Bispecific antibody that binds to EGFR and B7-H3

Abstract

The present invention provides a newly designed type of bispecific antibody molecule, particularly an anti-B7-H3 / EGFR bispecific antibody molecule that can simultaneously bind to B7-H3 and EGFR.

Description

Technical Field

[0001] The present invention generally relates to the fields of immunology and antibody engineering. Specifically, the present invention relates to a novel bispecific antibody molecule artificially designed, particularly a bispecific antibody that simultaneously binds to EGFR and B7-H3, a polynucleotide encoding the antibody molecule or each of its chains, a vector containing the polynucleotide, a host cell containing the polynucleotide or vector, an immune complex and a pharmaceutical composition containing the antibody molecule, and the use of the antibody molecule in the immunotherapy, prevention and / or diagnosis of diseases.

Background Art

[0002] Worldwide, lung cancer is one of the most common types of cancer tumors, and about 80% - 85% of lung cancer patients are non-small cell lung cancer (NSCLC) patients. NSCLC can be classified into common types such as lung adenocarcinoma, lung squamous cell carcinoma, and large cell lung cancer. Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor, a large transmembrane glycoprotein with a molecular weight of about 170 kDa, a member of the ErbB receptor family, and the most common cancer driver gene in NSCLC. In Asian NSCLC patients, about 40% are caused by EGFR mutations, and in the Caucasian population, about 15% are caused by EGFR mutations (Gower A, Wang Y, Giaccone G. Oncogenic drivers, targeted therapies, and acquired resistance in non-small-cell lung cancer. J. Mol. Med. 2014, 92: 697 - 707.). In other cancer tumor patients, such as epithelial-derived cancers of kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, and head and neck cancer, there are all overexpression and / or abnormal mutant activation of EGFR.

[0003] Regarding the abnormal activation and amplification of NSCLC-EGFR, currently, there are already targeted treatment methods with EGFR-TKI small molecule inhibitors (gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, almonertinib, etc.) and biological monoclonal antibody macromolecules (cetuximab, panitumumab, necitumumab, nimotuzumab). EGFR-TKI small molecule inhibitors are still the standard treatment method for current non-small cell lung cancer (NSCLC), but EGFR-TKI small molecules mainly target patients with tyrosine kinase active structure mutations and always cause drug resistance obstacles due to target gene mutations. Therefore, it is necessary to continuously develop new target drugs for new mutation sites, which greatly limits the clinical use of this class of drugs and is also a severe challenge faced by the entire EGFR-TKI industry.

[0004] The EGFR activation mutation region mainly occurs in the EGFR exon 18-21 tyrosine kinase domain (Jiyeon Yun, Soo-Hwan Lee, Seok-Young Kim, et al. Antitumor activity of Amivantamab (JNJ-61186372), an EGFR-MET bispecific antibody, in diverse models of EGFR exon 20 insertion-driven NSCLC. Cancer Discov 2020, 10:1194-209.). The binding region of the EGFR antibody is mainly located in the EGFR extracellular ligand domain, which can avoid the occurrence of drug resistance mutations. At the same time, the EGFR antibody can inhibit the growth of tumor cells by inhibiting the binding of EGFR and the ligand, and can also utilize its unique ADCC (antibody-dependent cell-mediated cytotoxicity) to kill tumors together with immune cells, thereby exerting an anti-tumor killing effect through various mechanisms of action.

[0005] B7-H3 (also known as CD276) is a type I transmembrane protein (Picarda E, Ohaegbulam KC, Zang X. Molecular pathways: targeting B7-H3 (CD276) for human cancer immunotherapy. Clin Cancer Res. 2016, 22:3425-31. and, Yang S, Wei W, Zhao Q. B7-H3, a checkpoint molecule, as a target for cancer immunotherapy. Int J Biol Sci. 202016:1767-73), which has a very similar structure to PD-L1 and belongs to the B7 / CD28 superfamily. It is expressed at low levels in most normal human tissues, but is abnormally highly expressed in tumor cells such as lung cancer, colon cancer, head and neck cancer, breast cancer, ovarian cancer, pancreatic cancer, etc. (Lee YH, Martin-Orozco N, Zheng P, Li J, Zhang P, Tan H, et al. Inhibition of the B7-H3 immune checkpoint limits tumor growth by enhancing cytotoxic lymphocyte function. Cell Res. 2017, 27:1034-45. and, Kontos F, Michelakos T, Kurokawa T, Sadagopan A, Schwab JH, Ferrone CR, et al. B7-H3: an attractive target for antibody-based immunotherapy. Clin Cancer Res. 2020. https: / / doi.org / 10.1158 / 1078-0432.CCR-20-2584. and, Seaman S, Zhu Z, Saha S, Zhang XM, Yang MY, Hilton MB, et al. Eradication of tumors through simultaneous ablation of CD276 / B7-H3-positive tumor cells and tumor vasculature. Cancer Cell. 2017, 31:501-15).Although the B7-H3 receptor has not yet been confirmed, in tumor immunity, B7-H3 may be involved in regulating the immune function of cytotoxic lymphocytes (Kraan J, van den Broek P, Verhoef C, Grunhagen DJ, Taal W, Gratama JW, et al. Endothelial CD276 (B7-H3) expression is increased in human malignancies and distinguishes between normal and tumour-derived circulating endothelial cells. Br J Cancer. 2014, 111: 149-56), and there is evidence that B7-H3 expression may be correlated with the EGFR gene expression status and the anti-PD-1 treatment effect (Yonesaka K, Haratani K, Takamura S, Sakai H, Kato R, Takegawa N, et al. B7-H3 negatively modulates CTL-mediated cancer immunity. Clin Cancer Res. 2018, 24: 2653-64). Because B7-H3 has a good selective expression profile, there are currently biopharmaceutical research and development companies developing B7-H3 monoclonal monomers or B7-H3-ADC drugs for research and treatment in the field of related tumor diseases.

[0006] Antibody molecules can specifically bind to corresponding antigens and have become important therapeutic, prophylactic and / or diagnostic agents for various diseases (such as cancer, autoimmune diseases, inflammatory diseases, infectious diseases, etc.). However, in clinical use, there are several limitations to monoclonal antibodies that target only one target. After a patient is treated with a monoclonal antibody, drug resistance or non-response may occur. With the research on cancer and other various diseases, it has been found that various signaling pathways are often involved in the occurrence and progression of diseases, and single-target immunotherapy usually does not fully exert the therapeutic effect of diseases in many diseases.

[0007] Multispecific antibodies (e.g., bispecific antibodies) can specifically bind to different antigens, and thus can be designed as signal transduction pathways that act on two or more different media simultaneously. Due to these advantages, the wide use of multispecific antibodies (e.g., bispecific antibodies) can be expected to have future potential.

[0008] In this field, bispecific antibodies with selectable improved properties are still needed. The above bispecific antibodies can simultaneously bind to different antigens, particularly EGFR and B7-H3, and retain the binding activity of different epitopes corresponding to each antigen-binding site and other properties. At the same time, it is necessary to obtain a form of bispecific antibody that has a certain stability and better productivity and developability. The form of bispecific antibody is physically and biologically stable, and thus the antibody is recognized to have better productivity and developability.

Summary of the Invention

[0009] The first aspect of the present invention relates to an antibody comprising an antigen-binding region that specifically recognizes EGFR (e.g., human EGFR) and an antigen-binding region that specifically binds to B7-H3. In some embodiments, the antibody is a multispecific antibody, e.g., a bispecific antibody.

[0010] The antibody or its antigen-binding fragment that binds to EGFR and B7-H3 provided by the present invention improves the medicinal efficacy and safety selectivity of the antibody molecule by reducing the affinity for EGFR and improving the affinity for B7-H3. At the same time, the GlymaxX low-fucosylation technology is adopted to enhance the antibody-dependent cell-mediated cytotoxicity (ADCC). Compared with amivantamab (JNJ-372) used for NSCLC-EGFR exon 20 insertion mutation (exon20ins), B7-H3 has a wider expression profile than cMET and a wider application range for tumor treatment. At the same time, under the introduction of the B7-H3 parent, not only the EGFR antibody blocking activity in the bispecific antibody is improved, but also the overall ADCC effect of the bispecific antibody is improved.

[0011] The bispecific antibody or antigen-binding fragment thereof that binds to EGFR and B7-H3 provided by the present invention has one or more of the following characteristics: (a) It binds specifically to one or two antigens with high affinity, (b) It is easily expressed in cultured cells in vitro, and the chains of the antibody molecule can be accurately coupled or paired with each other, (c) It has good physical stability, particularly good long-term thermal stability, and can maintain biological activity over a long period, (d) When it binds specifically to one or two antigens, it exerts biological functions by regulating (e.g., inhibiting or activating) the signal transduction pathways involved in each antigen, (e) It exerts effector functions, (f) It has better antitumor activity.

[0012] In one embodiment, the different antigen-binding sites bind to the same epitope or different epitopes on the same antigen.

[0013] In one embodiment, the first antigen-binding region or the second antigen-binding region is derived from a human antibody, a humanized antibody, or a chimeric antibody.

[0014] In some embodiments, the antibody of the present invention, such as a bispecific antibody, also includes a heavy chain constant region. In one embodiment, the heavy chain constant domain is derived from IgG1. It should be understood that mutations may be made to the Fc in the constant domain to achieve the effect of stabilizing the antibody or enhancing the effector function.

[0015] In one embodiment, the first antigen-binding region is specific for the first antigen, and in one embodiment, the first antigen is EGFR.

[0016] In one embodiment, the second antigen-binding region is specific for the second antigen, and in one embodiment, the second antigen is B7-H3.

[0017] The antibody of the present invention may also contain other antigen-binding regions that bind to other antigens in order to form a multispecific antibody. The types of other antigens to which the antibody molecule of the present invention specifically binds are not particularly limited, and the antigen may be, for example, a cytokine, a growth factor, a hormone, a signaling protein, an inflammatory mediator, a ligand, a cell surface receptor or a fragment thereof. In one embodiment, the other antigens to which the antibody molecule of the present invention specifically binds are selected from tumor-associated antigens, immune checkpoint molecules, angiogenesis-inducing factors, tumor necrosis factor receptor superfamily members and costimulatory molecules in the immune system, and ligands and / or receptors of these molecules.

[0018] In one aspect, the present invention provides a nucleic acid encoding any one or more polypeptide chains in the antibody molecule of the present invention, a vector containing the nucleic acid, and a host cell containing the nucleic acid or the vector.

[0019] In one aspect, the present invention provides a vector, preferably an expression vector such as pcDNA3.1, containing a polynucleotide encoding any one or more polypeptide chains in the antibody molecule of the present invention.

[0020] In one aspect, the present invention provides a method for producing the antibody molecule of the present invention or a fragment thereof.

[0021] In some embodiments, the present invention provides an immune complex, a pharmaceutical composition, a reagent kit, a combination product or a product containing the antibody of the present invention.

[0022] In some embodiments, the antibodies, pharmaceutical compositions, immunocomplexes, combination products, or reagent kits of the present invention are used, for example, to prevent or treat diseases such as acute and chronic inflammatory diseases, infections (e.g., chronic infections), tumors, etc. For example, the above diseases are tumors (e.g., cancer) or infections. In some embodiments, the tumor is tumor immune escape. Preferably, the tumor is a gastrointestinal tumor, a lung tumor, or a skin tumor. In some embodiments, the infection is a chronic infection.

[0023] In another aspect, the present invention relates to a method for preventing or treating a disease in a subject or individual, the method comprising administering to the subject an effective amount of any antibody or fragment thereof, pharmaceutical composition, immunocomplex, combination product, or reagent kit described in the present invention. For example, the above diseases are tumors (e.g., cancer) or infections. In some embodiments, the tumor is tumor immune escape. In one embodiment, the tumor is a gastrointestinal tumor, a lung tumor, or a skin tumor. In one embodiment, the infection is a chronic infection.

[0024] In another aspect, the present invention also relates to the use of any antibody or fragment thereof or immunocomplex described in the present invention for the manufacture of a drug, pharmaceutical composition, reagent kit, or combination product for the treatment of tumors (e.g., cancer) or infections in a subject. In some embodiments, the tumor is tumor immune escape. In one embodiment, the tumor is a gastrointestinal tumor, a lung tumor, or a skin tumor. In one embodiment, the infection is a chronic infection.

[0025] The present invention also relates to a method for detecting an antigen in a sample.

[0026] In another aspect, the present invention relates to the following specific embodiments: 1. A bispecific antibody that binds to EGFR and B7-H3, which comprises a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to EGFR and the second antigen-binding region specifically binds to B7H3.

[0027] 2. The bispecific antibody of Embodiment 1, wherein the second antigen-binding region comprises the HCDR1, HCDR2, and HCDR3 sequences of the heavy-chain variable region shown in SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, and the LCDR1, LCDR2, and LCDR3 sequences of the light-chain variable region shown in SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. Preferably, the HCDR1 adopts the Abm scheme, and the HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 adopt the Kabat scheme.

[0028] 3. The bispecific antibody according to Embodiment 1, wherein the second antigen-binding region comprises the HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region VH and the LCDR1, LCDR2, and LCDR3 of the light-chain variable region VL, where (i) the HCDR1, HCDR2, and HCDR3 are the three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH shown in SEQ ID NO: 3, and the LCDR1, LCDR2, and LCDR3 are the three complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in VL shown in SEQ ID NO: 4; (ii) the HCDR1, HCDR2, and HCDR3 are the three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH shown in SEQ ID NO: 5, and the LCDR1, LCDR2, and LCDR3 are the three complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in VL shown in SEQ ID NO: 6, or (iii) the HCDR1, HCDR2, and HCDR3 are the three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH shown in SEQ ID NO: 7, and the LCDR1, LCDR2, and LCDR3 are the three complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in VL shown in SEQ ID NO: 8. Preferably, the HCDR1 adopts the Abm scheme, and the HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 adopt the Kabat scheme.

[0029] 4. The bispecific antibody according to Embodiment 1, wherein the second antigen-binding region comprises HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region VH, and LCDR1, LCDR2, and LCDR3 of the light-chain variable region VL, where (i) the HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 15, the HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, the HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, the LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 18, the LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19, the LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 20, or (ii) the HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 21, the HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 22, the HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 23, the LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 24, the LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 25, the LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 26, or (iii) the HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 28, the HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 29, the LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 30, the LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 31, and the LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 32.

[0030] 5. The bispecific antibody according to any one of Embodiments 1 to 4, wherein the second antigen-binding region contains a heavy-chain variable region VH, and the VH contains the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or consists of such amino acid sequences.

[0031] 6. The bispecific antibody according to any one of Embodiments 1 to 5, wherein the second antigen-binding region contains a light-chain variable region VL, and the VL contains the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, or consists of such amino acid sequences.

[0032] 7. The bispecific antibody according to any one of Embodiments 1 to 6, wherein the second antigen-binding region contains a heavy-chain variable region VH and a light-chain variable region VL, and (i) the VH contains the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 3, or consists of such amino acid sequences, and the VL contains the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 4, or consists of such amino acid sequences, (ii) The VH contains, or consists of, the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 5, and the VL contains, or consists of, the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 6, or (iii) The VH contains, or consists of, the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 7, and the VL contains, or consists of, the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 8.

[0033] 8. A bispecific antibody according to any one of Embodiments 1 to 7, wherein the second antigen-binding region contains, or consists of, a heavy chain variable region VH and a light chain variable region VL, and wherein the VH and the VL are each SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or contain, or consist of, the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8.

[0034] 9. A bispecific antibody according to any one of Embodiments 1 to 7, wherein the first antigen-binding region contains the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region VH, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region VL, where The above HCDR1, HCDR2, and HCDR3 are three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH shown in SEQ ID NO: 1, and the above LCDR1, LCDR2, and LCDR3 are three complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in VL shown in SEQ ID NO: 2. Preferably, the above CDR is determined by adopting the Kabat method.

[0035] 10. The bispecific antibody according to Embodiment 9, wherein the HCDR1 of the above first antigen-binding region comprises, or consists of, the amino acid sequence of SEQ ID NO: 9, the above HCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 10, the above HCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 11, and the LCDR1 of the above first antigen-binding region comprises, or consists of, the amino acid sequence of SEQ ID NO: 12, the above LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 13, and the above LCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 14.

[0036] 11. The bispecific antibody according to Embodiment 9 or 10, wherein the above first antigen-binding region comprises a heavy chain variable region VH, and the above VH comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 1, or consists of them.

[0037] 12. The bispecific antibody according to any one of Embodiments 9 to 11, wherein the above first antigen-binding region comprises a light chain variable region VL, and the above VL comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 2, or consists of them.

[0038] 13. A bispecific antibody according to any one of Embodiments 9 to 12, wherein the first antigen-binding region comprises a heavy-chain variable region VH and a light-chain variable region VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 1, or consists of these, and the VL comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 2, or consists of these.

[0039] 14. A bispecific antibody according to any one of Embodiments 9 to 13, wherein the first antigen-binding region comprises or consists of a heavy-chain variable region VH and a light-chain variable region VL, wherein the VH and the VL each comprise or consist of the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0040] 15. A bispecific antibody according to any one of Embodiments 1 to 14, wherein the first antigen-binding region specifically binds to EGFR and comprises HCDR1, HCDR2 and HCDR3 of the heavy-chain variable region VH and LCDR1, LCDR2 and LCDR3 of the light-chain variable region VL, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 9, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 10, the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 11, the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 12, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 13, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14, and The second antigen-binding region specifically binds to B7H3 and includes HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region VH, and LCDR1, LCDR2, and LCDR3 of the light-chain variable region VL, where (i) the HCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 15, the HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 16, the HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 17, the LCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 18, the LCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 19, and the LCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 20, (ii) the HCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 21, the HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 22, the HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 23, the LCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 24, the LCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 25, and the LCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 26, or (iii) the HCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 27, the HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 28, the HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 29, the LCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 30, the LCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 31, and The above LCDR3 contains or consists of the amino acid sequence of SEQ ID NO: 32.

[0041] 16. The bispecific antibody according to Embodiment 15, wherein the first antigen-binding region comprises a VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 1 and a VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2, and the second antigen-binding region respectively SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or comprises or consists of a VH and a VL comprising the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8.

[0042] 17. The bispecific antibody according to any one of Embodiments 1 to 16, which comprises an Fc region, and preferably, the Fc region has hypofucosylation, for example, hypofucosylation obtained after treatment by the GlymaxX technology.

[0043] 18. The bispecific antibody according to Embodiment 17, which comprises a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region are the same or different.

[0044] 19. The bispecific antibody according to Embodiment 17 or 18, wherein the first Fc region and the second Fc region are each a human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, and for example, comprise or consist of the amino acid sequences shown in SEQ ID NO: 46 or SEQ ID NO: 47, or amino acid sequences having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity therewith.

[0045] 20. The bispecific antibody according to Embodiment 18 or 19, wherein mutations that promote heterodimerization of the first Fc region and the second Fc region are introduced into the first Fc region and the second Fc region.

[0046] 21. The bispecific antibody according to embodiment 20, wherein the mutation is introduced based on the Innobody technology.

[0047] 22. The bispecific antibody according to embodiment 21, wherein CH3 of one Fc region contains S364R and D399K mutations, and CH3 of another Fc region contains Y349T, K370S and K409D mutations.

[0048] 23. The bispecific antibody according to embodiment 22, wherein a) one Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 49 or SEQ ID NO: 50, and another Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 52 or SEQ ID NO: 53, b) one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 49 or SEQ ID NO: 50, and another Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 52 or SEQ ID NO: 53, or c) one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 49 or SEQ ID NO: 50 and contains the mutations Y349T, K370S and K409D, and another Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 52 or SEQ ID NO: 53 and contains the mutations S364R and D399K.

[0049] 24. The bispecific antibody according to Embodiment 20, wherein the mutation is introduced based on the Knob-into-Hole technology, and the corresponding Knob mutation and Hole mutation are introduced into the first Fc region and the second Fc region.

[0050] 25. The bispecific antibody according to Embodiment 24, wherein a) one Fc region polypeptide contains the mutation T366W and the other Fc region polypeptide contains T366S, L368A and Y407V (numbering according to the EU index), or b) one Fc region contains the amino acid substitutions S354C and T366W, and the other Fc region contains the amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the EU index).

[0051] 26. The bispecific antibody according to any one of Embodiments 1 to 25, wherein the first and / or second antigen-binding region (e.g., the heavy chain variable region herein) may be further linked to one or two heavy chain constant regions (e.g., the heavy chain constant regions of human IgG1, human IgG2, human IgG3, or human IgG4), and the heavy chain constant region includes a CH1 and an Fc region and is linked with or without a hinge region, for example, the C-terminus of the heavy chain variable region is linked to the N-terminus of the CH1 of the heavy chain constant region.

[0052] 27. The bispecific antibody according to Embodiment 26, wherein the CH1 contains the amino acid sequence shown in SEQ ID NO: 42, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 42, or consists of them.

[0053] 28. A bispecific antibody according to any one of Embodiments 1 to 27, wherein the first and / or second antigen-binding region (e.g., the light chain variable region herein) may be further linked to the light chain constant region. For example, the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region.

[0054] 29. The bispecific antibody according to Embodiment 28, wherein the light chain constant region is a kappa light chain constant region or a lambda light chain constant region.

[0055] 30. The bispecific antibody according to Embodiment 20, wherein the light chain constant region comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 54, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 54.

[0056] 31. A bispecific antibody according to any one of Embodiments 1 to 30, wherein the bispecific antibody is an IgG-like antibody having a configuration as shown in FIG. 1.

[0057] 32. The bispecific antibody according to Embodiment 31, which comprises the heavy chain 1 and the light chain 1, and the heavy chain 2 and the light chain 2, wherein the heavy chain 1 and the light chain 1 constitute the first half-antibody, and the heavy chain 2 and the light chain 2 constitute the second half-antibody, wherein the heavy chain 1 comprises the heavy chain variable region of the first antigen-binding region and the first heavy chain constant region, the light chain 1 comprises the light chain variable region of the first antigen-binding region and the first light chain constant region, the heavy chain 2 comprises the heavy chain variable region of the second antigen-binding region and the second heavy chain constant region, and the light chain 2 comprises the light chain variable region of the second antigen-binding region and the second light chain constant region.

[0058] 33. The bispecific antibody according to Embodiment 32, wherein the heavy chain 1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 33.

[0059] 34. The bispecific antibody according to Embodiment 32 or 33, wherein the light chain 1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 34.

[0060] 35. The bispecific antibody according to any one of Embodiments 32 to 34, wherein the heavy chain 1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 33, and the light chain 1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 34.

[0061] 36. The bispecific antibody according to any one of Embodiments 32 to 35, wherein the heavy chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO: 39, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO: 39.

[0062] 37. A bispecific antibody according to any one of Embodiments 32 to 36, wherein the light chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40.

[0063] 38. A bispecific antibody according to any one of Embodiments 32 to 37, wherein (1) the heavy chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 35, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 35, and the light chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 36, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 36, (2) the heavy chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 37, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 37, and the light chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 38, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 38, (3) The heavy chain 2 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 39, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 39, and the light chain 2 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 40, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 40.

[0064] 39. A bispecific antibody according to any one of Embodiments 32 to 38, wherein the heavy chain 1 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 33, and the light chain 1 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 34, and the heavy chain 2 and the light chain 2 are each of the following SEQ ID NOs., i) SEQ ID NO: 35 and SEQ ID NO: 36, ii) SEQ ID NO: 37 and SEQ ID NO: 38, iii) the amino acid sequences shown in SEQ ID NO: 39 and SEQ ID NO: 40, or contain, or consist of, an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequences shown above.

[0065] 40. A bispecific antibody according to any one of Embodiments 32 to 39, wherein (i) The heavy chain 1 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 33, and the light chain 1 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 34, The heavy chain 2 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 35, and the light chain 2 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 36, or (ii) The heavy chain 1 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 33, and the light chain 1 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 34, The heavy chain 2 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 37, and the light chain 2 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 38, or (iii) The heavy chain 1 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 33, and the light chain 1 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 34, The heavy chain 2 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 39, and the light chain 2 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 40.

[0066] 41. A bispecific antibody or an antigen-binding fragment thereof that binds to EGFR and B7-H3 according to any one of Embodiments 1 to 40, wherein the antibody or the antigen-binding fragment thereof has one or more of the following characteristics: (i) On the one hand, the antibody can block the binding of an EGFR ligand to EGFR, inhibit biological signal transduction, and block the corresponding biological activity of the tumor. On the other hand, the antibody can stimulate EGFR endocytosis, and ultimately, EGFR is degraded by intracellular lysosomes, etc., (ii) The antibody adopts a low-affinity EGFR antibody parent sequence for EGFR to significantly reduce the toxicity and side effects of a series of EGFR monoclonal antibodies against normal epithelial tissues such as the skin, (iii) Based on the low affinity of the above antibody for EGER, it cites the B7-H3 high-affinity antibody parental sequence, significantly improving EGFR signal blockade activity, and enhancing the pharmacodynamic biological activity and pharmacodynamic safety window of the bispecific antibody of the present invention. (iv) The above antibody is a low-fucosylated antibody. (ii) The above antibody has relatively high pharmacodynamic biological activity and safety. (v) The above antibody has excellent tumor killing and inhibitory effects. (vi) The above antibody has excellent ADCC in vitro and in vivo pharmacodynamic activities. (vii) The combination of the above antibody and a KRAS small molecule inhibitor has excellent antitumor synergistic effects.

[0067] 42. An isolated nucleic acid encoding any one of the chains of a bispecific antibody that binds to EGFR and B7-H3 according to any one of Embodiments 1 to 41.

[0068] 43. A vector containing the nucleic acid according to Embodiment 42, preferably the above vector is an expression vector, and preferably the above expression vector is pcDNA, such as pcDNA3.1.

[0069] 44. A host cell containing the nucleic acid according to Embodiment 42 or the vector according to Embodiment 43, preferably the above host cell is a prokaryote or a eukaryote, more preferably a yeast cell or a mammalian cell (for example, 293 cells or CHO cells, such as 293F cells or 293T cells or CHO-S cells).

