, an anti-human B7-H3 antibody or its fragment is The heavy chain variable region shown in Sequence ID No. 1 and the light chain variable region shown in Sequence ID No. 2, The heavy chain variable region shown in Sequence ID No. 3 and the light chain variable region shown in Sequence ID No. 4, or The heavy chain variable region shown in Sequence ID No. 5 and the light chain variable region shown in Sequence ID No. 6, It holds.

[0030] According to yet another aspect of the present invention, the disclosure provides an anti-human B7-H3 humanized antibody or a fragment thereof. According to an embodiment of the present invention, the anti-human B7-H3 humanized antibody or a fragment thereof is obtained by CDR grafting based on the above anti-human B7-H3 antibody or a fragment thereof.

[0031] According to embodiments of the present invention, an anti-human B7-H3 humanized antibody or a fragment thereof is A heavy chain variable region indicated by any one sequence selected from the group consisting of SEQ ID NOs: 11 and 17, or their heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, A light chain variable region indicated by any one sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15, and 16, or light chain CDR1, CDR2, and CDR3 thereof. It holds.

[0032] According to embodiments of the present invention, an anti-human B7-H3 humanized antibody or a fragment thereof is A heavy chain variable region indicated by any one sequence selected from the group consisting of SEQ ID NOs: 18, 21, 22, and 23, or their heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, A light chain variable region indicated by any one sequence selected from the group consisting of sequence numbers 19 and 20, or light chain CDR1, light chain CDR2, and light chain CDR3 thereof. It holds.

[0033] According to embodiments of the present invention, an anti-human B7-H3 humanized antibody or a fragment thereof is A heavy chain variable region indicated by any one sequence selected from the group consisting of SEQ ID NOs. 24, 34, 35, 36, and 37, or their heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, A light chain variable region represented by any one sequence selected from the group consisting of SEQ ID NOs. 25, 26, 27, 28, 29, 30, 31, 32, and 33, or their light chain CDR1, CDR2, and CDR3, It holds.

[0034] In yet another aspect of the present invention, the disclosure provides a method for producing a humanized anti-human B7-H3 antibody or fragment thereof. According to an embodiment of the present invention, a non-human anti-B7-H3 antibody or fragment thereof is used as a basis, and humanization of the heavy chain variable region and / or the light chain variable region is achieved using CDR grafting technology to obtain the above-mentioned anti-human B7-H3 antibody or fragment thereof.

[0035] In a method for producing a humanized anti-human B7-H3 antibody or fragment thereof according to an embodiment of the present invention, a non-human monoclonal antibody is humanized by manipulating it using gene cloning, DNA recombination technology, etc. After this operation, the expressed antibody that binds to a human target has most of its amino acid sequence replaced with a human sequence, substantially retaining the affinity and specificity of the non-human monoclonal parent antibody, and also exhibiting lower heterogeneity, making it suitable for application to the human body. For example, the antibody or antigen-binding fragment according to an embodiment of the present invention can specifically bind to human B7-H3 after humanization.

[0036] According to embodiments of the present invention, the non-human anti-B7-H3 antibody or its fragment is a mouse anti-B7-H3 antibody or its fragment.

[0037] According to an embodiment of the present invention, the process of achieving humanization of the heavy chain variable region using CDR grafting technology is: (1) Perform homology alignment using the heavy chain variable region sequence of a non-human anti-B7-H3 antibody or its fragment, and select a similar human antibody heavy chain template. (2) Grafting the VH CDR1, CDR2, and CDR3 of a non-human anti-B7-H3 antibody or its fragment onto a human antibody heavy chain template, (3) Select the J region sequence according to the sequence of VH's CDR3 to achieve humanization of the heavy chain variable region and obtain the above-mentioned anti-human B7-H3 antibody or its fragment, Includes.

[0038] According to an embodiment of the present invention, the process of achieving humanization of the light chain variable region using CDR grafting technology is: (1) Perform homology alignment using the light chain variable region sequence of a non-human anti-B7-H3 antibody or its fragment, and select a similar human antibody light chain template. (2) Grafting CDR1, CDR2, and CDR3 of the VL of a non-human anti-B7-H3 antibody or its fragment onto a human antibody light chain template, (3) Select the JK region sequence according to the sequence of the VL CDR3 to achieve humanization of the light chain variable region and obtain the above anti-human B7-H3 antibody or its fragment, Includes.

[0039] According to yet another aspect of the present invention, the disclosure provides a method for producing an anti-human B7-H3 humanized antibody or fragment thereof. According to an embodiment of the present invention, the method for producing an anti-human B7-H3 humanized antibody or fragment thereof comprises obtaining the above-mentioned anti-human B7-H3 antibody or fragment by using CDR grafting technology to achieve humanization of the heavy chain variable region and / or the light chain variable region, based on the above-mentioned non-human anti-B7-H3 antibody or fragment thereof.

[0040] According to an embodiment of the present invention, the step of achieving humanization of the heavy chain variable region using the above-described CDR grafting technology is: (1) Perform homology alignment using the heavy chain variable region sequence of the non-human anti-B7-H3 antibody or its fragment, and select a similar human antibody heavy chain template. (2) Grafting the VH CDR1, CDR2, and CDR3 of a non-human anti-B7-H3 antibody or its fragment onto a human antibody heavy chain template, (3) Select the J region sequence according to the sequence of VH's CDR3 to achieve humanization of the heavy chain variable region and obtain the above-mentioned anti-human B7-H3 antibody or its fragment, Includes.

[0041] According to an embodiment of the present invention, the process of achieving humanization of the light chain variable region using the above-described CDR grafting technology is: (1) Perform homology alignment using the light chain variable region sequence of the non-human anti-B7-H3 antibody or its fragment, and select a similar human antibody light chain template. (2) Grafting CDR1, CDR2, and CDR3 of the VL of a non-human anti-B7-H3 antibody or its fragment onto a human antibody light chain template, (3) Select the JK region sequence according to the sequence of the VL CDR3 to achieve humanization of the light chain variable region and obtain the above anti-human B7-H3 antibody or its fragment, Includes.

[0042] According to embodiments of the present invention, a method for producing an anti-human B7-H3 humanized antibody or a fragment thereof further comprises affinity maturation, site-directed mutagenesis in the CDR sequence, and / or chemical modification of the humanized antibody after CDR grafting.

[0043] Use of antibodies According to another aspect of the present invention, the present disclosure provides a fusion molecule. According to an embodiment of the present invention, the fusion molecule is (1) A first active element which is a first targeting portion containing the above-mentioned anti-human B7-H3 antibody or a fragment thereof, (2) One or more second active elements including an effector element and / or a second targeting portion, This includes, where the first active element is linked to the second active element via a bond or linker, and one or more of the second active elements may be the same or different.

[0044] According to another aspect of the present invention, the disclosure provides a polynucleotide. According to embodiments of the present invention, this polynucleotide encodes the above-mentioned anti-human B7-H3 antibody or a fragment thereof, or the above-mentioned fusion molecule. Therefore, the polypeptide encoded by this polynucleotide can specifically bind to B7-H3.

[0045] According to another aspect of the present invention, the disclosure provides a nucleic acid construct. According to an embodiment of the present invention, this nucleic acid construct comprises the polynucleotide described above in the disclosure.

[0046] According to another aspect of the present invention, the disclosure provides a host cell. According to embodiments of the present invention, the host cell comprises the polynucleotide or nucleic acid construct described above in the disclosure.

[0047] According to another aspect of the present invention, the disclosure provides a conjugate. According to an embodiment of the present invention, the conjugate comprises a first active element which is a first targeting moiety comprising the above-mentioned anti-human B7-H3 antibody or a fragment thereof.

[0048] According to embodiments of the present invention, the conjugate further comprises at least one second active element, wherein the first active element is covalently linked to the second active element, and the second active element comprises one or more effector portions.

[0049] According to embodiments of the present invention, the effector portion is one or more effector molecules or derivatives thereof from the following groups (1) to (3): (1) Small molecule compounds that are tubulin inhibitors: meitansinoids, monomethyl auristatin E, monomethyl auristatin F, monomethyl dorastatin 10, tubulicin and its derivatives, cryptophycin and its derivatives, and tartobrin, (2) Small molecule compounds that are topoisomerase inhibitors: PNU-159682 (metabolite of doxorubicin) and its derivatives, SN38 (metabolite of irinotecan) and its derivatives, and exatecan, (3) Small molecule compounds that act as DNA binders: PBD and its derivatives, and duocalmycin and its derivatives, It is a small molecule drug conjugate selected from the group consisting of the following:

[0050] According to another aspect of the present invention, the disclosure provides a composition. According to embodiments of the present invention, the composition comprises the above-mentioned anti-human B7-H3 antibody or fragment thereof, the above-mentioned fusion molecule, the above-mentioned polynucleotide, the above-mentioned nucleic acid construct, the above-mentioned host cell, or the above-mentioned conjugate, and optionally, pharmaceutically acceptable additives (may be more than one). Thus, the composition can specifically bind to B7-H3 and has strong specificity and good efficacy.

