Bispecific antibody binding to EGFR and b7-h3

Abstract

This invention provides a novel, artificially designed bispecific antibody molecule, particularly an anti-B7-H3 / EGFR bispecific antibody molecule that can bind to both B7-H3 and EGFR simultaneously.

Description

Technical Field

[0001] The present invention generally relates to the fields of immunology and antibody engineering. Specifically, the present invention relates to novel, artificially designed bispecific antibody molecules, particularly bispecific antibodies that simultaneously bind to EGFR and B7-H3, polynucleotides encoding such antibody molecules or individual chains thereof, vectors comprising such polynucleotides, host cells comprising such polynucleotides or vectors, immunoconjugates and pharmaceutical compositions comprising such antibody molecules, and the use of such antibody molecules in the immunotherapy, prevention, and / or diagnosis of diseases. Prior Art

[0002] Globally, lung cancer is one of the most common types of cancer tumors. Approximately 80%-85% of lung cancer patients are non-small cell lung cancer (NSCLC). NSCLC can be divided 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 approximately 170 kDa. It is a member of the ErbB receptor family and is the most common driver of NSCLC. EGFR mutations are responsible for approximately 40% of NSCLC cases in Asians and 15% in Caucasians (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.). Overexpression and / or abnormal mutational activation of EGFR are also common in other epithelial cancers, such as renal, prostate, pancreatic, breast, colon, and head and neck cancers.

[0003] Targeted therapies for abnormal EGFR activation and amplification in NSCLC include small molecule EGFR-TKI inhibitors (such as gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, and ametinib) and large biopharmaceutical monoclonal antibodies (cetuximab, panitumumab, nexitozumab, and nimotuzumab). While small molecule EGFR-TKI inhibitors remain the standard treatment for non-small cell lung cancer (NSCLC), they primarily target patients with structural mutations in tyrosine kinase activity. These EGFR-TKIs often lead to drug resistance and ineffectiveness due to target gene mutations. This necessitates the continuous development of new targeted drugs targeting novel mutation sites, significantly limiting their clinical application and presenting a significant challenge for the entire EGFR-TKI industry.

[0004] The EGFR activating 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 EGFR antibodies is mainly located in the extracellular ligand domain of EGFR, which can avoid the occurrence of drug-resistant mutations. At the same time, EGFR antibodies can inhibit the growth of tumor cells by inhibiting the binding of EGFR to ligands, and can also use their own specific ADCC (antibody-dependent cell-mediated cytotoxicity) to jointly kill tumors with immune cells. In this way, they can jointly exert anti-tumor killing effects through multiple 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) with a structure very similar 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, and pancreatic cancer (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).The B7-H3 receptor has not yet been identified, but in tumor immunity, B7-H3 may participate in the regulation of 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 tumor-derived circulating endothelial cells. Br J Cancer. 2014;111: 149-56). There is also evidence that B7-H3 expression may be associated with EGFR gene expression and the efficacy of anti-PD-1 therapy (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 well-selected expression profile, some biotechnology companies have developed B7-H3 monoclonal antibodies or B7-H3-ADC drugs for research and treatment of related tumor diseases.

[0006] Antibodies, capable of targeted and specific binding to their corresponding antigens, are increasingly becoming important therapeutic, preventative, and / or diagnostic agents for a variety of diseases (e.g., cancer, autoimmune diseases, inflammatory diseases, infectious diseases, etc.). However, monospecific antibodies that target only a single target have limitations in clinical application. Patients may develop drug resistance or non-response after monospecific antibody treatment. Research on cancer and various other diseases has recognized that multiple signal transduction pathways are often involved in the development and progression of diseases, and single-target immunotherapy is often insufficient to effectively treat many diseases.

[0007] Because multispecific antibodies (e.g., bispecific antibodies) can specifically bind to different antigens and can be designed to simultaneously act on two or more different signal transduction pathways, these advantageous properties have opened up broad application prospects for multispecific antibodies (e.g., bispecific antibodies).

[0008] There remains a need in the art for alternative bispecific antibodies with improved performance, capable of simultaneously binding to different antigens, particularly EGFR and B7-H3, while maintaining the binding activity of each antigen-binding site to its corresponding distinct epitope, as well as other properties. Furthermore, there is a need for bispecific antibody formats that are physically and biologically stable, allowing for improved manufacturability and scalability. Summary of the Invention [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, such as a bispecific antibody.

[0010] The antibodies or antigen-binding fragments thereof provided herein that bind to EGFR and B7-H3 improve the antibody's efficacy, safety, and selectivity by reducing EGFR affinity and increasing B7-H3 affinity. Furthermore, the use of GlymaxX low-fucose technology enhances antibody-dependent cell-mediated cytotoxicity (ADCC). Compared to Amivantamab (JNJ-372), used for NSCLC-EGFR exon 20 insertion mutations (exon20ins), B7-H3 has a broader expression spectrum than cMET and a wider range of tumor therapeutic applications. Furthermore, the introduction of the B7-H3 parental protein not only enhances the EGFR blocking activity of the bispecific antibody but also improves the overall ADCC activity of the bispecific antibody.

[0011] The bispecific antibodies or antigen-binding fragments thereof that bind to EGFR and B7-H3 provided by the present invention have one or more of the following characteristics: (a) specifically binds to one or two antigens with high affinity; (b) It is easy to express in cultured cells in vitro, and the chains of the antibody molecules can be correctly coupled or paired; (c) having good physical stability, in particular, good long-term thermal stability; and being able to maintain biological activity for a long time; (d) After specifically binding to one or two antigens, it exerts its biological function by regulating (e.g., inhibiting or activating) the signal transduction pathways involved in each antigen; (e) exerting effector functions; (f) Has better anti-tumor 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 from a human, or humanized, or chimeric antibody.

[0014] In some embodiments, antibodies of the present invention, such as bispecific antibodies, further comprise a heavy chain constant region. In one embodiment, the heavy chain constant domain is derived from IgG1. It should be understood that mutations to the Fc region of the constant domain can be performed to stabilize the antibody or enhance effector function.

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

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

[0017] The antibodies of the present invention may also contain other antigen-binding regions that bind to other antigens to form multispecific antibodies. The types of other antigens specifically bound by the antibody molecules of the present invention are not particularly limited, and the antigens may be, for example, cytokines, growth factors, hormones, signaling proteins, inflammatory mediators, ligands, cell surface receptors, or fragments thereof. In one embodiment, the other antigens specifically bound by the antibody molecules of the present invention are selected from tumor-associated antigens, immune checkpoint molecules, angiogenesis-inducing factors, members of the tumor necrosis factor receptor superfamily, and co-stimulatory molecules in the immune system, as well as the 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 comprising the nucleic acid, and a host cell comprising the nucleic acid or the vector.

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

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

[0021] In some embodiments, the present invention provides immunoconjugates, pharmaceutical compositions, kits, combination products or articles of manufacture comprising an antibody of the present invention.

[0022] In some embodiments, the antibodies, pharmaceutical compositions, immunoconjugates, combination products, or kits of the present invention are used to prevent or treat diseases, such as acute and chronic inflammatory diseases, infections (e.g., chronic infections), tumors, and the like. For example, the disease is a tumor (e.g., cancer) or an infection. In some embodiments, the tumor is a tumor that has escaped immune function. 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, comprising administering to the subject an effective amount of any of the antibodies or fragments thereof, pharmaceutical compositions, immunoconjugates, combination products, or kits described herein. For example, the disease is a tumor (e.g., cancer) or an infection. In some embodiments, the tumor is a tumor that has escaped immune function. 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 of the antibodies, fragments thereof, or immunoconjugates described herein for the preparation of a medicament, pharmaceutical composition, kit, or combination product for treating a tumor (e.g., cancer) or infection in a subject. In some embodiments, the tumor is an immune evasion tumor. 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:

[0027] 1. A bispecific antibody that binds to EGFR and B7-H3, 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.

[0028] 2. The bispecific antibody of embodiment 1, wherein the second antigen-binding region comprises the HCDR1, 2, and 3 sequences of the heavy chain variable region as shown in SEQ ID NO: 3, 5, or 7, and the LCDR1, 2, and 3 sequences of the light chain variable region as shown in SEQ ID NO: 4, 6, or 8; preferably, the HCDR1 adopts the Abm scheme, and the HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 adopt the Kabat scheme.

[0029] 3. The bispecific antibody of 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, wherein (i) the HCDR1, HCDR2, and HCDR3 are the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO: 3; and the LCDR1, LCDR2, and LCDR3 are the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO: 4; (ii) the HCDR1, HCDR2 and HCDR3 are the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 5; and the LCDR1, LCDR2 and LCDR3 are the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as 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 the VH as shown in SEQ ID NO: 7; and the LCDR1, LCDR2, and LCDR3 are the three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in the VL as 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.

[0030] 4. The bispecific antibody of 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, wherein (i) HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 15, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 18, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 20; or,

[0031] (ii) HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 21, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 22, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 23, LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 24, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 25, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 26; or,

[0032] (iii) HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 27, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 28, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 29, LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 30, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 31, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 32.

[0033] 5. The bispecific antibody of any one of embodiments 1 to 4, wherein the second antigen-binding region comprises a heavy chain variable region VH, wherein the VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 3, 5 or 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, 5 or 7.

[0034] 6. The bispecific antibody of any one of embodiments 1 to 5, wherein the second antigen-binding region comprises a light chain variable region VL, wherein the VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 4, 6 or 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, 6 or 8.

[0035] 7. The bispecific antibody of any one of embodiments 1 to 6, wherein the second antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein (i) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:3, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:3, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:4; (ii) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:5, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:6, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:6; or (iii) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:7, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:8, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:8.

[0036] 8. The bispecific antibody according to any one of embodiments 1 to 7, wherein the second antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL each comprise or consist of the amino acid sequence shown below: SEQ ID NO:3 and SEQ ID NO:4; SEQ ID NO:5 and SEQ ID NO:6; or SEQ ID NO:7 and SEQ ID NO:8.

[0037] 9. The bispecific antibody of any one of embodiments 1 to 7, wherein the first 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 The HCDR1, HCDR2 and HCDR3 are the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as 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 as shown in SEQ ID NO: 2; Preferably, the CDRs are determined using the Kabat scheme.

[0038] 10. The bispecific antibody of embodiment 9, wherein HCDR1 of the first antigen-binding region 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; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 11; and LCDR1 of the first antigen-binding region comprises or consists of the amino acid sequence of SEQ ID NO: 12; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 13; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14.

[0039] 11. The bispecific antibody of embodiment 9 or 10, wherein the first antigen-binding region comprises a heavy chain variable region VH, wherein the VH comprises or consists of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1.

[0040] 12. The bispecific antibody of any one of embodiments 9 to 11, wherein the first antigen-binding region comprises a light chain variable region, VL, wherein the VL comprises or consists of the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2.

[0041] 13. The bispecific antibody of 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 or consists of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1, and the VL comprises or consists of the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2.

[0042] 14. The bispecific antibody of any one of embodiments 9 to 13, wherein the first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL respectively comprise or consist of the amino acid sequences shown below: SEQ ID NO: 1 and SEQ ID NO: 2.

[0043] 15. The bispecific antibody of any one of embodiments 1 to 14, wherein the first antigen-binding region specifically binds to EGFR, comprising HCDR1, HCDR2, and HCDR3 of the heavy chain variable region VH, and LCDR1, LCDR2, and LCDR3 of the light chain variable region VL, wherein 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; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 11; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 12; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 13; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14; and

[0044] The second antigen binding region specifically binds to B7H3, which 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) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 15; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 16; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 17; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 18; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 19; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 20;

[0045] (ii) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 21; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 22; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 23; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 24; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 25; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 26; or

[0046] (iii) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 27; HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 28; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 29; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 30; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 31; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 32.