[0070] 45. The host cell according to Embodiment 44, which is glycoengineered to express the RMD enzyme, preferably the above host cell is a CHO cell.

[0071] 46. The host cell according to Embodiment 45, which contains a nucleic acid encoding the RMD enzyme.

[0072] 47. The host cell according to Embodiment 46, wherein the RMD enzyme comprises, or consists of, an amino acid sequence shown in SEQ ID NO: 41 or an amino acid sequence having at least 90% identity therewith, and preferably, the RMD enzyme is derived from Pseudomonas aeruginosa.

[0073] 48. A method for producing a bispecific antibody that binds to EGFR and B7-H3, the method comprising culturing a host cell according to any one of Embodiments 44 to 47 under conditions suitable for the expression of a nucleic acid encoding the bispecific antibody according to any one of Embodiments 1 to 41, and optionally isolating the antibody or an antigen-binding fragment thereof, and optionally, the method further comprises recovering the antibody or an antigen-binding fragment thereof from the host cell (or the host cell culture medium).

[0074] 49. An immune complex comprising the bispecific antibody according to any one of Embodiments 1 to 41, which is bound to a therapeutic or diagnostic agent.

[0075] 50. A pharmaceutical composition comprising the bispecific antibody according to any one of Embodiments 1 to 41 or the immune complex according to Embodiment 49, and optionally a pharmaceutically acceptable excipient.

[0076] 51. The pharmaceutical composition according to Embodiment 50, which further comprises a second therapeutic agent, and preferably, the second therapeutic agent is selected from an anti-angiogenic agent, a chemotherapeutic agent, another antibody, a cytotoxic agent, a vaccine, an anti-infective agent, a small molecule drug, or an immunomodulatory agent (e.g., an activator of a costimulatory molecule or an inhibitor of an immune checkpoint molecule), and preferably, the second therapeutic agent is selected from a KRAS small molecule inhibitor, such as a KRAS G12C inhibitor (e.g., AMG510 (Sotorasib) or GFH925), a KRAS G12D (e.g., MRTX1133), or a KRAS G12S inhibitor.

[0077] 52. A pharmaceutical combination product, which comprises the bispecific antibody according to any one of Embodiments 1 to 41, or the immune complex according to Embodiment 49, or the pharmaceutical composition according to Embodiment 50, and one or more second therapeutic agents. Preferably, the second therapeutic agent is selected from an anti-angiogenic agent, a chemotherapeutic agent, another antibody, a cytotoxic agent, a vaccine, an anti-infective agent, a small molecule drug or an immunomodulatory agent (for example, an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule). Preferably, the second therapeutic agent is selected from a KRAS small molecule inhibitor, for example, a KRAS G12C inhibitor (for example, AMG510 (Sotorasib) or GFH925), a KRAS G12D (for example, MRTX1133) or a KRAS G12S inhibitor.

[0078] 53. A method for preventing or treating a tumor or an infectious disease in a subject, the method comprising administering to the subject an effective amount of the bispecific antibody according to any one of Embodiments 1 to 41, or the immune complex according to Embodiment 49, or the pharmaceutical composition according to Embodiment 50.

[0079] 54. The method according to Embodiment 53, further comprising co-administering to the subject one or more other therapies, the therapies including, for example, treatment modalities and / or other therapeutic agents. Preferably, the treatment modality includes surgical treatment and / or radiotherapy, or the therapeutic agent is selected from an anti-angiogenic agent, a chemotherapeutic agent, another antibody, a cytotoxic agent, a vaccine, an anti-infective agent, a small molecule drug or an immunomodulatory agent (for example, an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule). Preferably, the second therapeutic agent is selected from a KRAS small molecule inhibitor, for example, a KRAS G12C inhibitor (for example, AMG510 (Sotorasib) or GFH925), a KRAS G12D (for example, MRTX1133) or a KRAS G12S inhibitor.

[0080] 55. A method for preventing or treating a tumor or an infectious disease in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition according to embodiment 51 or the pharmaceutical combination product according to embodiment 52.

[0081] 56. The method according to any one of embodiments 53 to 55, wherein the tumor is cancer, such as a solid tumor or a hematological tumor, including cancers of epithelial origin, such as gastrointestinal tumors, lung tumors, or skin tumors, such as skin cancer (e.g., cutaneous squamous cell carcinoma, head and neck cancers such as head and neck squamous cell carcinoma), esophageal cancer (e.g., esophageal squamous cell carcinoma), intestinal cancer (e.g., colon cancer, rectal cancer, colorectal cancer), or lung cancer (e.g., non-small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma).

[0082] 57. The method according to any one of embodiments 53 to 56, wherein in the tumor cells of the tumor, (i) compared with normal cells of adjacent tissues or compared with normal cells of the same tissue in a healthy subject, overexpresses wild-type EGFR (e.g., wild-type EGFR with increased nucleic acid or protein levels) and / or expresses mutant EGFR, preferably the mutant EGFR comprises one or more mutations selected from R521K, L858R, T790M, G719X, C797S, Y1069C, Exon19 deletion (Del19), Exon20ins (e.g., S768_D770dup), preferably the mutant EGFR comprises R521K / Y1069C, R521K, L858R / T790M / C797S, Del19 / T790M / C797S or S768_D770dup, (ii) compared with normal cells of adjacent tissues or compared with normal cells of the same tissue in a healthy subject, overexpresses wild-type KRAS (e.g., having wild-type KRAS with increased nucleic acid or protein levels) or expresses mutant KRAS, preferably the mutant KRAS comprises a G12 or G13 mutation, such as G12D or G12C, (iii) having B7-H3 with elevated nucleic acid or protein levels compared to normal cells of adjacent tissues or compared to normal cells of the same tissue in a healthy subject, and / or (iv) the tumor cells are drug resistant to tyrosine kinase inhibitors such as first generation (erlotinib) and third generation (osimertinib).

[0083] 58. The method according to embodiment 57, wherein mutated EGFR and mutated KRAS are expressed in the tumor cells of the tumor, for example, including mutated EGFR having R521K and mutated KRAS having G120D.

[0084] 59. A method for detecting antigen EGFR and / or B7-H3 in a sample, the method comprising: (a) contacting the sample with the bispecific antibody according to any one of embodiments 1 to 41, and (b) detecting the formation of a complex between the antibody or its antigen-binding fragment and EGFR and / or B7-H3, and optionally the antibody is detectably labeled.

[0085] 60. Use of the antibody or its antigen-binding fragment according to any one of embodiments 1 to 41, and / or the isolated nucleic acid according to embodiment 42, and / or the vector according to embodiment 43, and / or the host cell according to any one of embodiments 44 to 47, and / or the immune complex according to embodiment 49, and / or the pharmaceutical composition according to embodiment 50 or 51, or the pharmaceutical combination product according to embodiment 52, in the manufacture of a drug for preventing and / or treating a subject's disease.

[0086] The present invention further includes any combination of any of the embodiments described herein. Any of the embodiments described herein or any combination thereof applies to any and all antibodies or fragments thereof, immune complexes, pharmaceutical compositions, combination products, or reagent kits, methods and uses of the invention described herein.

[0087] The preferred embodiments of the present invention described in detail below will be better understood when read in conjunction with the following drawings. For the purpose of explaining the present invention, the drawings show the current preferred embodiments. However, it should be understood that the present invention is not limited to the embodiments shown in the drawings.

Brief Description of the Drawings

[0088]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7.1

Figure 7.2

Figure 7.3

Figure 7.4

Figure 7.5

[0089] Here, the antibody or small molecule TKI (Osimertinib or Erlotinib) was diluted as follows: Figure 7.1 NCI-292, NCI-H322: The final dilution concentration of the antibody was up to 300 nM, with a 3.16-fold dilution. Figure 7.2 NCI-H1650, NCI-H1975: The final dilution concentration of the antibody was up to 300 nM, with a 3.16-fold dilution. Figure 7.3 NCI-H1703, SK-MES-1: For the antibody molecule, the final dilution concentration was up to 300 nM, with a 3.16-fold dilution, and for the small molecule TKI (Osimertinim and Erlotinib), the final dilution concentration was up to 1000 nM, with a 3.16-fold dilution. Figure 7.4 H1975 (EGFR L858R / T790M / C797S ): The final dilution concentration of various antibody molecules and Osimertinib was up to 300 nM, with a 3.16-fold dilution. For H322 (EGFR S768-D77dup ): The final dilution concentration of various antibody molecules and Osimertinib was up to 300 nM, with a 4-fold dilution. For PC9 + B7H3 (EGFR Del19 / T790M / C79S ): The final dilution concentration of various antibody molecules and Osimertinib was up to 100 nM, with a 4-fold dilution. Figure 7.5 LS180 and H292: The final dilution concentration of the antibody is up to 300 nM, with a 4-fold dilution, and for SK-MES-1: the final concentration is up to 300 nM, with a 3.16-fold dilution.

Figure 8

Figure 8(A)

Figure 8(B)

Figure 9

Figure 9(A)

Figure 10

Figure 11

Figure 12.1

Figure 12.2

Figure 12.3

Figure 12.4

Figure 13.1

Figure 13.1(A)

Figure 13.1(B)

Figure 13.1(C)

Figure 13.2

Figure 13.3

Figure 13.3(A)

Figure 13.3(B)

Figure 13.3(C)

Figure 13.4

Figure 14

Figure 15A

Figure 15B

Figure 15C

Figure 16A

Figure 16B

Figure 17A

Figure 17B

Figure 18A

Figure 18B

Modes for Carrying Out the Invention

[0090] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples described herein are illustrative only and not intended to be limiting. Other features, objects, and advantages of the invention will be apparent from the specification, drawings, and appended claims.

[0091] I. Definitions It should be understood that the invention is not limited to the specific methodologies, forms, or reagents described herein, as they may be modified. Also, the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the invention, which is to be understood as being limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0092] The following definitions are used to interpret this specification, and where appropriate, terms used in the singular may include the plural and vice versa. It must be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0093] The term "about," when used in conjunction with a numerical value, means a value within a range that is 5% less than the specified lower limit and 5% greater than the specified upper limit of the specified numerical value.

[0094] As used herein, the term "and / or" refers to any one of the selectable options, or two or more of the selectable options.

[0095] As used herein, the terms "comprising" or "including" refer to including the recited element, integer, or step without excluding any other element, integer, or step. When used herein, the terms "comprising" or "including" cover, unless otherwise specified, the case of consisting of the recited element, integer, or step. For example, when an antibody variable region "comprising" a specific sequence is recited, it is also intended to cover an antibody variable region consisting of the specific sequence.

[0096] As used herein, the references to "first" and "second" are for the sole purpose of distinguishing between two domains or two chains, and do not in any way indicate the position of the two domains.

[0097] As used herein, the amino acid positions of all variable regions of heavy and light chains are numbered based on the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "Kabat numbers".

[0098] As used herein, when used to refer to amino acid positions in domains of an antibody other than the variable regions (e.g., constant regions, e.g., the Fc region), they are numbered based on the EU numbering system described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "EU numbers". As is known to those of skill in the art, when position numbers and / or amino acid residues are assigned to a particular antibody isotype, they are intended to be applicable to the corresponding positions and / or amino acid residues of any other antibody isotype.

[0099] General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0100] The term "antibody" is used herein in the broadest sense and refers to a protein containing an antigen-binding site, covering natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, whole antibodies, and antibody fragments.

[0101] The terms "whole antibody", "full-length antibody", "complete antibody", and "complete antibody" are used interchangeably herein to refer to a naturally occurring glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected via disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH region and VL region can be further divided into hypervariable regions (complementary determining regions (CDR)) with relatively conserved regions (framework regions (FR)) intervening therebetween. Each VH and VL consists of three CDRs and four FRs, and are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions.

[0102] "Half antibody" or "half polymer" refers to a monovalent antigen-binding polypeptide. In some embodiments, the half antibody or half polymer comprises a VH / VL unit and optionally at least a portion of an immunoglobulin constant domain. In some embodiments, the half antibody or half polymer comprises one immunoglobulin heavy chain associated with one immunoglobulin light chain, or an antigen-binding fragment thereof. In some embodiments, the half antibody or half polymer is monospecific, i.e., binds to a single antigen or epitope. In some specific embodiments, the half antibody binds to EGFR and does not bind to B7-H3. In some specific embodiments, the half antibody binds to B7-H3 and does not bind to EGFR. Those skilled in the art will readily understand that the half antibody may have an antigen-binding domain consisting of a single variable domain (e.g., derived from Camelidae).

[0103] The term "antigen-binding fragment" of an antibody is a molecule distinct from the full-length antibody, which includes a part of the full-length antibody but can bind to the antigen of the full-length antibody or can bind to the antigen in competition with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived). Antigen-binding fragments can be produced by recombinant DNA technology or by cleaving a complete antibody by enzymatic or chemical means. Antigen-binding fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv, single-chain Fv, diabody, single-domain antibody (sdAb), nanobody.

[0104] "Fab fragment" or "Fab" can be used interchangeably herein and refers to an immunoglobulin fragment consisting of two polypeptide chains and containing the immunoglobulin heavy-chain variable region VH, heavy-chain constant domain CH1, light-chain variable region VL, and light-chain constant domain CL, where one polypeptide chain contains VH from the N-terminus to the C-terminus and one constant domain selected from CH1 and CL, and the other polypeptide chain contains VL from the N-terminus to the C-terminus and another constant domain selected from CL and CH1, where the VH domain and the VL domain pair to form an antigen-binding site. The Fab’ fragment is different from the Fab fragment because several residues (including one or more cysteines derived from the antibody hinge region) are added to the carboxy-terminal of the heavy-chain CH1 domain. Fab’-SH is the designation for a Fab’ in which the cysteine residue of the constant domain carries a free thiol group. The F(ab’)2 antibody fragment is first produced as a pair of Fab’ fragments with hinge cysteines between the Fab’ fragments. Other chemical couplings of antibody fragments are also known.

[0105] As used herein, the term "bispecific antibody" includes antigen-binding domains that specifically bind to two antigens or two epitopes. Unless otherwise specified, the order of the antigens to which the bispecific antibodies are named is arbitrary. That is, in some embodiments, the terms "anti-EGFR / B7-H3 bispecific antibody" and "anti-B7-H3 / EGFR bispecific antibody" can be used interchangeably. In some embodiments, the bispecific antibody includes two half-antibodies, where each half-antibody includes a single-chain variable region and optionally at least a portion of the heavy-chain constant region, and a single light-chain variable region and optionally at least a portion of the light-chain constant region. In some embodiments, the bispecific antibody includes two half-antibodies, where each half-antibody includes a single-chain variable region and a single light-chain variable region, and does not include more than one single-chain variable region and does not include more than one single light-chain variable region. In some embodiments, the bispecific antibody includes two half-antibodies, where each half-antibody includes a single-chain variable region and a single light-chain variable region, and the first half-antibody binds to the first antigen / epitope and does not bind to the second antigen, and the second half-antibody binds to the second antigen / epitope and does not bind to the first antigen.

[0106] The multispecific antibodies of the present invention may contain a linker. As used herein, the term "linker" refers to any molecule that can be directly linked to different parts of the multispecific antibody. Examples of linkers that establish covalent bonds between different parts of the multispecific antibody include peptide linkers and non-protein polymers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol. In some embodiments, the term "peptide linker" according to the present invention refers to an amino acid sequence that links together the amino acid sequences of different parts of the multispecific antibody. Preferably, the peptide linker has a length such that it sufficiently links the two entities so that they maintain their conformations relative to each other without interfering with the desired activity. The peptide linker may or may not mainly contain amino acid residues such as Gly, Ser, Ala, or Thr. Useful linkers include, for example, glycine-serine polymers including (GS)n, (GSGGS)n, (GGGGS)n, (GGGS)n, and (GGGGS)nG, where n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, or 10). Useful linkers further include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.

[0107] The terms "Fc domain" or "Fc region" are used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. The native immunoglobulin "Fc domain" includes two or three constant domains, namely, the CH2 domain, the CH3 domain, and an optional CH4 domain. For example, in a native antibody, the immunoglobulin Fc domain includes the second and third constant domains (CH2 domain and CH3 domain) derived from the two heavy chains of IgG, IgA, and IgD class antibodies, or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) derived from the two heavy chains of IgM and IgE class antibodies. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or the heavy chain constant region is numbered based on the EU numbering system (also referred to as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Two Fc regions can dimerize to form a dimeric Fc, and two different Fc regions can heterodimerize to form a heterodimeric Fc. As used herein, the terms "Fc region", "Fc portion", and "dimeric Fc (e.g., heterodimeric Fc)" do not include the heavy chain variable region VH and the light chain variable region VL, and the heavy chain constant region CH1 and the light chain constant region CL, but in some situations may include the hinge region at the N-terminus of the heavy chain constant region. In one embodiment, the human IgG heavy chain Fc region extends from Asp221 or Cys226 or Asp231 to the carboxyl terminus of the heavy chain. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region belongs to the human IgG4 subclass. In one embodiment, the Fc region belongs to the human IgG1 subclass.

[0108] As used herein, "Fc dimerization" refers to the dimerization of two Fc regions to form a dimer. "Fc heterodimerization" refers to the dimerization of two different Fc regions to form a dimer. The heterodimerized Fc regions constitute the Fc scaffold of a bispecific or multispecific antibody. Thus, a "heterodimer Fc scaffold" is a scaffold that can be linked at its N-terminus or C-terminus to a domain that binds an antigen (e.g., the heavy and / or light chain variable regions of an antibody that can bind a target molecule, or an antigen-binding fragment of an antibody, or a soluble portion of a ligand or receptor that can bind a target molecule) to form a multispecific antibody, such as a bispecific antibody, and that contains two different Fc regions or is the scaffold formed after dimerization of two different Fc regions.

[0109] An amino acid mutation is indicated by (original amino acid, amino acid position, mutated amino acid). For example, when the mutation site is located in the Fc region, "T366W" refers to the substitution of T at EU numbering position 366 with W. When referring to a combination of mutations, the combined mutations are linked by "and" or " / ". "R521K / Y1069C" indicates the simultaneous inclusion of mutations R521K and Y1069C. When describing a mutation, it should be noted that the specific position simultaneously covers the corresponding amino acid position on other polypeptide chains. For example, when referring to C220, this covers the amino acid at position 220 according to the EU numbering of the IgG1 heavy chain and the corresponding amino acids on other heavy chains, such as the amino acid at position 131 in IgG2, IgG3, or IgG4. When describing a mutation, the original amino acid at the specific position may be the amino acid described or may be another amino acid at the corresponding position.

[0110] "Complementary determining region", "CDR region" or "CDR" refers to a region within an antibody variable domain that is hypervariable in sequence and forms loops (the "hypervariable loops") determined in structure and / or contains antigen - contacting residues (the "antigen - contact points"). CDRs are mainly responsible for binding to the antigen epitope. The CDRs of the heavy and light chains are usually called CDR1, CDR2, and CDR3 and are numbered in order from the N - terminus. The CDRs located in the heavy - chain variable region of an antibody are called HCDR1, HCDR2, and HCDR3, and the CDRs located in the light - chain variable region of an antibody are called LCDR1, LCDR2, and LCDR3. In the amino - acid sequence of a given light - chain variable region or heavy - chain variable region, the precise amino - acid sequence boundaries of each CDR can be determined using any one or a combination of many known antibody CDR assignment systems. The above - mentioned assignment systems include, for example, Chothia based on the three - dimensional structure of the antibody and the topology of the CDR loops (Chothia et al., (1989) Nature 342:877 - 883, Al - Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927 - 948 (1997)), Kabat based on the variability of the antibody sequence (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (available at imgt.cines.fr / on the World Wide Web), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0111] For example, depending on different determination schemes of CDRs, the residues of each CDR are as follows.

[0112] [Table 1]

[0113] The CDR may be determined based on having the same Kabat numbering positions as the sequence of the reference CDR (for example, any one of the exemplary CDRs of the present invention).

[0114] Unless otherwise specified, in the present invention, the terms "CDR" or "CDR sequence" cover CDR sequences determined by any one of the above methods.

[0115] Unless otherwise specified, in the present invention, when referring to residue positions in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering positions based on the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0116] In one embodiment, the CDRs of the antibody of the present invention are determined by the Kabat rules, or the AbM rules, or a combination of those rules.

[0117] In one embodiment of the present invention, HCDR1 of VH in the antigen-binding region that binds to B7-H3 in the present invention is determined by the AbM rule, HCDR2 and HCDR3 are determined by the Kabat rule, and the CDRs of VL are determined by the Kabat rule.

[0118] In one embodiment of the present invention, the CDRs of VH and VL in the antigen-binding region that binds to EGFR in the present invention are determined by the Kabat law.

[0119] The term "hinge region" refers to a portion of an antibody heavy chain polypeptide that in a wild-type antibody heavy chain connects the CH1 region and the CH2 region and that, for example, in the IgG1 hinge region, has the sequence D221 - P230 according to EU numbering. The hinge regions of other IgG subclasses can be determined by aligning with the hinge region cysteine residues of the IgG1 subclass sequence.

[0120] As used herein, the terms "binding site" or "antigen-binding site" or "antigen-binding region" refer to any portion of an antibody molecule, such as a multispecific antibody, such as a bispecific antibody, that binds to a particular target or antigen. The antigen-binding region can be, for example, an antibody or immunoglobulin itself, or an antibody fragment. Such an antigen-binding region may or may not have a tertiary structure independent of the remainder of the BsAB and may or may not bind to its antigen / epitope as a single entity. In some embodiments, the antigen-binding region comprises a VH / VL pair consisting of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), a heavy chain variable region derived from a camelid heavy chain antibody, a VH-like single domain (v-NAR) derived from shark IgNAR, a camelized human VH domain, or a heavy chain variable region of a humanized camelid antibody.

[0121] When referring to the "first antigen-binding region" in a multispecific antibody or bispecific antibody, it refers to the binding region that binds to the first antigen and is not intended to limit the number of such antigen-binding regions included in the antibody. For example, a multispecific antibody or bispecific antibody may comprise one or more first antigen-binding regions. For example, a bispecific antibody comprises a first antigen-binding region and a second antigen-binding region, but may also comprise one or more first antigen-binding regions and one or more second antigen-binding regions.

[0122] When referring to "the antigen-binding region is derived from an antibody", the binding domain constituting the antigen-binding region refers to the binding domain of the antibody that specifically binds to the antigen or is derived therefrom. For example, a fragment that specifically binds to the antigen of the antigen-binding region, such as Fab, is the corresponding fragment of the antibody, such as Fab, or is derived therefrom, or the heavy chain variable region and / or light chain variable region of the antigen-binding region is the heavy chain variable region and / or light chain variable region of the antibody, or is derived therefrom, or one, two, three, four, five, or six CDRs of the antigen-binding region are the CDRs of the antibody. In some embodiments, the antigen-binding region is an antigen-binding fragment of an antibody.

[0123] As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen-binding sites, and each antigen-binding site of the at least two antigen-binding sites binds to a different epitope of the same antigen or a different epitope of a different antigen. The antibodies provided herein are typically multispecific antibodies, e.g., bispecific antibodies. A multispecific antibody is an antibody having binding specificity for at least two different antigen epitopes. In one embodiment, the present specification provides such a bispecific antibody having binding specificity for a first antigen and a second antigen.

[0124] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, an immunoglobulin of the IgG class is a heterotetrameric glycoprotein of approximately 150,000 daltons consisting of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has one heavy chain variable region (VH), also called the variable heavy domain, followed by three heavy chain constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each immunoglobulin light chain has one light chain variable region (VL), also called the variable light domain, followed by one light chain constant domain (CL). The heavy chains of immunoglobulins can be classified into one of five classes called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), where some classes can be further divided into subclasses such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can be classified into one of two types called κ and λ based on the amino acid sequence of their constant domains. An immunoglobulin basically consists of two Fab molecules and one Fc domain joined via the immunoglobulin hinge region.

[0125] The term "effector function" refers to a biological activity resulting from the immunoglobulin Fc region that is altered by the immunoglobulin isotype. Examples of immunoglobulin effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), cytokine secretion, uptake of antigen by antigen-presenting cells mediated by immune complexes, downregulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0126] The term "n-valent" antibody refers to the number of antigen-binding sites present in an antibody molecule. "Bivalent", "trivalent", and "tetravalent" antibodies refer to the presence of two, three, and four antigen-binding sites, respectively, in an antibody molecule.

[0127] As used herein, the terms "bind" or "specifically bind" refer to a binding action that is selective for an antigen and distinguishable from unwanted or non-specific interactions. The ability of a particular antigen-binding site to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as radioimmunoassay (RIA), biolayer interferometry, MSD assay, or surface plasmon resonance (SPR).

[0128] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y is usually indicated by the dissociation constant (K D ), and the dissociation constant is the ratio of the dissociation rate constant to the association rate constant (K dis and K on ) respectively. Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay described herein.

[0129] The "knob-in-hole" mutation or "knob-in-hole" variation refers to introducing mutations into the first Fc polypeptide and the second Fc polypeptide respectively by the "knob-in-hole" technique in this specification to form a protrusion ("knob") and a complementary cavity ("hole") at the interface of the first Fc polypeptide and the interface of the second Fc polypeptide. As is known in the art, the "knob-in-hole" technique can promote the accurate association of each chain of an antibody molecule by modifying the interface between different chains of the antibody molecule. Generally, the technique involves introducing a "protrusion / knob" into the interface of one chain and a corresponding "cavity / hole" into the interface of the other chain to be paired so that the protrusion can be placed in the cavity. One preferred interface includes the CH3 domain of the heavy chain constant domain of one chain and the CH3 domain of the heavy chain constant domain of another chain to be paired. A protrusion can be constructed by replacing a small amino acid side chain at the interface of the CH3 domain of the heavy chain constant domain derived from one chain with a relatively large side chain (such as tyrosine or tryptophan). By replacing a relatively large amino acid side chain with a relatively small side chain (such as alanine or threonine), a compensatory cavity of the same or similar size as the protrusion is constructed at the interface of the CH3 domain of the heavy chain constant domain of the other chain to be paired. Another selectable interface includes the CL domain of the light chain and the CH1 domain of the heavy chain of the Fab fragment as described above, and promotes accurate heterodimerization between the two chains of the Fab fragment by constructing a protrusion-cavity interaction.