[0051] According to another aspect of the present invention, the present disclosure provides a method for producing an anti-human B7-H3 antibody or a fragment thereof. According to an embodiment of the present invention, the method for producing an anti-human B7-H3 antibody or a fragment thereof is: (1) Culturing the above host cells under appropriate conditions, (2) Separating and recovering anti-human B7-H3 monoclonal antibodies or their fragments, anti-human B7-H3 chimeric antibodies or their fragments, or anti-human B7-H3 humanized antibodies or their fragments, Includes.

[0052] Therefore, antibodies produced by the method according to the embodiments of the present invention can specifically bind to human B7-H3 and have strong specificity.

[0053] According to another aspect of the present invention, the present disclosure provides the use of the above-mentioned anti-human B7-H3 antibody or its fragment, the above-mentioned fusion molecule, the above-mentioned polynucleotide, the above-mentioned nucleic acid construct, the above-mentioned host cell, the above-mentioned conjugate, and the above-mentioned composition in the preparation of an active substance for diagnosing diseases associated with B7-H3 overexpression.

[0054] According to another aspect of the present invention, the present disclosure provides the use of the above-mentioned anti-human B7-H3 antibody or fragment thereof, the above-mentioned fusion molecule, the above-mentioned polynucleotide, the above-mentioned nucleic acid construct, the above-mentioned host cell, the above-mentioned conjugate, and the above-mentioned composition in the preparation of a pharmaceutical for treating diseases associated with B7-H3 overexpression.

[0055] According to embodiments of the present invention, the disease associated with B7-H3 overexpression is solid tumor cancer.

[0056] According to embodiments of the present invention, solid tumors include gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, esophageal cancer, non-small cell carcinoma, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, Wilms' tumor, and fibrinogenic small round cell tumors.

[0057] The anti-human B7-H3 antibody or fragment thereof according to embodiments of the present invention can specifically kill tumor cells and specifically bind to B7-H3 on the surface of recombinant cells and tumor cells, contributing to remarkable tumor-killing effects in cell and animal models, without the observation of any apparent adverse reactions. Thus, the drawbacks of conventional anti-human B7-H3 antibodies, which cause off-target effects and adverse reactions due to insufficient affinity and specificity for B7-H3 proteins on membrane surfaces, are overcome.

[0058] Furthermore, according to yet another aspect of the present invention, the disclosure provides a method for treating a subject having cancer. According to an embodiment of the present invention, the method comprises administering to the subject a therapeutically effective amount of the composition described above.

[0059] According to embodiments of the present invention, the cancer is a solid tumor carcinoma.

[0060] According to embodiments of the present invention, solid tumors include gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, esophageal cancer, non-small cell carcinoma, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, Wilms' tumor, and fibrinogenic small round cell tumors.

[0061] To better understand this invention, certain terms are defined first. Other definitions are described throughout the detailed explanation.

[0062] The term "B7-H3" refers to B7 homolog 3. B7-H3 exists in two forms: 2Ig-B7-H3 and 4Ig-B7-H3. In 2001, Chapoval et al. searched the databases of the National Center for Biotechnology Information (NCBI) and Human Genome Sciences Inc. using the extracellular domains of all publicly available B7 family members. Chapoval et al. identified a B7-like molecule in a human dendritic cell-derived cDNA library and confirmed its nucleic acid sequence using RT-PCR in a human tumor cell lineage THP-1 library and also in a DC library. The B7-H3 gene was found to encode a 316-amino acid protein containing a signal peptide, a single variable-like and constant-like immunoglobulin domain, a transmembrane region, and a 45-amino acid cytoplasmic tail. Steinberger et al. recognized the molecule induced on monocytes differentiated into dendritic cells in vitro by producing a monoclonal antibody against human dendritic cells. Retroviral expression cloning identified this molecule as B7-H3, a type I membrane protein with four Ig-like domains, and the identified molecule was therefore named 4Ig-B7-H3. RT-PCR analysis and Western blotting experiments demonstrated that 4Ig-B7-H3 is a common form of B7-H3. As discussed by Steinberger et al., human B7-H3 is encoded on chromosome 15, and 4Ig-B7-H3 appears to be the result of duplication of the loci encoding the B7-H3-IgV and B7-H3-IgC exons.

[0063] The term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. The term "immunological binding" refers to a specific binding reaction that occurs between an antibody molecule and the specific antigen of that antibody. The strength or affinity of an immunological binding interaction can be measured in terms of the equilibrium dissociation constant (KD) of the interaction, with a smaller KD value indicating higher affinity. The immunological binding properties between two molecules can be quantified using methods commonly known in the art. One method involves measuring the formation and dissociation rates of the antigen-binding site / antigen complex. The "association rate constant" (Ka or Kon) and "dissociation rate constant" (Kd or Koff) of a particular antibody-antigen interaction can both be calculated from the concentration and the actual association and dissociation rates (see Malmqvist M, 1993, Nature, 361: 186-187). The Kd / Ka ratio is equal to the dissociation constant KD (see Davies DR et al., 1990, Annual Rev Biochem., 59: 439-473). The KD, Ka, and Kd values ​​can be measured by any useful method. In a preferred embodiment, the dissociation constant is measured using bioluminescence interferometry. In another preferred embodiment, the dissociation constant can be measured using surface plasmon resonance techniques (e.g., Biacore) or KinExa.

[0064] As used herein, the term “antibody” is intended to encompass full-length antibodies and any antigen-binding fragment (i.e., antigen-binding portion) or single chain thereof. A full-length antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, with the heavy and light chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, namely CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further divided into a hypervariable region called the complementarity-determining region (CDR) and a more conserved framework region (FR) separating the CDR. VH and VL each consist of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to various immune system cells (e.g., effector cells) and host tissues or factors, including the first component (C1q) of the classical complement system.

[0065] The terms "monoclonal antibody," "mAb," or "monoclonal antibody composition" refer to antibody molecular preparations consisting of a single molecule. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

[0066] As used herein, the term “antigen-binding fragment” (or “antibody fragment”) refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen. It has been demonstrated that the antigen-binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of binding fragments contained within the “antigen-binding moiety” of an antibody include: (i) the Fab fragment, a monovalent fragment composed of VL, VH, CL, and CH1; (ii) the F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by disulfide crosslinking in a hinge region; (iii) the Fd fragment, composed of VH and CH1; (iv) the Fv fragment, composed of VL and VH of a single arm of the antibody; (v) the dAb fragment, composed of VH (Ward et al, (1989) Nature 341: 544-546); (vi) an isolated complementarity-determining region (CDR); and (vii) a nanobody, a heavy-chain variable region containing a single variable domain and two constant domains. Furthermore, the two domains VL and VH of the Fv fragment, although encoded by different genes, can be recombined via a synthetic linker to form a single protein chain where the VL and VH regions pair up to form a monovalent molecule (referred to as single-chain Fc(scFv); see, e.g., Bird et al, (1988) Science 242: 423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). These single-chain antibodies are also intended to be included in the meaning of this term. These antibody fragments can be obtained using conventional methods known to those skilled in the art and can be functionally screened in the same way as intact antibodies.

[0067] The antigen-binding fragments of the present invention include those that can specifically bind to antigen molecules. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single-stranded Fv(scFv) fragments, and single-domain fragments.

[0068] The Fab fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that several residues containing one or more cysteines from the antibody hinge region are attached to the carboxyl terminus of the heavy chain CH1 domain. The F(ab') fragment is produced by cleaving the disulfide bond at the hinge cysteine ​​of the F(ab')2 pepsin digest product. The additional chemical binding of antibody fragments is known to those skilled in the art. The Fab and F(ab')2 fragments lack the fragment crystallizable (Fc) region of intact antibodies, are removed more rapidly from animal circulation, and may exhibit less nonspecific tissue binding than intact antibodies (see, e.g., Wahl et al, 1983, J. Nucl. Med. 24:316).

[0069] As is generally understood in the art, the "Fc" region is the constant region of an antibody fragment that does not contain an antigen-specific binding region. In the IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments derived from the second and third constant domains (CH2 and CH3 domains, respectively) of the two heavy chains of the antibody. In IgM and IgE, the Fc region contains three heavy chain constant domains (CH2, CH3, and CH4 domains) in each polypeptide chain.