[0047] 16. The bispecific antibody of 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 comprises a VH and a VL comprising or consisting of the amino acid sequence shown below, respectively: SEQ ID NO:3 and SEQ ID NO:4; SEQ ID NO:5 and SEQ ID NO:6; or SEQ ID NO:7 and SEQ ID NO:8.

[0048] 17. The bispecific antibody according to any one of embodiments 1 to 16, comprising an Fc region, preferably, the Fc region has low fucosylation, such as low fucosylation obtained by treatment with GlymaxX technology.

[0049] 18. The bispecific antibody of embodiment 17, 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.

[0050] 19. The bispecific antibody of embodiment 17 or 18, wherein the first Fc region and the second Fc region are respectively human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc, e.g., comprising or consisting of the amino acid sequence of SEQ ID NO: 46 or 47 or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or higher identity thereto.

[0051] 20. The bispecific antibody of 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.

[0052] 21. The bispecific antibody of embodiment 20, wherein the mutation is introduced based on Innobody technology.

[0053] 22. The multispecific antibody of embodiment 21, wherein the CH3 of one Fc region comprises S364R and D399K mutations, and the CH3 mutations of another Fc region comprise Y349T, K370S and K409D mutations.

[0054] 23. The bispecific antibody of embodiment 22, wherein a) one Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 49 or 50, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 52 or 53; b) one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 49 or 50, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 52 or 53; or c) one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 49 or 50 and comprises the mutations Y349T, K370S, and K409D, and the other Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 52 or 53 and comprises the mutations S364R and D399K.

[0055] 24. The bispecific antibody of embodiment 20, wherein the mutation is introduced based on the Knob-into-Hole technology, wherein corresponding Knob mutations and Hole mutations are introduced into the first Fc region and the second Fc region.

[0056] 25. The bispecific antibody of embodiment 24, wherein a) one Fc region polypeptide comprises the mutation T366W and the other Fc region polypeptide comprises T366S, L368A and Y407V (numbering according to the EU index), or b) One Fc region comprises amino acid substitutions S354C and T366W, and the other Fc region comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering is according to the EU index).

[0057] 26. The bispecific antibody of any one of embodiments 1 to 25, wherein the first and / or second antigen binding region (e.g., the heavy chain variable region therein) can also be connected to one or two heavy chain constant regions (e.g., the heavy chain constant region of human IgG1, human IgG2, human IgG3 or human IgG4), which heavy chain constant region comprises CH1 and Fc region, connected via or not via a hinge region, for example, the C-terminus of the heavy chain variable region is connected to the N-terminus of CH1 of the heavy chain constant region.

[0058] 27. The bispecific antibody of embodiment 26, wherein the CH1 comprises or consists of the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 42.

[0059] 28. The bispecific antibody of any one of embodiments 1 to 27, wherein the first and / or second antigen binding region (e.g., the light chain variable region therein) can also be connected to the light chain constant region, for example, the C-terminus of the light chain variable region is connected to the N-terminus of the light chain constant region.

[0060] 29. The bispecific antibody of embodiment 28, wherein the light chain constant region is a kappa light chain constant region or a lambda light chain constant region.

[0061] 30. The bispecific antibody of embodiment 20, wherein the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54 or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 54.

[0062] 31. The bispecific antibody of any one of embodiments 1 to 30, wherein the bispecific antibody is an IgG-like antibody having the configuration shown in Figure 1.

[0063] 32. The bispecific antibody of embodiment 31, comprising 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 a heavy chain variable region of a first antigen-binding region and a first heavy chain constant region; light chain 1 comprises a light chain variable region of a first antigen-binding region and a first light chain constant region; and heavy chain 2 comprises a heavy chain variable region of a second antigen-binding region and a second heavy chain constant region; light chain 2 comprises a light chain variable region of a second antigen-binding region and a second light chain constant region.

[0064] 33. The bispecific antibody of embodiment 32, wherein heavy chain 1 comprises or consists of the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 33.

[0065] 34. The bispecific antibody of embodiment 32 or 33, wherein light chain 1 comprises or consists of the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 34.

[0066] 35. The bispecific antibody of any one of embodiments 32 to 34, wherein heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 33, and light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 34.

[0067] 36. The bispecific antibody of any one of embodiments 32 to 35, wherein heavy chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 35, 37 or 39, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 35, 37 or 39.

[0068] 37. The bispecific antibody of any one of embodiments 32 to 36, wherein light chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 36, 38 or 40, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 36, 38 or 40.

[0069] 38. The bispecific antibody of any one of embodiments 32 to 37, wherein (1) Heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:35, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:35; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO:36, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:36; (2) heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 37; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 38; (3) Heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 39; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 40.

[0070] 39. The bispecific antibody of any one of embodiments 32 to 38, wherein Heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33, and light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 34; and heavy chain 2 and light chain 2, respectively, comprise, or consist of, the amino acid sequence of SEQ ID NO: i) SEQ ID NO:35 and SEQ ID NO:36; ii) SEQ ID NO:37 and SEQ ID NO:38; iii) SEQ ID NO: 39 and SEQ ID NO: 40.

[0071] 40. The bispecific antibody of any one of embodiments 32 to 39, wherein:

[0072] (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, Heavy chain 2 comprises or consists of 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,

[0073] (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, Heavy chain 2 comprises or consists of 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,

[0074] (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.

[0075] 41. The bispecific antibody or antigen-binding fragment thereof that binds to EGFR and B7-H3 according to any one of embodiments 1 to 40, wherein the antibody or antigen-binding fragment thereof has one or more of the following characteristics: (i) On the one hand, the antibody can block the binding of EGFR ligand to EGFR, inhibiting biological signal transmission and blocking the corresponding biological activity of tumors; on the other hand, it can stimulate the endocytosis of EGFR and ultimately be degraded by intracellular lysosomes; (ii) This antibody uses the EGFR antibody parent sequence with low affinity for EGFR, which greatly reduces the toxic side effects of the series of EGFR monoclonal antibodies on normal epithelial tissues such as the skin; (iii) This antibody, while having low affinity for EGFR, incorporates the high-affinity antibody parent sequence of B7-H3, significantly enhancing its EGFR signal-blocking activity and improving the pharmacodynamic bioactivity and safety window of the bispecific antibody of the present invention; (iv) the antibody is a hypofucosylated antibody; (ii) the antibody has high pharmacodynamic bioactivity and safety; (v) The antibody has excellent tumor killing and inhibitory effects; (vi) The antibody has excellent ADCC activity in vitro and in vivo; (vii) The antibody has excellent synergistic anti-tumor effect when used in combination with KRAS small molecule inhibitors.

[0076] 42. An isolated nucleic acid encoding any one chain of the bispecific antibody that binds to EGFR and B7-H3 according to any one of embodiments 1 to 41.

[0077] 43. A vector comprising the nucleic acid of embodiment 42, preferably the vector is an expression vector, preferably the expression vector is pcDNA, such as pcDNA3.1.

[0078] 44. A host cell comprising the nucleic acid of embodiment 42 or the vector of embodiment 43. Preferably, the host cell is prokaryotic or eukaryotic, more preferably a yeast cell or a mammalian cell (e.g., a 293 cell or a CHO cell, such as a 293F cell, a 293T cell, or a CHO-S cell).

[0079] 45. The host cell of embodiment 44, which is glycoengineered to express RMD enzyme, preferably, the host cell is a CHO cell.

[0080] 46. ​​The host cell of embodiment 45, comprising a nucleic acid encoding an RMD enzyme.

[0081] 47. The host cell of embodiment 46, wherein 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% identity thereto, preferably, the RMD enzyme is from Pseudomonas aeruginosa.

[0082] 48. A method for preparing a bispecific antibody that binds to EGFR and B7-H3, the method comprising culturing the host cell of any one of embodiments 44 to 47 under conditions suitable for expressing a nucleic acid encoding the bispecific antibody of any one of embodiments 1 to 41, isolating the antibody or its antigen-binding fragment as needed, and optionally recovering the antibody or its antigen-binding fragment from the host cell (or host cell culture medium).

[0083] 49. An immunoconjugate comprising the bispecific antibody of any one of the preceding embodiments 1 to 41 conjugated to a therapeutic or diagnostic agent.

[0084] 50. A pharmaceutical composition comprising the bispecific antibody of any one of embodiments 1 to 41 or the immunoconjugate of embodiment 49, and optionally a pharmaceutical excipient.

[0085] 51. The pharmaceutical composition of embodiment 50 further comprises a second therapeutic agent; preferably, the second therapeutic agent is selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective active agents, small molecule drugs or immunomodulators (such as activators of co-stimulatory molecules or inhibitors of immune checkpoint molecules); preferably, the second therapeutic agent is selected from KRAS small molecule inhibitors, such as KRAS G12C inhibitors (such as AMG510 (Sotorasib) or GFH925), KRAS G12D (such as MRTX1133) or KRAS G12S inhibitors.

[0086] 52. A pharmaceutical combination product comprising the bispecific antibody of any one of embodiments 1 to 41, the immunoconjugate of embodiment 49, or the pharmaceutical composition of embodiment 50, and one or more second therapeutic agents, preferably, the second therapeutic agent is selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, small molecule drugs or immunomodulators (such as activators of costimulatory molecules or inhibitors of immune checkpoint molecules); preferably, the second therapeutic agent is selected from KRAS small molecule inhibitors, such as KRAS G12C inhibitors (such as AMG510 (Sotorasib) or GFH925), KRAS G12D (such as MRTX1133) or KRAS G12S inhibitors.

[0087] 53. A method for preventing or treating a tumor or infectious disease in a subject, the method comprising administering to the subject an effective amount of the bispecific antibody of any one of embodiments 1 to 41, or the immunoconjugate of embodiment 49, or the pharmaceutical composition of embodiment 50.

[0088] 54. The method described in embodiment 53 further comprises administering one or more other therapies to the subject in combination, the therapies comprising, for example, treatment modalities and / or other therapeutic agents. Preferably, the treatment modalities comprise surgical treatment and / or radiotherapy, or the therapeutic agents are selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, small molecule drugs or immunomodulators (such as activators of co-stimulatory molecules or inhibitors of immune checkpoint molecules); preferably, the second therapeutic agent is selected from KRAS small molecule inhibitors, such as KRAS G12C inhibitors (such as AMG510 (Sotorasib) or GFH925), KRAS G12D (such as MRTX1133) or KRAS G12S inhibitors.

[0089] 55. A method for preventing or treating a tumor or infectious disease in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 51 or the pharmaceutical combination product of embodiment 52.

[0090] 56. The method of any one of embodiments 53 to 55, wherein the tumor is a cancer, such as a solid tumor or a blood tumor, including cancers of epithelial origin, such as gastrointestinal tumors or lung tumors or skin tumors, such as skin cancer (e.g., squamous cell carcinoma of the skin, head and neck cancer 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, squamous cell lung carcinoma, adenocarcinoma of the lung).

[0091] 57. The method of any one of embodiments 53 to 56, wherein the tumor cells of the tumor (i) overexpressing wild-type EGFR (e.g., wild-type EGFR with elevated nucleic acid or protein levels) and / or expressing mutated EGFR compared to normal cells in adjacent tissues or normal cells in the same tissue in healthy subjects, 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) overexpressing wild-type KRAS (e.g., having elevated nucleic acid or protein levels of wild-type KRAS) compared to normal cells in adjacent tissues or compared to normal cells in the same tissue in healthy subjects, or expressing mutated KRAS, preferably, the mutated KRAS comprises a G12 or G13 mutation, such as G12D or G12C; (iii) having elevated nucleic acid or protein levels of B7-H3 compared to normal cells in adjacent tissue or compared to normal cells in the same tissue in healthy subjects; and / or (iv) The tumor cells are resistant to tyrosine kinase inhibitors, such as first-generation (erlotinib) and third-generation (osimertinib), for example, resistant to osimertinib.