[0130] As used herein, an antibody constant region or antibody constant domain includes CH1, CL, and Fc domains, as well as CH2, CH3, and optional CH4 domains that make up the Fc domain, and can be selected according to the expected function of the antibody molecule. For example, the constant region can be an IgA, IgD, IgE, IgG or IgM region, particularly the immunoglobulin constant domain of human IgG, such as the constant domain of human IgG1, IgG2, IgG3 or IgG4, preferably the constant domain of human IgG1. Further by way of example, the Fab fragment of an antibody may include CH and CL constant regions derived from IgG1. Further by way of example, the Fc region of an antibody may include CH2 and CH3 domains derived from IgG1. The immunoglobulin constant region can have a native sequence or a variant sequence.

[0131] The term "antigen" refers to a molecule that elicits an immune response. Such an immune response may relate to the production of antibodies or the activation of specific immune cells, or both. Those skilled in the art will understand that essentially any macromolecule, including all proteins or peptides, can be utilized as an antigen. Note that the antigen may be derived from recombinant or genomic DNA. In some embodiments herein, the first antigen and the second antigen are two different antigens.

[0132] The terms "tumor-associated antigen" or "cancer antigen" are used interchangeably to refer to a molecule (generally a protein, carbohydrate or lipid) that is expressed as a complete or fragment (e.g., MHC / peptide) on the surface of cancer cells, preferably as compared to normal cells, and such molecule can be used in the targeting of cancer cells preferentially by a drug.

[0133] The term "immune checkpoint molecule" refers to an inhibitory signaling molecule present in the immune system that is involved in avoiding tissue damage by regulating the persistence and intensity of the immune response in peripheral tissues and maintaining tolerance to its own antigens.

[0134] The term "cytokine" is a general term for proteins released from cell populations that act on other cells as intercellular mediators.

[0135] An "immune complex" is an antibody that binds to one or more other substances (including, but not limited to, cytotoxic agents or labels).

[0136] As used herein, the term "EGFR" refers to the epidermal growth factor receptor, a tyrosine kinase receptor, a large transmembrane glycoprotein with a molecular weight of approximately 170 kDa, belonging to the ErbB receptor family, the most common cancer driver gene in NSCLC. The EGFR activation mutation region mainly occurs in the EGFR exon 18-21 tyrosine kinase domain. The binding region of the EGFR antibody is mainly located in the EGFR extracellular ligand domain. The occurrence of drug resistance mutations can be avoided. At the same time, the EGFR antibody can inhibit the growth of tumor cells by inhibiting the binding of EGFR and the ligand, and can also utilize its own ADCC (antibody-dependent cell-mediated cytotoxicity) to kill tumors together with immune cells, thereby exerting an anti-tumor killing effect through various mechanisms of action.

[0137] The terms "B7-H3", "B7H3" and "CD276" can be used interchangeably herein. B7-H3 is a type I transmembrane glycoprotein belonging to the B7 / CD28 superfamily member, and its extracellular domain has a similar sequence to that of PD-L1. B7-H3 has 316 amino acids including a putative signal peptide consisting of 28 amino acids, an extracellular region and transmembrane region consisting of 217 amino acids, and a cytoplasmic domain consisting of 45 amino acids, with a molecular weight of approximately 45 kDa to 66 kDa. In the human body, due to exon duplication, the extracellular structure of B7-H3 may be an IgV-IgC-like domain (2Ig-B7-H3) or an IgV-IgC-IgV-IgC-like domain (4Ig-B7-H3). The sequence of cynomolgus monkey B7-H3 has approximately 90% homology with its human counterpart.

[0138] The term "effector function" refers to biological activities that are attributable to the Fc region of an antibody and vary depending on the isotype of the antibody. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0139] The term "GlymaxX technology" is a technology platform developed by ProBioGen to enhance the effect of bispecific antibody ADCC. It is a CHO host cell that stably overexpresses the bacterial RMD protein, and finally blocks the fucose modification of the Fc region by interfering with the formation of the substrate GDP-fucose, thereby enhancing the ADCC effect (WO2011035884A1).

[0140] The term "effective amount" refers to the amount or dosage that, after administration of one or more doses of the antibody or fragment or complex or composition of the present invention to a patient, results in the desired effect in a patient in need of treatment or prevention. The effective amount can be readily determined by a treating physician, who is skilled in the art, taking into account various factors such as, for example, the mammalian species, its body weight, age and health status, the specific disease involved, the degree or severity of the disease, the response of the individual patient, the specific antibody being administered, the administration form, the characteristics of the bioavailability of the dosage formulation, the dosing schedule selected, and the use of any concomitant therapies.

[0141] "Therapeutically effective amount" refers to the amount that, over a necessary period and at a necessary dosage, effectively achieves a desired therapeutic result. The therapeutically effective amount of an antibody or antibody fragment or its conjugate or composition may be varied by, for example, various factors such as the state of the disease, the age, sex and weight of the individual, and the ability of the antibody or antibody moiety to elicit a desired response in the individual. A therapeutically effective amount is also an amount at which any toxic or adverse effects of the antibody or antibody fragment or its conjugate or composition do not outweigh the beneficial effects of the treatment. Compared to an untreated subject, a "therapeutically effective amount" preferably inhibits at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, at least about 60% or at least about 70%, even more preferably at least about 80% of a measurable parameter (e.g., tumor growth rate). In an animal model system showing efficacy in human tumors, the ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated. Optionally, such properties of a composition can be evaluated by verifying the inhibitory ability of the compound, and the above inhibition is measured in vitro by methods known to those skilled in the relevant art.

[0142] "Prophylactically effective amount" refers to the amount that, over a necessary period and at a necessary dosage, effectively achieves a desired prophylactic result. Usually, since prophylactic dosages are used before or at the initial stage of a disease in a subject, a prophylactically effective amount is less than a therapeutically effective amount.

[0143] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain involved in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of a natural antibody usually have a similar structure, where each domain contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (e.g., Kindt et al., Kuby Immunology, 6 th(See ed., W.H.Freeman and Co.p91(2007)). A single VH domain or VL domain can sufficiently confer antigen-binding specificity. In addition, by isolating an antibody that binds to the antigen using a VH domain or VL domain derived from an antibody that binds to a specific antigen, a library of complementary VL domains or VH domains can be screened, respectively.

[0144] The term "host cell" refers to a cell into which an exogenous polynucleotide has already been introduced and includes the progeny of such a cell. Host cells include "transformants" and "transformed cells" and include cells transformed from primary cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly the same as the parental cell in terms of nucleic acid content and may contain mutations. As used herein, it includes mutant progeny having the same function or biological activity screened or selected from primary transformed cells. The host cell may be any type of cell line that can be used for the production of the antibody molecules of the present invention and includes eukaryotic cells such as mammalian cells, insect cells, yeast cells, and prokaryotic cells such as Escherichia coli cells. Host cells include cultured cells and cells inside genetically modified animals, genetically modified plants, or cultured plant or animal tissues.

[0145] The term "anti-tumor effect" refers to a biological effect that can be manifested by various means and includes, but is not limited to, a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in the survival rate of tumor cells.

[0146] The terms "tumor" and "cancer" are used interchangeably herein and cover solid tumors and liquid tumors.

[0147] The terms "cancer" and "cancerous" typically refer to or describe a physiological disorder in mammals characterized by unregulated cell growth. In some embodiments, cancers suitable for treatment with the antibodies of the present invention include cancers of epithelial origin, such as skin cancer (e.g., squamous cell carcinoma of the skin, head and neck cancers such as squamous cell carcinoma of the head and neck), esophageal cancer (e.g., squamous cell carcinoma of the esophagus), intestinal cancer (e.g., colon cancer, rectal cancer, colorectal cancer), or lung cancer (e.g., non-small cell lung cancer, squamous cell carcinoma of the lung, adenocarcinoma of the lung), including metastatic forms of these cancers.

[0148] The term "tumor" refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous" and "tumor" are not mutually exclusive when referred to in this specification.

[0149] The term "infectious disease" refers to a disease caused by a pathogen, including, for example, viral infections, bacterial infections, fungal infections, or protozoal infections such as those caused by parasites.

[0150] The term "chronic infection" refers to an infection in which the immune response has already been induced in a host infected with an infectious agent (e.g., a pathogen such as a virus, bacterium, protozoan such as a parasite, fungus), but has not been eliminated or removed from the host as in the case of an acute infection.

[0151] As used herein, the term "label" refers to a compound or composition that is directly or indirectly bound or fused to a reagent (e.g., a polynucleotide probe or an antibody) and facilitates the detection of that bound or fused reagent. The label may itself be detectable (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzyme label, be capable of catalyzing a chemical change in a detectable substrate compound or composition. The term is intended to cover directly labeling a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, and indirectly labeling a probe or antibody by reaction with another reagent that is directly labeled. Examples of indirect labeling include the detection of a primary antibody by a fluorescently labeled secondary antibody, and the use of terminal labeling of a DNA probe having biotin, detectable with a fluorescently labeled streptavidin protein.

[0152] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, goats, cats, dogs, and horses), primates (e.g., non-human primates such as humans and monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is human.

[0153] An "isolated" antibody is one that is separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing electrophoresis (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). An overview of methods for assessing antibody purity is provided, for example, in Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0154] "Isolated" nucleic acid refers to a nucleic acid molecule that is separated from the components of its natural environment. Isolated nucleic acids usually contain nucleic acid molecules that are contained within a cell that contains the nucleic acid molecule, but the nucleic acid molecule is present in vitro in a stained form or at a chromosomal location different from its natural chromosomal location.

[0155] The sequence identity between sequences is calculated as follows. To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced into one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison). In one preferred embodiment, for comparison, the length of the reference sequence to be aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, still more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. Next, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position.

[0156] A mathematical algorithm can be used to perform array comparison between two arrays and calculate the percentage of identity. In one preferred embodiment, the percentage of identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithms incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com), using the Blossum 62 matrix or PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6. In yet another preferred embodiment, the percentage of identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), using the NWSgapdna.CMP matrix, and gap weights of 40, 50, 60, 70 or 80 and length weights of 1, 2, 3, 4, 5 or 6. A particularly preferred parameter set (and the one to be used unless otherwise specified) employs the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. Also, the percentage of identity between two amino acid sequences or nucleotide sequences may be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) incorporated into the ALIGN program (version 2.0), using the PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4. Additionally or alternatively, by using the nucleic acid and protein sequences described herein as "query sequences" to perform searches against public databases, for example, sequences of other family members or related sequences can be identified.

[0157] The term "pharmaceutical composition" refers to a composition that can present the biological activity of the active ingredient(s) contained therein in an effective form and that does not contain other components that are toxic to the subject to which the composition is administered.

[0158] The term "pharmaceutical additive" refers to diluents, adjuvants (e.g., Freund's adjuvant (complete and incomplete)), vectors, excipients, or stabilizers, etc. that are administered together with the active substance.

[0159] As used herein, "treatment" refers to reducing, interrupting, delaying, remitting, halting, decreasing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequence of the disease, preventing metastasis, decreasing the rate of disease progression, improving or alleviating the disease state, and remission or improvement of the prognosis. In some embodiments, the antibody molecules of the present invention are used to delay the onset of a disease or to reduce the progression of a disease.

[0160] As used herein, "prevention" includes inhibiting the occurrence or progression of a disease, disorder, or symptoms of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for a prevention program. Typically, in the context of cancer, the term "prevention" refers to drug administration before the onset of cancer signs or symptoms, particularly in subjects at risk of developing cancer.

[0161] The term "effective amount" refers to an amount or dosage of one or more administrations of an antibody or fragment or composition or combination of the present invention to a patient after which the desired effect is obtained in a patient in need of treatment or prevention.

[0162] "Therapeutically effective amount" refers to an amount that, over a required period and at a required dosage, effectively achieves a desired therapeutic result. A therapeutically effective amount is also an amount such that any toxic or harmful effects of the antibody or antibody fragment or composition or combination do not outweigh the beneficial effects of the treatment. Compared to an untreated subject, a "therapeutically effective amount" preferably inhibits or improves at least about 40%, more preferably at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and even 100% of a measurable parameter.

[0163] "Prophylactically effective amount" refers to an amount that, over a required period and at a required dosage, effectively achieves a desired prophylactic result. Usually, since prophylactic dosages are used before or at the initial stages of a disease in a subject, a prophylactically effective amount is less than a therapeutically effective amount.

[0164] As used herein, the term "therapeutic agent" covers any substance effective in the prevention or treatment of diseases such as tumors (e.g., cancer) and infections (e.g., chronic infections), and includes angiogenesis agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, small molecule drugs, or immunomodulatory agents.

[0165] "Chemotherapeutic agent" includes chemical compounds useful in the treatment of cancer, including but not limited to anti-tumor agents, alkylating agents, antimetabolites, natural products, antibiotics, enzymes, heterocides, hormones and antagonists, anti-estrogens, anti-androgens, and non-steroidal anti-androgens, etc.

[0166] As used herein, the term "immunomodulatory agent" refers to a natural or synthetic active agent or drug that inhibits or modulates an immune response. The immune response may be a humoral response or a cellular response. Immunomodulatory agents include inhibitors of immune checkpoint molecules and co-stimulatory molecule activators.

[0167] The term "small molecule drug" refers to a low molecular weight organic compound capable of regulating a biological process.

[0168] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or blocks cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes, chemotherapeutic agents or drugs, growth inhibitors, enzymes such as nuclease and fragments thereof, antibiotics, small molecule toxins or enzymatically active toxins derived from bacteria, fungi, plants or animals including fragments and / or variants thereof.

[0169] "Tumor immune escape" refers to the process by which a tumor evades immunological recognition and elimination. Thus, as a therapeutic concept, tumor immunity is "treated" when such escape is weakened, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor elimination.

[0170] "Immunogenicity" refers to the ability of a particular substance to elicit an immune response. Tumors are immunogenic, and enhancing the immunogenicity of tumors contributes to the elimination of tumor cells through the immune response.

[0171] As used herein, "agonist activity of an antibody" refers to the ability of an antibody to activate the biological activity of the antigen to which it binds.

[0172] "Anti-angiogenic agent" refers to a compound that blocks or in some way interferes with the development of blood vessels. Anti-angiogenic agents may be, for example, small molecules or antibodies that bind to growth factors or growth factor receptors involved in the promotion of angiogenesis.

[0173] The term "drug combination or combination product" refers to non-fixed combination products or fixed combination products, including but not limited to drug kits, pharmaceutical compositions. The term "non-fixed combination" refers to those in which the active ingredients (e.g., (i) the immune complexes of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously as separate entities without specific time limitations or at the same or different time intervals, where such administration provides two or more active agents at prophylactically or therapeutically effective levels in the patient's in vivo. The term "fixed combination" refers to those in which two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, by selecting the dosages and / or time intervals of two or more active agents, the combined use of each part can achieve a better effect in the treatment of a disease or medical condition than when any one component is used alone. Each component may be in a separate formulation form, which may be the same or different.

[0174] The term "combination therapy" or "combined therapy" refers to the administration of two or more therapeutic agents for treating cancer or infection as described in the present disclosure. Such administration basically includes administering these therapeutic agents together, for example, in a single capsule having a certain proportion of active ingredients simultaneously. Alternatively, such administration includes administering together, separately, or sequentially each active ingredient (e.g., tablets, capsules, powders, and liquids) in multiple or separate containers. The powders and / or liquids can be reconstituted or diluted to the desired dosage before administration. In some embodiments, the administration also includes continuously administering each type of therapeutic agent at approximately the same time or at different times. In any case, the treatment program provides the beneficial effects of the pharmaceutical combination in the treatment of the medical conditions or diseases described herein.

[0175] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid linked thereto. The term includes vectors that are self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which they have been introduced. Some vectors can direct the expression of a nucleic acid effectively linked thereto. Such vectors are referred to herein as "expression vectors".

[0176] A "subject / patient sample" is a collection of cells or fluids obtained from a patient or subject. Tissue or cell samples can be derived from fresh, frozen and / or preserved organ or tissue samples, biopsy samples, aspiration samples, solid tissues such as biopsy samples, blood or any blood component, cerebrospinal fluid, amniotic fluid (amniotic water), peritoneal fluid (ascites), interstitial fluid and other body fluids, and cells derived at any time during pregnancy or development in the subject. Tissue samples may contain compounds that are not naturally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. Examples of tumor samples herein include tumor biopsies, fine needle aspirates, bronchoalveolar lavage fluids, pleural effusions (pleural effusions), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like characteristics, and preserved tumor samples such as tumor samples fixed with formalin and embedded in paraffin or frozen tumor samples, but are not limited thereto.

[0177] II. Antibody Molecules of the Invention The present invention provides novel antibody molecules for use in immunotherapy, prevention and / or diagnosis of various diseases. The antibody molecules of the present invention contain at least two, three or four antigen-binding regions and can function as bispecific or multispecific antibodies, preferably as bispecific antibodies.

[0178] In some embodiments, the bispecific or multispecific antibodies of the present invention include a first binding specificity for EGFR and a second binding specificity for B7H3, and optionally other binding specificities.

[0179] Accordingly, one aspect of the present invention relates to a bispecific antibody, which comprises a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to EGFR and / or the second antigen-binding region binds to B7-H3.

[0180] The bispecific antibodies of the present invention can be produced using formats or techniques of bispecific antibodies known in the art. Specific and exemplary bispecific formats that can be used in the context of the present invention are referred to in Labrijn et al. Bispecific antibodies: a mechanistic review of the pipeline. Nature Reviews Drug Discovery, 2019, 18(8): 1-24. In one embodiment, the bispecific antibody format comprises an IgG-like antibody (Fan et al. (2015) Journal of Hematology & Oncology. 8: 130). The most common type of IgG-like antibody comprises two Fab regions and two Fc regions, and the heavy and light chains of each Fab can be derived from a single monoclonal antibody. In some embodiments, the bispecific antibody of the present invention is an IgG-like bispecific antibody and comprises a Fab fragment that specifically binds to EGFR as one antigen-binding region and a Fab fragment that specifically binds to B7H3 as another antigen-binding region.

[0181] > Antigen-binding region that specifically binds to EGFR In some embodiments, the antigen-binding region that specifically binds to EGFR is derived from an antibody that specifically binds to EGFR, for example, the EGFR antibody disclosed in WO02100348A2, such as the zalutumumab monoclonal antibody.

[0182] In some embodiments, the antigen-binding region that specifically binds to EGFR comprises one, two, three, four, five, or six CDRs of a known antibody that specifically binds to EGFR, for example, the EGFR antibodies disclosed in WO02100348A2, such as one of the Zalutumumab monoclonal antibodies.

[0183] In some embodiments, the antigen-binding region that specifically binds to EGFR comprises one, two, and three heavy chain variable region CDRs, namely HCDR1, HCDR2, and HCDR3, of a known antibody that specifically binds to EGFR, for example, the EGFR antibodies disclosed in WO02100348A2, such as the Zalutumumab monoclonal antibody.

[0184] In some embodiments, the antigen-binding region that specifically binds to EGFR comprises one, two, and three light chain variable region CDRs, namely LCDR1, LCDR2, and LCDR3, of a known antibody that specifically binds to EGFR, for example, the EGFR antibodies disclosed in WO02100348A2, such as the Zalutumumab monoclonal antibody.

[0185] In some embodiments, the antigen-binding region that specifically binds to EGFR comprises three heavy chain variable region CDRs and three light chain variable region CDRs of a known antibody that specifically binds to EGFR, for example, the EGFR antibodies disclosed in WO02100348A2, such as the Zalutumumab monoclonal antibody.

[0186] In some embodiments, the antigen-binding region that specifically binds to EGFR comprises the heavy chain variable region and the light chain variable region of a known antibody that specifically binds to EGFR, for example, the EGFR antibodies disclosed in WO02100348A2, such as the Zalutumumab monoclonal antibody.

[0187] In some embodiments, the antigen-binding region that specifically binds to EGFR comprises the Fab of an antibody known to specifically bind to EGFR, such as the EGFR antibodies disclosed in WO02100348A2, such as the Zalutumumab monoclonal antibody.

[0188] In some embodiments, the antigen-binding region that specifically binds to EGFR comprises HCDR1, HCDR2, and HCDR3, which are three complementarity-determining regions (HCDRs) from the heavy-chain variable region. In some embodiments, the antigen-binding region that specifically binds to EGFR comprises LCDR1, LCDR2, and LCDR3, which are three complementarity-determining regions (LCDRs) from the light-chain variable region. In some embodiments, the antigen-binding region that specifically binds to EGFR comprises three complementarity-determining regions (HCDRs) from the heavy-chain variable region and three complementarity-determining regions (LCDRs) from the light-chain variable region.

[0189] In some aspects, the antigen-binding region that specifically binds to EGFR comprises a heavy-chain variable region (VH). In some aspects, the antigen-binding region that specifically binds to EGFR comprises a light-chain variable region (VL). In some aspects, the antigen-binding region that specifically binds to EGFR comprises a heavy-chain variable region (VH) and a light-chain variable region (VL). In some embodiments, the heavy-chain variable region comprises HCDR1, HCDR2, and HCDR3, which are three complementarity-determining regions (CDRs) from the heavy-chain variable region. In some embodiments, the light-chain variable region comprises LCDR1, LCDR2, and LCDR3, which are three complementarity-determining regions (CDRs) from the light-chain variable region.

[0190] In some embodiments, the heavy-chain variable region of the antigen-binding region that specifically binds to EGFR is (i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 1, or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 1, or (iii) An amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2, or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 1, or consisting of the same, preferably, the above amino acid changes do not occur in the CDR region.

[0191] In some embodiments, the light chain variable region of the antigen-binding region that specifically binds to EGFR (i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 2, or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 2, or (iii) An amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2, or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 2, or consisting of the same, preferably, the above amino acid changes do not occur in the CDR region.

[0192] In some embodiments, the three complementarity-determining regions (HCDR) HCDR1, HCDR2, and HCDR3 from the heavy chain variable region of the antigen-binding region that specifically binds to EGFR (i) are the three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH shown in SEQ ID NO: 1, or (ii) are selected from sequences having a total of at least 1 and 5, 4, 3, 2, or 1 or less amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the above three HCDR regions for any one of the sequences in (i), For example, the above CDRs are determined by the Kabat method.

[0193] In some embodiments, LCDR1, LCDR2, and LCDR3, which are three complementarity determining regions (LCDRs) from the light chain variable region of an antigen-binding region that specifically binds to EGFR, are (i) LCDR1, LCDR2, and LCDR3, which are three complementarity determining regions contained in VL shown in SEQ ID NO: 2, or (ii) selected from the sequences that have a total of at least 1 and 5, 4, 3, 2, or 1 or fewer amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three LCDR regions with respect to any one of the sequences in (i), For example, the above CDRs are determined by the Kabat method.

[0194] In some embodiments, the antigen-binding region that specifically binds to EGFR includes three complementarity determining regions (HCDRs) contained in the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1 and three complementarity determining regions (HCDRs) contained in the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2.

[0195] In some embodiments, HCDR1 includes or consists of the amino acid sequence of SEQ ID NO: 9, or HCDR1 includes an amino acid sequence having 1, 2, or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 9.

[0196] In some embodiments, HCDR2 includes or consists of the amino acid sequence of SEQ ID NO: 10, or HCDR2 includes an amino acid sequence having 1, 2, or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 10.

[0197] In some embodiments, HCDR3 includes or consists of the amino acid sequence of SEQ ID NO: 11, or HCDR3 includes an amino acid sequence having 1, 2, or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) as compared to the amino acid sequence of SEQ ID NO: 11.

[0198] In some embodiments, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 12, or alternatively, LCDR1 comprises an amino acid sequence having 1, 2 or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 12.

[0199] In some embodiments, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 13, or alternatively, LCDR2 comprises an amino acid sequence having 1, 2 or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 13.

[0200] In some embodiments, LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14, or alternatively, LCDR3 comprises an amino acid sequence having 1, 2 or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 14.

[0201] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to EGFR comprises the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3.

[0202] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to EGFR comprises the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0203] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to EGFR comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 9, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 10, The HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 11, The LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 12, The LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 13, and The LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14.

[0204] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to EGFR comprises HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 10, HCDR3 shown in SEQ ID NO: 11, LCDR1 shown in SEQ ID NO: 12, LCDR2 shown in SEQ ID NO: 13, and LCDR3 shown in SEQ ID NO: 14.

[0205] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to EGFR comprises VH and VL, wherein the above VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the above VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0206] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to EGFR comprises VH and VL, wherein VH and VL each comprise or consist of the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0207] > The antigen-binding region that specifically binds to B7-H3 In some embodiments, the antigen-binding region that specifically binds to B7-H3 is derived from an antibody that specifically binds to B7-H3, such as the B7-H3 antibodies described in PCT / CN2021 / 140449, such as the monoclonal antibody with the number Hz20G5 (for example, Hz20G5.26 mentioned in the examples of the present invention).

[0208] In some embodiments, the antigen-binding region that specifically binds to B7-H3 comprises one, two, three, four, five, or six CDRs of a known antibody that specifically binds to B7-H3, such as the B7-H3 antibodies described in PCT / CN2021 / 140449, such as the monoclonal antibody with the number Hz20G5.

[0209] In some embodiments, the antigen-binding region that specifically binds to B7-H3 comprises the one, two, and three heavy-chain variable region CDRs, namely HCDR1, HCDR2, and HCDR3, of a known antibody that specifically binds to B7-H3, such as the B7-H3 antibodies described in PCT / CN2021 / 140449, such as the monoclonal antibody with the number Hz20G5.