[0070] The term "humanized antibody" primarily refers to antibodies expressed after manipulating mouse monoclonal antibodies using gene cloning and DNA recombination techniques. These antibodies have a large portion of their amino acid sequence replaced with human sequences, substantially retaining the affinity and specificity of the mouse monoclonal parent antibody, while also exhibiting lower heterogeneity.

[0071] The term "chimeric antibody" refers to an antibody in which part of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, but the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and insofar as it exhibits the desired biological activity (see, for example, U.S. Patent No. 4,816,567 and Morrison SL et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855, 1984). For example, the term "chimeric antibody" may include an antibody in which the heavy chain variable region and light chain variable region of the antibody originate from a first antibody (e.g., a mouse antibody), but the heavy chain constant region and light chain constant region of the antibody originate from a second antibody (e.g., a human antibody) (e.g., a human-mouse chimeric antibody).

[0072] Regarding the term "heavy chain," a heavy chain (H chain) is approximately twice the size of a light chain, contains 450 to 550 amino acid residues, and has a molecular weight of approximately 55 kDa or 75 kDa. Each H chain contains a cyclic peptide formed via four or five intrachain disulfide bonds. Different H chains have different antigenicity depending on the order of amino acid residues, the number and position of disulfide bonds, and the type and number of disulfide bonds. Based on differences in H chain antigenicity, they can be classified into five types: μ chains, γ chains, α chains, δ chains, and ε chains. The entire immunoglobulin molecule composed of different H chains and L chains (κ chains or λ chains) is called IgM, IgG, IgA, IgD, and IgE, respectively. The γ, α, and δ chains contain four peptides, while the μ and ε chains contain five cyclic peptides.

[0073] Regarding the term "light chain," a light chain (L chain) refers to a polypeptide chain in an immunoglobulin monomer molecule that has a smaller molecular weight compared to the heavy chain. The variable light chain region (VL) is a part of the 1 / 2 region near the amino terminus (N terminus) of each light chain that has an even more diverse amino acid composition, and it is one of the regions where the Ig molecule binds to the antigen.

[0074] The remaining half of the region, which has a relatively stable amino acid composition and arrangement, is the light chain constant region (CL). The light chain exists in two forms, κ and λ, due to several differences in the amino acid sequence within the light chain constant region.

[0075] The term "germ cell lineage" is also known as the germ cell system. Antibody-forming cells possess all the genes that encode the Ig molecule (i.e., a limited number of C genes and an unknown number of V genes). Germ cell lineages are formed through long-term evolution and are inherited from parent to offspring by germ cells. The heavy chain genes of human immunoglobulins are encoded by the VDJC gene segment, and the light chain genes of human immunoglobulins are encoded by the VJC gene segment, with varying numbers of gene segments. The human heavy chain gene is located on the long arm of chromosome 14, spans approximately 1100 kb, and consists of four gene segments V, D, J, and C, and includes approximately 95 Va gene segments (belonging to seven families, i.e., VH1-VH7) containing 65 functional gene segments, 27 D gene segments, 6 JH gene segments, and 9 CH gene segments. The Va gene segment is located upstream, the D gene segment is located between the VH gene cluster and the JH gene cluster, the JH gene is located downstream of DH, about 7kb away from the downstream C gene region, the CH gene is clustered and spans about 200kb, the P and S genes are located immediately downstream of the JH gene segment, and downstream of C8 are Cγ, Cα, and Cε in that order. Human light chain genes are grouped into λ genes and x genes, located on the long arm of chromosome 22 and the short arm of chromosome 2, respectively. There are about 40 functional VK gene segments, and following the Vc gene segment are 5 functional J and 1 Cκ, and about 30 Vλ gene segments, 4 Jλ gene segments, and 4 Cλ gene segments.

[0076] The term "EC50," also known as the median effective concentration, refers to the antibody concentration that produces 50% of the maximum effect.

[0077] The terms “vector” or “nucleic acid construct” refer to nucleic acid molecules capable of transporting another ligated nucleic acid. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which an additional DNA segment can be ligated. Another type of vector is a viral vector, where the additional DNA segment can be ligated within the viral genome. Certain vectors can autonomously replicate within the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may be integrated into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. Furthermore, certain vectors can induce the expression of genes to which they are operably ligated. Such vectors are referred to herein as “recombinant expression vectors” (or simply “recombinant vectors”). Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. However, other forms of expression vectors may be included, such as viral vectors that perform equivalent functions (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0078] The term "nucleic acid molecule" is intended to include both DNA and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, and may be cDNA.

[0079] The term "polypeptide" refers to a chain containing at least two consecutively linked amino acid residues, with no upper limit on chain length. One or more amino acid residues within a protein may, but are not limited to, undergo modifications such as glycosylation, phosphorylation, or disulfide bonding. A "protein" may contain one or more polypeptides.

[0080] The term "host cell" refers to a cell that can amplify a vector and express its DNA, and this cell may be a prokaryotic or eukaryotic cell. The term also encompasses all offspring of the host cell in question. It is understood that not all offspring need to be identical to the parent cell, as mutations can occur during replication, and such offspring are included.

[0081] The terms "cancer" and "tumor" refer to or describe a physiological condition in mammals characterized by uncontrolled cell growth of a population of cells. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, leukemias, and benign or malignant tumors. More specific examples of cancer include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, brain cancer, liver cancer, and various types of head and neck cancers, as well as neurofibromatosis type I or neurofibromatosis type II. Other examples of cancer include refractory, intractable, or metastatic cancers.

[0082] The term “antibody-drug conjugate” is abbreviated as “conjugate” and consists of an antibody, a linker, and a drug, where the linker is either cleavable or non-cleavable. The antibody is a globular protein containing a set of amino acid sites that can be used for drug-linker coupling. Due to their tertiary and quaternary structures, only amino acids that can reach the solvent are available for coupling. In fact, high-yield coupling usually occurs with the ε-amino group of a lysine residue or the sulfhydryl group of a cysteine ​​residue. The numerous lysine side chains on the surface of the antibody protein provide numerous sites available for drug conjugation, resulting in the production of antibody-drug conjugates, which are mixtures containing various numbers of drug conjugates (drug / antibody ratio, DAR) and various conjugate sites. The conjugate products provided by this disclosure, although still mixtures, have a narrower range of DAR values ​​compared to antibody-drug conjugates obtained in conventional conjugate methods. The average DAR value is close to 4, which is close to the optimal average range of DAR values ​​for antibody-drug conjugates (2-4). Furthermore, the conjugate products rarely contain naked antibodies (DAR=0), which are components that are not effective in killing cells. Also, the conjugate products do not contain highly coupled products (DAR=8), which are rapidly removed in vivo compared to components with low DAR values. Therefore, the antibody-drug conjugate products provided by this disclosure exhibit significantly improved heterogeneity.

[0083] The antibody constituting the antibody-drug conjugate provided by the present disclosure will better retain its original antigen-binding ability in the native state. Therefore, the antibody in the present disclosure can preferably specifically bind to its antigen. In order to develop cell-level targets effective for cancer diagnosis and treatment, researchers have tried to find transmembrane polypeptides or other tumor-related polypeptides. These targets can be specifically expressed on the surface of one or more cancer cells, while not expressed or rarely expressed on the surface of one or more non-cancer cells. Typically, such tumor-related polypeptides are overexpressed on the surface of cancer cells compared to the surface of non-cancer cells. Identification of such tumor-related factors can greatly improve the specific targeting characteristics of antibody-based cancer therapy.

[0084] The antibody provided by the present disclosure has a relatively higher binding ability with respect to B7-H3 and has an affinity constant KD value of less than 5×10 -10 M. The anti-B7-H3 humanized antibodies hz9B11, hz10B4, and hz17A3 can specifically bind to the human B7-H3 protein, and their affinities (KD) are 2.19×10 -10 M, 1.42×10 -10 M, and 1.77×10 -10 M, respectively, while the affinities (KD) of the control antibodies MGC-018 and enoblituzumab are 5.97×10 -10 M and 9.50×10 -10 M, respectively. The binding ability provides a molecular basis for their excellent efficacy.

[0085] The antibodies provided in this disclosure exhibit good biological activity. Antibodies hz9B11, hz10B4, and hz17A3 were able to effectively bind to recombinant human B7-H3 on the cell surface, with half maximal effective binding concentrations (EC50) values ​​of 0.08277 μg / ml, 0.1183 μg / ml, and 0.04407 μg / ml, respectively, which were superior to the control antibody enobrituzumab (EC50 value: 0.1906 μg / ml). The EC50 values ​​of hz9B11 and hz17A3 were close to the EC50 value of the control antibody MGC-018 (EC50 value: 0.05649 μg / ml). Furthermore, hz9B11, hz10B4, and hz17A3 exhibited good intracellular translocation capabilities.