[0092] 58. The method of embodiment 57, wherein the tumor cells express mutant EGFR and mutant KRAS, such as EGFR with a mutation of R521K and KRAS with a mutation of G120D.

[0093] 59. A method for detecting antigen EGFR and / or B7-H3 in a sample, the method comprising (a) contacting a sample with the bispecific antibody of any one of embodiments 1 to 41; and (b) detecting the formation of a complex between the antibody or antigen-binding fragment thereof and EGFR and / or B7-H3, wherein the antibody is detectably labeled.

[0094] 60. Use of the antibody or antigen-binding fragment thereof of any one of embodiments 1 to 41, and / or the isolated nucleic acid of embodiment 42, and / or the vector of embodiment 43, and / or the host cell of any one of embodiments 44 to 47, and / or the immunoconjugate of embodiment 49, and / or the pharmaceutical composition of embodiment 50 or 51 or the pharmaceutical combination product of embodiment 52 in the preparation of a medicament for preventing and / or treating a disease in a subject.

[0095] The present invention also encompasses any combination of any of the embodiments described herein. Any of the embodiments described herein or any combination thereof is applicable to any and all antibodies or fragments thereof or immunoconjugates or pharmaceutical compositions or combination products or kits, methods and uses of the invention described herein. Simple diagram description

[0096] 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 illustrating the present invention, the drawings show the presently preferred embodiments. However, it should be understood that the present invention is not limited to the embodiments shown in the drawings.

[0097] FIG1 shows a schematic structural diagram of the anti-B7-H3 / EGFR bispecific antibody of the present invention.

[0098] FIG2 shows a graph of a proliferation inhibition experiment for screening the anti-B7-H3 / EGFR bispecific antibody of the present invention.

[0099] FIG3 shows a screen ADCC experiment diagram of the anti-B7-H3 / EGFR bispecific antibody of the present invention.

[0100] FIG4 shows the expression detection diagram of B7H3 and EGFR of Hz20G5.26 / Zalu bsAb of the present invention in different cell lines.

[0101] FIG5 shows the experimental results of the Hz20G5.26 / Zalu bsAb of the present invention for inhibiting proliferation in CRC tumor cell lines CCK-81, NCI-H508, HT-55 and LS180, wherein the antibody dilutions are as follows: CCK-81, NCI-H508, HT-55: The maximum final concentration of the antibody is 125 nM, 4-fold dilution; LS180: The maximum final concentration of the antibody dilution is 300 nM, 4-fold dilution.

[0102] Figure 6 shows the experimental results of the Hz20G5.26 / Zalu bsAb of the present invention inhibiting proliferation in HNSCC tumor cell lines TE-1 and Colo680, wherein the antibody dilutions are as follows: TE-1, Colo680: the highest final antibody dilution concentration is 300 nM, 3.16-fold dilution.

[0103] Figure 7.1 shows the experimental results of the Hz20G5.26 / Zalu bsAb of the present invention inhibiting proliferation in different NSCLC-EGFRWT tumor cell lines;

[0104] Figure 7.2 shows the experimental results of the Hz20G5.26 / Zalu bsAb of the present invention inhibiting proliferation in NSCLC-EGFR classic mutation tumor cell lines;

[0105] Figure 7.3 shows the experimental results of the Hz20G5.26 / Zalu bsAb of the present invention inhibiting proliferation in NSCLC-EGFR abnormally amplified tumor cell lines;

[0106] Figure 7.4 shows the experimental results of the Hz20G5.26 / Zalu bsAb of the present invention inhibiting proliferation in rare NSCLC-EGFR mutant tumor cell lines;

[0107] Figure 7.5 shows the results of a comparative experiment on the efficacy of Hz20G5.26 / Zalu bsAb of the present invention in combination with gp120 / Zalu and gp120 / hz20G5.26; The antibody or small molecule TKI (Osimertinim or Erlotinib) was diluted as follows: Figure 7.1: NCI-292, NCI-H322: The highest final antibody concentration was 300 nM, 3.16-fold dilution; Figure 7.2: NCI-H1650, NCI-H1975: The highest final antibody concentration was 300 nM, 3.16-fold dilution; Figure 7.3: NCI-H1703, SK-MES-1: Antibody molecules, the highest final concentration was 300 nM, 3.16-fold dilution; small molecule TKIs (osimertinim and erlotinib), the highest final concentration was 1000 nM, 3.16-fold dilution; Figure 7.4: H1975 (EGFRL858R / T790M / C797S): The highest final concentration of various antibody molecules and osimertinib was 300 nM, 3.16-fold dilution; H322 (EGFR S768-D77dup): The highest final concentration of various antibody molecules and osimertinib was 300 nM, 4-fold dilution; PC9+B7H3 (EGFRDel19 / T790M / C79S): The highest final concentration of various antibody molecules and osimertinib was 100 nM, 4-fold dilution; Figure 7.5: LS180 and H292: The highest final antibody concentration was 300 nM, with a 4-fold dilution; SK-MES-1: The highest final concentration was 300 nM, with a 3.16-fold dilution.

[0108] FIG8 shows the comparative experimental results of the synergistic combination of Hz20G5.26 / Zalu bsAb of the present invention and AMG510 small molecule inhibitor in H358 NSCLC cell line, wherein Figure 8 (A): H358 in vitro proliferation inhibition assay: The final concentration of each antibody molecule was 300 nM, with a 4-fold dilution; Figure 8(B): Combination study of Hz20G5.26 / Zalu bsAb and KRAS G12C inhibitor AMG510 (MedChem Expresses, HY-114277): AMG510+bsAb@10nm means that a fixed 10nM Hz20G5.26 / Zalu bsAb was added to each well, and AMG510 was gradiently diluted in each well (final concentration, 1000nM, 4-fold dilution); AMG510 only: maximum final concentration 1000nM, 4-fold dilution; Hz20G5.26 / Zalu bsAb only: maximum final concentration 10nM, 5-fold dilution; a total of 5 dilution gradients.

[0109] Figure 9 shows the results of a comparative experiment of the synergistic combination of Hz20G5.26 / Zalu bsAb of the present invention and the KRAS G12D small molecule inhibitor MRTX1133 in the LS180 CRC cell line, wherein Figure 9 (A): Combination drug experiment of Hz20G5.26 / Zalu bsAb and G12D inhibitor MRTX1133 (MCE, cat:HY-134813A-10mg): MRTX1133+bsAb@100nm means that a fixed 100nM Hz20G5.26 / Zalu bsAb was added to each well, and MRTX1133 was gradiently diluted in each well (final concentration, 1000nM, 4-fold dilution); MRTX1133 only: maximum final concentration 1000nM, 4-fold dilution; Hz20G5.26 / Zalu bsAb only: maximum final concentration 100nM, 4-fold dilution; a total of 6 dilution gradients.

[0110] FIG10 shows the experimental results of the Hz20G5.26 / Zalu bsAb of the present invention for blocking EGFR phosphorylation signals in the H358 cell line.

[0111] FIG11 shows the experimental results of the Hz20G5.26 / Zalu bsAb of the present invention blocking the EGFR phosphorylation signal induced by EGFR ligands (EGF or TGF-α) in the H358 cell line.

[0112] Figure 12.1 shows the ADCC reporter assay results of the Hz20G5.26 / Zalu bsAb of the present invention in NSCLC-EGFRWT tumor cell lines; in the three cell lines, the highest final concentration of the antibody molecule was 6.25 nM, with a 4-fold dilution;

[0113] Figure 12.2 shows the ADCC reporter assay results of the Hz20G5.26 / Zalu bsAb of the present invention in NSCLC-EGFR mutant tumor cell lines. In the H1975 cell line, the maximum final concentration of the antibody molecule was 10 nM with a 4-fold dilution; and in the H1975 cell line, the maximum final concentration of the antibody molecule was 6.25 nM with a 4-fold dilution.

[0114] Figure 12.3 shows the ADCC reporter assay results of the Hz20G5.26 / Zalu bsAb of the present invention in NSCLC-EGFR amplified tumor cell lines. In the SK-MES-1 cell line, the maximum final concentration of the antibody molecule was 6.25 nM after 4-fold dilution; and in the H1703 cell line, the maximum final concentration of the antibody molecule was 100 nM after 4-fold dilution.

[0115] Figure 12.4 shows the results of an ADCC reporter assay of the Hz20G5.26 / Zalu bsAb of the present invention in an NSCLC-KRAS mutant tumor cell line. In the H358 cell line, the maximum final concentration of the antibody molecule was 25 nM with a 4-fold dilution ( Figure 12.4(A) ); and in the H358 cell line, the maximum final concentration of the antibody molecule was 1.56 nM with a 4-fold dilution ( Figure 12.4(B) );

[0116] Figure 13.1 shows the results of the huPBMCADCC reporter experiment of the Hz20G5.26 / Zalu bsAb of the present invention in the NSCLC-EGFR wild-type tumor cell line; wherein, Figure 13.1(A), in the H292 cell line, the highest final concentration of the antibody molecule was 2.5 nM, with 4-fold dilution; Figure 13.1(B): In the H322 cell line, the maximum final concentration of the antibody molecule was 100 nM, with a 4-fold dilution; Figure 13.1(C): In the H292 cell line, the maximum final concentration of the antibody molecule was 100 nM, with a 4-fold dilution;

[0117] Figure 13.2 shows the results of a huPBMC ADCC reporter assay using the Hz20G5.26 / Zalu bsAb of the present invention in NSCLC-EGFR mutant tumor cell lines. In the H1975 cell line, the maximum final concentration of the antibody molecule was 35 nM with a 4-fold dilution; in the H1650 cell line, the maximum final concentration of the antibody molecule was 100 nM with a 4-fold dilution.

[0118] Figure 13.3 shows the results of the huPBMC ADCC reporter experiment of the Hz20G5.26 / Zalu bsAb of the present invention in NSCLC-EGFR abnormally amplified tumor cell lines; Figure 13.3(A), in the SK-MES-1 cell line, the highest final concentration of the antibody molecule was 35 nM, with a 4-fold dilution; Figure 13.3(B): In the H1703 cell line, the highest final concentration of the antibody molecule was 25 nM, with a 4-fold dilution; Figure 13.3(C): In the SK-MES-1 cell line, the maximum final concentration of the antibody molecule was 25 nM, with a 4-fold dilution;

[0119] Figure 13.4 shows the results of the huPBMC ADCC reporter experiment of the Hz20G5.26 / Zalu bsAb of the present invention in the NSCLC-KRAS mutant tumor cell line, wherein in the two cell lines, the highest final concentration of the antibody molecule dilution was 35nM, and the dilution was 4 times.

[0120] Figure 14 shows the results of an in vitro study of the simulated skin toxicity of the Hz20G5.26 / Zalu bsAb drug of the present invention, in which the antibody molecules were diluted in cell culture medium and the final concentration of the antibody molecules was up to 300 nM in two human skin cell lines, a 3.16-fold dilution.

[0121] Figure 15A shows the experimental results of the effect of Hz20G5.26 / Zalu bsAb of the present invention on the anti-tumor effect of NCI-H292 tumor-bearing mice; Figure 15B shows the experimental results of the effect of Hz20G5.26 / Zalu bsAb of the present invention on the anti-tumor effect of NCI-H292 tumor-bearing mice - survival curve; Figure 15C shows the experimental results of the effect of Hz20G5.26 / Zalu bsAb of the present invention on the body weight of NCI-H292 tumor-bearing mice.

[0122] Figure 16A shows the experimental results of the effect of Hz20G5.26 / Zalu bsAb of the present invention on the anti-tumor effect of SK-MES-1 tumor-bearing mice; Figure 16B shows the experimental results of the effect of Hz20G5.26 / Zalu bsAb of the present invention on the body weight of SK-MES-1 tumor-bearing mice.