[0210] In some embodiments, the antigen-binding region that specifically binds to B7-H3 comprises the one, two, and three light-chain variable region CDRs, namely LCDR1, LCDR2, and LCDR3, of a known antibody that specifically binds to B7-H3, such as the B7-H3 antibodies described in PCT / CN2021 / 140449, such as the monoclonal antibody with the number Hz20G5.

[0211] In some embodiments, the antigen-binding region that specifically binds to B7-H3 comprises the three heavy-chain variable region CDRs and the three light-chain variable region CDRs of a known antibody that specifically binds to B7-H3, such as the B7-H3 antibodies described in PCT / CN2021 / 140449, such as the monoclonal antibody with the number Hz20G5.

[0212] In some embodiments, the antigen-binding region that specifically binds to B7-H3 comprises the heavy chain variable region and the light chain variable region of a known antibody that specifically binds to B7-H3, such as the B7-H3 antibody described in PCT / CN2021 / 140449, such as the monoclonal antibody with the number Hz20G5.

[0213] In some embodiments, the antigen-binding region that specifically binds to B7-H3 comprises the Fab of a known antibody that specifically binds to B7-H3, such as the B7-H3 antibody described in PCT / CN2021 / 140449, such as the monoclonal antibody with the number Hz20G5.

[0214] In some embodiments, the antigen-binding region that specifically binds to B7-H3 comprises HCDR1, HCDR2, and HCDR3, which are three complementarity-determining regions (HCDRs) from the heavy chain variable region. In some embodiments, the antigen-binding region that specifically binds to B7-H3 comprises LCDR1, LCDR2, and LCDR3, which are three complementarity-determining regions (LCDRs) from the light chain variable region. In some embodiments, the antigen-binding region that specifically binds to B7-H3 comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region and three complementarity-determining regions (LCDRs) from the light chain variable region.

[0215] In some aspects, the antigen-binding region that specifically binds to B7-H3 comprises the heavy chain variable region (VH). In some aspects, the antigen-binding region that specifically binds to B7-H3 comprises the light chain variable region (VL). In some aspects, the antigen-binding region that specifically binds to B7-H3 comprises the heavy chain variable region (VH) and the light chain variable region (VL). In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, which are three complementarity-determining regions (CDRs) from the heavy chain variable region. In some embodiments, the light chain variable region comprises LCDR1, LCDR2, and LCDR3, which are three complementarity-determining regions (CDRs) from the light chain variable region.

[0216] In some embodiments, the heavy chain variable region of the antigen-binding domain that specifically binds to B7-H3 is (i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2 or 1 or fewer) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, and preferably, the above amino acid changes do not occur in the CDR region.

[0217] In some embodiments, the light chain variable region of the antigen-binding domain that specifically binds to B7-H3 is (i) comprises or consists of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, or (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2 or 1 or fewer) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, and preferably, the above amino acid changes do not occur in the CDR region.

[0218] In some embodiments, HCDR1, HCDR2, and HCDR3, which are three complementarity-determining regions (HCDRs) from the heavy-chain variable region of an antigen-binding region that specifically binds to B7-H3, are (i) HCDR1, HCDR2, and HCDR3, which are three complementarity-determining regions contained in the VH shown in SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or (ii) selected from the sequences that have a total of at least 1 and 5, 4, 3, 2, or 1 or fewer amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three HCDR regions relative to any one of the sequences in (i), For example, HCDR1 adopts the Abm scheme, and the above HCDR2 and HCDR3 are determined by the Kabat scheme.

[0219] In some embodiments, LCDR1, LCDR2, and LCDR3, which are three complementarity-determining regions (LCDRs) from the light-chain variable region of an antigen-binding region that specifically binds to B7-H3, are (i) LCDR1, LCDR2, and LCDR3, which are three complementarity-determining regions contained in the VL shown in SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, or (ii) selected from the sequences that have a total of at least 1 and 5, 4, 3, 2, or 1 or fewer amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three LCDR regions relative to any one of the sequences in (i), For example, the above CDRs are determined by the Kabat scheme.

[0220] In some embodiments, an antigen-binding region that specifically binds to B7-H3 includes three complementarity-determining regions (HCDRs) contained in a heavy-chain variable region consisting of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, and three complementarity-determining regions (HCDRs) contained in a light-chain variable region consisting of the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0221] In some embodiments, HCDR1 comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 21, or SEQ ID NO: 27, or HCDR1 comprises an amino acid sequence having 1, 2, or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 21, or SEQ ID NO: 27.

[0222] In some embodiments, HCDR2 comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 22, or SEQ ID NO: 28, or HCDR2 comprises an amino acid sequence having 1, 2, or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 22, or SEQ ID NO: 28.

[0223] In some embodiments, HCDR3 comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 23, or SEQ ID NO: 29, or HCDR3 comprises an amino acid sequence having 1, 2, or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 23, or SEQ ID NO: 29.

[0224] In some embodiments, LCDR1 comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 24, or SEQ ID NO: 30, or LCDR1 comprises an amino acid sequence having 1, 2, or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 24, or SEQ ID NO: 30.

[0225] In some embodiments, LCDR2 comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 25, or SEQ ID NO: 31, or LCDR2 comprises an amino acid sequence having 1, 2, or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 25, or SEQ ID NO: 31.

[0226] In some embodiments, LCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 26, or SEQ ID NO: 32, or LCDR3 comprises an amino acid sequence having 1, 2, or 3 changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 26, or SEQ ID NO: 32.

[0227] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to B7-H3 comprises the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3.

[0228] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to B7-H3 comprises the above HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0229] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to B7-H3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein (i) the above HCDR1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 15, the above HCDR2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 16, the above HCDR3 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 17, the above LCDR1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 18, the above LCDR2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 19, the above LCDR3 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 20, or (ii) the above HCDR1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 21, the above HCDR2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 22, the above HCDR3 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 23, The above LCDR1 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 24, the above LCDR2 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 25, the above LCDR3 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 26, or (iii) the above HCDR1 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 27, the above HCDR2 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 28, the above HCDR3 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 29, The above LCDR1 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 30, the above LCDR2 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 31, and the above LCDR3 contains, or consists of, the amino acid sequence shown in SEQ ID NO: 32.

[0230] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to B7-H3 is (i) HCDR1 shown in SEQ ID NO: 15, HCDR2 shown in SEQ ID NO: 16, HCDR3 shown in SEQ ID NO: 17, LCDR1 shown in SEQ ID NO: 18, LCDR2 shown in SEQ ID NO: 19, and LCDR3 shown in SEQ ID NO: 20, (ii) HCDR1 shown in SEQ ID NO: 21, HCDR2 shown in SEQ ID NO: 22, HCDR3 shown in SEQ ID NO: 23, LCDR1 shown in SEQ ID NO: 24, LCDR2 shown in SEQ ID NO: 25, and LCDR3 shown in SEQ ID NO: 26, or (iii) HCDR1 shown in SEQ ID NO: 27, HCDR2 shown in SEQ ID NO: 28, HCDR3 shown in SEQ ID NO: 29, LCDR1 shown in SEQ ID NO: 30, LCDR2 shown in SEQ ID NO: 31, and LCDR3 shown in SEQ ID NO: 32.

[0231] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to B7-H3 contains VH and VL, (i) The VH contains, or consists of, the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 3, and the VL contains, or consists of, the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 4. (ii) The VH contains, or consists of, the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 5, and the VL contains, or consists of, the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 6, or (iii) The VH contains, or consists of, the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 7, and the VL contains, or consists of, the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 8.

[0232] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to B7-H3 contains VH and VL, where VH and VL are respectively SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or Comprises or consists of the amino acid sequences shown in SEQ ID NO:7 and SEQ ID NO:8.

[0233] The present invention also relates to an antibody that specifically binds to B7-H3 and comprises an antigen-binding region that specifically binds to the above-mentioned B7-H3, for example, an antibody comprising HCDR1, HCDR2, and HCDR3 defined in the present invention, and LCDR1, LCDR2, and LCDR3, or an antibody comprising VH or VL defined in the present invention, or an antibody comprising HC and LC defined in the present invention.

[0234] > FC region In some embodiments, the antibody molecule of the present invention, for example, a multispecific antibody (e.g., a bispecific antibody), further comprises an Fc region, where the Fc regions included may be the same or different.

[0235] In some embodiments, the above Fc region has hypofucosylation, for example, hypofucosylation obtained after treatment by the GlymaxX technology.

[0236] In some embodiments, the antibody molecule of the present invention comprises a first Fc region and a second Fc region, where the first Fc region and the second Fc region are the same or different.

[0237] In some embodiments, the first Fc region and the second Fc region are different and can dimerize to form a heterodimeric Fc scaffold.

[0238] As used herein, the Fc region refers to the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region, and may include a native sequence Fc region and a variant Fc region. The native sequence Fc region covers various naturally occurring immunoglobulin Fc sequences, such as the Fc regions of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). In some embodiments, the Fc region of the invention includes antibody CH2 and CH3. In some embodiments, the antibody Fc region may also have an IgG hinge region or a partial IgG hinge region, such as an IgG1 hinge region or a partial IgG1 hinge region, e.g., the sequence of D221 - P230 according to EU numbering, at the N-terminus. Mutations may be included in the above hinge region.

[0239] Unless otherwise specified herein, the numbering of amino acid residues in the Fc region is according to the EU numbering system, also referred to as the EU index, e.g., as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91 - 3242.

[0240] In some embodiments, the Fc region is a human IgG Fc, such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. In one embodiment, the Fc region includes, or consists of, the amino acid sequence of SEQ ID NO: 46 or 47, or an amino acid sequence having at least 90%, such as 95%, 96%, 97%, 99% or more identity thereto.

[0241] As will be understood by those skilled in the art, in order to promote the formation of the multispecific antibody of the present invention as a heterodimer, the Fc regions included in the multispecific antibody of the present invention may include mutations that promote the heterodimerization of the first Fc region and the second Fc region. In one embodiment, mutations are introduced into the CH3 regions of the two Fc regions.

[0242] This is a method for promoting the heterodimerization of Fc regions known in the art. For example, the CH3 region of the first Fc region and the CH3 region of the second Fc region are such that each CH3 region (or the heavy chain containing it) cannot homodimerize with itself, but is engineered to heterodimerize with other CH3 regions that are forced to be complementary (so that the CH3 regions of the first and second Fc regions heterodimerize and no homodimer is formed between the two first CH3 regions or the two second CH3 regions), and are engineered to be complementary.

[0243] Preferably, based on the Knob-in-Hole technology, Knob mutations and Hole mutations corresponding to the first Fc region and the second Fc region are introduced. This technology is referred to, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).

[0244] In one particular embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) (Knob mutation), while in the CH3 region of another Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (Hole mutation), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (numbering according to the EU index).

[0245] In yet another embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C), while in the CH3 region of another Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (Hole mutation), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering according to EU index), and optionally the tyrosine residue at position 349 is replaced with a cysteine residue (Y349C) (numbering according to EU index).

[0246] In one specific embodiment, one Fc region contains the amino acid substitution T366W, and another Fc region contains the amino acid substitutions T366S, L368A, and Y407V (numbering according to EU index).

[0247] In one specific embodiment, one Fc region contains the amino acid substitutions S354C and T366W, and another Fc region contains the amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to EU index).

[0248] Also, based on the Innobody technology, mutations corresponding to the first Fc region and the second Fc region can also be introduced. This technology is referred to, for example, in PCT / CN2021 / 143141.

[0249] In one particular embodiment, The first CH3 region described above includes the S364R / K mutation (preferably S364R) and optionally one or more other mutations. In some embodiments, the second CH3 region includes the K370S / T / A / V mutation (preferably K370S) and optionally one or more other mutations. In some embodiments, the first CH3 region includes the S364R / K mutation and the second CH3 region includes the K370S / T / A / V mutation. In some embodiments, the first CH3 region includes the S364R mutation and the second CH3 region includes the K370S mutation.

[0250] In some embodiments, the first CH3 region includes the S364R / K (preferably S364R) and D399K / R (preferably D399K) mutations. In some embodiments, the second CH3 region includes the K370S / T / A / V (preferably K370S) mutation and the K409D / E (preferably K409D) mutation. In some embodiments, the first CH3 region includes S364R / K + D399K / R and the second CH3 region includes K370S / T / A / V + Y349T / S / A / V. In some embodiments, the first CH3 region includes S364R + D399K and the second CH3 region includes K370S + Y349T. In some embodiments, the first CH3 region further includes E375N / Q (preferably E375N) and / or T350V / A (preferably T350V). In some embodiments, the second CH3 region further includes K409D / E (preferably K409D), Q347D / E (preferably Q347D) and / or T350V / A (preferably T350V).

[0251] In some embodiments, the first CH3 region includes S364R + D399K, and the second CH3 region includes K370S + Y349T + K409D. In some embodiments, the first CH3 region further includes E357N. In some embodiments, the second CH3 region further includes Q347D. In some embodiments, the first CH3 region further includes E357N, and the second CH3 region further includes Q347D. In some embodiments, the first CH3 region and the second CH3 region each further include T350V, or both include T350V.

[0252] Accordingly, in some embodiments, the first CH3 region includes S364R + D399K, and the second CH3 region includes K370S + Y349T + K409D + Q347D. In some embodiments, the first CH3 region includes S364R + D399K + E357N, and the second CH3 region includes K370S + Y349T + K409D + Q347D. In some embodiments, the first CH3 region includes S364R + D399K + E357N + T350V, and the second CH3 region includes K370S + Y349T + K409D + Q347D + T350V.

[0253] In some embodiments, the first CH3 region contains K409E / D (preferably K409E). In some embodiments, the second CH3 region contains D399K / R (preferably D399K) or K370T / S / A / V (preferably K370T). In some embodiments, the first CH3 region contains K409E / D (preferably K409E), and the second CH3 region contains D399K / R (preferably D399K). In some embodiments, the first CH3 region further contains T411R / K (preferably T411R). In some embodiments, the second CH3 region further contains K370T / S / A / V (preferably K370T). In some embodiments, the first CH3 region contains K409E / D + T411R / K, and the second CH3 region contains D399K / R + K370T / S / A / V. In some embodiments, the first CH3 region contains K409E + T411R, and the second CH3 region contains D399K + K370T.

[0254] In some specific embodiments, the first and second CH3 regions have the following combination of mutations:

[0255] [Table 2]

[0256] In one embodiment, the CH3 of one Fc region contains S364R and D399K mutations, and the CH3 of another Fc region contains Y349T, K370S and K409D mutations.

[0257] Thus, in one specific embodiment, the two Fc regions contained in the multispecific antibody of the present invention heterodimerize, where a) one Fc region polypeptide contains the mutation T366W, and another Fc region polypeptide contains T366S, L368A and Y407V, or b) One Fc region polypeptide contains the mutations S354C and T366W, and another Fc region polypeptide contains the mutations Y349C, T366S, L368A, and Y407V, or c) One Fc region polypeptide contains the mutations S364R and D399K, and another Fc region polypeptide contains the mutations Y349T, K370S, and K409D.

[0258] Thus, in one specific embodiment, the two Fc regions contained in the multispecific antibody of the present invention heterodimerize, wherein one Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 49 or SEQ ID NO: 50, and another Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 52 or SEQ ID NO: 53.

[0259] Thus, in one specific embodiment, the two Fc regions contained in the multispecific antibody of the present invention heterodimerize, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 49 or SEQ ID NO: 50, and another Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 52 or SEQ ID NO: 53.

[0260] Thus, in one specific embodiment, the two Fc regions contained in the multispecific antibody of the present invention heterodimerize, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 49 or SEQ ID NO: 50 and contains the mutations Y349T, K370S, and K409D, and another Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 52 or SEQ ID NO: 53 and contains the mutations S364R and D399K.

[0261] In some embodiments, the Fc region further comprises other mutations that are advantageous for the purification of the heterodimer.

[0262] > Antibody modification In one embodiment of the invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0263] In preferred embodiments, the amino acid changes described in the present invention occur in regions outside the CDRs (e.g., FRs). More preferably, the amino acid changes described in the present invention occur in regions outside the heavy chain variable region and / or outside the light chain variable region.

[0264] In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to the substitution of one amino acid by another amino acid of the same type, for example, one acidic amino acid is substituted by another acidic amino acid, one basic amino acid is substituted by another basic amino acid, or one neutral amino acid is substituted by another neutral amino acid. Exemplary substitutions are as shown in Table A below.

[0265] [Table 3]

[0266] In some embodiments, the antibodies provided herein are modified such that the degree of glycosylation of the antibody is increased or decreased.

[0267] Glycosylated antibodies with modifications, such as afucosylated antibodies with reduced amounts of fucosyl residues or antibodies with added bisecting GlcNac structures, can be produced. For example, fucosylation in the constant region (e.g., the Fc region) can be removed, for example, to obtain afucosylation or non-fucosylation.

[0268] Such altered glycosylation patterns indicate the ability to improve the ADCC ability of antibodies. For example, such glycan modifications can be achieved by expressing an antibody in a host cell with a modified glycosylation system. For example, afucosylated antibodies are expressed in host cells knocked out with α-1,6-fucosyltransferase 8 (Fut8) (WO2000061739). For example, hypofucosylated or afucosylated antibodies can be obtained by introducing host cells encoding the enzyme RMD related to glycan modification (see GlymaxX technology, ProBioGen AG, Patent Publication No.: WO2011035884A1). Alternatively, fucosidase may be used to excise the fucosyl residues of the antibody. For example, α-L-fucosidase is used to remove fucosyl residues from the antibody (Tarentino et al. (1975) Biochem. 14:5516-23).

[0269] > Exemplary bispecific antibody molecules In some embodiments, the anti-B7-H3 / EGFR bispecific antibody of the present invention has one or more of the following properties: (1) On the one hand, the bispecific antibody of the present invention can block the binding of EGFR ligands to EGFR, inhibit biological signal transduction, and block the corresponding biological activities of tumors. On the other hand, it can stimulate EGFR endocytosis and ultimately be degraded by intracellular lysosomes, etc., (2) The bispecific antibody of the present invention adopts an EGFR antibody parent sequence with low affinity for EGFR to significantly reduce the toxicity and side effects of a series of EGFR monoclonal antibodies against normal epithelial tissues such as the skin, (3) Based on the low affinity of EGER, the bispecific antibody of the present invention quotes a B7-H3 high-affinity antibody parent sequence to significantly improve the EGFR signal blocking activity, and improve the pharmaceutical bioactivity and pharmaceutical safety window of the bispecific antibody of the present invention, (4) The bispecific antibody of the present invention is a hypofucosylated antibody, (5) The bispecific antibody of the present invention has relatively high pharmaceutical bioactivity and safety, (6) The bispecific antibody of the present invention has excellent tumor killing and inhibitory effects, (7) The bispecific antibody of the present invention has excellent ADCC in vitro and in vivo pharmacological activities, (8) The combination of the bispecific antibody of the present invention and a KRAS small molecule inhibitor has excellent anti-tumor effects, particularly a synergistic effect.

[0270] In some embodiments, in the antibody molecule of the present invention, the antigen-binding region that specifically binds to EGFR is linked to the heavy chain constant region CH. For example, the heavy chain variable region here is linked to the heavy chain constant region CH. For example, the C-terminus of the heavy chain variable region here is linked to the N-terminus of the heavy chain constant region CH. In some embodiments, in the antibody molecule of the present invention, the antigen-binding region that specifically binds to EGFR is linked to the light chain constant region. For example, the light chain variable region here is linked to the light chain constant region CL. For example, the C-terminus of the light chain variable region here is linked to the N-terminus of the light chain constant region CL. In some embodiments, in the antibody molecule of the present invention, in the antigen-binding region that specifically binds to EGFR, the heavy chain variable region is linked to the heavy chain constant region CH, and the light chain variable region is linked to the light chain constant region CL.

[0271] In some embodiments, in the antibody molecule of the present invention, the antigen-binding region that specifically binds to B7-H3 is linked to the heavy chain constant region CH. For example, the heavy chain variable region here is linked to the heavy chain constant region CH. For example, the C-terminus of the heavy chain variable region here is linked to the N-terminus of the heavy chain constant region CH. In some embodiments, in the antibody molecule of the present invention, the antigen-binding region that specifically binds to B7-H3 is linked to the light chain constant region. For example, the light chain variable region here is linked to the light chain constant region CL. For example, the C-terminus of the light chain variable region here is linked to the N-terminus of the light chain constant region CL. In some embodiments, in the antibody molecule of the present invention, in the antigen-binding region that specifically binds to B7-H3, the heavy chain variable region is linked to the heavy chain constant region CH, and the light chain variable region is linked to the light chain constant region CL.

[0272] In some embodiments, the heavy chain constant region includes a CH1 and an Fc region linked, either through or without a hinge region.

[0273] In some embodiments, the heavy chain constant region is the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, such as the heavy chain constant region of human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the CH1 includes, or consists of, the amino acid sequence shown in SEQ ID NO: 42, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 42.

[0274] In some embodiments, the light chain constant region is a kappa light chain constant region or a lambda light chain constant region, such as a human kappa or human lambda light chain constant region. In some embodiments, the light chain constant region includes, or consists of, the amino acid sequence shown in SEQ ID NO: 54, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 54.

[0275] In some preferred embodiments, the present invention provides a bispecific antibody molecule comprising a Fab fragment that specifically binds to EGFR, a Fab fragment that specifically binds to B7-H3, and an Fc dimer, wherein the Fab fragment that specifically binds to EGFR forms a half-antibody that specifically binds to EGFR with one Fc, and the Fab fragment that specifically binds to B7-H3 forms a half-antibody that specifically binds to B7-H3 with one Fc.

[0276] In some embodiments, the bispecific antibody is an IgG-like antibody having a configuration as shown in FIG. 1.

[0277] In some possible embodiments, the bispecific antibody comprises heavy chain 1 and light chain 1, and heavy chain 2 and light chain 2, wherein heavy chain 1 and light chain 1 constitute a first half-antibody, and heavy chain 2 and light chain 2 constitute a second half-antibody, wherein heavy chain 1 comprises the heavy chain variable region and the first heavy chain constant region of the first antigen-binding region, light chain 1 comprises the light chain variable region and the first light chain constant region of the first antigen-binding region, and heavy chain 2 comprises the heavy chain variable region and the second heavy chain constant region of the second antigen-binding region, and light chain 2 comprises the light chain variable region and the second light chain constant region of the second antigen-binding region.

[0278] In some possible embodiments, in the bispecific antibody, heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 33, or consists of these.

[0279] In some possible embodiments, in the bispecific antibody, light chain 1 comprises the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 34, or consists of these.

[0280] In some possible embodiments, in the bispecific antibody, heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 33, or consists of these, and light chain 1 comprises the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 34, or consists of these.

[0281] In some possible embodiments, in the above bispecific antibody, heavy chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO: 39, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO: 39.

[0282] In some possible embodiments, in the above bispecific antibody, light chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 38 or SEQ ID NO: 40, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 38 or SEQ ID NO: 40.

[0283] In some possible embodiments, in the above bispecific antibody, (1) heavy chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 35, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 35, and light chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 36, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 36, (2) heavy chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 37, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 37, and light chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 38, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 38, (3) The heavy chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 39, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 39, and the light chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 40, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 40.

[0284] In some possible embodiments, in the above bispecific antibody, The heavy chain 1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 33, and the light chain 1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 34, and the heavy chain 2 and the light chain 2 are each the following SEQ ID NOs, i) SEQ ID NO: 35 and SEQ ID NO: 36, ii) SEQ ID NO: 37 and SEQ ID NO: 38, iii) comprise the amino acid sequences shown in SEQ ID NO: 39 and SEQ ID NO: 40, or comprise, or consist of, an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequences shown above.

[0285] In some possible embodiments, in the above bispecific antibody, (i) The heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO: 33, and the light chain 1 comprises the amino acid sequence shown in SEQ ID NO: 34, Heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO: 35, and light chain 2 comprises the amino acid sequence shown in SEQ ID NO: 36, or (ii) Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO: 33, and light chain 1 comprises the amino acid sequence shown in SEQ ID NO: 34, Heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO: 37, and light chain 2 comprises the amino acid sequence shown in SEQ ID NO: 38, or (iii) Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO: 33, and light chain 1 comprises the amino acid sequence shown in SEQ ID NO: 34, Heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO: 39, and light chain 2 comprises the amino acid sequence shown in SEQ ID NO: 40.

[0286] III. Immune Complex In some embodiments, the invention further comprises an antibody bound to another substance. Accordingly, the invention relates to an immune complex comprising an antibody bound to another substance. ("immune complex") In some embodiments, the other substance is, for example, a therapeutic agent or a marker, such as a cytotoxic agent, an immunomodulatory agent (e.g., an immune agonist), or a chemotherapeutic agent. A cytotoxic agent includes any agent that is harmful to cells. Examples of cytotoxic agents (e.g., chemotherapeutic agents) suitable for the production of immune complexes are known in the art. For example, cytotoxic agents include radioisotopes, growth inhibitors, enzymes such as nucleases and fragments thereof, antibiotics, small molecule toxins including fragments and / or variants thereof, toxins such as enzyme-active toxins derived from bacteria, fungi, plants or animals, and various known anti-tumor or anti-cancer agents, but are not limited thereto.

[0287] Also, the antibody molecules of the invention are easy to purify by binding to a labeled sequence (e.g., a peptide).

[0288] In other embodiments, the antibody molecules of the invention are conjugated with a diagnostic agent or a detectable reagent. Such antibodies can be used as part of a clinical assay (e.g., to confirm the effectiveness of a particular therapy) to monitor or predict the occurrence, establishment, progression, and / or severity of a disease or medical condition. Such diagnosis and testing can be achieved by coupling the antibody with a detectable substance, which includes, but is not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, radioactive substances, positron-emitting metals used in various positron emission tomography methods, and non-radioactive paramagnetic metal ions.

[0289] In addition, the antibody molecules of the invention can be conjugated with a therapeutic or drug moiety that modulates a given biological response. The therapeutic or drug moiety includes, but is not limited to, classical chemotherapeutic agents. For example, the drug moiety may be a protein, peptide, or polypeptide having a desired biological activity.