[0086] The antibodies provided in this disclosure exhibit good efficacy in vivo (in mice). In a subcutaneous tumor model of A431 cells in nude mice, the anti-B7-H3 antibody-drug conjugate (ADC) named hz10B4-M exhibited better efficacy than the control antibody-drug conjugates MGC-018-M and enobrituzumab-M, and hz17A3-M exhibited better efficacy than the control antibody-drug conjugate enobrituzumab-M. hz9B11-M showed certain efficacy, but it was weaker than the efficacy of hz10B4-M and hz17A3-M. Furthermore, no apparent toxic effects of the small molecule MMAE used in the preparation of the ADCs were observed, and animal body weight steadily increased in each experimental group, with no significant difference in body weight between the experimental and control groups.

[0087] The above and / or additional aspects and advantages of the present invention will become apparent and readily apparent from the description of the embodiments with reference to the following accompanying drawings. [Brief explanation of the drawing]

[0088] [Figure 1] This figure shows the binding of hybridoma supernatant to the B7-H3 antigen detected by ELISA. [Figure 2] This figure shows the binding of anti-human B7-H3 chimeric antibodies to various recombinant B7-H3 protein domains detected by ELISA. [Figure 3] This figure shows the binding of anti-human B7-H3 chimeric antibodies to B7-H3 on the surface of 293 / hB7-H3 cells detected by FACS. [Figure 4] This figure shows the binding of anti-human B7-H3 chimeric antibodies to B7-H3 on the surface of A431 cells, as detected by FACS. [Figure 5] This figure shows the ELISA curves illustrating the binding of humanized antibodies to recombinant human B7-H3. [Figure 6] This figure shows the ELISA curves illustrating the binding of humanized antibodies to recombinant monkey B7-H3. [Figure 7] This figure shows the ELISA curves illustrating the binding of humanized antibodies to recombinant mouse B7-H3. [Figure 8A] This figure shows the analysis of the affinity of hz10B4 for recombinant human B7-H3 extracellular domain protein. [Figure 8B] This figure shows the analysis of the affinity of hz17A3 to recombinant human B7-H3 extracellular domain protein. [Figure 8C] This figure shows the analysis of the affinity of hz9B11 to recombinant human B7-H3 extracellular domain protein. [Figure 8D] This figure shows the analysis of the affinity of MGC-018 to recombinant human B7-H3 extracellular domain protein. [Figure 8E] This figure shows the analysis of the affinity of enobrituzumab for recombinant human B7-H3 extracellular domain protein. [Figure 9] This figure shows the binding of anti-human B7-H3 humanized antibodies to SKVO3 cells detected by FACS. [Figure 10] This figure shows the intracellular translocation rate of anti-human B7-H3 humanized antibodies mediated by B7-H3 on the cell surface. [Figure 11]This figure shows the detected killing activity of the anti-human B7-H3 antibody drug conjugate against A431 cells. [Figure 12] This figure shows the detected killing activity of the anti-human B7-H3 antibody drug conjugate against SKVO3 cells. [Figure 13] This figure shows the tumor volume change curve in a subcutaneous tumor model of A431 cells in nude mice. [Figure 14] This figure shows the weight change curve of mice in a subcutaneous tumor model using A431 cells in nude mice. [Modes for carrying out the invention]

[0089] Embodiments of the present invention will be described in detail below and illustrated in the accompanying drawings. Here, the same or similar reference numerals refer to the same or similar elements, or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative and are used only to describe the present invention, and should not be construed as limiting the invention.

[0090] It should be noted that the terms “First” and “Second” are used solely for descriptive purposes and should not be understood as indicating or implying relative importance, or implicitly indicating the number of technical features being described. Therefore, features defined in “First” and “Second” may explicitly or implicitly include one or more features. Furthermore, in this disclosure, “multiple” means two or more unless otherwise indicated.

[0091] Solutions of the present invention are illustrated below with reference to examples. Those skilled in the art will understand that the following examples are merely illustrative of the present invention and should not be considered limiting the scope of the invention. Unless specific techniques or conditions are shown in the examples, the techniques or conditions or product instructions described in the literature of the art (e.g., Molecular Cloning: A Laboratory Manual (3rd edition), edited by J. Sambrook and translated by Peitang HANG et al., Scientific Press) shall apply. Reagents or equipment used without manufacturer's instructions are, for example, commonly available products from Sigma Inc. [Examples]

[0092] Example 1: Production and screening of hybridoma cells that produce anti-human B7-H3 antibodies 1.1 Construction of Hybridomas Immunization: Balb / c mice were immunized with recombinant human B7-H3 extracellular domain protein (accession number: UniProtKB-Q5ZPR3, amino acids 1-461), and serum titers were detected by ELISA in 96-well ELISA plates coated with recombinant human B7-H3-his protein (accession number: UniProtKB-Q5ZPR3, amino acids 1-461). Mice that met the fusion requirements were used for subsequent cell fusions.

[0093] Cell fusion and hybridoma generation: Mice with titers meeting the requirements were selected and subjected to a final boost. Three days later, the mouse spleens were aseptically harvested, a suspension of B lymphocytes was prepared, and these were fused with SP2 / 0 myeloma cells. The fused cells were resuspended in HAT medium and then seeded into 96-well cell culture plates, which were then cultured in an incubator at 37°C and 5% CO2.

[0094] 1.2 Binding Screening for Positive Hybridomas Ten to fourteen days after fusion, ELISA plates were coated overnight at 4°C with recombinant human B7-H3-his extracellular domain protein (accession number: UniProtKB-Q5ZPR3, amino acids 1 to 461) (20 ng / ml). The plates were washed three times with PBS and blocked at room temperature for 1 hour with 4% skim milk powder-PBS. Subsequently, the plates were washed three times with PBS, the culture supernatant of the hybridoma clones was added to the plates, and the plates were incubated at room temperature for 1 hour. The following controls were prepared: (1) Positive control (PC): Immunized mouse serum (diluted with PBS at 1:1000), and (2) Negative control (NC): Unimmunized mouse serum (diluted with PBS at 1:1000). The plates were washed three times with PBST (0.05% Tween 20-PBS) and twice with PBS. HRP-conjugated goat anti-mouse IgG (Fcγ) was then added to the plates, and incubated at 37°C for 0.5 hours. The plates were again washed three times with PBST (0.05% Tween 20-PBS), TMB substrate was added, and the plates were incubated in the dark for 15–30 minutes. The reaction was stopped by adding ELISA stop solution, and the absorbance at 450 nm was read using a microplate reader. The top 16 clones with ordered readings (highest to lowest) were selected, and their culture supernatants were subjected to a second ELISA for confirmation. Finally, clones 445 (9B11), 485 (10B4), and 870 (17A3) were selected as candidate clones for sequence cloning. The ELISA results are shown in Figure 1.

[0095] Example 2: Sequencing of mouse anti-human B7-H3 antibody Monoclonal hybridoma cells 9B11, 10B4, and 17A3 secreting anti-human B7-H3 antibody were subjected to expansion culture. Total RNA from the cells was extracted using the TRIzol kit (catalog: 15596026, Invitrogen) according to the instructions provided with the kit. The resulting total RNA from the hybridoma cells was reverse transcribed into cDNA using M-MuLV reverse transcriptase (catalog: M0253S, NEB). The sequences of the antibody's light chain variable region IgVL(κ) and heavy chain variable region VH were amplified using degenerate primers and the Phusion kit (catalog: E0553L, NEB). The PCR amplification products were purified using a gel recovery kit (catalog: AP-GX-250, Axygen), ligated to a T-vector using a T-vector cloning kit (catalog: ZC205, ZOMANBIO) according to the instructions provided with the kit, and transformed into competent E. coli cells. After strain amplification and plasmid extraction, the variable region sequences of the monoclonal antibodies were obtained by DNA sequencing. The heavy chain variable region sequence of mouse antibody 9B11 is shown in SEQ ID NO: 1, and the light chain variable region sequence of mouse antibody 9B11 is shown in SEQ ID NO: 2. The heavy chain variable region sequence of mouse antibody 10B4 is shown in SEQ ID NO: 3, and the light chain variable region sequence of mouse antibody 10B4 is shown in SEQ ID NO: 4. The heavy chain variable region sequence of mouse antibody 17A3 is shown in SEQ ID NO: 5, and the light chain variable region sequence of mouse antibody 17A3 is shown in SEQ ID NO: 6.