[0123] Figure 17A shows the experimental results of the effect of Hz20G5.26 / Zalu bsAb of the present invention on the anti-tumor effect of SK-MES-1 tumor-bearing mice; Figure 17B shows the experimental results of the effect of Hz20G5.26 / Zalu bsAb of the present invention on the body weight of SK-MES-1 tumor-bearing mice.

[0124] Figure 18A shows the experimental results of the effect of the combination of Hz20G5.26 / Zalu bsAb of the present invention and AMG510 on the anti-tumor effect of NCI-H358 tumor-bearing mice; Figure 18B shows the experimental results of the effect of the combination of Hz20G5.26 / Zalu bsAb of the present invention and AMG510 on the body weight of NCI-H358 tumor-bearing mice. Implementation Method [Detailed description of the invention]

[0125] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the present invention will become apparent from the present specification and drawings, as well as from the appended claims. [I. Definition]

[0126] It should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present invention, which will be limited solely by the appended 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 to which this invention belongs.

[0127] For the purpose of interpreting this specification, the following definitions will apply, and terms used in the singular may also include the plural, and vice versa, whenever appropriate. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0128] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0129] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0130] As used herein, the terms "comprising" or "including" mean including the elements, integers, or steps specified, but not excluding any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, they also encompass situations consisting of the stated elements, integers, or steps. For example, when referring to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0131] When "first" and "second" are mentioned herein, it is only to distinguish the two domains or two chains, and does not indicate the positions of the two domains in any way.

[0132] As used herein, the amino acid positions of all variable regions of heavy and light chains are numbered according to the Kabat numbering system described in Kabat 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 "Kabat numbers."

[0133] As used herein, when referring to amino acid positions in antibody domains other than the variable region (e.g., constant region, e.g., Fc region), the positions are numbered according to the EU numbering system described in Kabat, EA 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 numbering." When position numbers and / or amino acid residues are assigned to a particular antibody isotype, it is intended to apply to the corresponding positions and / or amino acid residues of any other antibody isotype, as known to those of ordinary skill in the art.

[0134] General information regarding the nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0135] The term "antibody" is used herein in the broadest sense to refer to a protein that contains an antigen binding site and encompasses 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, intact antibodies, and antibody fragments.

[0136] The terms "whole antibody," "full-length antibody," "complete antibody," and "intact antibody" are used interchangeably herein to refer to naturally occurring glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions, known as complementarity-determining regions (CDRs), interspersed with more conserved regions, known as framework regions (FRs). Each VH and VL region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region is not directly involved in binding the antibody to the antigen but exhibits various effector functions.

[0137] "Half-antibody" or "hemimer" refers to a monovalent antigen-binding polypeptide. In some embodiments, a half-antibody or hemimer comprises a VH / VL unit and, optionally, at least a portion of an immunoglobulin constant domain. In some embodiments, a half-antibody or hemimer comprises a single immunoglobulin heavy chain, or an antigen-binding fragment thereof, associated with a single immunoglobulin light chain. In some embodiments, a half-antibody or hemimer is monospecific, i.e., binds to a single antigen or epitope. In some specific embodiments, a half-antibody binds to EGFR and does not bind to B7-H3. In some specific embodiments, a half-antibody binds to B7-H3 and does not bind to EGFR. A person of ordinary skill in the art will readily appreciate that a half-antibody can have an antigen-binding domain composed of a single variable domain (e.g., derived from a Camelidae species).

[0138] The term "antigen-binding fragment" of an antibody refers to a molecule, distinct from a full-length antibody, that comprises a portion of a full-length antibody but is capable of binding to the full-length antibody's antigen or competing for antigen binding with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived). Antigen-binding fragments can be prepared using recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabodies, single-domain antibodies (sdAbs), and nanobodies.

[0139] "Fab fragment" or "Fab" are used interchangeably herein to refer to an immunoglobulin fragment composed of two polypeptide chains comprising an immunoglobulin heavy chain variable region (VH), a heavy chain constant domain (CH1), a light chain variable region (VL), and a light chain constant domain (CL). One polypeptide chain comprises, from N-terminus to C-terminus, VH and a constant region selected from CH1 and CL; the other polypeptide chain comprises, from N-terminus to C-terminus, VL and another constant region selected from CL and CH1. The VH and VL domains pair to form an antigen-binding site. Fab' fragments differ from Fab fragments by the addition of residues to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation for Fab' fragments in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0140] As used herein, the term "bispecific antibody" comprises an antigen-binding domain that specifically binds to two antigens or two epitopes. Unless otherwise specified, the order of the antigens bound by the bispecific antibody in the listed bispecific antibody names 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 comprises two half antibodies, each of which comprises a single heavy chain variable region and, optionally, at least a portion of a heavy chain constant region, and a single light chain variable region and, optionally, at least a portion of a light chain constant region. In some embodiments, the bispecific antibody comprises two half antibodies, each of which comprises a single heavy chain variable region and a single light chain variable region, and does not comprise more than one single heavy chain variable region and does not comprise more than one single light chain variable region. In some embodiments, the bispecific antibody comprises two half antibodies, wherein each half antibody comprises a single heavy chain variable region and a single light chain variable region, and wherein the first half antibody binds to a first antigen / epitope and does not bind to a second antigen and the second half antibody binds to the second antigen / epitope and does not bind to the first antigen.

[0141] The multispecific antibodies of the present invention may include a linker. As used herein, the term "linker" refers to any molecule that enables direct connection of the different parts of a multispecific antibody. Examples of linkers that establish a covalent connection between the different parts of a multispecific antibody include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, 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 connects the amino acid sequences of the various parts of the multispecific antibody. Preferably, the peptide linker is of a length sufficient to connect the two entities in a manner that allows them to maintain their conformation relative to each other so as not to interfere with the desired activity. The peptide linker may or may not primarily consist of the following amino acid residues: Gly, Ser, Ala, or Thr. Useful linkers include glycine-serine polymers, including, for example, (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, 10). Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.

[0142] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely, a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in a native antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 and CH3 domains) derived from two heavy chains of antibodies of the IgG, IgA, and IgD classes; or the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) derived from two heavy chains of antibodies of the IgM and IgE classes. Unless otherwise specified herein, the amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Two Fc regions can dimerize to form a dimeric Fc, and two different Fc regions heterodimerize to form a heterodimeric Fc. Herein, the terms "Fc region," "Fc portion," and "dimeric Fc (e.g., heterodimeric Fc)" do not include the heavy chain variable region VH and light chain variable region VL of an immunoglobulin, as well as the heavy chain constant region CH1 and light chain constant region CL, but may include the hinge region at the N-terminus of the heavy chain constant region in some cases. In one embodiment, the human IgG heavy chain Fc region extends from Asp221, or from Cys226, or from 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 is of the human IgG4 subclass. In one embodiment, the Fc region is of the human IgG1 subclass.

[0143] 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. Heterodimerized Fc regions constitute the Fc scaffold in bispecific or multispecific antibodies. Therefore, a "heterodimer Fc scaffold" refers to a scaffold comprising or formed by the dimerization of two different Fc regions, which can be linked to an antigen-binding domain (e.g., an antibody heavy and / or light chain variable region or an antibody antigen-binding fragment that can bind to a target molecule, or a soluble portion of a ligand or receptor that can bind to a target molecule) at its N-terminus or C-terminus to form a multispecific antibody, such as a bispecific antibody.

[0144] Amino acid mutations are indicated using (original amino acid, amino acid position, mutated amino acid). For example, when the mutation site is located in the Fc region, "T366W" means that the T at position 366 in the EU numbering is replaced by W. When describing a combination of mutations, the mutations in the combination are separated by "and" or " / ". "R521K / Y1069C" indicates that both the R521K and Y1069C mutations are included. It should be noted that when describing a mutation, the specific position also includes the corresponding amino acid positions in other polypeptide chains. For example, reference to C220 encompasses amino acid position 220 in the IgG1 heavy chain by EU numbering, as well as the corresponding amino acid in other heavy chains, such as amino acid position 131 in IgG2, IgG3, or IgG4. When describing a mutation, the original amino acid at a specific position may be the amino acid being described, or it may be another amino acid at the corresponding position.

[0145] "Complementarity determining regions" or "CDR regions" or "CDRs" are regions of an antibody variable domain that are highly variable in sequence and form structurally defined loops ("hypervariable loops") and / or contain antigen-contacting residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The CDRs located within the variable region of the antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable region of the antibody light chain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, 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 antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0146] For example, according to different CDR definition schemes, the residues of each CDR are as follows.

[0147] A CDR can also be identified based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).

[0148] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

[0149] Unless otherwise indicated, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), the numbering refers to the position according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0150] In one embodiment, the CDRs of an antibody of the invention have boundaries defined by Kabat's rules, or by AbM's rules, or by a combination thereof.

[0151] In one embodiment of the present invention, the HCDR1 of VH in the antigen-binding region that binds to B7-H3 of the present invention is determined by the AbM rule, HCDR2 and HCDR3 are determined by the Kabat rule, and the CDR of VL is determined by the Kabat rule.

[0152] In one embodiment of the present invention, the CDRs of VH and VL in the antigen-binding region that binds to EGFR of the present invention are determined by the Kabat rule.

[0153] The term "hinge region" refers to the portion of an antibody heavy chain polypeptide that connects the CH1 and CH2 regions in a wild-type antibody heavy chain, for example, the IgG1 hinge region, such as the sequence from D221 to P230 according to EU numbering. Hinge regions of other IgG subclasses can be identified by aligning the hinge region cysteine ​​residues with those of the IgG1 subclass sequence.

[0154] As used herein, the term "binding site" or "antigen binding site" or "antigen binding region" refers to any portion of an antibody molecule, such as a multispecific antibody, such as a bispecific antibody, that binds to a specific 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 rest of the BsAB and may or may not bind its antigen / epitope as a separate 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 of an IgNAR from a shark (v-NAR), a camelized human VH domain, or a humanized camelid antibody heavy chain variable region.

[0155] When referring to a "first antigen-binding region" in a multispecific antibody or bispecific antibody, it refers to the region that binds to the first antigen, and is not intended to limit the number of such antigen-binding regions contained in the antibody. For example, a multispecific antibody or bispecific antibody may contain one or more first antigen-binding regions. For example, a bispecific antibody contains a first antigen-binding region and a second antigen-binding region, but may also contain one or more first antigen-binding regions and one or more second antigen-binding regions.

[0156] When referring to an "antigen-binding region derived from an antibody," it means that the binding domain constituting the antigen-binding region is or is derived from the binding domain of the antibody that specifically binds to the antigen. For example, the antigen-binding fragment of the antigen-binding region, such as Fab, is or is derived from the corresponding fragment, such as Fab, of the antibody, or the heavy chain variable region and / or light chain variable region of the antigen-binding region is or is derived from the heavy chain variable region and / or light chain variable region of the antibody, or one, two, three, four, five, or six CDRs of the antigen-binding region are CDRs of the antibody. In some embodiments, the antigen-binding region is an antigen-binding fragment of an antibody.

[0157] As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. The antibodies provided herein are generally multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are antibodies that have binding specificities for at least two different antigenic epitopes. In one embodiment, provided herein are bispecific antibodies that have binding specificities for a first antigen and a second antigen.

[0158] The term "immunoglobulin molecule" refers to a protein with the structure of a naturally occurring antibody. For example, IgG-class immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 daltons composed of two disulfide-bonded light chains and two heavy chains. From N-terminus to C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3). Similarly, from N-terminus to C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, followed by a light chain constant domain (CL). Immunoglobulin heavy chains can be assigned to one of five classes, designated α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM). Some classes are further divided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains can be assigned to one of two types, designated κ and λ, based on the amino acid sequence of their constant domains. Immunoglobulins are essentially composed of two Fab molecules and an Fc domain connected by the immunoglobulin hinge region.