[0290] Furthermore, the antibody molecules of the invention can be conjugated to a therapeutic moiety such as a radioactive metal ion.

[0291] The antibody may be attached to a solid support, which is particularly useful in immunoassays or for the purification of the target antigen.

[0292] In some embodiments, the immune complex is used, for example, to prevent or treat diseases such as acute and chronic inflammatory diseases, infections (e.g., chronic infections), tumors, etc. For example, the disease is a tumor (e.g., cancer) or an infection. In some embodiments, the tumor is immune escape of the tumor. Preferably, the tumor is an epithelium-derived cancer, such as a gastrointestinal tumor, a lung tumor, or a skin tumor, such as skin cancer (e.g., cutaneous squamous cell carcinoma, head and neck squamous cell carcinoma such as head and neck cancer), esophageal cancer (e.g., esophageal squamous cell carcinoma), intestinal cancer (e.g., colon cancer, rectal cancer, colorectal cancer), or lung cancer (e.g., non-small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma). In some embodiments, the infection is a chronic infection. In some embodiments, the infection is, for example, a bacterial infection, a viral infection, a fungal infection, a protozoal infection, etc.

[0293] IV. Nucleic Acids of the Present Invention and Host Cells Containing the Same In one aspect, the present invention provides a nucleic acid encoding any of the above antibodies, fragments thereof, or any one of its chains. In one embodiment, a vector containing the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell containing the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells), or other cells suitable for the production of antibodies or antigen-binding fragments thereof. In another embodiment, the host cell is prokaryotic.

[0294] For example, the nucleic acid of the present invention includes a nucleic acid encoding an amino acid sequence shown in any one of SEQ ID NOs: 3 to 8 and 33 to 40, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in any one of SEQ ID NOs: 3 to 8 and 33 to 40.

[0295] As will be apparent to those skilled in the art, due to the degeneracy of the genetic code, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences. The nucleic acid sequences encoding the molecules of the present invention can be produced using methods well known in the art, for example, by novel solid-phase DNA synthesis or by PCR amplification.

[0296] In one aspect, the present invention provides a nucleic acid encoding any of the above antibodies or antibody chains. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit human EGFR and / or B7-H3 antigen-binding ability.

[0297] In yet another aspect, the present invention provides a nucleic acid encoding any of the above bispecific antibodies. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit human EGFR and / or B7-H3 antigen-binding ability. In one embodiment, the nucleic acids encoding each chain of the bispecific antibody may be on the same vector or on different vectors. In another embodiment, the nucleic acids encoding each chain of the bispecific antibody can be introduced into the same or different host cells for expression. Thus, in some embodiments, the method for producing the bispecific antibody of the present invention comprises culturing a host cell containing the nucleic acids encoding each chain under conditions suitable for the expression of each chain of the molecule to produce the bispecific antibody of the present invention.

[0298] In one embodiment, one or more vectors containing the above nucleic acid are provided. In one embodiment, the vector is an expression vector, for example, a eukaryotic expression vector. The vector includes, but is not limited to, a virus, plasmid, cosmid, λ phage or yeast artificial chromosome (YAC). In one embodiment, the vector is a pcDNA vector such as pcDNA3.1.

[0299] Once an expression vector or DNA sequence for expression is produced, the expression vector can be transfected or introduced into a suitable host cell. To achieve this purpose, for example, various techniques can be utilized such as protoplast fusion, calcium phosphate coprecipitation, electroporation, retroviral transduction, viral transfection, particle gun, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, the cells are cultured in a medium to screen for appropriate activity. Methods and conditions for culturing the produced transfected cells and recovering the produced antibody molecules are known to those skilled in the art and can be modified or optimized by the specific expression vector and mammalian host cell used, by the methods known in this specification and the prior art.

[0300] Also, by introducing one or more selectable markers into the transfected host cell, cells in which DNA has been stably introduced into their chromosomes can be selected.

[0301] In one embodiment, a host cell containing one or more polynucleotides of the present invention is provided. In some embodiments, a host cell containing the expression vector of the present invention is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells (e.g., CHO-S, e.g., ExpiCHO-S) or 293 cells (e.g., 293F, HEK293 cells), or other cells applicable to the production of antibodies or fragments thereof). In one embodiment, the host cell is prokaryotic, e.g., a bacterium, e.g., an E. coli cell.

[0302] Suitable host cells include prokaryotic microorganisms such as Escherichia coli. The host cells may be eukaryotic microorganisms such as filamentous fungi or yeasts, or various eukaryotic cells such as insect cells. Vertebrate cells may also be used as hosts. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Examples of mammalian host cell lines that can be used include monkey kidney CV1 cells transformed by SV40 (COS-7), human fetal kidney cell lines (HEK293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), dog kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), Chinese hamster ovary cells (CHO cells), CHOK1SV cells, CHOK1SV GS-KO cells, CHOS cells, NSO cells, myeloma cell lines such as YO, NS0, P3X63, and Sp2 / 0. A review of mammalian host cell lines suitable for protein production can be found, for example, in Yazaki & Wu, Methods in Molecular Biology, Volume 248 (edited by B.K.C. Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003). In one preferred embodiment, the host cell is a CHO cell, such as a CHOS cell, a CHOK1SV cell, or a CHOK1SV GS-KO, or the host cell is a 293 cell, such as a HEK293 cell. In some preferred embodiments, the host cell is a CHO cell. In one embodiment, the host cell of the present invention is a glycosylation-engineered host cell, preferably a glycosylation-engineered CHO cell. In one embodiment, the host cell is engineered to express the RMD enzyme. In one embodiment, the host cell contains a nucleic acid encoding the RMD enzyme. In one embodiment, the RMD enzyme comprises or consists of the amino acid sequence shown in SEQ ID NO: 41 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and preferably, the RMD enzyme is derived from Pseudomonas aeruginosa.

[0303] In one embodiment, the host cell of the present invention comprises a nucleic acid encoding one or more or all of the chains of the antibody molecule of the present invention, and a nucleic acid encoding an RMD enzyme.

[0304] V. Production and purification of the antibody molecule of the present invention In one embodiment, the present invention provides a method for producing the antibody molecule of the present invention, wherein the method comprises culturing the host cell under conditions suitable for the expression of the nucleic acid encoding the antibody molecule of the present invention, and optionally isolating the antibody. In some embodiments, the method further comprises recovering the antibody molecule of the present invention from the host cell.

[0305] In one embodiment, a method for producing the antibody molecule of the present invention is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector of the nucleic acid under conditions suitable for the expression of the antibody as described above, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0306] To recombinantly produce the antibody molecule of the present invention, a nucleic acid encoding the antibody (e.g., the above antibody, e.g., any one polypeptide chain and / or multiple polypeptide chains) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be easily isolated and sequenced by conventional processes (e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy and light chains of the antibody).

[0307] The antibody molecules produced as described in this specification can be purified by known prior art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions for purifying a specific protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these are obvious to those skilled in the art. The purity of the antibody molecules of the present invention can be determined by any one of various well-known analytical methods, and the well-known analytical methods described above include size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

[0308] VI. Measurement Methods Using various measurement methods known in the art, identification, screening, or characterization of the physical / chemical properties and / or biological activities of the antibody molecules of the present application can be performed. In one aspect, measurement of the antigen-binding activity of the antibody of the present invention is performed by known methods such as ELISA, Western blotting, etc. Binding to a target antigen can be measured using methods known in the art, and methods such as biolayer interferometry and SPR are exemplified and disclosed herein.

[0309] The present invention further provides a measurement method for identifying an antibody having biological activity. The biological activity may include, for example, antigen binding, binding to cell surface antigens, inhibitory or activating effects on antigens, etc. In vivo and / or in vitro, an antibody having such biological activity is further provided.

[0310] In certain embodiments, this biological activity is tested against the antibodies of the present invention.

[0311] The present invention further provides a method for identifying properties of an antibody, such as properties related to drug development potential. The properties related to drug development potential include, for example, thermal stability, such as long-term thermal stability.

[0312] Cells used in any of the above in vitro measurement methods include cell lines that naturally express an antigen or have been modified to express an antigen. Such cells further include cell lines that express an antigen and cell lines that do not normally express an antigen but have been transfected with DNA encoding the antigen.

[0313] Note that the antibody molecules of the present invention can be replaced or supplemented using the immune complexes of the present invention to implement any of the above measurement methods.

[0314] Note that any of the above measurement methods can be implemented using the antibody molecules of the present invention and other active agents.

[0315] In some embodiments, the antigen is EGFR (e.g., human EGFR) and / or B7-H3 (e.g., human B7-H3).

[0316] VII. Pharmaceutical Compositions and Pharmaceutical Preparations In some embodiments, the present invention provides a composition comprising any antibody molecule or fragment thereof (preferably, its antigen-binding fragment) or its immune complex described herein. Preferably, the composition is a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical adjuvant. In one embodiment, the composition, e.g., the pharmaceutical composition, comprises a combination of the antibody molecule or fragment thereof or its immune complex of the present invention and one or more other therapeutic agents.

[0317] In some embodiments, the other therapeutic agent is selected from an anti-angiogenic agent, a chemotherapeutic agent, another antibody, a cytotoxic agent, a vaccine, an anti-infective agent, a small molecule drug, or an immunomodulatory agent (e.g., an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule), preferably, the second therapeutic agent is a small molecule drug, preferably, the small molecule drug is selected from a KRAS small molecule inhibitor, e.g., a KRAS G12C inhibitor (e.g., AMG510 (Sotorasib) or GFH925), a KRAS G12D (e.g., MRTX1133), or a KRAS G12S inhibitor.

[0318] In some embodiments, the composition is used, for example, to prevent or treat diseases such as acute and chronic inflammatory diseases, infections (e.g., chronic infections), tumors, etc. For example, the disease is a tumor (e.g., cancer) or an infection. In some embodiments, the tumor is tumor immune escape. Preferably, the tumor is an epithelium-derived cancer, such as a gastrointestinal tumor or a lung tumor or a skin tumor, such as skin cancer (e.g., cutaneous squamous cell carcinoma, head and neck squamous cell carcinoma such as head and neck cancer), esophageal cancer (e.g., esophageal squamous cell carcinoma), intestinal cancer (e.g., colon cancer, rectal cancer, colorectal cancer), or lung cancer (e.g., non-small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma). In some embodiments, the infection is a chronic infection. In some embodiments, the infection is, for example, a bacterial infection, a viral infection, a fungal infection, a protozoal infection, etc.

[0319] The present invention further includes a composition (including a pharmaceutical composition or a pharmaceutical preparation) containing the antibody of the present invention or its immune complex, and / or a composition (including a pharmaceutical composition or a pharmaceutical preparation) containing a polynucleotide encoding the antibody of the present invention. In some embodiments, the composition contains one or more antibodies of the present invention or fragments thereof, or one or more polynucleotides encoding one or more antibodies of the present invention or fragments thereof.

[0320] These compositions may also include suitable pharmaceutical adjuvants, such as pharmaceutical carriers known in the art, pharmaceutical excipients containing buffers.

[0321] As used herein, "pharmaceutical carrier" includes any or all of physiologically compatible solvents, dispersion media, isotonic agents, absorption delaying agents, etc. Pharmaceutically acceptable carriers suitable for the present invention may be sterile liquids such as water and oils, including those derived from animals or plants such as petroleum, peanut oil, soybean oil, mineral oil, sesame oil, or synthesized ones. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Further, aqueous saline solutions and aqueous dextrose and glycerin solutions may be used as liquid carriers, particularly for injectable solutions.

[0322] Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, diol, water, ethanol, etc. Reference is also made to "Handbook of Pharmaceutical Excipients", 5th edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago for the use and selection of excipients.

[0323] Optionally, the above compositions may contain small amounts of wetting agents, emulsifying agents, or pH buffering agents. These compositions can take forms such as solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. Oral formulations may contain standard pharmaceutically acceptable carriers and / or excipients such as pharmaceutical mannitol, lactose, starch, magnesium stearate, saccharin, etc.

[0324] The compositions of the present invention may be in various forms. These forms include liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injection solutions and infusion solutions), dispersions or suspensions, liposome formulations and suppositories. The preferred form is determined by the desired mode of administration and therapeutic use. A generally preferred composition is in the form of an injection solution or an infusion solution. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal (i.p.), intramuscular) injection. In one preferred embodiment, the antibody molecule is administered by intravenous infusion or injection. In another preferred embodiment, the antibody molecule is administered by intramuscular, intraperitoneal or subcutaneous injection.

[0325] By mixing the antibody of the present invention having a predetermined purity with one or more optional pharmaceutical additives (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. (1980)), a pharmaceutical formulation containing the antibody described herein, preferably a lyophilized formulation or an aqueous formulation, can be produced.

[0326] The pharmaceutical composition or formulation of the present invention may further comprise one or more active ingredients, which are necessary for the specific indication to be treated and preferably have complementary activities that do not adversely affect each other. For example, it is desirable to provide other therapeutic agents as well. In some embodiments, the other therapeutic agent is selected from an anti-angiogenic agent, a chemotherapeutic agent, another antibody, a cytotoxic agent, a vaccine, an anti-infective agent, a small molecule drug or an immunomodulatory agent (e.g., an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule), preferably, the second therapeutic agent is a small molecule drug, preferably, the small molecule drug is selected from a KRAS small molecule inhibitor, such as a KRAS G12C inhibitor (e.g., AMG510 (Sotorasib) or GFH925), a KRAS G12D (e.g., MRTX1133) or a KRAS G12S inhibitor.

[0327] Sustained release formulations can be manufactured. Suitable examples of sustained release formulations include a semipermeable matrix of a hydrophobic solid polymer containing an antibody, and the matrix is a shaped article such as in the form of a film or microcapsules.

[0328] The pharmaceutical compositions of the present invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., injection or infusion).

[0329] Therapeutic compositions are generally sterile and should be stable under the conditions of manufacture and storage. The compositions can be manufactured in solution, microemulsion, dispersion, liposome or lyophilized form. A sterile injectable solution can be prepared by adding the active compound (i.e., the antibody molecule) in a predetermined amount to a suitable solvent and then filtering and disinfecting. Generally, a dispersion is prepared by adding the active compound to a sterile solvent, and the sterile solvent contains a basic dispersion medium and other components. Coating agents such as lecithin can be used. In the case of a dispersion, a surfactant can be used to maintain the appropriate fluidity of the solution. The absorption of the injectable composition can be extended by including substances that delay absorption, such as monostearate and gelatin, in the composition.

[0330] Reagent kits containing the antibody molecules described herein are also within the scope of the present invention. The reagent kit may contain one or more other elements, such as other reagents like a package insert, marker or coupling reagent, a pharmaceutically acceptable carrier, a device or other material for administration to a subject.

[0331] VIII. Combination Products or Reagent Kits In some embodiments, the present invention further provides a combination product comprising an antibody of the present invention or an antigen-binding fragment thereof, or an immune complex thereof, and one or more other therapeutic agents. In some embodiments, the other therapeutic agent is selected from an anti-angiogenic agent, a chemotherapeutic agent, another antibody, a cytotoxic agent, a vaccine, an anti-infective agent, a small molecule drug or an immunomodulatory agent (e.g., an activator of a costimulatory molecule or an inhibitor of an immune checkpoint molecule), preferably, the second therapeutic agent is a small molecule drug, preferably, the small molecule drug is selected from a KRAS small molecule inhibitor, such as a KRAS G12C inhibitor (e.g., AMG510 (Sotorasib) or GFH925), a KRAS G12D (e.g., MRTX1133) or a KRAS G12S inhibitor.

[0332] In some embodiments, the combination product is used, for example, to prevent or treat diseases such as acute and chronic inflammatory diseases, infections (e.g., chronic infections), tumors, etc. For example, the disease is a tumor (e.g., cancer) or an infection. In some embodiments, the tumor is immune evasion of the tumor. Preferably, the tumor is an epithelium-derived cancer, such as a gastrointestinal tumor or a lung tumor or a skin tumor, such as a skin cancer (e.g., cutaneous squamous cell carcinoma, head and neck cancers such as head and neck squamous cell carcinoma), esophageal cancer (e.g., esophageal squamous cell carcinoma), intestinal cancer (e.g., colon cancer, rectal cancer, colorectal cancer), or lung cancer (e.g., non-small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma). In some embodiments, the infection is a chronic infection. In some embodiments, the infection is, for example, a bacterial infection, a viral infection, a fungal infection, a protozoal infection, etc.

[0333] In some forms, two or more components in the combination product may be administered to a subject sequentially, individually, or simultaneously.

[0334] In some embodiments, the present invention further provides a reagent kit comprising an antibody, a pharmaceutical composition, an immune complex or a combination product of the present invention, and an optional package insert for administration instructions.

[0335] In some embodiments, the present invention further provides a drug product comprising the antibody, pharmaceutical composition, immune complex, and combination product of the present invention. Optionally, the drug product further comprises a package insert for administration instructions.

[0336] IX. Use of the Antibody Molecule of the Present Invention In another aspect, the present invention relates to a method for preventing or treating tumors (e.g., cancers) in a subject. The method comprises administering to the subject an effective amount of the antibody molecule, pharmaceutical composition, immune complex, combination product, or reagent kit disclosed herein. In some embodiments, the tumors in the subject of the present invention include solid tumors and hematological tumors. In some embodiments, the tumor is tumor immune escape. In some embodiments, the tumor is cancer.

[0337] In some embodiments, the tumor cells of the tumor have one or more of the following characteristics compared to normal cells of the same tissue adjacent to the same subject or normal cells adjacent to normal tissue, or compared to normal cells of the same tissue in a healthy subject: (i) Overexpress wild-type EGFR (e.g., wild-type EGFR with increased nucleic acid or protein levels) and / or express mutated EGFR compared to normal cells of adjacent tissue or normal cells of the same tissue in a healthy subject, for example, including the mutated EGFR listed in Passaro A et al., Nat Cancer. 2021. Preferably, the mutated EGFR comprises one or more mutations selected from R521K, L858R, T790M, G719X, C797S, Y1069C, Exon19 deletion (Del19), Exon20ins (e.g., S768_D770dup). Preferably, the mutated EGFR comprises R521K / Y1069C, R521K, L858R / T790M / C797S, Del19 / T790M / C797S, or S768_D770dup. (ii) overexpress wild-type KRAS (e.g., having wild-type KRAS with increased nucleic acid or protein levels) or express mutated KRAS as compared to normal cells of adjacent tissues or normal cells of the same tissue in a healthy subject, preferably, the mutated KRAS includes mutations at position G12 or G13, e.g., G12D or G12C, (iii) have increased levels of B7-H3 at the nucleic acid or protein level as compared to normal cells of adjacent tissues or normal cells of the same tissue in a healthy subject, and / or (iv) the tumor cells are drug-resistant to tyrosine kinase inhibitors, e.g., first-generation (erlotinib) and third-generation (osimertinib).

[0338] In another aspect, the present invention relates to a method for preventing or treating an infectious disease in a subject. The method includes administering an effective amount of an antibody molecule, pharmaceutical composition, immune complex, combination product or reagent kit of the present invention to the subject. In one embodiment, the infectious disease is a chronic infection.

[0339] The subject may be a mammal such as a primate, preferably a higher primate such as a human (e.g., a patient suffering from or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from a disease described herein (e.g., a tumor, infection or autoimmune disease described herein) or is at risk of suffering from a disease described herein. In some embodiments, the subject has received or is receiving other treatments such as chemotherapy and / or radiotherapy.

[0340] In another aspect, the present invention provides the use of an antibody molecule, fragment thereof, immune complex thereof, composition, combination product or reagent kit for producing or manufacturing a drug. The drug is used for treating related diseases or conditions mentioned herein.

[0341] In some embodiments, the antibodies, antibody fragments, immunocomplexes, compositions, combination products or reagent kits of the present invention can delay the onset of a disease condition and / or symptoms associated with the disease condition.

[0342] In some embodiments, the antibodies, pharmaceutical compositions, immunocomplexes, combination products or reagent kits of the present invention can be administered in combination with one or more other therapies, such as therapeutic methods and / or other therapeutic agents, for the prevention and / or treatment according to the present invention.

[0343] In some embodiments, the therapeutic method includes surgery (e.g., tumor resection) or radiotherapy.

[0344] In some embodiments, the therapeutic agent is selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, small molecule drugs, or immunomodulatory agents.

[0345] In some embodiments, the small molecule drug is selected from KRAS small molecule inhibitors, such as KRAS G12C inhibitors (e.g., AMG510 (Sotorasib) or GFH925), KRAS G12D (e.g., MRTX1133) or KRAS G12S inhibitors.

[0346] Immunomodulatory agents include inhibitors of immune checkpoint molecules and activators of costimulatory molecules.

[0347] In still some further embodiments, the antibody or fragment thereof of the present invention is used in combination with a KRAS small molecule inhibitor, and the small molecule inhibitor is, for example, a KRAS G12C inhibitor (e.g., AMG510 (Sotorasib) or GFH925), KRAS G12D (e.g., MRTX1133) or KRAS G12S inhibitor.

[0348] In some embodiments, the antibody or fragment thereof of the present invention may be administered in combination with a treatment comprising T cells (e.g., cytotoxic T cells or CTLs) that express a chimeric antigen receptor (CAR) by adoptive transfer.

[0349] In some embodiments, the antibody or fragment thereof of the present invention may be administered in combination with an anti-tumor agent.

[0350] In some embodiments, the antibody or fragment thereof of the present invention may be administered in combination with a cytokine. The cytokine may be administered as a fusion molecule with the antibody molecule of the present invention, or as a separate composition. In one embodiment, the antibody of the present invention is administered in combination with one, two, or more than three cytokines (e.g., as a fusion molecule or as a separate composition).

[0351] In some embodiments, the antibody or fragment thereof of the present invention can be combined with conventional cancer therapies in the art, which include (i) radiotherapy, (ii) chemotherapy, or the use of cytotoxic drugs, which generally affect rapidly dividing cells, (iii) targeted therapy, or drugs that specifically affect cancer cell proteins that are deregulated, (iv) immunotherapy, or enhancement of the host immune response (e.g., vaccines), (v) hormonal therapy, or blockade of hormones (e.g., if the tumor is hormone-sensitive), (vi) angiogenesis inhibitors, or blockade of blood vessel formation and growth, and (vii) palliative care, but are not limited thereto.

[0352] In some embodiments, the antibody or fragment thereof of the present invention can be combined with conventional methods for enhancing the host immune function.

[0353] The combinations of the various therapies described above can be further combined during treatment.

[0354] Such combinations of therapies include co - administration (where two or more therapeutic agents are included in the same formulation or in separate formulations), and separate administrations. In the latter case, administration of the antibody of the present invention is carried out before, simultaneously with, and / or after another therapy, e.g., a treatment method and / or a therapeutic agent. Administration of the antibody molecule and / or another therapy, e.g., administration of a therapeutic agent or a treatment method, is carried out in any of the active phase, remission phase, or low - activity inactive phase of the active disease. The antibody molecule may be administered before, simultaneously with, after another treatment, or during the remission phase of the disease.

[0355] The antibodies of the present invention (and pharmaceutical compositions or immune complexes containing the same, as well as any other therapeutic agent) may be administered by any suitable method including parenteral administration, intratracheal administration, and intranasal administration, and, if necessary for local treatment, may be administered into the lesion. Parenteral infusion includes intramuscular administration, intravenous administration, intra - arterial administration, intraperitoneal administration, or subcutaneous administration. To some extent, whether the administration is short - term or long - term determines the route, and it can be administered by injection through any suitable route, e.g., intravenous injection or subcutaneous injection. Various dosing schedules are included herein, including, but not limited to, single - dose or multiple - dose administrations at multiple time points, bolus administration, and pulse infusion.

[0356] To prevent or treat a disease, an appropriate dosage of the antibody of the present invention (either alone or when combined with one or more other therapeutic agents) is determined by the type of the disease to be treated, the type of the antibody, the severity and progression of the disease, whether the above - mentioned antibody is administered for preventive purposes or therapeutic purposes, past treatments, the patient's clinical history and response to the above - mentioned antibody, and the judgment of the attending physician. The above - mentioned antibody is appropriately administered to the patient in a single treatment or through a series of treatments.

[0357] The dosage and dosing schedule of the antibody molecule of the present invention may be determined by those skilled in the art. In some embodiments, the dosing schedule is adjusted so as to obtain a desired response (e.g., a therapeutic response).

[0358] In some embodiments, the antibodies of the present invention (and pharmaceutical compositions or immune complexes containing the same) can be administered twice a week, or once a week, or once every two weeks (either alone or in combination with other therapeutic agents).

[0359] Furthermore, the antibodies of the present invention can be replaced or supplemented using the immune complexes, compositions, combination products or reagent kits of the present invention to achieve any treatment.

Examples

[0360] Example 1. Construction of anti-B7-H3 / EGFR bispecific antibody The anti-B7-H3 / EGFR bispecific antibody molecule of the present invention was assembled in IgG1 antibody form from the anti-B7-H3 antibody parent and the anti-EGFR antibody parent by the Protein Science Innobody technology platform of Innovent Biologics (Suzhou) Co., Ltd. (Application No.: PCT / CN2021 / 143141, Invention Title: Heterodimeric antibody Fc-containing protein and its production method), or by adopting conventional methods in the art (as shown in Figure 1). The bispecific antibody form contains four polypeptide chains and can bind to two antigens, where antigen A is EGFR and antigen B is B7-H3.