[0096] Example 3: Preparation of anti-human B7-H3 chimeric antibody and control antibody The light and heavy chain sequences of the control antibodies enobrituzumab and MGC-018 were completely synthesized and cloned into eukaryotic transient expression vectors to obtain plasmids expressing the light and heavy chains of the control antibodies. The plasmids were transformed into E. coli cells and amplified, and numerous plasmids containing the light and heavy chains of the control antibodies were recovered. These plasmids containing the light and heavy chains of the control antibodies were then transfected into HEK293 cells using 293fectin (catalog: 12347019, Gibco) transfection reagent, according to the manufacturer's instructions for recombinant expression. Five to six days after cell transfection, the culture supernatant was collected and purified through a ProA affinity chromatography column to obtain the control antibody. The amino acid sequence of the control antibody enobrituzumab was derived from WHO Drug Information (Vol. 30, No. 4, 2016), with the heavy chain amino acid sequence shown in SEQ ID NO: 7 and the light chain amino acid sequence shown in SEQ ID NO: 8. The amino acid sequence of the control antibody MGC-018 was derived from the patent document International Publication No. 2017 / 180813, with the heavy chain amino acid sequence shown in SEQ ID NO: 9 and the light chain amino acid sequence shown in SEQ ID NO: 10.

[0097] The light chain variable region genes and heavy chain variable region genes of mouse antibodies 9B11, 10B4, and 17A3 obtained by the cloning described above were introduced at restriction enzyme cleavage sites by PCR and cloned upstream of the gene encoding the human κ light chain constant region and the gene encoding the human IgG1 heavy chain constant region, respectively, contained in a eukaryotic transient expression vector. Plasmids expressing human-mouse chimeric light chains (pKN019-ch9B11L, pKN019-ch10B4L, and pKN019-ch17A3L) and human-mouse chimeric heavy chains (pKN025-ch9B11H, pKN025-ch10B4H, and pKN025-ch17A3H) were obtained, transformed into E. coli cells, amplified, and numerous plasmids containing human-mouse chimeric light and heavy chains were recovered. Plasmids containing the light and heavy chains of the chimeric antibodies 9B11, 10B4, and 17A3 were then transfected into HEK293 cells using 293fectin (catalog: 12347019, Gibco) transfection reagent, according to the manufacturer's instructions for recombinant expression. Five to six days after cell transfection, the culture supernatant was collected and purified through a ProA affinity chromatography column to obtain the chimeric antibodies 9B11, 10B4, and 17A3 (ch9B11, ch10B4, and ch17A3).

[0098] Example 4: Analysis of the affinity of chimeric antibodies Antibody affinity was determined using a Fortebio Octet QKe system instrument by an assay that included capturing antibody Fc fragments with an anti-human IgG Fc capture (AHC) biosensor. For the assay, the chimeric antibodies ch9B11, ch10B4, and ch17A3, and the control antibody enobrituzumab were each diluted to 4 μg / ml in PBS and passed over the surface of an AHC biosensor (catalog: 18-0015, PALL) for 120 seconds. Recombinant human B7-H3 extracellular domain protein (accession number: UniProtKB-Q5ZPR3, amino acids 1 to 461, C-his tag) (60 nM) was used as the mobile phase. The association time was 300 seconds and the dissociation time was 300 seconds. After the assay was complete, the data, after subtracting the response values ​​of the blank control, were fitted to a 1:1 Langmuir binding model using software, and the rate constant for antigen-antibody binding was then calculated. The rate constants are shown in Table 1 below. The results in Table 1 indicate that all clones were successfully cloned and able to bind to recombinant human B7-H3 protein.

[0099] [Table 1]

[0100] Example 5: Conjugation of various recombinant B7-H3 proteins and chimeric antibodies to various domains detected by ELISA. Plates were coated overnight at 4°C with recombinant human B7-H3-his protein (accession number: UniProtKB-Q5ZPR3, amino acids 1 to 461), recombinant human B7-H3 IgV1-IgV2-his extracellular domain protein (accession number: UniProtKB-Q5ZPR3, amino acids 1 to 362), recombinant human B7-H3 IgV1-IgC1-his extracellular domain protein (accession number: UniProtKB-Q5ZPR3, amino acids 1 to 238), recombinant human B7-H3 IgC2-mFc extracellular domain protein (accession number: UniProtKB-Q5ZPR3, amino acids 363 to 456), and recombinant monkey B7-H3-his extracellular domain protein (accession number: XP-005560056.1, amino acids 1 to 465). Here, each protein was present at a concentration of 0.2 μg / ml per 100 μl. The plate was then blocked in a 37°C incubator with 5% BSA for 60 minutes, followed by three washes with PBST. 100 μl each of ch9B11, ch10B4, and ch17A3 at concentrations of 0.5 μg / ml, along with the control antibody enobrituzumab and isotype control antibody (NC), were added and reacted in a 37°C incubator for 60 minutes. After incubation, the plate was washed four times with PBST, and HRP-anti-human Fc (catalog: 109-035-098, Jackson Immuno Research), diluted 1:5000, was added to the plate and reacted for 45 minutes. TMB (catalog: ME142, GalaxyBio, Beijing) substrate was added and allowed to develop for 15 minutes, after which the reaction was stopped by adding 2M HCl. The absorbance was read at 450 nm and 630 nm (as reference wavelengths), and the values ​​for A in the wells of the plate from 450 nm to 630 nm were recorded.

[0101] As detected by ELISA, ch9B11, ch10B4, and ch17A3, as well as the control antibody enobrituzumab, all bound to recombinant human B7-H3 domain protein and recombinant monkey B7-H3 extracellular domain protein (Figure 2). Among these antibodies, the binding activity of ch10B4 to monkey B7-H3 was significantly weaker than that of the other antibodies, indicating weak cross-binding activity. The binding of all antibodies to B7H3-IgC2 was slightly weaker than their binding to other domains, with ch9B11 being the most evident antibody.

[0102] Example 6: Binding of anti-human B7-H3 chimeric antibody to recombinant human B7-H3 protein on the surface of 293 cells detected by FACS. Construction of a 293 cell line transiently expressing human B7-H3: HEK293 cells were expressed in a 10 ml volume at a rate of 6 × 10⁶ cells per ml. 5 Cells were inoculated into a shaking flask at a density of 1 cell, and then subjected to shaking culture at 130 rpm for 24 hours in a shaking incubator at 37°C and 5% CO2. The expression plasmid encoding full-length human B7-H3 (accession number: UniProtKB-Q5ZPR3, amino acids 1 to 534) and 293fectin (catalog: 12347019, Gibco) were incubated in a 1:1.2 ratio in 0.5 mL of Opti-MEM (catalog: 11058021, Gibco) medium for 5 minutes each, then mixed and left at room temperature for 15 minutes. The mixture was then added to HEK293 cells, and these were subjected to shaking culture at 130 rpm for 48 hours in a shaking incubator at 37°C and 5% CO2. The resulting cells, i.e., 293 / hB7-H3 cells, were detected via FACS as follows.

[0103] A suspension of 293 cells (293 / hB7-H3 cells) recombinantly expressing human B7-H3 was incubated with chimeric antibodies (ch9B11, ch10B4, and ch17A3 at concentrations of 1 μg / ml, 0.1 μg / ml, and 0.01 μg / ml, respectively) at 37°C for 30 minutes. The following controls were prepared: (1) Positive control (PC): enobrituzumab control antibody, (2) Negative control (NC): isotype control antibody. The cells were washed three times with PBS, and goat anti-human IgG-FITC (catalog: F9512, Sigma) diluted 1:200 was added to the cells, followed by incubation for 30 minutes. Next, the cells were washed three more times with PBS, and the mean fluorescence intensity (MFI) of the cells was measured using a flow cytometer (model B49007AD, SNAW31211, BECKMAN COULTER) to detect the binding ability of the chimeric antibodies to human B7-H3 protein on the surface of 293 cells. As detected by FACS, ch9B11, ch10B4, and ch17A3 all bound to recombinant human B7-H3 protein on the surface of 293 cells with superior binding ability compared to the control antibody enobrituzumab (Figure 3).