[0159] The term "effector function" refers to those biological activities attributable to the Fc region of an immunoglobulin that vary with 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 cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0160] The term "...valent" antibody refers to the number of antigen binding sites present in the antibody molecule. "Bivalent," "trivalent," and "tetravalent" antibodies refer to antibodies with 2, 3, and 4 antigen binding sites, respectively.

[0161] As used herein, the term "binding" or "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as radioimmunoassay (RIA) or thin-layer interferometry or MSD assay or surface plasmon resonance (SPR).

[0162] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (Kdis and Kon, 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.

[0163] "Knob-in-hole" mutations, or "knob-in-knob" mutations, are used herein to refer to the introduction of mutations into a first Fc polypeptide and a second Fc polypeptide, respectively, using the "knob-in-knob" technique to create a protrusion ("knob") and a complementary cavity ("hole") at the interface of the first Fc polypeptide and at the interface of the second Fc polypeptide. The "knob-in-knob" technique is known in the art to modify the interface between different chains of an antibody molecule to promote proper association of the antibody chains. Generally, this technique involves introducing a "knob / knob" at the interface of one chain and a corresponding "hole / knob" at the interface of the other intended pairing chain, such that the protrusion fits within the cavity. A preferred interface comprises the CH3 domain of the heavy chain constant domain of one chain and the CH3 domain of the heavy chain constant domain of the other intended pairing chain. The protrusion can be constructed by replacing small amino acid side chains from the interface of the CH3 domains of the heavy chain constant domains of one chain with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory cavity of the same or similar size as the knob is constructed at the interface of the CH3 domain of the heavy chain constant domain of the other chain to be paired. Alternatively, the aforementioned interface, comprising the light chain CL domain and the heavy chain CH1 domain of the Fab fragment, promotes proper heterodimerization between the two chains of the Fab fragment by establishing knob-cavity interactions.

[0164] As used herein, antibody constant regions or antibody constant domains, including the CH1, CL, and Fc domains, as well as the CH2, CH3, and optional CH4 domains that comprise the Fc domain, can be selected based on the intended function of the antibody molecule. For example, the constant region can be an IgA, IgD, IgE, IgG, or IgM region, particularly an immunoglobulin constant domain of human IgG, such as a constant domain of human IgG1, IgG2, IgG3, or IgG4, preferably a constant domain of human IgG1. For another example, an antibody Fab fragment can comprise the CH and CL constant regions from IgG1. For another example, an antibody Fc region can comprise the CH2 and CH3 domains from IgG1. Immunoglobulin constant regions can have native sequences or variant sequences.

[0165] The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. Those skilled in the art will appreciate that any macromolecule, including essentially any protein or peptide, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. In some embodiments herein, the first and second antigens are two different antigens.

[0166] The terms "tumor-associated antigen" or "cancer antigen" interchangeably refer to molecules (usually proteins, carbohydrates, or lipids) that are preferentially expressed, either in their entirety or as fragments (e.g., MHC / peptides), on the surface of cancer cells compared to normal cells, and which can be used in the preferential targeting of pharmaceutical agents to cancer cells.

[0167] The term "immune checkpoint molecules" refers to a class of inhibitory signaling molecules present in the immune system that prevent tissue damage by regulating the persistence and intensity of immune responses in peripheral tissues and participate in maintaining tolerance to self-antigens.

[0168] The term "cytokine" is a general term for proteins released by one cell population that act as intercellular mediators on another cell.

[0169] An "immunoconjugate" is an antibody conjugated to one or more other substances, including but not limited to a cytotoxic agent or a label.

[0170] As used in this article, the term "EGFR" refers to epidermal growth factor receptor, a tyrosine kinase receptor, a large transmembrane glycoprotein with a molecular weight of approximately 170KDa. It is a member of the ErbB receptor family and is the most common oncogenic driver gene in NSCLC. The EGFR activating mutation region mainly occurs in the EGFR exon 18-21 tyrosine kinase domain. The binding region of EGFR antibodies is mainly located in the EGFR extracellular ligand domain, which can avoid the occurrence of drug-resistant mutations. At the same time, EGFR antibodies can inhibit the growth of tumor cells by inhibiting the binding of EGFR to ligands, and can also use their own specific ADCC (antibody-dependent cell-mediated cytotoxicity) to kill tumors together with immune cells. In this way, they can jointly exert anti-tumor killing effects through multiple mechanisms of action.

[0171] The terms "B7-H3," "B7H3," and "CD276" are used interchangeably herein. B7-H3 is a type I transmembrane glycoprotein belonging to the B7 / CD28 superfamily, and shares sequence similarity with the extracellular domain of PD-L1. B7-H3 consists of 316 amino acids, including a putative 28-amino acid signal peptide, a 217-amino acid extracellular region, a transmembrane region, and a 45-amino acid cytoplasmic domain, with a molecular weight of approximately 45-66 kDa. In humans, due to exon duplication, the extracellular structure of B7-H3 can be either 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 shares approximately 90% homology with its human counterpart.

[0172] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0173] The term "GlymaxX technology" refers to a technology platform developed by ProBioGen to increase the ADCC effect of bispecific antibodies. It is a CHO host cell that stably overexpresses bacterial RMD protein. By interfering with the formation of substrate GDP-fucose, it ultimately blocks the fucose modification of the Fc region, thereby enhancing the ADCC effect (WO2011035884A1).

[0174] The term "effective amount" refers to that amount or dosage of an antibody, fragment, conjugate, or composition of the invention which, when administered to a patient in single or multiple doses, produces the desired effect in a patient in need of treatment or prevention. An effective amount can be readily determined by the attending physician, who is a person of ordinary skill in the art, by considering a variety of factors, such as the species of mammal; its size, age, and general health; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific antibody to be administered; the mode of administration; the bioavailability characteristics of the administered formulation; the dosing regimen selected; and the use of any concomitant therapy.

[0175] A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome, at the required dosage and for the required period of time. A therapeutically effective amount of an antibody or antibody fragment, or conjugate or composition thereof, can vary depending on a variety of factors, such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or deleterious effects of the antibody or antibody fragment, or conjugate or composition thereof, are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80% relative to an untreated subject. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in animal model systems predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the compound's ability to inhibit in vitro by assays known to those skilled in the art.

[0176] A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result, at the dosages and for the period of time necessary. Generally, because a prophylactic dose is used in a subject prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0177] The term "variable region" or "variable domain" refers to the domain of an antibody's heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain can be sufficient to confer antigen-binding specificity. Furthermore, VH or VL domains from antibodies that bind to a specific antigen can be used to isolate antibodies that bind to that antigen, allowing for screening of libraries of complementary VL or VH domains, respectively.

[0178] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny screened or selected for the same function or biological activity as the initially transformed cell are included herein. Host cells are any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells, and prokaryotic cells, such as E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0179] The term "anti-tumor effect" refers to a biological effect that can be demonstrated by various means, including but not limited to, for example, a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.

[0180] The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid and liquid tumors.

[0181] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include cancers of epithelial origin, such as skin cancer (e.g., squamous cell carcinoma of the skin, head and neck cancer, 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, squamous cell lung carcinoma, adenocarcinoma of the lung), including metastatic forms of those cancers.

[0182] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" as used herein are not mutually exclusive.

[0183] The term "infectious disease" refers to a disease caused by a pathogen, including, for example, viral infection, bacterial infection, fungal infection, or protozoan infection such as a parasite.

[0184] The term "chronic infection" refers to an infection in which the infectious agent (e.g., a pathogen such as a virus, bacteria, protozoa such as a parasite, fungus, or the like) has induced an immune response in the infected host but has not been cleared or eliminated from the host as during an acute infection.

[0185] As used herein, the term "label" refers to a compound or composition that is conjugated or fused, directly or indirectly, to a reagent (such as a polynucleotide probe or antibody) and facilitates detection of the reagent to which it is conjugated or fused. The label can itself be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of enzymatic labeling, can catalyze a detectable chemical alteration of a substrate compound or composition. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of a probe or antibody by reaction with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin.

[0186] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0187] An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0188] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0189] Carry out the calculating of sequence identity between sequence as follows.For determining the identity percentage of two amino acid sequences or two nucleotide sequences, this sequence is compared for the best comparison purpose (for example, can introduce room or can abandon non-homologous sequence for comparison purpose in one or both of the first and second amino acid sequences or nucleotide sequences for the best comparison). In a preferred embodiment, for comparison purpose, the length of the compared reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80%, 90%, 100% reference sequence length.Compare subsequently at the amino acid residue or the nucleotide at corresponding amino acid position or nucleotide position.When the position in the first sequence was occupied by the identical amino acid residue or the nucleotide at corresponding position in the second sequence, then this molecule was identical at this position.

[0190] Mathematical algorithms can be utilized to realize the sequence comparison between two sequences and the calculation of percent identity. In a preferred embodiment, use the Needlema and Wunsch ((1970) J.Mol.Biol.48:444-453) algorithm (available at http: / / www.gcg.com) that has been integrated into the GAP program of the GCG software package, use Blossum 62 matrix or PAM250 matrix and gap weight 16,14,12,10,8,6 or 4 and length weight 1,2,3,4,5 or 6, determine the percent identity between two amino acid sequences. In another preferred embodiment, use the GAP program (available at http: / / www.gcg.com) in the GCG software package, use NWSgapdna.CMP matrix and gap weight 40,50,60,70 or 80 and length weight 1,2,3,4,5 or 6, determine the percent identity between two nucleotide sequences. The parameter set that is particularly good (and the parameter set that should be used unless otherwise specified) is the Blossum 62 scoring matrix that adopts gap penalty 12, gap extension penalty 4 and frameshift gap penalty 5.Can also use PAM120 weighted remainder table, gap length penalty 12, gap penalty 4), utilize E.Meyers and W.Miller algorithm ((1989) CABIOS, 4:11-17) that has been incorporated into ALIGN program (version 2.0) to determine the identity percentage between two amino acid sequences or nucleotide sequences.Additionally or alternatively, can further use nucleotide sequence and protein sequence as described herein as " query sequence " to perform search for public database, to for example identify other family member sequences or related sequences.

[0191] The term "pharmaceutical composition" refers to a composition that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition is administered.

[0192] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), carrier, excipient, stabilizer, or the like, which is administered together with the active substance.

[0193] As used herein, "treat" refers to slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desirable therapeutic effects include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some embodiments, the antibody molecules of the invention are used to delay disease development or to slow the progression of a disease.

[0194] As used herein, "prevention" includes the inhibition of the development or progression of a disease or condition, or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before signs or symptoms of cancer develop, particularly in a subject at risk for cancer.

[0195] The term "effective amount" refers to an amount or dosage of an antibody, fragment, composition or combination of the present invention that produces the desired effect in a patient in need of treatment or prevention after administration in single or multiple doses to the patient.

[0196] A "therapeutically effective amount" is an amount effective to achieve the desired therapeutic outcome, at the required dosage and for the required period of time. A therapeutically effective amount is also one in which any toxic or deleterious effects of the antibody, antibody fragment, composition, or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter or improves a measurable parameter by at least about 40%, and more preferably, by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%, relative to an untreated subject.

[0197] A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result, at the dosages and for the period of time necessary. Generally, because a prophylactic dose is used in a subject prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0198] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating a disease, such as a tumor (e.g., cancer) and an infection (e.g., a chronic infection), including anti-angiogenic agents, chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators.

[0199] "Chemotherapeutic agents" include chemical compounds useful in the treatment of cancer, including but not limited to antineoplastic agents, including alkylating agents; antimetabolites; natural products; antibiotics; enzymes; miscellaneous agents; hormones and antagonists; antiestrogens; antiandrogens; and nonsteroidal antiandrogens, among others.