[0361] Here, the parent antibody used to construct the bispecific antibody is the anti-EGFR monoclonal antibody anti-Zalutumumab (hereinafter abbreviated as Zalu, Publication No.: WO02100348A2, Invention Name: Human monoclonal antibodies to epidermal growth factor receptor (EGFR) receptor (EGFR)) and anti-B7-H3 monoclonal antibody, which is derived from Cinda Bio's hybridoma technology screening platform and obtained the antigen-binding region sequences of anti-B7-H3 monoclonal antibodies Hz20G5, Hz19A2 by antibody humanization (Application No.: PCT / CN2021 / 140449, Invention Name: Hz20G5, Hz19A2 in Anti-B7-H3 Antibody and Use Thereof), and derived from Cinda Bio's hybridoma technology screening platform and obtained the antigen-binding region sequence of anti-B7-H3 monoclonal antibody Hz5C2.9 by antibody humanization, and at the same time, mutations were made in the Fc region using Innobody technology to enhance the formation of specific antibody heterodimers (Application No.: PCT / CN2021 / 143141, Invention Name: Heterodimeric Antibody Fc-Containing Protein and Its Preparation Method). Three bispecific antibodies that simultaneously bind to B7H3 and EGFR were screened and obtained, which were numbered Hz5C2.9 / Zalu bsAb, Hz19A2.25 / Zalu bsAb and Hz20G5.26 / Zalu bsAb bispecific antibody molecules, respectively, with the specific amino acid sequence numbers shown in Table 1 and Table A-Sequence Information. All three bispecific antibodies adopt an IgG-type structure, with the specific structural diagram shown in Figure 1.

[0362] [Table 4]

[0363] 1Zhou, T., Xu, L., Dey, B. et al. Structural definition of a conserved neutralization epitope on HIV-1 gp120. Nature 445, 732 - 737 (2007) Example 2. Production of anti - B7 - H3 / EGFR bispecific antibody molecule 1. Construction of bispecific antibody molecule plasmid The heavy - chain sequence of the anti - EGFR antibody, the light - chain sequence of the anti - EGFR antibody, the heavy - chain sequence of the anti - B7 - H3 antibody, and the light - chain sequence of the anti - B7 - H3 antibody described in Table 1 were inserted into the vector pcDNA3.1 (Invitrogen, V790 - 20) respectively, to obtain the heavy - chain plasmid and light - chain plasmid of the anti - EGFR terminus, and the heavy - chain plasmid and light - chain plasmid of the anti - B7 - H3 terminus respectively.

[0364] 2. Production process of bispecific antibody Using the GlymaxX technology (see ProBioGen AG, Publication No. WO2011035884A1, the entire content of which is incorporated herein by reference for the purpose of the present invention), by PEI MAX (Polysciences, A804355), the plasmid of RMD enzyme (GDP - 6 - deoxy - D - lyxo - 4 - hexylose reductase, sequence: SEQ ID NO: 41) was transiently transfected into ExpiCHO (Invitrogen, A29133) cells together with the heavy - chain plasmid and light - chain plasmid of the anti - EGFR parental antibody, and the heavy - chain plasmid and light - chain plasmid of the anti - B7 - H3 antibody, to express the antibody parents of anti - EGFR and anti - B7 - H3 termini with low fucose content. After 7 days, the cell fermentation broth was collected, filtered and clarified, and captured by Hitrap Mabselect Sure chromatography column (GE Healthcare, 11 - 0034 - 95) respectively to obtain the antibodies of EGFR parent and B7H3 parent.

[0365] After detecting the concentration by the A280 method, the antibody parents were mixed at a molar ratio of 1:1, an appropriate amount of the reducing agent GSH was added, and the reaction was carried out overnight at room temperature. The reducing agent was removed by ultrafiltration to stop the reaction. It was precisely purified by MonoS cation exchange chromatography (GE Healthcare, 17-5168-01). The anti-B7-H3 parent solution was 20 mM sodium phosphate buffer (pH 6.6), and the anti-EGFR parent solution was 20 mM sodium phosphate buffer (pH 6.6) containing 1 M sodium chloride, and the elution gradient was 0% to 50% (30 column volumes). The protein solution obtained by elution was ultrafiltered and buffer-exchanged into PBS (Gibco, 70011-044), and the purity was detected by SEC-HPLC.

[0366] Example 3. Detection of the affinity of the Hz20G5.26 / Zalu bsAb bispecific antibody molecule The Biolayer Interferometry (BLI) was used to measure the affinity (KD) of the bispecific antibody of the present invention for binding to B7H3 and EGFR.

[0367] Half an hour before the start of the experiment, according to the number of samples, an appropriate number of AHC sensors (18-5060, Sartorius) were immersed in SD buffer (1xPBS, 0.1% BSA, 0.05% Tween-20). The antibodies and human B7H3 (B73-H52E2, Acro Biosystem), human EGFR (EGR-H5222, Acro Biosystem) prepared above were each diluted to 100 nM.

[0368] SD buffer, antibody solution, human B7H3, and human EGFR were each added to a 96-well black polystyrene microplate (Greiner, 655209). Detection was performed using a Fortebio Octet Red96e. The plate was laid out according to the position of the samples, and the position of the sensor was selected. The instrument setting parameters were as follows: execution steps: perform a baseline equilibrium for 120 seconds, add the immobilized antibody for 100 seconds, perform a baseline equilibrium for 120 seconds, antigen binding for 100 seconds, and dissociation for 120 seconds. The rotation speed was 1000 rpm, and the temperature was 30°C. After the experiment was completed, the KD value was analyzed using ForteBio Octet analysis software, and the results are shown in Table 2 below.

[0369]

Table 5

[0370] Example 4. Screening of anti-B7-H3 / EGFR bispecific antibody molecules Using the Innobody technology platform, the anti-B7-H3 and anti-EGFR antibody parental sequences were expressed and assembled into Hz5C2.9 / Zalu bsAb, Hz19A2.25 / Zalu bsAb, and Hz20G5.26 / Zalu bsAb bispecific antibody molecules. Through the growth inhibition assay of the antibody against cells and the antibody-dependent cell-mediated cytotoxicity assay (ADCC cell report), the B7H3 / Zalu bsAb bispecific antibody molecules were detected for their in vitro activity in non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), and at the same time, the safety verification was performed in the human skin squamous cell carcinoma cell line (A431).

[0371] Source and medium of cell lines: PC9 (NSCLC): Shanghai Yubo Biotechnology, YB-H3210D; medium: MEM + 10% FBS + 1% Pen / strep TE-1 (HNSCC): CoBioer, CBP60655; Medium: RPMI 1640 + 10% FBS + 1% Pen / strep SK-MES-1 (NSCLC): CoBioer, CBP60152; Medium: MEM + 1% NEAA + 1 mM Sodium Pyruvate + 10% FBS + 1% Pen / strep A431: Squamous cell carcinoma cells of the skin, ATCC, CRL-1555; Medium: DMEM + 10% FBS + 1% Pen / strep Construction of PC9 + hB7H3 and CHO-S + hB7H3 cell lines: Lentvirush + B7H3 lentivirus (hB7H3 (UniProt, Q5ZPR3-1), Lentvirus (PPL plasmid and protein sharing library, BC000141)) was constructed and packaged, and PC9 (NSCLC, Shanghai Yubo Biotechnology, YB-H3210) and CHO-S (Thermo) were infected with Lentvirus + hB7H3 respectively, and PC9 + hB7H3 and CHO-S + hB7H3 stable transformed cell lines were obtained by pressure screening and selection.

[0372] Experimental methods: 1. Growth inhibition assay (1) NSCLC: Plated at 1500 - 2500 cells / 100 μL in a 96-well low-attachment plate (Corning, CLS7007-24EA) for 3D cell culture.

[0373] HNSCC: Plated at 1500 - 2000 cells / 100 μL in a 96-well white-bottom plate (NUNC, 136101) for 2D cell culture.

[0374] (2) The pre-diluted antibody molecules (maximum concentration 300 nM, 3.16-fold dilution) were added to the corresponding cell well plates, mixed uniformly, and cultured at 37 °C and 5% CO2 for 5 days.

[0375] (3) After continuously culturing the growth inhibition experiment for 5 days, the pre-prepared Cell-Titer reagent (Promega, G7572) was added to the corresponding cell wells, and the mixture was left standing at room temperature in the dark for 15 to 25 minutes. The HNSCC cells were directly detected using a multifunctional microplate reader (Molecular Devices, SpectraMAXi3).

[0376] (4) NSCLC: The Cell-Titer and cell mixture were transferred to a 96-well white-bottom plate (NUNC, 136101) and detected using a multifunctional microplate reader (Molecular Devices, SpectraMAXi3).

[0377] The results are shown in Figure 2. Here, the percentage of surviving cells (% of surviving cell) (Figure 2A, B, C, D, and F) is the relative survival rate of the detected cells. The labels and categories of the corresponding cells are shown in Figure 2 and the figure legend. For example, PC9 is a type of NSCLC, and TE-1 is a type of HNSCC. Here, A431 is a cutaneous squamous cell carcinoma cell that does not respond to antibody treatment, and the purpose was to measure the drug side effects.

[0378] The growth inhibition percentage (Growth inhibition%) in Figure 2E was obtained after analysis and statistics based on the results (% of surviving cell) in Figure 2. The details are as follows: At the highest antibody concentration of 300 nM, the maximum cell inhibition rate in each cell of the drug, calculation method: 300 nM, (1 - % of surviving cell) × 100%.

[0379] In HNSCC and NSCLC cell lines, the results of the growth inhibition experiments (Figures 2A, B, and C) of three bispecific antibody molecules, Hz5C2.9 / Zalu bsAb, Hz19A2.25 / Zalu bsAb, and Hz20G5.26 / Zalu bsAb, showed that all three bispecific antibody molecules had a significant tumor-killing effect in both HNSCC and NSCLC. Here, the Hz20G5.26 / Zalu bsAb bispecific antibody molecule not only had excellent drug efficacy but also had low in vitro drug efficacy in A431 (Figure 2D), indicating that Hz20G5.26 / Zalu bsAb was expected to reduce or mitigate side effects such as skin toxicity in the clinical administration of the EGFR antibody series.

[0380] 2. ADCC Report Experiment (ADCC cell report assay) (1) According to the effector-to-target ratio (10:1), target cells (CHO-S+hB7H3 or NCI-H522 constructed above (CoBioer, CBP60140)) and ADCC effector cells (Promega, G7102) were uniformly mixed in a 96-well white-bottom plate (NUNC, 136101).

[0381] (2) The pre-diluted and prepared antibody molecules (for CHO-S+HB7H3 detection: at the final concentration after antibody dilution, up to 6.25 nM, 4-fold serial dilution; for NCI-H522 detection: at the final concentration after antibody dilution, up to 100 nM, 4-fold serial dilution) were added to the corresponding cell well plates, uniformly mixed, and cultured at 37°C and 5% CO2 for 20 hours.

[0382] (3) The pre-prepared Bio-glo reagent (Promega, G755B) was added to the cell well plates, left to stand at room temperature in the dark for 10 to 15 minutes, and detected using a multifunctional microplate reader (Molecular Devices, SpectraMAXi3).

[0383] The experimental results are shown in Figure 3, where the vertical axis was the fold change relative to IgG1 obtained using IgG1 as a control.

[0384] In NSCLC and HNSCC tumor cell lines, through cell growth inhibition (Figure 2) and ADCC reporting experiments (Figure 3), from the experimental results, Hz20G5.26 / Zalu bsAb has the overall optimal drug efficacy in vitro. In the A431 squamous cell carcinoma cell line, it was discovered that the in vitro toxic activity of Hz20G5.26 / Zalu bsAb is the lowest (used to measure the toxicity of EGFR inhibitors to human skin). Therefore, the bispecific antibody of the present invention, especially Hz20G5.26 / Zalu bsAb, has low toxicity to human skin and has more effective safety drug dosages when it does not cause or weakly causes toxic side effects, that is, it has a wider drug efficacy selection window and the drug efficacy is safer.

[0385] Example 5. In vitro drug efficacy activity of Hz20G5.26 / Zalu bsAb bispecific antibody molecule growth inhibition After the EGFR ligand binds to EGFR on the tumor cell surface, EGFR forms a homodimer and transmits signals into the cell, causing biological activities such as tumor cell proliferation, invasion, metastasis, and anti-apoptosis through various cascade reactions.

[0386] The anti-EGFR antigen-binding portion in Hz20G5.26 / Zalu bsAb, after binding to EGFR on the surface of tumor cells, on the one hand, blocks the binding of EGFR ligand to EGFR, inhibits biological signal transduction, and can block the corresponding biological activities of tumors. On the other hand, it stimulates EGFR endocytosis and is finally degraded by intracellular lysosomes and the like. EGFR is an epithelial-derived broad-spectrum tyrosine kinase receptor. Hz20G5.26 / Zalu bsAb adopts a low-affinity EGFR antibody parental sequence for EGFR, significantly reducing the toxicity and side effects of EGFR monoclonal antibodies on normal epithelial tissues such as the skin. B7-H3 is highly expressed in various tumor cell lines, has low expression in normal tissues and organs, is a TAA with extremely high selectivity and a wide expression spectrum. Hz20G5.26 / Zalu bsAb quotes the B7-H3 high-affinity Hz20G5.26 parent based on the low affinity of EGER, greatly improving the EGFR signal blocking activity and improving the pharmacodynamic biological activity and pharmacodynamic safety window of Hz20G5.26 / Zalu bsAb.

[0387] Example 5.1. Detection of Tumor Cell Line B7-H3 and EGFR Expression The drug sensitivity of Hz20G5.26 / Zalu bsAb to tumor cell lines was positively correlated with the receptor expression level on the surface of tumor cells. A part of the cell lines were screened from NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), CRC (colorectal cancer) and normal cell lines to detect the relative expression of B7-H3 and EGFR. According to the expression levels of B7-H3 and EGFR on the cell surface, the pharmacodynamic activity of Hz20G5.26 / Zalu bsAb was detected and verified in vitro, and the experimental results are shown in Figure 4.

[0388] Experimental method: 1. Resuspend various cells in FACS buffer, plate them in a 96-well plate (Corning, CLS3799-50EA) at 100,000 to 200,000 cells, and add 10 μg / mL of EGFR antibody (Cetuximab), 10 μg / mL of B7-H3 antibody (Hz20G5.26mAb), and 10 μg / mL of IgG to the cell well plate, and incubate at 4°C for 1 hour.

[0389] 2. Wash twice with PBS, add the prepared APC-anti human Fc antibody (Biolegend, 410712) to the corresponding cell well plate, and incubate at 4°C for 30 to 40 minutes.

[0390] 3. Wash twice with PBS, perform FACS detection (BD, Celesta), and the results are shown in Figure 4.

[0391] Figure 4 shows the expression status of B7H3 and EGFR in different cell lines. Here, cell lines with high B7H3 expression and dependent on the EGFR signaling pathway (for example, cell lines containing EGFR mutations obtained by subsequent sequence analysis (results not shown) or EGFR overexpressing cell lines) could be selected for the activity detection of the bispecific antibody.

[0392] Example 5.2. Proliferation inhibition experiment of the Hz20G5.26 / Zalu bsAb bispecific antibody molecule in various tumor cell lines EGFR is highly expressed on the cell surface of various solid tumors such as lung cancer (30% - 80%), head and neck squamous cell carcinoma (36% - 100%), colorectal cancer (25% - 77%), esophageal cancer (43% - 89%), etc., and is a broad-spectrum anti-tumor tyrosine kinase target protein. As a broad-spectrum anti-tumor target protein, EGFR shows the highest mutation rate among the mutation types of non-small cell lung cancer patients (the world average mutation rate is about 35%, and in China it reaches 40%). Among NSCLC patients with EGFR mutations, classical mutations (L858R, T790M or Exon19 deletion) account for 85% - 90%, and rare mutations such as EGFR exon 20ins, T790M primary point mutations and compound mutations, as well as other point mutations located between exons 18 - 21 represented by G719X and their sequence duplication mutations account for about 10% of the EGFR mutation rate.

[0393] H1975-EGFR L858R / T790M / C797S 、PC9+B7H3-EGFR Del19 / T790M / C797S およびH322-EGFR S768_D770dup Cell line construction: Lentvirus+EGFR respectively L858R / T790M / C797S 、Lentvirus+EGFR Del19 / T790M / C797S 、およびLentvirus+EGFR S768_D770dup Construct and package the virus, and use Lentvirus+EGFR respectively L858R / T790M / C797S 、Lentvirus+EGFR Del19 / T790M / C797S およびLentvirus+EGFR S768_D770du The viruses were used to infect NCI-H1975 (ATCC, CRL-5908), PC9h+B7H3 (constructed as above) and H322, and through pressure screening, H1975-EGFR L858R / T790M / C797S 、PC9+B7H3-EGFR Del19 / T790M / C797S およびH322-EGFR S768_D770dup Stable transformed cell lines were obtained.

[0394] Experimental methods: Proliferation inhibition experiment (1) NSCLC: Plated at 1500 cells - 2500 cells / 100 μL in a 96 - well low - attachment plate (Corning, CLS7007 - 24EA) for 3D cell culture. Here, the following cell lines and specific amounts were used.

[0395] NSCLC: 2000 cells / 100 μL / well: NCI - H292: Cell Bank of the Chinese Academy of Sciences, SCSP - 582 NCI - H322: Cobioer, CBP60134 NCI - H1650: ATCC, CRL - 5883 NCI - H1975: ATCC, CRL - 5908 SK - MES - 1: CoBioer, CBP60152 NCI - H1703: ATCC, HTB - 43 PC9 + hB7H3: Constructed as described above CRC and HNSCC: Plated at 1500 cells - 2000 cells / 100 μL in a 96 - well white - bottom plate (NUNC, 136101) for 2D cell culture. Here, the following cell lines and specific amounts were used.

[0396] CRC: 1500 cells / 100 μL / well CCK - 81: CoBioer, CBP60581 HT - 55: CoBioer, CBP60012 H508: CoBioer, CBP60795 LS180: CoBioer, CBP60034 HNSCC: 1500 cells / 100 μL / well TE - 1: CoBioer, CBP60655 Colo680: CoBioer, CBP60452. (2) The pre - diluted antibody molecules were added to the corresponding cell well plates, mixed uniformly, and cultured in a 37°C, 5% CO₂ incubator for 5 days.

[0397] (3) After continuously culturing the growth inhibition experiment for 5 days, the pre-prepared Cell-Titer reagent (Promega, G7572) was added to the cell wells, left standing at room temperature in the dark for 15 to 25 minutes, and the HNSCC cells were directly detected using a multifunctional microplate reader (Molecular Devices, SpectraMAXi3).

[0398] (4) NSCLC: The Cell-Titer and cell mixture were transferred to a 96-well white-bottom plate (NUNC, 136101) and detected using a multifunctional microplate reader (Molecular Devices, SpectraMAXi3).

[0399] EGFR is a broad-spectrum anti-tumor target protein. From the anti-tumor efficacy of growth inhibition in vitro, Hz20G5.26 / Zalu bsAb has an anti-tumor effect in esophageal cancer tumor cell lines (as shown in Figure 6). Here, in the TE-1 esophageal cancer tumor cell line, it was discovered that Hz20G5.26 / Zalu bsAb has a better anti-tumor effect than JNJ372. In tumor cell lines such as colon cancer (as shown in Figure 5), Hz20G5.26 / Zalu bsAb can achieve the anti-tumor effect of EGFR monoclonal antibodies. Especially in the LS180 colon cancer tumor cell line, its in vitro anti-tumor effect is superior to that of EGFR monoclonal antibodies. In the NCI-H508 colon cancer tumor cell line, the maximum anti-tumor killing rate reached 84%.

[0400] In the case of lung cancer, not all cancer tumor patients developed due to EGFR primary gene mutations. The average mutation rate of EGFR in non-small cell lung cancer patients is about 35%. The EGFR-TKI small molecules used as treatment criteria (for example, Erlotinib (Selleck Chemicals, cat: S7786) or Osimertinib (Selleck Chemicals, cat: S7297)) are mainly targeted at patients with sensitive gene mutations. However, drug resistance disorders caused by gene mutations often occur, and the development of new target drugs for new mutation sites has always been needed. In lung cancer, Hz20G5.26 / Zalu bsAb is not only effective against NSCLC tumor cell lines with EGFR mutations, but also has an anti-tumor in vitro effect on wild-type EGFR and abnormally amplified EGFR non-small cell lung cancer tumor cell lines. In NSCLC tumor cell lines, the in vitro anti-tumor effect of Hz20G5.26 / Zalu bsAb was far superior to that of JNJ-372 in terms of its overall in vitro anti-tumor drug efficacy when compared with JNJ-372.

[0401] NSCLC-EGFR WT In tumor cell lines (Figure 7.1), the maximum in vitro anti-tumor killing rate of NCI-H292 was 74.2 ± 2.8%. In NSCLC-EGFR classical mutation (L858R, T790M or Exon19 deletion) tumor cell lines (Figure 7.2), NCI-H1975 (EGFR L858R / T790M) (Figure 7.2B), the maximum in vitro antitumor killing rate for SK-MES-1 was 51.2 ± 1.4%, with an IC50 of 0.99 nM, and the in vitro efficacy was superior to that of JNJ-372 and EGFR monoclonal antibodies, and in NSCLC-EGFR amplified tumor cell lines (Figure 7.3), the maximum in vitro antitumor killing rate for SK-MES-1 was 60.7 ± 15.9%, with an IC50 of 0.40 nM, and the overall in vitro efficacy was superior to that of JNJ-372 and EGFR monoclonal antibodies, and the amplified NSCLC-EGFR tumor cell lines SK-MES-1 and NCI-H1703 were insensitive and resistant to first generation (Erlotinib) and third generation (Osimertinib) drugs. Osimertinib, a third-generation EGFR-TKI small molecule inhibitor used in the marketed T790M mutation therapy, exon20ins Low efficacy against rare mutations, EGFR exon20ins To investigate the efficacy of Hz20G5.26 / Zalu bsAb against tumors, we used NCI-H322-EGFR WT Cell-based H322-EGFR S768_D770dup Stable transformed cell lines were established, and the in vitro growth inhibitory and antitumor drug efficacy was demonstrated (Figure 7.4). exon20ins The in vitro pharmacological activity of Hz20G5.26 / Zalu bsAb against tumors was demonstrated. With the administration and treatment of EGFR-TKI small molecule inhibitors, NSCLC patients continue to develop double (e.g., Del19 / T790M, L858R / T790M) or triple (e.g., Del19 / T790M / C797S, L858R / T790M / C797S) drug resistance mutations, which is also an important direction for the development of next-generation EGFR-TKIs. To verify the pharmacodynamic activity of Hz20G5.26 / Zalu bsAb against EGFR-TKI triple resistance mutations, H1975-EGFR L858R / T790M / C797S and PC9+B7H3-EGFR Del19 / T790M / C797S Stable transformed cell lines were constructed and in vitro proliferation and antitumor efficacy were evaluated (Figure 7.4). L858R / T790M / C797S and EGFR Del19 / T790M / C797S The results showed that Hz20G5.26 / Zalu bsAb had a certain in vitro therapeutic activity against

[0402] To further verify the in vitro pharmacological activity of Hz20G5.26 / Zalu bsAb, a comparative analysis of the in vitro growth inhibitory efficacy was performed by combining Hz20G5.26 / Zalu bsAb with gp120 / Zalu and gp120 / Hz20G5.26 (Figure 7.5). The results showed that in NCI-H292 (Lung adeno, EGFR WT ) and SK-MES-1 (sqNSCLC, EGFR Amp ) NSCLC tumor cell lines and LS180 CRC tumor cell lines, the in vitro pharmacological efficacy of Hz20G5.26 / Zalu bsAb was found to be far superior to the combined administration of gp120 / Zalu and gp120 / Hz20G5. The experimental results of Hz20G5.26 / Zalu bsAb growth inhibition and tumor killing are shown in Table 3:

[0403]

Table 6

[0404] Example 5.3. Synergistic effect of combined use of Hz20G5.26 / Zalu bsAb bispecific antibody molecule and KRAS small molecule inhibitor In cancer tumor patients, in addition to the occurrence of cancer due to abnormal activation of EGFR, the activation of KRAS gene mutation was also a common driving cause of cancer tumors. Approximately 25% of human cancers were KRAS mutations, and KRAS mutations were greatly involved in the prognosis and treatment of cancer. In CRC tumors and NSCLC tumors, KRAS-G12D and KRAS-G12C were common abnormal mutations. Currently, with the use of KRAS small molecule inhibitors, it was expected that KRAS acquired drug resistance would occur later. KRAS small molecule inhibitors were used in combination with other drugs (such as immunotherapeutic drugs or targeted therapeutic drugs), or it was a direction for new drug research and development.

[0405] Experimental method: Proliferation inhibition experiment (1) NSCLC (H358) (Cobioer, CBP60136) and CRC (LS180) (Cobioer, CBP60034): Plated at 1500 cells to 2500 cells / 100 μL in a 96-well low-attachment plate (Corning, CLS7007-24EA) and performed 3D cell culture.

[0406] (2) Added the antibody molecules and small molecule inhibitors prepared by pre-dilution to the corresponding cell well plates, mixed uniformly, and cultured at 37 °C and 5% CO2 for 5 days.

[0407] (3) After continuing the growth inhibition experiment for 5 days of culture, added the pre-prepared Cell-Titer reagent (Promega, G7572) to the cell wells and allowed to stand at room temperature in the dark for 15 minutes to 25 minutes.

[0408] (4) Transferred the Cell-Titer and the above cell mixture to a 96-well white-bottom plate (NUNC, 136101) and detected with a multi-functional microplate reader (Molecular Devices, SpectraMAXi3).

[0409] NCI-H358 (EGFR WT , KRAS G12C ) Results of NSCLC tumor cell lines (Figure 8A) show that the in vitro maximum anti-tumor killing rate of Hz20G5.26 / Zalu bsAb in H358 is 66.9 ± 1.9%, and IC 50At 0.48 nM, its anti-tumor efficacy is far superior to that of the EGFR monoclonal antibody Zalu monoclonal antibody and JNJ-372, and it has been found to be superior to the combined administration of Gp120 / Zalu and Gp120 / Hz20G5.26. As a result of the combined administration with AMG510 (KRAS-G12C small molecule inhibitor) (MedChem Express, HY-114277), Hz20G5.26 / Zalu and AMG510 have a synergistic effect. It has been found that under the action of 0.98 nM AMG510 with 10 nM Hz20G5.26 / ZalubsAb, the killing rate of H358 cells increases from 22.51% to 67.12%, and the anti-tumor effect is enhanced in H358 cells (Figure 8B). To prove the synergistic effect, as further shown in Figure 8B, the drug efficacy synergistic effect score was calculated with SynergyFinder (https: / / synergyfinder.fimm.fi / synergy / 20210817124619827928 / ), and the results are shown in Figures 8C and 8D. Figure 8D shows the total score in various dimensions. Here, the total score is 11.895, which is greater than 10, indicating a synergistic effect between the two drugs.