[0104] Example 7: Binding of anti-human B7-H3 chimeric antibody to A431 cells (human cutaneous squamous cell carcinoma cells) detected by FACS. A suspension of A431 cells, which naturally express human B7-H3, was incubated at 37°C for 30 minutes with chimeric antibodies (ch9B11, ch10B4, and ch17A3; diluted in a 3-fold gradient from a concentration of 10 μg / ml, for a total of 11 concentrations). The following controls were prepared: (1) Positive control (PC): enobrituzumab control antibody, (2) Negative control (NC): isotype control antibody. The cells were washed three times with PBS, and goat anti-human IgG-FITC (catalog: F9512, Sigma) diluted 1:200 was added to the cells, followed by incubation for 30 minutes. The cells were then washed three more times with PBS, and the mean fluorescence intensity (MFI) of the cells was measured using a flow cytometer (model B49007AD, SNAW31211, BECKMAN COULTER) to detect the binding ability of the chimeric antibodies to the B7-H3 protein on the surface of A431 cells. As detected by FACS, ch9B11, ch10B4, and ch17A3 all bound to the B7-H3 protein on the surface of A431 cells with superior binding ability compared to the control antibody enobrituzumab. The half-effect concentration (EC50) values ​​for each antibody are shown in Table 2, and binding is shown in Figure 4.

[0105] [Table 2]

[0106] Example 8: Humanization and Mutation Design of Anti-Human B7-H3 Monoclonal Antibodies 8.1 Humanization of mouse monoclonal antibody 9B11 (1) CDR grafting First, the heavy chain sequences of mouse antibodies were comprehensively analyzed to determine the complementarity-determining region (CDR) responsible for antigen-antibody binding and the framework region supporting the conserved three-dimensional structure of the antibody. Subsequently, based on the homology alignment results, the most similar human template VH1 (1-03) was selected as the basic template, and CDR grafting was performed in combination with the results of the whole-sequence BLAST. For CDR grafting, JH6 (wgqgtTvtvss) was selected as the J region sequence based on the CDR3 sequence (STTTATFYWYFDV; SEQ ID NO: 42), and then humanization was achieved in the framework region of the heavy chain variable region (VH) at 9B11. Similarly, the most similar human templates VK III (A27) and VK VI (A26) were selected as base templates, and CDR grafting was performed in combination with the results of whole-sequence BLAST. For CDR grafting, JK2 (FGQGTKLEIK) was selected as the JK region sequence based on the CDR3 sequence (QQSHSWPYT; SEQ ID NO: 45), and then humanization was achieved in the light chain framework region. The amino acid sequence of the humanized heavy chain variable region hz9B11_VH1 with a CDR grafted from antibody 9B11 is shown in SEQ ID NO: 11, the amino acid sequence of the humanized light chain variable region hz9B11_VL1 with a CDR grafted from antibody 9B11 is shown in SEQ ID NO: 12, and the amino acid sequence of the humanized light chain variable region hz9B11_VL2 with a CDR grafted from antibody 9B11 is shown in SEQ ID NO: 13.

[0107] (2) Mutation design in CDR Mutations were designed in the sequences of the humanized heavy chain variable region and humanized light chain variable region containing grafted CDRs according to the sequence characteristics of mouse antibody 9B11, and the mutation sites are shown in Table 3 below.

[0108] [Table 3]

[0109] 8.2 Humanization of mouse monoclonal antibody 10B4 (1) CDR grafting First, the heavy chain sequences of mouse antibodies were comprehensively analyzed to determine the complementarity-determining region (CDR) responsible for antigen-antibody binding and the framework region supporting the conserved three-dimensional structure of the antibody. Subsequently, VH1 (1-03) was selected as the basic template according to the homology alignment results, and CDR grafting was performed in combination with the results of the whole-sequence BLAST. For CDR grafting, JH6 (WGQGTTVTVSS) was selected as the J region sequence based on the CDR3 sequence (KGKDYFDWYFDV; SEQ ID NO: 48), and then humanization was achieved in the framework region of the heavy chain variable region (VH) of 10B4. Based on the homology alignment results, the most similar human template VK I(O12) was selected as the base template, and CDR grafting was performed in combination with the results of the whole sequence BLAST. For CDR grafting, JK4(FGGGTKVEIK) was selected as the JK region sequence based on the CDR3 sequence (QQWSSNPLT; SEQ ID NO: 51), and then humanization was achieved in the light chain framework region. The amino acid sequence of the humanized heavy chain variable region hz10B4_VH1 with a CDR grafted from antibody 10B4 is shown in SEQ ID NO: 18, and the amino acid sequence of the light chain variable region hz10B4_VL1 with a CDR grafted from antibody 10B4 is shown in SEQ ID NO: 19.

[0110] (2) Mutation design in CDR Mutations were designed in the sequences of the humanized heavy chain variable region and humanized light chain variable region containing grafted CDRs according to the sequence characteristics of mouse antibody 10B4, and the mutation sites are shown in Table 4 below.

[0111] [Table 4]

[0112] 8.3 Humanization of mouse monoclonal antibody 17A3 (1) CDR grafting First, the heavy chain sequences of mouse antibodies were comprehensively analyzed to determine the complementarity-determining region (CDR) responsible for antigen-antibody binding and the framework region supporting the conserved three-dimensional structure of the antibody. Subsequently, based on the homology alignment results, the most similar human template VH1 (1-02) was selected as the basic template, and CDR grafting was performed in combination with the results of the whole-sequence BLAST. For CDR grafting, JH4 (WGQGTLVTVSS) was selected as the J region sequence based on the CDR3 sequence (GITAVVGTRYYAMDY), and then humanization was achieved in the framework region of the heavy chain variable region (VH) of 17A3. Based on the homology alignment results, VK III (A27) and VK VI (A26) were selected as basic templates, and CDR grafting was performed in combination with the results of whole-sequence BLAST. For CDR grafting, JK4 (FGGGTKVEIK) was selected as the JK region sequence based on the CDR3 sequence (QQSYSWPLT), and then humanization was achieved in the light chain framework region. The amino acid sequence of the humanized heavy chain variable region hz17A3_VH1 with a CDR grafted from antibody 17A3 is shown in SEQ ID NO: 24, the amino acid sequence of the light chain variable region hz17A3_VL1 with a CDR grafted from antibody 17A3 is shown in SEQ ID NO: 25, and the amino acid sequence of the light chain variable region hz17A3_VL2 with a CDR grafted from antibody 17A3 is shown in SEQ ID NO: 26.

[0113] (2) Mutation design in CDR Mutations were designed in the sequences of the humanized heavy chain variable region and humanized light chain variable region containing grafted CDRs according to the sequence characteristics of mouse antibody 17A3, and the mutation sites are shown in Table 5 below.

[0114] [Table 5]

[0115] Example 9: Recombinant expression of anti-human B7-H3 humanized monoclonal antibody The sequences of the light chain variable region and heavy chain variable region (hz9B11_VL1, hz9B11_VL2, hz9B11_VH1, hz10B4_VL1, hz10B4_VH1, hz17A3_VL1, hz17A3_VL2, and hz17A3_VH1), obtained by humanization design based on antibodies 9B11, 10B4, and 17A3, were completely synthesized. The humanized heavy chain variable region was cloned into the eukaryotic transient expression vector pKN041 upstream of the gene encoding the human IgG1 heavy chain constant region by enzymatic digestion (the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 38), and the humanized light chain variable region was cloned into the eukaryotic transient expression vector pKN019 upstream of the gene encoding the human Cκ light chain by enzymatic digestion (the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 39). Plasmids expressing the humanized light chain and humanized heavy chain based on antibodies 9B11, 10B4, and 17A3 were constructed. Site-directed mutagenesis was performed on the plasmids expressing the humanized light chain and humanized heavy chain, respectively, according to the mutation design, using the StarMut gene site-directed mutagenesis kit (catalog: T111-01, GenStar). Mutant plasmids were transformed into E. coli cells and amplified to obtain plasmids expressing humanized and mutant light and heavy chains based on antibodies 9B11, 10B4, and 17A3. All plasmids containing various humanized light and heavy chains based on antibodies 9B11, 10B4, and 17A3 were combined as shown in Tables 6-8 and transfected into HEK293 cells using 293fectin (catalog: 12347019, Gibco) transfection reagent according to the manufacturer's instructions for recombinant expression. Five to six days after cell transfection, the culture supernatant was purified through a ProA affinity chromatography column to obtain various humanized antibodies. Antibody affinity was determined using a Fortebio Octet QKe system instrument by assays including capturing antibody Fc fragments with an anti-human IgG Fc capture (AHC) biosensor.For the assay, humanized antibodies and chimeric antibodies based on antibodies 9B11, 10B4, and 17A3 were each diluted to 4 μg / ml in PBS and passed over the surface of an AHC biosensor (catalog: 18-0015, PALL) for 300 seconds. Recombinant human B7-H3-his protein (accession number: UniProtKB-Q5ZPR3, amino acids 1 to 461) was used as the mobile phase at a concentration of 60 nM. The association time was 100 seconds and the dissociation time was 300 seconds. After the assay was complete, the data, after subtracting the response value of the blank control, were fitted to a 1:1 Langmuir binding model using software, and the rate constant for antigen-antibody binding was calculated. The light and heavy chain combinations and affinity constant KD values ​​for the humanized antibodies and mutant antibodies based on antibodies 9B11, 10B4, and 17A3 are shown in Tables 6 to 8, respectively.