[0200] The term "immunomodulator" as used herein refers to a natural or synthetic agent or drug that inhibits or modulates an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immune checkpoint molecule inhibitors and co-stimulatory molecule activators.

[0201] The term "small molecule drug" refers to low molecular weight organic compounds that are capable of modulating biological processes.

[0202] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular 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 and their fragments, such as nucleolytic enzymes; antibiotics; and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof.

[0203] "Tumor immune escape" refers to the process by which tumors evade immune recognition and elimination. Thus, as a therapeutic concept, tumor immunity is "treated" when this evasion is compromised, allowing the tumor to be recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor elimination.

[0204] "Immunogenicity" refers to the ability of a substance to elicit an immune response. Tumors are immunogenic, and enhancing tumor immunogenicity helps eliminate tumor cells through immune responses.

[0205] 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.

[0206] "Anti-angiogenic agent" refers to a compound that blocks or interferes to some extent with the development of blood vessels. An anti-angiogenic agent can be, for example, a small molecule or an antibody that binds to a growth factor or growth factor receptor involved in promoting angiogenesis.

[0207] The term "pharmaceutical combination" or "combination product" refers to either a non-fixed combination or a fixed combination, including, but not limited to, kits and pharmaceutical compositions. The term "non-fixed combination" means that the active ingredients (e.g., (i) the immunoconjugate of the invention, and (ii) the other therapeutic agent) are administered to a patient as separate entities simultaneously, without specific time limits, or sequentially, at equal or different time intervals, wherein such administration provides prophylactically or therapeutically effective levels of the two or more active agents in the patient. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously as a single entity. The dosages and / or time intervals of the two or more active agents are preferably selected so that the combined use of the ingredients produces a greater effect in treating the disease or condition than that achieved by either ingredient alone. Each ingredient may be in the form of a separate formulation, which may be the same or different.

[0208] The term "combination therapy" or "combination therapy" refers to the administration of two or more therapeutic agents to treat a cancer or infection as described herein. Such administration includes co-administration of the therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of the active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids), separate administration, or sequential administration. The powders and / or liquids can be reconstituted or diluted to the desired dose prior to administration. In some embodiments, administration further includes administering each type of therapeutic agent at approximately the same time, or at different times in a sequential manner. In either case, the treatment regimen will provide the beneficial effects of the drug combination in treating the disorders or conditions described herein.

[0209] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0210] "Subject / patient sample" refers to a collection of cells or fluids obtained from a patient or subject. Tissue or cell samples can be derived from solid tissues, such as fresh, frozen, and / or preserved organ or tissue samples or biopsy or aspirate samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; or cells from a subject at any time during gestation or development. Tissue samples may contain compounds that are not naturally contaminated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage fluid, pleural fluid (pleural effusion), 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 properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples. [II. Antibody molecules of the present invention]

[0211] The present invention provides a novel antibody molecule that can be used for immunotherapy, prevention, and / or diagnosis of a variety of diseases. The antibody molecule of the present invention comprises at least two, three, or four antigen-binding regions and can function as a bispecific antibody or a multispecific antibody. Preferably, it can function as a bispecific antibody.

[0212] In some embodiments, the bispecific or multispecific antibodies of the invention comprise a first binding specificity for EGFR and a second binding specificity for B7H3, and optionally additional binding specificities.

[0213] Therefore, one aspect of the present invention relates to 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 / or the second antigen binding region specifically binds to B7-H3.

[0214] The bispecific antibodies of the present invention can be prepared using bispecific antibody formats or technologies known in the art. Specific exemplary bispecific formats that can be used in the context of the present invention are described, for example, 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 IgG-like antibody type comprises two Fab regions and two Fc regions, and the heavy chain and light chain of each Fab can be derived from separate monoclonal antibodies. In some embodiments, the bispecific antibody of the present invention is an IgG-like bispecific antibody comprising 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.

[0215] 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, such as the EGFR antibody disclosed in WO02100348A2, such as Zalutumumab monoclonal antibody.

[0216] In some embodiments, the antigen binding region that specifically binds to EGFR comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to EGFR, such as the EGFR antibody disclosed in WO02100348A2, such as zalutumumab monoclonal antibody.

[0217] In some embodiments, the antigen binding region that specifically binds to EGFR comprises 1, 2, and 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of a known antibody that specifically binds to EGFR, such as the EGFR antibody disclosed in WO02100348A2, such as zalutumumab monoclonal antibody.

[0218] In some embodiments, the antigen binding region that specifically binds to EGFR comprises 1, 2, and 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of a known antibody that specifically binds to EGFR, such as the EGFR antibody disclosed in WO02100348A2, such as zalutumumab monoclonal antibody.

[0219] 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, such as the EGFR antibody disclosed in WO02100348A2, such as Zalutumumab monoclonal antibody.

[0220] In some embodiments, the antigen binding region that specifically binds to EGFR comprises the heavy chain variable region and light chain variable region of a known antibody that specifically binds to EGFR, such as the EGFR antibody disclosed in WO02100348A2, such as the zalutumumab monoclonal antibody.

[0221] In some embodiments, the antigen binding region that specifically binds to EGFR comprises a known antibody that specifically binds to EGFR, such as the EGFR antibody disclosed in WO02100348A2, such as the Fab of Zalutumumab monoclonal antibody.

[0222] In some embodiments, the antigen binding region that specifically binds to EGFR comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen binding region that specifically binds to EGFR comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. 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.

[0223] 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 (VH). 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 three complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0224] In some embodiments, the heavy chain variable region specifically binds to the antigen binding region of EGFR (i) comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 1; or (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 1, and preferably, the amino acid changes do not occur in the CDR region.

[0225] In some embodiments, the light chain variable region specifically binds to the antigen binding region of EGFR (i) comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 2; or (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 2, and preferably, the amino acid changes do not occur in the CDR region.

[0226] In some embodiments, the three complementary determining regions (HCDRs) of the heavy chain variable region that specifically bind to the EGFR antigen binding region, HCDR1, HCDR2, and HCDR3, are selected from (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 1, or (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three HCDR regions relative to the sequence of any one of (i), For example, the CDRs are determined by the Kabat scheme.

[0227] In some embodiments, the three complementarity determining regions (LCDRs) of the light chain variable region that specifically bind to the antigen binding region of EGFR, LCDR1, LCDR2, and LCDR3 are selected from (i) three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 2, or (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three LCDR regions relative to the sequence of any one of (i), For example, the CDRs are determined by the Kabat scheme.

[0228] In some embodiments, the antigen binding region that specifically binds to EGFR comprises three complementarity determining regions (HCDRs) contained in a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1 and three complementarity determining regions (LCDRs) contained in a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2.

[0229] In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 9, or consists of the amino acid sequence, or HCDR1 comprises an amino acid sequence having one, two, or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 9.

[0230] In some embodiments, HCDR2 comprises the amino acid sequence of SEQ ID NO: 10, or consists of the amino acid sequence, or HCDR2 comprises an amino acid sequence having one, two, or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 10.

[0231] In some embodiments, HCDR3 comprises the amino acid sequence of SEQ ID NO: 11, or consists of the amino acid sequence, or HCDR3 comprises an amino acid sequence having one, two, or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 11.

[0232] In some embodiments, LCDR1 comprises the amino acid sequence of SEQ ID NO: 12, or consists of the amino acid sequence, or LCDR1 comprises an amino acid sequence having one, two, or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 12.

[0233] In some embodiments, LCDR2 comprises the amino acid sequence of SEQ ID NO: 13, or consists of the amino acid sequence, or LCDR2 comprises an amino acid sequence having one, two, or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 13.

[0234] In some embodiments, LCDR3 comprises the amino acid sequence of SEQ ID NO: 14, or consists of the amino acid sequence, or LCDR3 comprises an amino acid sequence having one, two, or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 14.

[0235] In some embodiments of the present invention, the antigen binding region that specifically binds to EGFR comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 as described above.

[0236] In some embodiments of the present invention, the antigen binding region that specifically binds to EGFR comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as described above.

[0237] In some embodiments of the present invention, the antigen binding region that specifically binds to EGFR comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein 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; HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 11; LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 12; LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 13; and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 14.

[0238] In some specific embodiments of the present invention, the antigen binding region that specifically binds to EGFR comprises HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, HCDR3 as shown in SEQ ID NO:11; LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13 and LCDR3 as shown in SEQ ID NO:14.

[0239] In some embodiments of the present invention, the antigen binding region that specifically binds to EGFR comprises VH and VL, wherein The VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0240] 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 comprise or consist of the amino acid sequences shown below: SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0241] 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 antibody described in PCT / CN2021 / 140449, such as the monoclonal antibody numbered Hz20G5 (e.g., Hz20G5.26 mentioned in the Examples of the present invention).

[0242] In some embodiments, the antigen binding region that specifically binds to B7-H3 comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to B7-H3, such as the B7-H3 antibody described in PCT / CN2021 / 140449, for example, the monoclonal antibody numbered Hz20G5.

[0243] In some embodiments, the antigen binding region that specifically binds to B7-H3 comprises 1, 2, and 3 heavy chain variable region CDRs, i.e., HCDR1, HCDR2, and HCDR3, of a known antibody that specifically binds to B7-H3, such as the B7-H3 antibody described in PCT / CN2021 / 140449, for example, the monoclonal antibody numbered Hz20G5.

[0244] In some embodiments, the antigen binding region that specifically binds to B7-H3 comprises 1, 2, and 3 light chain variable region CDRs, i.e., LCDR1, LCDR2, and LCDR3, of a known antibody that specifically binds to B7-H3, such as the B7-H3 antibody described in PCT / CN2021 / 140449, for example, the monoclonal antibody numbered Hz20G5.

[0245] In some embodiments, the antigen binding region that specifically binds to B7-H3 comprises three heavy chain variable region CDRs and three light chain variable region CDRs of a known antibody that specifically binds to B7-H3, such as the B7-H3 antibody described in PCT / CN2021 / 140449, for example, the monoclonal antibody numbered Hz20G5.

[0246] In some embodiments, the antigen binding region that specifically binds to B7-H3 comprises the heavy chain variable region and 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 numbered Hz20G5.

[0247] In some embodiments, the antigen binding region that specifically binds to B7-H3 comprises a known antibody that specifically binds to B7-H3, such as the B7-H3 antibody described in PCT / CN2021 / 140449, such as the Fab of the monoclonal antibody numbered Hz20G5.

[0248] 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, namely HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen binding region that specifically binds to B7-H3 comprises three complementarity determining regions (LCDRs) from the light chain variable region, namely LCDR1, LCDR2, and LCDR3. 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.

[0249] In some aspects, the antigen-binding region that specifically binds to B7-H3 comprises a heavy chain variable region (VH). In some aspects, the antigen-binding region that specifically binds to B7-H3 comprises a light chain variable region (VH). In some aspects, the antigen-binding region that specifically binds to B7-H3 comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementary determining regions (CDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementary determining regions (CDRs) from the light chain variable region: LCDR1, LCDR2, and LCDR3.

[0250] In some embodiments, the heavy chain variable region specifically binds to the antigen binding region of B7-H3 (i) comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 3, 5 or 7; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 3, 5 or 7; or (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 3, 5, or 7, consisting of the amino acid sequence, preferably, the amino acid changes do not occur in the CDR region.

[0251] In some embodiments, the light chain variable region specifically binds to the antigen binding region of B7-H3 (i) comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 4, 6 or 8; or (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 4, 6 or 8; or (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 4, 6, or 8, consisting of the amino acid sequence, preferably, the amino acid changes do not occur in the CDR region.

[0252] In some embodiments, the three complementarity determining regions (HCDRs) of the heavy chain variable region that specifically bind to the antigen binding region of B7-H3, HCDR1, HCDR2, and HCDR3 are selected from (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 3, 5 or 7, or (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three HCDR regions relative to the sequence of any one of (i), For example, the HCDR1 is determined using the Abm scheme, and the HCDR2 and HCDR3 are determined using the Kabat scheme.