[0410] LS180 (EGFR R521K , KRAS G12D ) From the results of the CRC tumor cell line, the in vitro anti-tumor efficacy of Hz20G5.26 / Zalu bsAb is not only superior to that of the EGFR monoclonal antibody Zalu, JNJ372, and the combined administration of Gp120 / Zalu and gp120 / Hz20G5.26 (Figure 7.5), but also has an obvious synergistic effect when combined with MRTX1133 (KRAS-G12D small molecule inhibitor). Under the condition of 1.56 nM Hz20G5.26 / Zalu bsAb, the anti-LS180 tumor efficacy of 15.62 nM MRTX1133 increases from 36.45% to 61.56%, and the anti-tumor effect is significantly enhanced (Figure 9A). In Figures 9B and 9C, the SynergyFinder was further used to calculate the synergistic effect score, and the total score was 37.714, indicating a synergistic effect between the two drugs.

[0411] In vitro study on the mechanism of action of the growth inhibitory effect of the 5.4.Hz20G5.26 / Zalu bsAb bispecific antibody molecule In the growth inhibition experiment, Hz20G5.26 / Zalu bsAb, with the help of the B7H3 antibody, further enhanced EGFR signal blockade and achieved a more potent anti-tumor killing effect. In NCI-H358 cells, studies were conducted in two directions: inhibition and blockade of the EGFR signal, and blockade of the EGFR ligand, using Western blotting, to explore the reasons why the in vitro anti-tumor effect of Hz20G5.26 / Zalu bsAb is superior to that of the EGFR monoclonal antibody and the combined administration of Gp120 / Zalu and Gp120 / Hz20G5.26.

[0412] Experimental method: 1. EGFR signal blockade experiment (Signal blocking) (1) Cell treatment <1> NCI-H358 (Cobioer, CBP60136) was taken and plated in a 6-well plate (NEST, 703011) at 7.0E5 cells / well and cultured overnight.

[0413] <2> The medium (RPMI1640 complete medium (RPMI1640 + 10% FBS)) was removed, and serum-free medium RPMI1640 (assay medium) was added for overnight starvation treatment.

[0414] Remove the medium in <3><2>, and add serum-free assay medium containing 200 nM of Hz20G5.26 / Zalu bsAb, 200 nM of Gp120 / Zalu, 200 nM of Gp120 / Hz20G5.26, 200 nM of Zalu mAb, and 200 nM of Gp120 / Zalu + 200 nM of Gp120 / Hz20G5.26 to the corresponding cell well plates, and set up the Blank group (medium), bsAb group (200 nM of Hz20G5.26 / Zalu bsAb), Gp120 / Zalu group (200 nM of Gp120 / Zalu), Gp120 / B7H3 group (200 nM of Gp120 / Hz20G5.26), Zalu group (200 nM of Zalu mAb), and Gp120 / Zalu + Gp120 / B7H3 group (200 nM of Gp120 / Zalu + 200 nM of Gp120 / Hz20G5.26), and leave them untreated at 37°C and 5% CO2 for 2 hours.

[0415] (2) Protein extraction and total protein quantification <1> Prepare cell lysate in advance, and the composition of the cell lysate: 100 μL / tube - 200 μL / tube of RIPA (Thermo, 89900) + 1:100 phosphatase inhibitor (abcam, ab201112) + 1:20 protease inhibitor (Roche, 11836170001).

[0416] <2> Add the pre-prepared cell lysate to the cells obtained in (1) above to digest the cells and dissolve them on ice for 20 minutes.

[0417] <3> Pre-cool the centrifuge at 4°C in advance, centrifuge at 12000 rpm / min at 4°C for 15 minutes, and collect the supernatant.

[0418] <4> Use BCA reagent (Beyotime, P0012) to calibrate the concentration of the extracted total protein.

[0419] <5>The corresponding total protein was diluted with LDS Sample buffer (Invitrogen, 2201446), denatured at 70 °C for 10 minutes, and stored at -40 °C for later use.

[0420] (3) Western blotting <1>The protein sample obtained in (3) and the protein marker (Prestained Protein Ladder (Thermo, 26620)) were added to a precast gel (Thermo, NP0321BOX), and electrophoresis was performed using an electrophoresis apparatus (BIO-RAD, TRANSSD) at a voltage of 200 V for about 50 minutes.

[0421] <2>The film was taken out, and membrane transfer was performed using a transfer apparatus (Invitrogen, IBCOT2).

[0422] <3>The prepared blocking solution (containing 5% non-fat dry milk in TBST) was used to slowly block for 1 to 2 hours, and EGF Receptor Rabbit mAb (CST, 4267), pEGF Receptor Rabbit mAb (CST, 3777), and GAPDH Rabbit mAb (CST, 2118) antibodies were added and incubated overnight at 4 °C.

[0423] <4>Washing was performed 3 times with TBST for 8 to 10 minutes, and the shaker speed was controlled at 80 rpm / min to 100 rpm / min.

[0424] <5>Incubation was performed at room temperature for 1 to 2 hours with the labeled HRP-Goat Anti-Rabbit IgG (abcam, Ab205718).

[0425] <6>Washing was performed 3 times with TBST for 8 to 10 minutes, and the shaker speed was controlled at 80 rpm / min to 100 rpm / min.

[0426] <7>Developed with ECL luminescent solution (Beyotime, P0018AM) and exposed with a developing device (BIO-RAD, chemiDocMP).

[0427] The results are shown in Figure 10.

[0428] 2. EGFR Ligand Blocking Experiment (Signal blocking) (1) Cell treatment <1>Took NCI-H358 (H358), plated it in a 6-well plate (NEST, 703011) at 7.0E5 cells / well, and cultured it overnight.

[0429] <2>Removed the medium (RPMI1640 complete medium (RPMI1640 + 10% FBS)), added serum-free medium RPMI1640, and performed overnight starvation treatment.

[0430] <3>Removed the medium, and added medium (2-cell wells), medium containing 200 nM of Hz20G5.26 / Zalu bsAb, 200 nM of Gp120 / Zalu, 200 nM of Gp120 / Hz20G5.26, 200 nM of Zalu mAb, 200 nM of Gp120 / Zalu + 200 nM of Gp120 / Hz20G5.26 to the corresponding cell well plates, and set up Blank group (medium), Control group (medium), bsAb group (200 nM of Hz20G5.26 / Zalu bsAb), Gp120 / Zalu group (200 nM of Gp120 / Zalu), Gp120 / B7H3 group (200 nM of Gp120 / Hz20G5.26), Zalu group (200 nM of Zalu mAb), Gp120 / Zalu + Gp120 / B7H3 group (200 nM of Gp120 / Zalu + 200 nM of Gp120 / Hz20G5.26), and left it untreated at 37°C and 5% CO2 for 1 hour.

[0431] <4>After 1 hour, the Blank group (medium), Control group (medium), bsAb group (200 nM of Hz20G5.26 / Zalu bsAb), Gp120 / Zalu group (200 nM of Gp120 / Zalu), Gp120 / B7H3 group (200 nM of Gp120 / Hz20G5.26), Zalu group (200 nM of Zalu mAb), and Gp120 / Zalu + Gp120 / B7H3 group (200 nM of Gp120 / Zalu + 200 nM of Gp120 / Hz20G5.26) were each treated with 30 nM of EGF (ACRO, EGF-H52b) (upper figure in Figure 11) and 30 nM of TGF-α (R&D, 239-A-100) (lower figure in Figure 11) at 37°C and 5% CO2 for 1 hour.

[0432] (2) Protein extraction and total protein quantification <1>Prepare cell lysate in advance. Composition of cell lysate: 100 μL / tube - 200 μL / tube of RIPA (Thermo, 89900) + 1:100 phosphatase inhibitor (abcam, ab201112) + 1:20 protease inhibitor (Roche, 11836170001).

[0433] <2>Add the pre-prepared cell lysate to the cells obtained in (1) above to digest the cells and dissolve them on ice for 20 minutes.

[0434] <3>Pre-cool the centrifuge at 4°C in advance, centrifuge at 12000 rpm / min at 4°C for 15 minutes, and collect the supernatant.

[0435] <4>Use the BCA reagent (Beyotime, P0012) to calibrate the concentration of the extracted total protein.

[0436] <5>Dilute the corresponding total protein with LDS Sample buffer (Invitrogen, 2201446), denature it at 70°C for 10 minutes, and store it at -40°C for later use.

[0437] (3) Western blotting <1>The protein sample and the protein marker (Prestained Protein Ladder (Thermo, 26620)) were added to a precast gel (Thermo, NP0321BOX), and electrophoresis was performed for approximately 50 minutes using an electrophoresis apparatus (BIO-RAD, TRANS SD CELL) under a voltage of 200V.

[0438] <2>The film was taken out and membrane transfer was performed using a transfer apparatus (Invitrogen, IBCOT2).

[0439] <3>The prepared blocking solution (containing 5% non-fat dry milk in TBST) was used for slow blocking for 1 to 2 hours, and the EGF Receptor Rabbit mAb (CST, 4267), pEGF Receptor Rabbit mAb (CST, 3777), and GAPDH Rabbit mAb (CST, 2118) antibodies were added and incubated overnight at 4 degrees.

[0440] <4>Washing was performed 3 times with TBST for 8 to 10 minutes, and the shaker speed was controlled at 80 rpm / min to 100 rpm / min.

[0441] <5>Incubation was performed for 1 to 2 hours at room temperature with the labeled HRP-Goat Anti-Rabbit IgG (abcam, Ab205718).

[0442] <6>Washing was performed 3 times with TBST for 8 to 10 minutes, and the shaker speed was controlled at 80 rpm / min to 100 rpm / min.

[0443] <7>Development was carried out with ECL luminescent solution (Beyotime, P0018AM), and exposure was performed with a developing apparatus (BIO-RAD, chemiDocMP).

[0444] The results are shown in Figure 11.

[0445] From the results of the study on the Signal blocking mechanism (Figures 10 and 11), it was discovered that Hz20G5.26 / Zalu bsAb was stronger than the combined effects of the EGFR monoclonal antibodies Zalu and Gp120 / Zalu and Gp120 / Hz20G5.26 in both signal blocking and ligand blocking. This result also clarified the reason why the in vitro anti-CRC, NSCLC, and HNSCC tumor effects of Hz20G5.26 / Zalu bsAb were superior to the combination of the EGFR monoclonal antibodies Zalu and Gp120 / Zalu and Gp120 / Hz20G5.26.

[0446] Example 6. In vitro pharmacological activity of the ADCC of the Hz20G5.26 / Zalu bsAb bispecific antibody molecule Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism for anti-tumor antibodies to exert anti-tumor effects. Its principle is to bind to the antigen epitope on the surface of tumor cells using the Fab fragment of the antibody, and its Fc fragment binds to the FcR on the surface of killing immunity (NK cells, macrophages, neutrophils, etc.) to directly kill tumor cells through immune cell mediation. The ADCC effect was mainly realized by the antibody Fc and the FcRIIIa receptor on the NK surface.

[0447] The introduction of the Hz20G5.26 parent not only improved the EGFR antibody blocking activity in Hz20G5.26 / Zalu bsAb, but also overall improved the ADCC effect of Hz20G5.26 / Zalu bsAb (Hz20G5.26 is speculated to bind to a specific epitope at the membrane proximal end of B7H3 and cause a strong ADCC function). At the same time, using the GlymaxX low-fucose technology, the ADCC effect of Hz20G5.26 / Zalu bsAb was further enhanced.

[0448] Therefore, the Hz20G5.26 / Zalu bsAb bispecific antibody molecule was able to jointly exert various anti-tumor pharmacological effects through two mechanisms of action: EGFR signal blocking and ADCC. The specific experimental steps were as follows: Example 6.1. ADCC Reporting Experiment In the ADCC reporting experiment, the ADCC effector cells ((Promega, G7102)) were engineered to overexpress the FcRIIIa (V158) receptor in Jurkat T cells, and were cells engineered to be able to rapidly respond to the strength of ADCC by the NFAT-RE driving element in Jurkat T cells.

[0449] Experimental method: (1) According to the effector-to-target ratio (10:1), tumor cells (cell lines listed in Table 3) and ADCC effector cells (Promega, G7102) were uniformly mixed in a 96-well white-bottom plate (NUNC, 136101) (1.5E4 cells: 1.5E5 cells / well / 100 μL (1.5E5: 1.5E6 cells / mL)).

[0450] (2) The antibody molecules prepared by pre-dilution were added to the corresponding cell well plates, uniformly mixed, and cultured at 37 °C and 5% CO2 for 20 hours.

[0451] (3) The pre-prepared Bio-glo reagent (Promega, G755B) was added to the cell well plates, left to stand at room temperature in the dark for 10 to 15 minutes, and detected by a multifunctional microplate reader (Molecular Devices, SpectraMAXi3).

[0452] The inventors selected different types of EGFR tumor cell lines (EGFR wild-type, EGFR-amplified, EGFR-mutated, EGFR wild-type and KRAS-mutated) in NSCLC tumor cell lines, verified the ADCC activity of Hz20G5.26 / Zalu bsAb through an ADCC reporter experiment, and from the ADCC reporter experiment results (Figure 12 and Table 4), it was discovered that in different types of NSCLC-EGFR tumor cell lines, the ADCC activity of Hz20G5.26 / Zalu bsAb was stronger than that of JNJ373 and EGFR monoclonal antibodies. At the same time, from the experimental results of H292 (NSCLC-EGFR wild-type) and H358 (NSCLC-EGFR wild-type and KRAS-mutated), it was discovered that the ADCC activity of Hz20G5.26 / Zalu bsAb was not only stronger than that of JNJ372 and EGFR monoclonal antibodies, but also stronger than the combined ADCC activity of Gp120 / Zalu and Gp120 / Hz20G5.26.

[0453]

Table 7

[0454] Example 6.2. huPBMC ADCC Experiment From the above ADCC reporter experiment results, it was discovered that the ADCC activity of Hz20G5.26 / Zalu bsAb was stronger than that of JNJ372, the EGFR monoclonal antibody Zalu, and the combination of Gp120 / Zalu and Gp120 / Hz20G5.26. In order to more accurately and effectively reflect the ADCC activity of Hz20G5.26 / Zalu bsAb, a huPBMC ADCC experiment was designed and ADCC activity verification was performed using normal human PBMC.

[0455] Experimental method: (1) Preheat CTS medium (Gibco, A3021002) at 37°C, take huPBMC (Miaotong Biotechnology, PB100C-W), quickly dissolve it in a water bath, and slowly add the cells to 8 mL of CTS medium (containing 1% DNA enzyme).

[0456] (2) Centrifuge at 300 g for 8 minutes, remove the supernatant, resuspend with 30 mL of CTS (containing 10 μL of DNA enzyme), transfer to a T75 culture flask, and culture overnight in an incubator at 37 °C.

[0457] (3) Take the overnight cultured suspension cells, centrifuge at 300 g / 8 minutes, remove the supernatant, adjust the cell density with CTS, and plate effector cells (huPBMC) and target cells (tumor cells in Table 5) at an effector-to-target ratio of 50:1, with huPBMC and pre-prepared tumor cells (Target:huPBMC = 1.5E4 / 7.5E5 cells / well / 100 μL) in a 96-well low-attachment plate (Corning, CLS7007-24EA).

[0458] (4) Add the diluted and prepared antibody drug to the corresponding cell well plate and culture at 37 °C and 5% CO2 for 8 hours.

[0459] (5) Centrifuge at 300 g for 5 minutes, transfer 50 μL of the supernatant to a 96-well clear flat-bottom plate (NUNC, 136101), add 50 μL of the pre-prepared LDH reagent (Promega, G1780) to the corresponding well plate, and let stand at room temperature in the dark for 15 to 30 minutes.

[0460] (6) Take 50 μL of LDH Stop Solution (Promega, G1780), read at 490 nm, and detect with a multifunctional microplate reader (Molecular Devices, SpectraMAXi3).

[0461] The results are shown in Figure 13 and Table 5. The HuPBMC ADCC experimental results of Hz20G5.26 / Zalu bsAb could more accurately and effectively reflect the actual drug efficacy results. From the HuPBMC ADCC results (Figure 13 and Table 5), the huPBMC ADCC activity results of Hz20G5.26 / Zalu bsAb basically coincide with the ADCC reported activity results. In many different types of NSCLC-EGFR tumor cell lines, the ADCC activity of Hz20G5.26 / Zalu bsAb is stronger than that of JNJ373 and EGFR monoclonal antibodies, and stronger than the combined ADCC activity of Gp120 / Zalu and Gp120 / Hz20G5.26. Due to the huPBMC ADCC effect, Hz20G5.26 / Zalu bsAb has the lowest anti-tumor killing rate in H1975, but its killing rate also has 36.53%. It was discovered that Hz20G5.26 / Zalu bsAb has the highest anti-tumor killing rate in H322, and its killing rate reaches 100%.

[0462] Therefore, in addition to enhancing EGFR signal blockade, Hz20G5.26 / Zalu bsAb can further exert its anti-tumor drug efficacy activity in many cancer tumors by participating in ADCC through immune cells.

[0463]

Table 8

[0464] Example 7. In vitro study on the safety of the Hz20G5.26 / Zalu bsAb bispecific antibody molecule drug EGFR is an epithelial-derived epidermal growth factor. In addition to causing the occurrence of cancer tumors due to overexpression or abnormal activation, it is also expressed in some epithelial cells and keratinocytes. Compared with the side effects of EGFR-TKI small molecule inhibitors such as rash, diarrhea, paronychia, oral mucositis, liver injury, and interstitial lung disease, EGFR monoclonal antibodies also have specific side effects mainly on the skin, such as pustular rash, perionychitis, and dryness and itching of the skin. This has a great impact on the quality of life and treatment compliance of cancer patients.

[0465] The inventors selected human cutaneous squamous cell carcinoma cell lines (A431, ATCC, CRL-1555) and human cutaneous keratinocytes (HaCat, Cell lines service, 300493) cells as the Hz20G5.26 / Zalu bsAb side effect research model and explored the resistance of Hz20G5.26 / Zalu bsAb in in vitro experiments.

[0466] Experimental method (1) A431 and HaCat cells were plated in a 96-well low-attachment plate (Corning, CLS7007-24EA) at 1500 cells to 2000 cells / 100 μL for 3D cell culture. Here, all the media used were DMEM + 10% FBS + 1% Pen / strep.

[0467] (2) The pre-diluted antibody molecules were added to the corresponding cell well plates, mixed uniformly, and cultured in a 37 °C, 5% CO2 incubator for 5 days.

[0468] (3) After continuing the growth inhibition experiment for 5 days of culture, the pre-prepared Cell-Titer reagent (Promega, G7572) was added to the cell wells and left standing at room temperature in the dark for 15 minutes to 25 minutes.

[0469] (4) The Cell-Titer and cell mixture were transferred to a 96-well white-bottom plate (NUNC, 136101) and detected using a multifunctional microplate reader (Molecular Devices, SpectraMAXi3).

[0470] The results are shown in Figure 14. From the experimental results, it was found that the in vitro drug efficacy of Hz20G5.26 / Zalu bsAb in A431 and HaCat cells is much lower than that of the EGFR monoclonal antibody, indicating that the cells have very high resistance to Hz20G5.26 / Zalu bsAb, the side effects are much lower than those of the EGFR monoclonal antibody, and at the same time, the resistance of the cells to Hz20G5.26 / Zalu bsAb is also superior to that of JNJ372.

[0471] Therefore, based on the reduced EGER affinity, Hz20G5.26 / Zalu bsAb uses the B7H3 high-affinity Hz20G5.26 parent to improve the pharmacodynamic bioactivity and pharmacodynamic safety window of Hz20G5.26 / Zalu bsAb.

[0472] Example 8. In vivo pharmacodynamic activity of the Hz20G5.26 / Zalu bsAb bispecific antibody molecule To demonstrate the pharmacodynamic effect of the Hz20G5.26 / Zalu bispecific antibody molecule in vivo, NSCLC-EGFR WT The tumor cell line NCI-H292 cells (ATCC) were used to inoculate Balb / c Nude mice, and the antitumor pharmacodynamic effect of the Hz20G5.26 / Zalu bispecific antibody of the present invention was measured. In the experiment, SPF-grade female Balb / c Nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used, and the certificate number was NO.110011211108430747.

[0473] The NCI-H292 cells were regularly passaged and then used in the in vivo experiment. The cells were collected by centrifugation, mixed with PBS (1×) and Matrigel Matix (Corning) at an equal ratio, and the NCI-H292 cells were resuspended to a cell concentration of 20×106 A cell suspension of 0.2 cells / mL was prepared. On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the right abdominal region of Balb / c nude mice to establish NCI-H292 tumor-bearing mouse models.

[0474] Four days after tumor cell inoculation, the tumor volume of each mouse was detected and grouped (5 mice per group), with the dosage and administration method shown in Table 6.

[0475] [Table 9]

[0476] h-IgG, JNJ372, Gp120 / Zalu, Hz20G5.26 / Zalu, and Zalu mAb were all used at a concentration of 0.5 mg / mL and were administered every 3 to 4 days for a total of four times (Q3-4Dx4). They were administered on the 4th, 8th, 12th, and 15th days after NCI-H292 cell inoculation, respectively, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figure 15A, and the monitoring was terminated after 59 days. Since some mice in the groups died early due to tumors, the relative tumor inhibition rate (TGI%) was calculated based on the tumor volume on the 22nd day after inoculation, and the calculation formula was: TGI% = 100% × (tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - tumor volume before administration of control group).

[0477] Measurement of tumor volume: The maximum long axis (L) and maximum width axis (W) of the tumor were measured using a caliper, and the tumor volume was calculated according to the following formula: V = L × W 2 Calculated according to / 2.

[0478] Body weight was measured using an electronic balance. 3 Mice exceeding 1 h or experiencing more than 20% weight loss were euthanized.

[0479] The results of the tumor inhibition rate are shown in Table 7: On the 22nd day after inoculation, compared with the h-IgG, 5 mg / kg group, the tumor inhibition rates of JNJ372, Gp120 / Zalu, Hz20G5.26 / Zalu, and Zalu mAb were 100%, 72%, 108%, and 107%, respectively. In each of the Hz20G5.26 / Zalu and Zalu mAb groups, there was one mouse that showed complete tumor remission. As shown in the mouse survival curve in Figure 15B, Hz20G5.26 / Zalu was able to significantly extend the survival period of the mice. In summary, the antitumor efficacy of Hz20G5.26 / Zalu against NCI-H292 tumor-bearing mice was equivalent to that of the parental Zalu mAb and superior to JNJ372 and non-target GP120 / Zalu.

[0480] From the results of simultaneously monitoring the body weight of the mice (Figure 15C), it was shown that within 59 days after inoculation, the body weight of the mice in the Hz20G5.26 / Zalu group did not significantly decrease.

[0481]

Table 10

[0482] To prove the in vivo efficacy of the Hz20G5.26 / Zalu bispecific antibody molecule against NSCLC with EGFR abnormal amplification, SK-MES-1 cells (Nanjing Kebai) were used to inoculate Balb / c Nude mice, and the antitumor efficacy of the Hz20G5.26 / Zalu bispecific antibody molecule of the present invention was measured. In the experiment, SPF-grade female Balb / c Nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used, and the certificate number was NO.110011211108966881.

[0483] SK-MES-1 cells were regularly passaged and then used in the in vivo experiment. The cells were collected by centrifugation, mixed with PBS (1×) and Matrigel Matix (Corning) at an equal ratio, and SK-MES-1 cells were resuspended, and the cell concentration was 20×10 6A cell suspension of cells / mL was prepared. On day 0, 0.2 mL of the cell suspension was taken and subcutaneously inoculated into the right abdominal region of Balb / c Nude mice to establish an SK-MES-1 tumor-bearing mouse model.

[0484] Six days after tumor cell inoculation, the tumor volume of each mouse was detected and grouped (7 mice per group), and the dosage and administration method were shown in Table 8.

[0485]

Table 11

[0486] h-IgG, Hz20G5.26 / Zalu, JNJ372, Zalu mAb, and Gp120 / Zalu were all used at a concentration of 0.1 mg / mL. They were administered once on the 6th day after SK-MES-1 cell inoculation. As shown in Figure 16A, the tumor volume and body weight of the mice were monitored twice a week, and the monitoring was terminated after 24 days.

[0487] On the 24th day after inoculation, the relative tumor inhibition rate (TGI%) was calculated, and the calculation formula was TGI% = 100% × (control group tumor volume - treatment group tumor volume) / (control group tumor volume - control group tumor volume before administration).

[0488] Measurement of tumor volume: The maximum major axis (L) and maximum minor axis (W) of the tumor were measured using calipers, and the tumor volume was calculated according to the following formula: V = L × W 2 / 2. The body weight was measured using an electronic balance.

[0489] The results of the tumor inhibition rate are shown in Table 9: On the 24th day after inoculation, compared with the h-IgG, 1 mg / kg group, the tumor inhibition rates of Hz20G5.26 / Zalu, JNJ372, Zalu mAb, and Gp120 / Zalu were 143%, 122%, 145%, and 113% respectively. At the same time, the result of monitoring the body weight of the mice (Figure 16B) showed that there was no significant difference in the body weight of the mice on the 24th day after inoculation.

[0490]

Table 12

[0491] To further prove the superiority of the efficacy of the Hz20G5.26 / Zalu molecule as a bispecific antibody in vivo, SSK-MES-1 cells (Nanjing Kebai) were used to inoculate Balb / c Nude mice, and the antitumor efficacy of the Hz20G5.26 / Zalu bispecific antibody molecule of the present invention was measured. In the experiment, SPF-grade female Balb / c Nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used, and the certificate number was NO.110011221103705231.