[0116] The detection results of the affinity of these mutant antibodies revealed that mutations at different sites significantly alter antibody affinity. Among the antibodies, 1.55 × 10⁶ -10 We selected the antibody hz9B11-6, which shows affinity (KD) for M, and named it "hz9B11". -10 We selected the antibody hz10B4-7, which shows affinity (KD) for M, and named it "hz10B4". 3.01 × 10 -10 We selected the antibody hz17A3-14, which showed affinity (KD) for M, and named it "hz17A3." We then selected these three antibodies and performed functional validation in subsequent studies.

[0117] [Table 6]

[0118] [Table 7]

[0119] [Table 8]

[0120] Example 10: Species specificity study of the binding of anti-B7-H3 humanized antibodies to B7-H3 detected by ELISA. Plates were coated overnight at 4°C with recombinant human B7-H3-his extracellular domain protein (accession number: UniProtKB-Q5ZPR3, amino acids 1-461), recombinant cynomolgus monkey B7-H3-his protein (catalog: 90806-C08H, Beijing Yiqiao Shenzhou Science and Technology Co., Ltd.), and recombinant mouse B7-H3-his protein (catalog: CM83, Beijing Yiqiao Shenzhou Science and Technology Co., Ltd.). Each protein had a coating concentration of 1 μg / ml. The plates were washed three times with PBS, blocked at 37°C for 60 minutes with the addition of 5% BSA PBS, and then washed three times with PBST. Various dilutions of hz10B4 (diluted in a 3-fold gradient from a starting concentration of 10 μg / ml, resulting in a total of 12 concentrations), hz17A3 (diluted in a 3-fold gradient from a starting concentration of 3 μg / ml, resulting in a total of 12 concentrations), hz9B11 (diluted in a 3-fold gradient from a starting concentration of 10 μg / ml, resulting in a total of 12 concentrations), enobrituzumab (diluted in a 3-fold gradient from a starting concentration of 10 μg / ml, resulting in a total of 12 concentrations), and MGC-018 (diluted in a 3-fold gradient from a starting concentration of 10 μg / ml, resulting in a total of 12 concentrations) were added to the plates. The plates were incubated at 37°C for 60 minutes and then washed four times with PBST. HRP-anti-human Fc (catalog: 109-035-098, Jackson Immuno Research), diluted 1:5000, was added to the plates and incubated at 37°C for 30 minutes. The plate was then washed four times with PBST, and TMB substrate was added to induce color development. After incubation at 37°C for 10 minutes, the reaction was stopped by adding 2M HCl, and the absorbance at 450nm and 630nm (as reference wavelengths) was read, recording the values ​​for A450nm~630nm in the wells of the plate.

[0121] These experimental results revealed that hz10B4, hz17A3, hz9B11, and the control antibody enobrituzumab all specifically bound to recombinant human B7H3 and cynomolgus monkey B7H3, but did not have binding activity to recombinant mouse B7H3 (Figures 5, 6, and 7). The half-effect concentration (EC50) values ​​for each antibody are shown in Table 9.

[0122] [Table 9]

[0123] Example 11: Analysis of affinity of anti-B7-H3 humanized antibody Antibody affinity was determined using a Fortebio Octet QKe system instrument by an assay that included capturing antibody Fc fragments with an anti-human IgG Fc capture (AHC) biosensor. For the assay, each antibody (hz10B4, hz17A3, and hz9B11, as well as the control antibodies MGC-018 and enobrituzumab) was diluted to 4 μg / ml in PBS and passed over the surface of an AHC biosensor (catalog: 18-0015, PALL) for 120 seconds. Recombinant human B7-H3-his extracellular domain protein (accession number: UniProtKB-Q5ZPR3, amino acids 1 to 461) was used as the mobile phase. The association time was 300 seconds and the dissociation time was 300 seconds. After the assay was complete, the data, after subtracting the response values ​​of the blank control, were fitted to a 1:1 Langmuir binding model using software, and the rate constant for antigen-antibody binding was then calculated.

[0124] The reaction curves between hz10B4, hz17A3, hz9B11, and the control antibody MGC-018, enobrituzumab, and recombinant human B7-H3 protein are shown in Figures 8A-E. The curves were fitted and affinity was calculated: the affinity (KD) of hz10B4 is 1.42 × 10⁻¹⁴. -10 The affinity (KD) of the Hz17A3 is M, and the affinity (KD) is 1.77 × 10⁻⁶. -10The affinity (KD) of hz9B11 is M, and the affinity (KD) is 2.19 × 10⁻⁶. -10 The affinity (KD) of MGC-018 is M, and the affinity (KD) is 5.97 × 10⁻⁶. -10 The M grade indicates that the affinity (KD) for enobrituzumab is 9.50 × 10⁻¹⁰. -10 The result was M. Detailed kinetic parameters are shown in Table 10 below. These results revealed that hz10B4, hz17A3, and hz9B11 have high affinity for human B7-H3, and are superior to the affinity of the control antibody.

[0125] [Table 10]

[0126] Example 12: Conjugation of anti-B7-H3 humanized antibody to cells naturally expressing human B7-H3 detected by FACS. A suspension of human ovarian cancer cells (SKVO3 cells) naturally expressing human B7-H3 was incubated at 37°C for 30 minutes with humanized antibodies (hz9B11, hz10B4, and hz17A3; diluted in a 3-fold gradient from a concentration of 10 μg / ml, for a total of 10 concentrations). The following controls were prepared: (1) Positive control (PC): control antibodies enobrituzumab and MGC-018, (2) Negative control (NC): isotype control antibody. The cells were washed three times with PBS, and goat anti-human IgG-FITC (catalog: F9512, Sigma) diluted 1:200 was added to the cells, followed by incubation for 30 minutes. Next, the cells were washed three more times with PBS, and the mean fluorescence intensity (MFI) of the cells was measured using a flow cytometer (model B49007AD, SNAW31211, BECKMAN COULTER) to detect the binding ability of humanized antibodies to the B7-H3 protein on the surface of SKVO3 cells. As detected by FACS, the humanized antibodies hz9B11, hz10B4, and hz17A3 all bound to the B7-H3 protein on the surface of SKVO3 cells with superior binding ability compared to the control antibody enobrituzumab. The half-effect concentration (EC50) values ​​for each antibody are shown in Table 11, and binding is shown in Figure 9.

[0127] [Table 11]

[0128] Example 13: Intracellular translocation activity of anti-human B7-H3 humanized antibody that binds to B7-H3 on the cell surface. Human breast cancer cells naturally expressing human B7-H3, MBA-MB-468, are placed in 3 x 10⁶ wells. 3Cells were inoculated at a density of 1 cell per well into a 96-well cell culture plate and cultured for 24 hours. The cells were washed once with PBS, and the supernatant was discarded. Humanized antibodies hz9B11, hz10B4, and hz17A3, as well as the positive control enobrituzumab and the negative control (isotype control antibody), were diluted to 10 μg / ml in RPMI 1640 (containing 10% FBS) and added to MBA-MB-468 cells. The plates were then divided into two groups: one group was placed in an electrically heated constant temperature incubator at 37°C, and the other group was placed in a refrigerator at 4°C as the negative control. The negative control plate was incubated for 1 hour, washed three times with PBS, and the cells were added with goat anti-human IgG Fc-FITC secondary antibody diluted 1:200, followed by incubation at 4°C for 30 minutes. Next, the cells were washed three times again with PBS, and the mean fluorescence intensity (MFI) of the cells was measured by FACS. The experimental group plates were incubated at 37°C for 5 hours, and after adding the 1:200 diluted goat anti-human IgG Fc-FITC secondary antibody to the cells, the mixture was incubated at 4°C for 30 minutes. Then, the cells were washed three times again with PBS, and the mean fluorescence intensity (MFI) of the cells was measured by FACS. The intracellular transfer efficiency of the antibody was calculated according to the formula: intracellular transfer rate % = 100 - (MFI of the sample incubated at 37°C × 100 / MFI of the control sample incubated at 4°C).

[0129] These experimental results (Figure 10) revealed that the humanized antibodies hz9B11, hz10B4, and hz17A3, as well as the control antibody enobrituzumab, can all be endocytotically mediated by B7-H3.