[0253] In some embodiments, the three complementarity determining regions (LCDRs) from the light chain variable region that specifically bind to the antigen binding region of B7-H3, LCDR1, LCDR2, and LCDR3 are selected from (i) three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 4, 6 or 8, or (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three LCDR regions relative to the sequence of any one of (i), For example, the CDRs are determined by the Kabat scheme.

[0254] In some embodiments, the antigen binding region that specifically binds to B7-H3 comprises three complementarity determining regions (HCDRs) contained in a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 3, 5, or 7, and three complementarity determining regions (LCDRs) contained in a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 4, 6, or 8.

[0255] In some embodiments, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 15, 21 or 27, or HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 15, 21 or 27.

[0256] In some embodiments, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 16, 22 or 28, or HCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 16, 22 or 28.

[0257] In some embodiments, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 17, 23 or 29, or HCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 17, 23 or 29.

[0258] In some embodiments, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 18, 24 or 30, or LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 18, 24 or 30.

[0259] In some embodiments, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 19, 25 or 31, or LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 19, 25 or 31.

[0260] In some embodiments, LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 20, 26 or 32, or LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 20, 26 or 32.

[0261] In some embodiments of the present invention, the antigen binding region that specifically binds to B7-H3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 as described above.

[0262] In some embodiments of the present invention, the antigen binding region that specifically binds to B7-H3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as described above.

[0263] In some 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) HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 15, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 18, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 20; or, (ii) HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 21, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 22, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 23, LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 24, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 25, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 26; or, (iii) HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 27, HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 28, and HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 29, LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 30, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 31, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 32.

[0264] In some embodiments of the present invention, the antigen binding region that specifically binds to B7-H3 comprises (i) HCDR1 as shown in SEQ ID NO: 15, HCDR2 as shown in SEQ ID NO: 16, HCDR3 as shown in SEQ ID NO: 17; LCDR1 as shown in SEQ ID NO: 18, LCDR2 as shown in SEQ ID NO: 19, and LCDR3 as shown in SEQ ID NO: 20; (ii) HCDR1 as set forth in SEQ ID NO:21, HCDR2 as set forth in SEQ ID NO:22, HCDR3 as set forth in SEQ ID NO:23; LCDR1 as set forth in SEQ ID NO:24, LCDR2 as set forth in SEQ ID NO:25, and LCDR3 as set forth in SEQ ID NO:26; or (iii) HCDR1 as shown in SEQ ID NO:27, HCDR2 as shown in SEQ ID NO:28, HCDR3 as shown in SEQ ID NO:29; LCDR1 as shown in SEQ ID NO:30, LCDR2 as shown in SEQ ID NO:31 and LCDR3 as shown in SEQ ID NO:32.

[0265] In some embodiments of the present invention, the antigen binding region that specifically binds to B7-H3 comprises VH and VL, wherein (i) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:3, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:3, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:4, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:4; (ii) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:5, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:6, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:6; or (iii) the VH comprises, or consists of, the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:7, and the VL comprises, or consists of, the amino acid sequence of SEQ ID NO:8, or an amino acid sequence that is at least 80%, 85%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:8.

[0266] In some embodiments of the present invention, the antigen-binding region that specifically binds to B7-H3 comprises VH and VL, wherein VH and VL each comprise or consist of the amino acid sequence shown below: SEQ ID NO:3 and SEQ ID NO:4; SEQ ID NO:5 and SEQ ID NO:6; or SEQ ID NO:7 and SEQ ID NO:8.

[0267] The present invention also relates to antibodies that specifically bind to B7-H3 and comprise the above-mentioned antigen-binding region that specifically binds to B7-H3, such as antibodies comprising HCDR1, HCDR2 and HCDR3, and LCDR1, LCDR2 and LCDR3 as defined in the present invention; or antibodies comprising VH or VL as defined in the present invention, or antibodies comprising VH and VL as defined in the present invention.

[0268] [Fc region] In some embodiments, the antibody molecules of the present invention, such as multispecific antibodies (such as bispecific antibodies), further comprise an Fc region, wherein the Fc regions comprised may be the same or different.

[0269] In some embodiments, the Fc region has hypofucosylation, such as that obtained by treatment with GlymaxX technology.

[0270] In some embodiments, the antibody molecules of the invention comprise a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region are the same or different.

[0271] In some embodiments, the first and second Fc regions are different and are capable of dimerizing to form a heterodimeric Fc scaffold.

[0272] As used herein, the Fc region refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region and can include native sequence Fc regions and variant Fc regions. Native sequence F regions encompass various naturally occurring immunoglobulin Fc sequences, such as the Fc regions of various Ig subclasses 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 present invention comprises antibody CH2 and CH3. In some embodiments, the antibody Fc region may also contain an IgG hinge region or a portion of an IgG hinge region at the N-terminus, such as an IgG1 hinge region or a portion of an IgG1 hinge region, such as the sequence D221 to P230 according to EU numbering. This hinge region may contain mutations.

[0273] Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region is according to the EU numbering system, also known as the EU index, as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0274] In some embodiments, the Fc region is a human IgG Fc, e.g., human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. In one embodiment, the Fc region comprises or consists of the amino acid sequence of SEQ ID NO: 46 or 47, or an amino acid sequence that is at least 90% identical thereto, e.g., 95%, 96%, 97%, 99% or more identical thereto.

[0275] As understood by those skilled in the art, to promote heterodimer formation of the multispecific antibodies of the present invention, the Fc regions of the multispecific antibodies of the present invention may include mutations that facilitate heterodimerization of the first and second Fc regions. In one embodiment, mutations are introduced into the CH3 regions of both Fc regions.

[0276] Methods for promoting Fc region heterodimerization are known in the art. For example, the CH3 region of a first Fc region and the CH3 region of a second Fc region are engineered in a complementary manner such that each CH3 region (or a heavy chain comprising it) is no longer able to homodimerize with itself but is forced to heterodimerize with the other complementarily engineered CH3 region (such that the CH3 regions of the first and second Fc regions heterodimerize and homodimers are not formed between the two first CH3 regions or the two second CH3 regions).

[0277] Preferably, corresponding knob mutations and hole mutations are introduced into the first and second Fc regions based on the Knob-in-Hole technique. This technique is described, for example, in US Pat. No. 5,731,168; US Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).

[0278] In a specific embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) (knob mutation); and 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).

[0279] In another embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced by a tryptophan residue (T366W) and the serine residue at position 354 is replaced by a cysteine ​​residue (S354C) or the glutamate residue at position 356 is replaced by a cysteine ​​residue (E356C) (particularly, the serine residue at position 354 is replaced by a cysteine ​​residue); and in the CH3 region of another Fc region, the tyrosine residue at position 407 is replaced by a valine residue (Y407V) (hole mutation), optionally the threonine residue at position 366 is replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by an alanine residue (L368A) (numbering according to the EU index), and optionally the tyrosine residue at position 349 is replaced by a cysteine ​​residue (Y349C) (numbering according to the EU index).

[0280] In a specific embodiment, one Fc region comprises the amino acid substitution T366W and the other Fc region comprises the amino acid substitutions T366S, L368A, and Y407V (numbering according to the EU index).

[0281] In a specific embodiment, one Fc region comprises amino acid substitutions S354C and T366W, and the other Fc region comprises amino acid substitutions Y349C, T366S, L368A, and Y407V (numbering according to the EU index).

[0282] Corresponding mutations can also be introduced into the first and second Fc regions based on the Innobody technology. For this technology, see, for example, PCT / CN2021 / 143141.

[0283] In a specific embodiment, The first CH3 region comprises an S364R / K mutation (preferably S364R), and optionally one or more other mutations. In some embodiments, the second CH3 region comprises a K370S / T / A / V mutation (preferably K370S), and optionally one or more other mutations. In some embodiments, the first CH3 region comprises an S364R / K mutation, and the second CH3 region comprises a K370S / T / A / V mutation. In some embodiments, the first CH3 region comprises an S364R mutation, and the second CH3 region comprises a K370S mutation.

[0284] In some embodiments, the first CH3 region comprises S364R / K (preferably S364R) and D399K / R (preferably D399K) mutations. In some embodiments, the second CH3 region comprises K370S / T / A / V (preferably K370S) mutations and K409D / E (preferably K409D) mutations. In some embodiments, the first CH3 region comprises S364R / K + D399K / R, and the second CH3 region comprises K370S / T / A / V + Y349T / S / A / V. In some embodiments, the first CH3 region comprises S364R + D399K, and the second CH3 region comprises K370S + Y349T. In some embodiments, the first CH3 region further comprises E375N / Q (preferably E375N) and / or T350V / A (preferably T350V). In some embodiments, the second CH3 region further comprises K409D / E (preferably K409D), Q347D / E (preferably Q347D) and / or T350V / A (preferably T350V).

[0285] In some embodiments, the first CH3 region comprises S364R + D399K, and the second CH3 region comprises K370S + Y349T + K409D. In some embodiments, the first CH3 region further comprises E357N. In some embodiments, the second CH3 region further comprises Q347D. In some embodiments, the first CH3 region further comprises E357N, and the second CH3 region further comprises Q347D. In some embodiments, the first CH3 region and the second CH3 region further comprise T350V, or both comprise T350V.

[0286] Thus, in some embodiments, the first CH3 region comprises S364R+D399K, and the second CH3 region comprises K370S+Y349T+K409D+Q347D. In some embodiments, the first CH3 region comprises S364R+D399K+E357N, and the second CH3 region comprises K370S+Y349T+K409D+Q347D. In some embodiments, the first CH3 region comprises S364R+D399K+E357N+T350V, and the second CH3 region comprises K370S+Y349T+K409D+Q347D+T350V.

[0287] In some embodiments, the first CH3 region comprises K409E / D (preferably K409E). In some embodiments, the second CH3 region comprises D399K / R (preferably D399K) or K370T / S / A / V (preferably K370T). In some embodiments, the first CH3 region comprises K409E / D (preferably K409E), and the second CH3 region comprises D399K / R (preferably D399K). In some embodiments, the first CH3 region further comprises T411R / K (preferably T411R). In some embodiments, the second CH3 region further comprises K370T / S / A / V (preferably K370T). In some embodiments, the CH3 region comprises K409E / D + T411R / K, and the second CH3 region comprises D399K / R + K370T / S / A / V. In some embodiments, the CH3 region comprises K409E+T411R, and the second CH3 region comprises D399K+K370T.

[0288] In some specific embodiments, the first and second CH3 regions have the following mutation combinations:

[0289] In one embodiment, the CH3 of one Fc region comprises S364R and D399K mutations, and the CH3 mutations of the other Fc region comprise Y349T, K370S, and K409D mutations.

[0290] Thus, in a specific embodiment, the multispecific antibody of the present invention comprises two Fc regions that are heterodimerized, wherein a) one Fc region polypeptide comprises the mutation T366W and the other Fc region polypeptide comprises T366S, L368A and Y407V, or b) one Fc-region polypeptide comprises the mutations S354C and T366W, and the other Fc-region polypeptide comprises the mutations Y349C, T366S, L368A and Y407V, or c) one Fc-region polypeptide comprises the mutations S364R and D399K, and the other Fc-region polypeptide comprises the mutations Y349T, K370S and K409D.

[0291] Therefore, in a specific embodiment, the multispecific antibody of the present invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 49 or 50, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 52 or 53.

[0292] Thus, in a specific embodiment, the multispecific antibody of the invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 49 or 50, and the other Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 52 or 53.

[0293] Thus, in a specific embodiment, the multispecific antibody of the invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 49 or 50 and comprises the mutations Y349T, K370S, and K409D, and the other Fc region comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 52 or 53 and comprises the mutations S364R and D399K.