[0492] SK-MES-1 cells were regularly passaged and used in subsequent in vivo experiments. The cells were collected by centrifugation, and SK-MES-1 cells were mixed with PBS (1×) and Matrigel Matix (Corning) at an equal ratio to prepare a cell suspension with a cell concentration of 25×10 6 cells / mL. On day 0, 0.2 mL of the cell suspension was taken and subcutaneously inoculated into the right abdominal region of Balb / c Nude mice to establish an SK-MES-1 tumor-bearing mouse model.

[0493] Fourteen days after tumor cell inoculation, the tumor volume of each mouse was detected and grouped (6 mice per group), and the dosage and administration method were shown in Table 10.

[0494]

Table 13

[0495] h-IgG was used at a concentration of 0.06 mg / mL, and Hz20G5.26 / Zalu, Gp120 / Zalu, and Gp120 / Hz20G5.26 were all used at a concentration of 0.03 mg / mL. They were administered once on the 14th day after SK-MES-1 cell inoculation. As shown in Figure 17A, the tumor volume and body weight of the mice were monitored twice a week, and the monitoring was terminated after 35 days.

[0496] On the 35th day after inoculation, the relative tumor inhibition rate (TGI%) was calculated, and the calculation formula was TGI% = 100% × (control group tumor volume - treatment group tumor volume) / (control group tumor volume - control group pre - administration tumor volume).

[0497] Measurement of tumor volume: Using calipers, the maximum major axis (L) and maximum minor axis (W) of the tumor were measured, and the tumor volume was calculated according to the following formula: V = L × W 2 / 2. The body weight was measured using an electronic balance.

[0498] The results of the tumor inhibition rate are shown in Table 11: On the 35th day after inoculation, compared with the 0.6 mg / kg group of h - IgG, the tumor inhibition rates of Hz20G5.26 / Zalu, Gp120 / Zalu, Gp120 / Hz20G5.26, and Gp120 / Zalu + Gp120 / Hz20G5.26 were 121%, 31%, 1%, and 16% respectively. The anti - tumor efficacy of Hz20G5.26 / Zalu was superior to that of the control non - target monoclonal antibodies Gp120 / Zalu, Gp120 / Hz20G5.26, or the combination of both, demonstrating the unique mechanism of Hz20G5.26 / Zalu, that is, the pulling effect of Hz20G5.26 on Zalu that blocks the EGFR signal. At the same time, from the results of monitoring the body weight of mice (Figure 17B), on the 24th day after inoculation, it was shown that there was no significant difference in the body weight of mice.

[0499]

Table 14

[0500] Example 9. In vivo pharmacodynamic activity of the combination of Hz20G5.26 / Zalu bsAb bispecific antibody molecule and KRAS small molecule inhibitor To prove the pharmacodynamic effect of the combination of the Hz20G5.26 / Zalu bispecific antibody molecule and the KRAS small molecule inhibitor in vivo, KRAS G12CThe NSCLC tumor cell line NCI-H358 (Nanjing Kebai) with mutations was used to inoculate NOG mice, and the antitumor efficacy of the combined use of the Hz20G5.26 / Zalu bispecific antibody of the present invention and the KRAS small molecule inhibitor was measured. In the experiment, SPF-grade female NOG mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used, and the certificate number was NO.1100112211001153625.

[0501] NCI-H358 cells were subcultured regularly and then used for subsequent in vivo experiments. The cells were collected by centrifugation, mixed with PBS (1×) and Matrigel Matix (Corning) at an equal ratio, and NCI-H358 cells were resuspended to produce a cell suspension with a cell concentration of 25×10 6 cells / mL. On day 0, 0.2 mL of the cell suspension was taken and subcutaneously inoculated into the right abdominal region of NOG mice to establish an NCI-H358 tumor-bearing mouse model.

[0502] Six days after tumor cell inoculation, the tumor volume of each mouse was detected and grouped (7 mice per group), and the dosage and administration method were shown in Table 12.

[0503]

Table 15

[0504] h-IgG was used at a concentration of 2 mg / mL, and Hz20G5.26 / Zalu, JNJ372, and ZalumAb were all used at a concentration of 1 mg / mL. They were administered once every 3 to 4 days for a total of 4 times (Q3-4D×4). Administration was carried out on the 6th, 9th, 12th, and 15th days after NCI-H358 cell inoculation, respectively. The tumor volume and body weight of the mice were monitored twice a week. AMG510 was used at a concentration of 1 mg / mL. Administration was started on the 6th day after NCI-H358 cell inoculation, once a day for a total of 14 times (QD×14). The tumor volume of the mice was monitored twice a week. During the dosing period, the body weight of the mice was monitored daily, and after the dosing was completed, the body weight of the mice was monitored twice a week. As shown in Figure 18A, the monitoring was completed after 33 days. On the 33rd day after inoculation, the relative tumor growth inhibition rate (TGI%) was calculated, and the calculation formula was TGI% = 100% × (control group tumor volume - treatment group tumor volume) / (control group tumor volume - control group pre-dose tumor volume).

[0505] Measurement of tumor volume: The maximum major axis (L) and maximum minor axis (W) of the tumor were measured using calipers, and the tumor volume was calculated according to the following formula: V = L×W 2 / 2. The body weight was measured using an electronic balance.

[0506] The results of the tumor growth inhibition rate are shown in Table 13: On the 33rd day after inoculation, compared with the h-IgG, 20 mg / kg group, the tumor growth inhibition rates of AMG510, Hz20G5.26 / Zalu, AMG510+Hz20G5.26 / Zalu, JNJ372, and ZalumAb were 89%, 110%, 130%, 82%, and 111%, respectively. In each group, the combination of Hz20G5.26 / Zalu and AMG510 showed the most excellent antitumor effect against NCI-H358 tumor-bearing mice and had a synergistic effect.

[0507] At the same time, from the results of monitoring the body weight of the mice (Figure 18B), it was shown that the body weight of the mice gradually decreased due to the administration of AMG510, recovered after the administration was stopped, and the mice in each group had normal body weight on the 33rd day.

[0508]

Table 16

[0509]

Table 17-1

[0510]

Table 17-2

[0511]

Table 17-3

[0512]

Table 17-4

[0513]

Table 17-5

[0514]

Table 17-6

[0515]

Table 17-7

[0516]

Table 17-8

Claims

**Claim 1** A bispecific antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region specifically binds to EGFR and the second antigen-binding region specifically binds to B7H3. A bispecific antibody that binds to EGFR and B7-H3. **Claim 2** The second antigen-binding region comprises the HCDR1, HCDR2, and HCDR3 sequences of the heavy-chain variable region shown in SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, and the LCDR1, LCDR2, and LCDR3 sequences of the light-chain variable region shown in SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO:

8. The bispecific antibody according to claim 1. **Claim 3** The second antigen-binding region comprises HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region VH and LCDR1, LCDR2, and LCDR3 of the light-chain variable region VL, wherein (i) the HCDR1, HCDR2, and HCDR3 are the three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH shown in SEQ ID NO: 3, and the LCDR1, LCDR2, and LCDR3 are the three complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in VL shown in SEQ ID NO: 4; (ii) the HCDR1, HCDR2, and HCDR3 are the three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH shown in SEQ ID NO: 5, and the LCDR1, LCDR2, and LCDR3 are the three complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in VL shown in SEQ ID NO: 6; or (iii) the HCDR1, HCDR2, and HCDR3 are the three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in VH shown in SEQ ID NO: 7, and the LCDR1, LCDR2, and LCDR3 are the three complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in VL shown in SEQ ID NO:

8. The bispecific antibody according to claim 1. **Claim 4** The second antigen-binding region comprises HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region VH and LCDR1, LCDR2, and LCDR3 of the light-chain variable region VL, wherein (i) The HCDR1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 15, the HCDR2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 16, and the HCDR3 comprises, or consists of, the amino acid sequence shown in SEQ ID NO:

17. The LCDR1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 18, the LCDR2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 19, and the LCDR3 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 20, or (ii) The HCDR1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 21, the HCDR2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 22, and the HCDR3 comprises, or consists of, the amino acid sequence shown in SEQ ID NO:

23. The LCDR1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 24, the LCDR2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 25, and the LCDR3 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 26, or (iii) The HCDR1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 28, and the HCDR3 comprises, or consists of, the amino acid sequence shown in SEQ ID NO:

29. The LCDR1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 30, the LCDR2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 31, and the LCDR3 comprises, or consists of, the amino acid sequence shown in SEQ ID NO:

32. The bispecific antibody according to claim 1.

5. The second antigen-binding region comprises a heavy chain variable region VH, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or consists of these. The bispecific antibody according to any one of claims 1 to 4. **Claim 6** The second antigen-binding region comprises a light chain variable region VL, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8, or consists of them. The bispecific antibody according to any one of claims 1 to 5. **Claim 7** The second antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein (i) the VH comprises the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 3, and the VL comprises the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 4, or consists of them; (ii) the VH comprises the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 5, and the VL comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 6, or consists of them, or (iii) The VH comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO: 7, and the VL comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO:

8. The bispecific antibody according to any one of claims 1 to 6.

8. The second antigen-binding region comprises, or consists of, a heavy-chain variable region VH and a light-chain variable region VL, wherein the VH and the VL are each SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or comprise, or consist of, the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO:

8. The bispecific antibody according to any one of claims 1 to 7.

9. The first antigen-binding region comprises the HCDR1, HCDR2, and HCDR3 of the heavy-chain variable region VH and the LCDR1, LCDR2, and LCDR3 of the light-chain variable region VL, wherein the HCDR1, HCDR2, and HCDR3 are the three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in the VH shown in SEQ ID NO: 1, and the LCDR1, LCDR2, and LCDR3 are the three complementarity-determining regions LCDR1, LCDR2, and LCDR3 contained in the VL shown in SEQ ID NO:

2. The bispecific antibody according to any one of claims 1 to 7.

10. The HCDR1 of the first antigen-binding region comprises, or consists of, the amino acid sequence of SEQ ID NO: 9, the HCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 10, the HCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 11, and the LCDR1 of the first antigen-binding region comprises, or consists of, the amino acid sequence of SEQ ID NO: 12, the LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 13, and the LCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO:

14. The bispecific antibody according to claim 9.

11. The first antigen-binding region comprises a heavy chain variable region VH, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 1, or consists of them, The bispecific antibody according to claim 9 or 10.

12. The first antigen-binding region comprises a light chain variable region VL, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 2, or consists of them, The bispecific antibody according to any one of claims 9 to 11.

13. The first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 1, or consists of them, and the VL comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 2, or consists of them, The bispecific antibody according to any one of claims 9 to 12.

14. The first antigen-binding region comprises or consists of a heavy chain variable region VH and a light chain variable region VL, wherein the VH and the VL respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, The bispecific antibody according to any one of claims 9 to 13.

15. The first antigen-binding region specifically binds to EGFR and comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH, and LCDR1, LCDR2 and LCDR3 of the light chain variable region VL, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 9, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 10, The HCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 11, the LCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 12, the LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 13, and the LCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 14, and the second antigen-binding region specifically binds to B7H3 and comprises HCDR1, HCDR2, and HCDR3 of the heavy chain variable region VH and LCDR1, LCDR2, and LCDR3 of the light chain variable region VL, where (i) the HCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 15, the HCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 16, the HCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 17, the LCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 18, the LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 19, and the LCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 20, (ii) the HCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 21, the HCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 22, the HCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 23, the LCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 24, the LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 25, and the LCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 26, or (iii) the HCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 27, the HCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 28, the HCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 29, the LCDR1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 30, the LCDR2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 31, and the LCDR3 comprises, or consists of, the amino acid sequence of SEQ ID NO: 32, The bispecific antibody according to any one of claims 1 to 14.

16. The first antigen-binding region comprises or consists of a VH comprising the amino acid sequence shown in SEQ ID NO: 1 and a VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2, and the second antigen-binding region respectively comprises SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or VH and VL comprising or consisting of the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, The bispecific antibody according to claim 15.

17. Comprising an Fc region, preferably, the Fc region has hypofucosylation, for example, hypofucosylation obtained after treatment by the GlymaxX technology, The bispecific antibody according to any one of claims 1 to 16.

18. Comprising a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region are the same or different, The bispecific antibody according to claim 17.

19. The first Fc region and the second Fc region are respectively human IgG Fc, such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, for example, the amino acid sequences shown in SEQ ID NO: 46 or SEQ ID NO: 47, or amino acid sequences having at least 90% identity with them, such as 95%, 96%, 97%, 99% or more identity, The bispecific antibody according to claim 17 or 18.

20. Mutations that promote heterodimerization of the first Fc region and the second Fc region are introduced into the first Fc region and the second Fc region, The bispecific antibody according to claim 18 or 19.

21. The mutation is introduced based on the Innobody technology, The bispecific antibody according to claim 20.

22. CH3 of one Fc region contains S364R and D399K mutations, and CH3 of another Fc region contains Y349T, K370S and K409D mutations, The bispecific antibody according to claim 21.

23. a) One Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 49 or SEQ ID NO: 50, and another Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 52 or SEQ ID NO: 53, b) One Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 49 or SEQ ID NO: 50, and another Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 52 or SEQ ID NO: 53, or c) One Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 49 or SEQ ID NO: 50, and contains mutations Y349T, K370S and K409D, and another Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 52 or SEQ ID NO: 53, and contains mutations S364R and D399K, The bispecific antibody according to claim 22.

24. The mutations are introduced based on the Knob-into-Hole technology, where the corresponding Knob mutation and Hole mutation are introduced into the first Fc region and the second Fc region. The bispecific antibody according to claim 20.

25. a) One Fc region polypeptide contains the mutation T366W, and another Fc region polypeptide contains T366S, L368A and Y407V (numbering according to EU index), or b) One Fc region contains the amino acid substitutions S354C and T366W, and another Fc region contains the amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to EU index). The bispecific antibody according to claim 24.

26. The first and / or second antigen-binding region (e.g., the heavy chain variable region herein) may be further linked to one or two heavy chain constant regions (e.g., the heavy chain constant regions of human IgG1, human IgG2, human IgG3, or human IgG4), and the heavy chain constant region contains CH1 and the Fc region, and is linked with or without a hinge region. For example, the C-terminus of the heavy chain variable region is linked to the N-terminus of CH1 of the heavy chain constant region. The bispecific antibody according to any one of claims 1 to 25.

27. The CH1 includes, or consists of, an amino acid sequence shown in SEQ ID NO: 42, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO:

42. The bispecific antibody according to claim 26.

28. The first and / or second antigen-binding region (e.g., the light chain variable region herein) may be further linked to the light chain constant region. For example, the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region. The bispecific antibody according to any one of claims 1 to 27.

29. The light chain constant region is a kappa light chain constant region or a lambda light chain constant region. The bispecific antibody according to claim 28.

30. The light chain constant region includes, or consists of, an amino acid sequence shown in SEQ ID NO: 54, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO:

54. The bispecific antibody according to claim 20.

31. The bispecific antibody is an IgG-like antibody having a structure as shown in FIG.

1. The bispecific antibody according to any one of claims 1 to 30.

32. It includes heavy chain 1 and light chain 1, and heavy chain 2 and light chain 2, wherein the heavy chain 1 and the light chain 1 constitute the first half antibody, and the heavy chain 2 and the light chain 2 constitute the second half antibody, where the heavy chain 1 includes the heavy chain variable region of the first antigen-binding region and the first heavy chain constant region, the light chain 1 includes the light chain variable region of the first antigen-binding region and the first light chain constant region, and the heavy chain 2 includes the heavy chain variable region of the second antigen-binding region and the second heavy chain constant region, and the light chain 2 includes the light chain variable region of the second antigen-binding region and the second light chain constant region. The bispecific antibody according to claim 31.

33. The heavy chain 1 includes, or consists of, an amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO:

33. The bispecific antibody according to claim 32.

34. The light chain 1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO:

34. The bispecific antibody according to claim 32 or 33.

35. The heavy chain 1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 33, and the light chain 1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO:

34. The bispecific antibody according to any one of claims 32 to 34.

36. The heavy chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO: 39, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 37 or SEQ ID NO:

39. The bispecific antibody according to any one of claims 32 to 35.

37. The light chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 38 or SEQ ID NO: 40, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 38 or SEQ ID NO:

40. The bispecific antibody according to any one of claims 32 to 36.

38. (1) The heavy chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 35, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 35, and the light chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 36, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:

36. (2) The heavy chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 37, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 37, and the light chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 38, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:

38. (3) The heavy chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 39, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 39, and the light chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 40, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:

40. The bispecific antibody according to any one of claims 32 to 37.

39. The heavy chain 1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 33, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 33, and the light chain 1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 34, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 34, and the heavy chain 2 and the light chain 2 are respectively the following SEQ ID NOs., i) SEQ ID NO: 35 and SEQ ID NO: 36, ii) SEQ ID NO: 37 and SEQ ID NO: 38, iii) the amino acid sequences shown in SEQ ID NO: 39 and SEQ ID NO: 40, or comprise, or consist of, an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequences shown above, The bispecific antibody according to any one of claims 32 to 38.

40. (i) The heavy chain 1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 33, and the light chain 1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 34, the heavy chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 35, and the light chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 36, or (ii) The heavy chain 1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 33, and the light chain 1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 34, the heavy chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 37, and the light chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 38, or (iii) The heavy chain 1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 33, and the light chain 1 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 34, the heavy chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 39, and the light chain 2 comprises, or consists of, the amino acid sequence shown in SEQ ID NO: 40, The bispecific antibody according to any one of claims 32 to 39.

41. A bispecific antibody or an antigen-binding fragment thereof that binds to EGFR and B7-H3 according to any one of Claims 1 to 40, wherein the antibody or the antigen-binding fragment thereof has one or more of the following characteristics: (i) The antibody can, on the one hand, block the binding of an EGFR ligand to EGFR, inhibit biological signal transduction, and block the corresponding biological activity of the tumor, and on the other hand, stimulate EGFR endocytosis, and ultimately, EGFR is degraded by intracellular lysosomes, etc. (ii) The antibody employs a low-affinity EGFR antibody parental sequence for EGFR to significantly reduce the toxicity and side effects of a series of EGFR monoclonal antibodies against normal epithelial tissues such as the skin. (iii) The antibody quotes a B7-H3 high-affinity antibody parental sequence based on the low affinity of EGER, significantly improves the EGFR signal blocking activity, and improves the pharmacodynamic biological activity and pharmacodynamic safety window of the bispecific antibody of the present invention. (iv) The antibody is a low-fucosylated antibody. (ii) The antibody has relatively high pharmacodynamic biological activity and safety. (v) The antibody has excellent tumor killing and inhibitory effects. (vi) The antibody has excellent ADCC in vivo and in vitro pharmacodynamic activities. (vii) The combination of the antibody and a KRAS small molecule inhibitor has excellent anti-tumor synergistic effects. Antibody or an antigen-binding fragment thereof.

42. An isolated nucleic acid encoding any one strand of a bispecific antibody that binds to EGFR and B7-H3 according to any one of Claims 1 to 41. Isolated nucleic acid.

43. A vector containing the nucleic acid according to Claim 42, preferably the vector is an expression vector, and preferably the expression vector is pcDNA, such as pcDNA3.

1. Vector.

44. A host cell containing the nucleic acid according to Claim 42 or the vector according to Claim 43, preferably the host cell is a prokaryote or a eukaryote, more preferably a yeast cell or a mammalian cell (for example, 293 cells or CHO cells, such as 293F cells or 293T cells or CHO-S cells). Host cell.

45. The host cell according to Claim 44, which is glycoengineered to express the RMD enzyme, and preferably the host cell is a CHO cell. Host cell according to Claim 44.

46. Containing a nucleic acid encoding the RMD enzyme. Host cell according to Claim 45.

47. The RMD enzyme comprises, or consists of, an amino acid sequence shown in SEQ ID NO: 41 or an amino acid sequence having at least 90% identity thereto, and preferably, the RMD enzyme is derived from Pseudomonas aeruginosa. The host cell according to claim 46.

48. A method for producing a bispecific antibody that binds to EGFR and B7-H3, the method comprising culturing a host cell according to any one of claims 44 to 47 under conditions suitable for the expression of a nucleic acid encoding the bispecific antibody according to any one of claims 1 to 41, and optionally isolating the antibody or an antigen-binding fragment thereof, and optionally the method further comprises recovering the antibody or an antigen-binding fragment thereof from the host cell (or host cell culture medium). Method.

49. An immunocomplex comprising the bispecific antibody according to any one of claims 1 to 41, bound to a therapeutic or diagnostic agent. Immunocomplex.

50. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 41 or the immunocomplex according to claim 49, and optionally a pharmaceutically acceptable excipient. Pharmaceutical composition.

51. Further comprising a second therapeutic agent, preferably, the second therapeutic agent is selected from an anti-angiogenic agent, a chemotherapeutic agent, another antibody, a cytotoxic agent, a vaccine, an anti-infective agent, a small molecule drug or an immunomodulatory agent (e.g., an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule), preferably, the second therapeutic agent is selected from a KRAS small molecule inhibitor, such as a KRAS G12C inhibitor (e.g., AMG510 (Sotorasib) or GFH925), a KRAS G12D (e.g., MRTX1133) or a KRAS G12S inhibitor. The pharmaceutical composition according to claim 50.

52. A pharmaceutical combination product comprising the bispecific antibody according to any one of claims 1 to 41, or the immune complex according to claim 49, or the pharmaceutical composition according to claim 50, and one or more second therapeutic agents, preferably, the second therapeutic agent is selected from an anti-angiogenic agent, a chemotherapeutic agent, another antibody, a cytotoxic agent, a vaccine, an anti-infective agent, a small molecule drug or an immunomodulatory agent (e.g., an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule), preferably, the second therapeutic agent is selected from a KRAS small molecule inhibitor, such as a KRAS G12C inhibitor (e.g., AMG510 (Sotorasib) or GFH925), a KRAS G12D (e.g., MRTX1133) or a KRAS G12S inhibitor. Pharmaceutical combination product.

53. A method for preventing or treating a tumor or an infectious disease in a subject, the method comprising administering to the subject an effective amount of the bispecific antibody according to any one of claims 1 to 41, or the immune complex according to claim 49, or the pharmaceutical composition according to claim 50. Method.

54. The method further comprises co-administering to the subject one or more other therapies, the therapies including, for example, treatment modalities and / or other therapeutic agents, preferably, the treatment modality includes surgical treatment and / or radiotherapy, or the therapeutic agent is selected from an anti-angiogenic agent, a chemotherapeutic agent, another antibody, a cytotoxic agent, a vaccine, an anti-infective agent, a small molecule drug or an immunomodulatory agent (e.g., an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule), preferably, the second therapeutic agent is selected from a KRAS small molecule inhibitor, such as a KRAS G12C inhibitor (e.g., AMG510 (Sotorasib) or GFH925), a KRAS G12D (e.g., MRTX1133) or a KRAS G12S inhibitor. The method according to claim 53.

55. A method for preventing or treating a tumor or an infectious disease in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition according to claim 51 or the pharmaceutical combination product according to claim 52. Method.

56. The tumor is cancer, such as a solid tumor or a hematological tumor, and includes cancers of epithelial origin, such as gastrointestinal tumors, lung tumors, or skin tumors, such as skin cancer (e.g., cutaneous squamous cell carcinoma, head and neck cancers such as head and neck squamous cell carcinoma), esophageal cancer (e.g., esophageal squamous cell carcinoma), intestinal cancer (e.g., colon cancer, rectal cancer, colorectal cancer), or lung cancer (e.g., non-small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma). The method according to any one of claims 53 to 55.

57. In the tumor cells of the tumor, (i) overexpress wild-type EGFR (e.g., wild-type EGFR with increased nucleic acid or protein levels) and / or express mutated EGFR, preferably the mutated EGFR includes one or more mutations selected from R521K, L858R, T790M, G719X, C797S, Y1069C, Exon19 deletion (Del19), Exon20ins (e.g., S768_D770dup), preferably the mutated EGFR includes R521K / Y1069C, R521K, L858R / T790M / C797S, Del19 / T790M / C797S or S768_D770dup, compared to normal cells of adjacent tissues or compared to normal cells of the same tissue in a healthy subject. (ii) overexpress wild-type KRAS (e.g., wild-type KRAS with increased nucleic acid or protein levels) or express mutated KRAS, preferably the mutated KRAS includes mutations at position G12 or G13, such as G12D or G12C, compared to normal cells of adjacent tissues or compared to normal cells of the same tissue in a healthy subject. (iii) have increased B7-H3 at the nucleic acid level or protein level compared to normal cells of adjacent tissues or compared to normal cells of the same tissue in a healthy subject, and / or (iv) the tumor cells are drug-resistant to tyrosine kinase inhibitors, such as first-generation (erlotinib) and third-generation (osimertinib). The method according to any one of claims 53 to 56.

58. Express mutated EGFR and mutated KRAS in the tumor cells of the tumor, for example, including mutated EGFR having R521K and mutated KRAS having G120D. The method according to claim 57. **Claim 59** A method for detecting antigen EGFR and / or B7-H3 in a sample, said method comprising: (a) contacting the sample with the bispecific antibody according to any one of claims 1 to 41; and (b) detecting the formation of a complex between the antibody or its antigen-binding fragment and EGFR and / or B7-H3, optionally wherein the antibody is detectably labeled. Method. **Claim 60** Use of the antibody or its antigen-binding fragment according to any one of claims 1 to 41, and / or the isolated nucleic acid according to claim 42, and / or the vector according to claim 43, and / or the host cell according to any one of claims 44 to 47, and / or the immune complex according to claim 49, and / or the pharmaceutical composition according to claim 50 or 51, or the pharmaceutical combination product according to claim 52, in the manufacture of a medicament for preventing and / or treating the disease of interest in a subject. Use.

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