[0130] Example 14: Detection of the killing activity of an anti-human B7-H3 antibody drug conjugate in tumor cells A suspension of SKVO3 cells and A431 cells that naturally express human B7-H3 was placed in 1 × 10⁶ wells. 3After inoculating 96-well cell culture plates with a cell density of 100 cells, these were incubated overnight in a 37°C, 5% CO2 incubator. Anti-human B7-H3 antibody drug conjugates named hz9B11-M, hz10B4-M, and hz17A3-M, prepared by conjugating MMAE, along with positive controls enobrituzumab-M and MGC-018-M, and the prepared ADC control MW14-M, were added to cells at various concentrations (from 11.1 μg / ml to 3-fold dilution, a total of 6 concentrations, each in a 3-well cell chain). These were then incubated in a 37°C, 5% CO2 incubator. After 4 days, the cytotoxic activity of the anti-B7-H3 antibody drug conjugates was detected using the Cell Counting Kit-8 (CCK-8 kit).

[0131] These results (Figures 11 and 12) revealed that each anti-B7-H3 antibody-drug conjugate could kill tumor cells in a dose-dependent manner. In A431 cells, hz10B4-M exhibited slightly better activity than hz9B11-M and hz17A3-M, was comparable to the activity of the control antibody-drug conjugate MGC-018-M, and was clearly superior to the activity of enobrituzumab-M. In SKOV-3 cells, low concentrations of hz9B11-M and hz17A3-M showed certain activities that were clearly superior to the activity of the control antibody-drug conjugate, while hz10B4-M showed activity comparable to the activity of the control antibody-drug conjugate MGC-018-M and was clearly superior to the activity of enobrituzumab-M.

[0132] Example 15: Pharmacodynamic evaluation of anti-B7-H3 antibody drug conjugate in a subcutaneous tumor model of A431 cells in nude mice. 3 x 10 oz. of 5-week-old male BALB / c nude mice 6 Individual A431 cells were subcutaneously inoculated, and the tumor grew to approximately 100 mm. 3When the mice reached maturity, they were randomly divided into groups of 5 per group. The group divisions, dosage, and frequency for each group are shown in Table 15. Each group of mice received intravenous injections twice a week for a total of 4 times, and the tumor volume and body weight of each mouse were measured simultaneously. After discontinuation of administration, the mice were continuously observed for 2 weeks, and if a nude mouse lost more than 15% of its body weight, or if a nude mouse exceeded 3000 mmHg, the tumor volume was reduced. 3 If the tumor volume exceeds 2000 mm, or if the entire group of nude mice exceeds 2000 mm 3 If the average tumor volume exceeded a certain value, the experiment was stopped and the mouse(s) were euthanized. The grouping and administration scheme of nude mice are shown in Table 12, and the tumor volume change curves and body weight change curves of nude mice are shown in Figures 13 and 14, respectively.

[0133] [Table 12]

[0134] As shown in Figures 13 and 14, in a subcutaneous tumor model of A431 cells in nude mice, hz10B4-M showed the best antitumor effect, which was essentially equivalent to or slightly better than the effect of MGC-018-M, hz17A3-M was second best in terms of antitumor effect, superior to the antitumor effect of enobrituzumab-M, and hz9B11-M showed certain efficacy, but was weaker than the efficacy of hz10B4-M and hz17A3-M. No apparent toxic effects of the small molecule MMAE used in the preparation of ADCs were observed, and the body weight of the animals in each experimental group steadily increased, with no significant difference in body weight between the experimental and control groups.

[0135] While specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to these details in accordance with all disclosed teachings, and that all such modifications remain within the scope of the present invention. The entire scope of the present invention is given by the appended claims and all their equivalents.

[0136] In this specification, descriptions of terms such as “one embodiment,” “several embodiments,” “exemplary embodiments,” “examples,” “specific examples,” and “several examples” mean that the specific features, structures, materials, or characteristics described in relation to an embodiment or example are included in at least one embodiment or example of this disclosure. In this specification, exemplary descriptions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be appropriately combined in any one or more embodiments or examples.

Claims

1. An anti-human B7-H3 antibody or a fragment thereof, a heavy chain variable region (VH) comprising complementarity determining region (CDR) 1, CDR2, and CDR3; a light chain variable region (VL) comprising CDR1, CDR2, and CDR3; Including, wherein the CDR1, CDR2, and CDR3 of the VH and the CDR1, CDR2, and CDR3 of the VL have the following amino acid sequence combinations: [Table 1] An anti-human B7-H3 antibody or a fragment thereof, comprising one of the following:

2. The VH and VL are a combination of the following amino acid sequences: [Table 2-1] [Table 2-2] The anti-human B7-H3 antibody or fragment thereof according to claim 1, characterized in that it comprises one of the following:

3. The anti-human B7-H3 antibody or a fragment thereof is It is capable of specifically binding to the human B7-H3 extracellular domain and has a specific binding affinity of 5×10 －１０ having an affinity constant (KD) less than M; having cross-reactivity with monkey B7-H3 and no binding activity to mouse B7-H3; has weak cross-reactivity with monkey B7-H3 and its binding activity to monkey B7-H3 is lower than its binding activity to human B7-H3; or Capable of specifically binding to B7-H3 on the cell surface and being internalized into said cell mediated by B7-H3. The anti-human B7-H3 antibody or a fragment thereof according to claim 1,

4. A fusion molecule comprising: (1) a first active element which is a first targeting moiety comprising an anti-human B7-H3 antibody or a fragment thereof according to any one of claims 1 to 3; (2) one or more second active elements comprising an effector element and / or a second targeting moiety; and wherein the first active element is linked to the second active element via a bond or a linker, and the one or more second active elements may be the same or different.

5. A polynucleotide, characterized in that it encodes the anti-human B7-H3 antibody or a fragment thereof according to any one of claims 1 to 3, or the fusion molecule according to claim 4.

6. A construct, characterized in that it comprises the polynucleotide of claim 5.

7. A host cell, characterized in that it comprises a polynucleotide according to claim 5 or a construct according to claim 6.

8. A conjugate, characterized in that it comprises a first active element which is a first targeting moiety comprising an anti-human B7-H3 antibody or a fragment thereof according to any one of claims 1 to 3.

9. The conjugate of claim 8, further comprising at least one second active element, wherein the first active element is covalently linked to the second active element, and the second active element comprises one or more effector moieties.

10. The effector moiety is one or more effector molecules in the following groups (1) to (3) or derivatives thereof: (1) Small molecule compounds that are tubulin inhibitors: maytansinoids, monomethylauristatin E, monomethylauristatin F, monomethyldolastatin 10, tubulysin and its derivatives, cryptophycin and its derivatives, and taltubulin; (2) Small molecule compounds that are topoisomerase inhibitors: PNU-159682 (a metabolite of doxorubicin) and its derivatives, SN38 (a metabolite of irinotecan) and its derivatives, and exatecan; (3) Small molecule compounds that are DNA binders: PBD and its derivatives, and duocarmycin and its derivatives; The conjugate of claim 9, which is a small molecule drug conjugate selected from the group consisting of:

11. An anti-human B7-H3 antibody or fragment thereof according to any one of claims 1 to 3, a fusion molecule according to claim 4, a polynucleotide according to claim 5, a construct according to claim 6, a host cell according to claim 7, or a conjugate according to any one of claims 8 to 10; optionally, a pharma- ceutically acceptable excipient(s); A composition comprising:

12. An agent for diagnosing a disease associated with B7-H3 overexpression, comprising an anti-human B7-H3 antibody or fragment thereof described in any one of claims 1 to 3, a fusion molecule described in claim 4, a polynucleotide described in claim 5, a construct described in claim 6, a host cell described in claim 7, a conjugate described in any one of claims 8 to 10, or a composition described in claim 11.

13. A pharmaceutical for treating a disease associated with B7-H3 overexpression, comprising an anti-human B7-H3 antibody or fragment thereof described in any one of claims 1 to 3, a fusion molecule described in claim 4, a polynucleotide described in claim 5, a construct described in claim 6, a host cell described in claim 7, a conjugate described in any one of claims 8 to 10, or a composition described in claim 11.

14. The agent according to claim 12 or the medicament according to claim 13, characterized in that the disease associated with B7-H3 overexpression is solid tumor cancer.

15. 15. The agent or medicament of claim 14, wherein the solid tumor cancer comprises gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, esophageal cancer, non-small cell carcinoma, prostate cancer, ovarian cancer, neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma, Wilms' tumor, and desmoplastic small round cell tumor.

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