[0294] In some embodiments, the Fc region further comprises other mutations that facilitate purification of the heterodimer.

[0295] Antibody modification In one embodiment of the present 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.

[0296] In a preferred embodiment, the amino acid changes described herein occur in regions outside of the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside of the heavy chain variable region and / or outside of the light chain variable region.

[0297] In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to the replacement of one amino acid with another amino acid within the same class, for example, an acidic amino acid with another acidic amino acid, a basic amino acid with another basic amino acid, or a neutral amino acid with another neutral amino acid. Exemplary substitutions are shown in Table A below:

[0298] Table A

[0299] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated.

[0300] Antibodies with altered glycosylation can be prepared, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. For example, fucosylation in a constant region (e.g., an Fc region) can be eliminated, for example, to obtain low or no fucosylation.

[0301] Such altered glycosylation patterns have been shown to increase the ADCC activity of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation machinery. For example, afucosylated antibodies are expressed in host cells deficient for α-1,6-fucosyltransferase 8 (Fut8) (WO2000061739). For example, low or no fucosylated antibodies can be obtained by introducing a host cell encoding the enzyme RMD, which is involved in sugar chain modification (GlymaxX technology, ProBioGen AG, see Patent Publication No. WO2011035884A1). Alternatively, fucose residues on antibodies can be removed using a fucosidase; for example, the fucosidase α-L-fucosidase removes fucosyl residues from antibodies (Tarentino et al. (1975) Biochem. 14:5516-23).

[0302] Exemplary bispecific antibody molecules

[0303] In some embodiments, the anti-B7-H3 / EGFR bispecific antibodies of the present invention have one or more of the following properties: (1) The bispecific antibody of the present invention can, on the one hand, block the binding of EGFR ligand to EGFR, inhibit biological signal transmission, and block the corresponding biological activity of tumors; on the other hand, it can stimulate the endocytosis of EGFR and ultimately be degraded by intracellular lysosomes; (2) The bispecific antibody of the present invention uses the EGFR antibody parent sequence with low affinity for EGFR, which greatly reduces the toxic side effects of the series of EGFR monoclonal antibodies on normal epithelial tissues such as skin; (3) The bispecific antibody of the present invention uses the antibody parent sequence of B7-H3 with high affinity on the basis of low affinity for EGFR, which greatly improves the EGFR signal blocking activity and enhances the pharmacodynamic biological activity and pharmacodynamic safety window of the bispecific antibody of the present invention; (4) The bispecific antibody of the present invention is a low-fucosylated antibody; (5) The bispecific antibodies of the present invention have high pharmacodynamic biological activity and safety; (6) The bispecific antibodies of the present invention have excellent tumor killing and inhibitory effects; (7) The bispecific antibody of the present invention has excellent ADCC pharmacodynamic activity in vitro and in vivo; (8) The bispecific antibody of the present invention combined with the KRAS small molecule inhibitor has excellent anti-tumor effect, especially synergistic effect.

[0304] In some embodiments, in the antibody molecules of the present invention, the antigen binding region that specifically binds to EGFR is connected to the heavy chain constant region CH, for example, the heavy chain variable region is connected to the heavy chain constant region CH, for example, the C-terminus of the heavy chain variable region is connected to the N-terminus of the heavy chain constant region CH. In some embodiments, in the antibody molecules of the present invention, the antigen binding region that specifically binds to EGFR is connected to the light chain constant region, for example, the light chain variable region is connected to the light chain constant region CL, for example, the C-terminus of the light chain variable region is connected to the N-terminus of the light chain constant region CL. In some embodiments, in the antibody molecules of the present invention, in the antigen binding region that specifically binds to EGFR, the heavy chain variable region is connected to the heavy chain constant region CH and the light chain variable region is connected to the light chain constant region CL.

[0305] In some embodiments, in an antibody molecule of the 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 is linked to the heavy chain constant region CH, for example, the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region CH. In some embodiments, in an antibody molecule of the 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 is linked to the light chain constant region CL, for example, the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region CL. In some embodiments, in an antibody molecule of the 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.

[0306] In some embodiments, the heavy chain constant region comprises a CH1 and an Fc region, connected with or without a hinge region.

[0307] In some embodiments, the heavy chain constant region is an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as a human IgG1, human IgG2, human IgG3, or human IgG4 heavy chain constant region. In some embodiments, the CH1 comprises or consists of the amino acid sequence of SEQ ID NO: 42, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 42.

[0308] 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 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 54.

[0309] 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 an Fc, and the Fab fragment that specifically binds to B7-H3 forms a half-antibody that specifically binds to B7-H3 with an Fc.

[0310] In some embodiments, the bispecific antibody is an IgG-like antibody having the configuration shown in FIG1 .

[0311] In some 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 a heavy chain variable region and a first heavy chain constant region of a first antigen-binding region; light chain 1 comprises a light chain variable region and a first light chain constant region of a first antigen-binding region; and heavy chain 2 comprises a heavy chain variable region and a second heavy chain constant region of a second antigen-binding region; and light chain 2 comprises a light chain variable region and a second light chain constant region of a second antigen-binding region.

[0312] In some embodiments, in the bispecific antibody, heavy chain 1 comprises or consists of the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33.

[0313] In some embodiments, in the bispecific antibody, light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 34.

[0314] In some embodiments, in the bispecific antibody, heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33, and light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 34.

[0315] In some embodiments, in the bispecific antibody, heavy chain 2 comprises or consists of the amino acid sequence of SEQ ID NO: 35, 37 or 39, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 35, 37 or 39.

[0316] In some embodiments, in the bispecific antibody, light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 36, 38, or 40, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 36, 38, or 40.

[0317] In some embodiments, the bispecific antibody (1) Heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 35, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 35; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 36, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 36; (2) heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 37; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 38; (3) Heavy chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 39; and light chain 2 comprises, or consists of, the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 40.

[0318] In some embodiments, the bispecific antibody Heavy chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33, and light chain 1 comprises, or consists of, the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 34; and heavy chain 2 and light chain 2 each comprise, or comprise, or consist of, the amino acid sequence of SEQ ID NO: i) SEQ ID NO:35 and SEQ ID NO:36; ii) SEQ ID NO:37 and SEQ ID NO:38; iii) SEQ ID NO: 39 and SEQ ID NO: 40.

[0319] In some embodiments, the 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) 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: 37, and light chain 2 comprises the amino acid sequence shown in SEQ ID NO: 38; or, (iii) 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, The heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO: 39, and the light chain 2 comprises the amino acid sequence shown in SEQ ID NO: 40. [III. Immunoconjugates]

[0320] In some embodiments, the present invention also encompasses antibodies conjugated to other substances. Thus, the present invention relates to immunoconjugates comprising antibodies conjugated to other substances ("immunoconjugates"). In some embodiments, the other substance is, for example, a therapeutic agent or label, such as a cytotoxic agent, an immunomodulator (e.g., an immunoagonist), or a chemotherapeutic agent. Cytotoxic agents include any agent that is detrimental to cells. Examples of cytotoxic agents (e.g., chemotherapeutic agents) suitable for forming immunoconjugates are known in the art. For example, cytotoxic agents include, but are not limited to: radioactive isotopes; growth inhibitors; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof; and various known anti-tumor or anti-cancer agents.

[0321] Additionally, the antibody molecules of the invention can be conjugated to a marker sequence (eg, a peptide) to facilitate purification.

[0322] In other embodiments, the antibody molecules of the present invention are conjugated to diagnostic or detectable agents. Such antibodies can be used as part of clinical testing methods (e.g., to determine the efficacy of a particular therapy) to monitor or predict the onset, development, progression, and / or severity of a disease or condition. Such diagnosis and detection can be achieved by coupling the antibody to a detectable substance, including but not limited to various enzymes; prosthetic groups; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metals and non-radioactive paramagnetic metal ions used in various positron emission tomography techniques.

[0323] In addition, the antibody molecules of the present invention can be conjugated to a therapeutic moiety or drug moiety that modulates a given biological response. Therapeutic moieties or drug moieties include, but are not limited to, classical chemotherapeutic drugs. For example, the drug moiety can be a protein, peptide, or polypeptide that possesses a desired biological activity.

[0324] Additionally, the antibody molecules of the invention can be conjugated to therapeutic moieties such as radioactive metal ions.

[0325] Antibodies can also be attached to a solid support, which is particularly useful in immunoassays or for purification of the target antigen.

[0326] In some embodiments, the immunoconjugates are used to prevent or treat diseases such as acute and chronic inflammatory diseases, infections (e.g., chronic infections), tumors, and the like. For example, the disease is a tumor (e.g., cancer) or an infection. In some embodiments, the tumor is a tumor that has escaped immune evasion. Preferably, the tumor is an epithelial cancer, such as a gastrointestinal tumor, a lung tumor, or a skin tumor, such as skin cancer (e.g., squamous cell carcinoma of the skin, head and neck cancer, 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, squamous cell lung 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, or a protozoan infection. [IV. Nucleic acid of the present invention and host cell containing the same]

[0327] In one aspect, the present invention provides nucleic acids encoding any of the above antibodies or fragments thereof, or any of their chains. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising 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 producing antibodies or antigen-binding fragments thereof. In another embodiment, the host cell is prokaryotic.

[0328] For example, the nucleic acids of the present invention include nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs: 3-8 and 33-40, or nucleic acids encoding an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 3-8 and 33-40.

[0329] As is apparent to those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences. Nucleic acid sequences encoding the molecules of the present invention can be generated using methods well known in the art, such as de novo solid-phase DNA synthesis or PCR amplification.

[0330] In one aspect, the present invention provides nucleic acids encoding any of the above antibodies or any antibody chains. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit human EGFR / or B7-H3 antigen binding ability.

[0331] In yet another aspect, the present invention provides nucleic acids encoding any of the above bispecific antibodies. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid is capable of exhibiting binding ability to human EGFR and / or B7-H3 antigens. In one embodiment, the nucleic acids encoding each chain of the bispecific antibody can be in the same vector or in 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. Therefore, in some embodiments, the method for producing the bispecific antibody of the present invention comprises the steps of culturing host cells containing nucleic acids encoding each chain under conditions suitable for expression of each chain of the molecule to produce the bispecific antibody of the present invention.

[0332] In one embodiment, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In one embodiment, the vector is, for example, a pcDNA vector, such as pcDNA3.1.

[0333] Once an expression vector or DNA sequence for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, the cells are grown in culture medium and screened for appropriate activity. The methods and conditions for culturing the resulting transfected cells and for recovering the produced antibody molecules are known to those of ordinary skill in the art and can be varied or optimized based on this specification and methods known in the art, depending on the specific expression vector and mammalian host cell used.

[0334] Additionally, cells that have stably incorporated the DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells.

[0335] In one embodiment, a host cell comprising one or more polynucleotides of the present invention is provided. In some embodiments, a host cell comprising an 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 such as CHO cells (e.g., CHO-S, such as ExpiCHO-S) or 293 cells (e.g., 293F or HEK293 cells), or other cells suitable for producing antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, for example, a bacterium, such as Escherichia coli.

[0336] Suitable host cells include prokaryotic microorganisms such as Escherichia coli. Host cells can also be eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells, such as insect cells. Vertebrate cells can also be used as hosts. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (HEK 293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine 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 Y0, NSO, P3X63, and Sp2 / 0, etc. For a review of mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Volume 248 (BKCLo, ed., Humana Press, Totowa, NJ), pages 255-268 (2003). In a preferred embodiment, the host cell is a CHO cell, such as a CHOS cell, CHOK1SV cell, or 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 glycosylated host cell, preferably a glycosylated CHO cell. In one embodiment, the host cell is engineered to express an RMD enzyme. In one embodiment, the host cell comprises a nucleic acid encoding an 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 from Pseudomonas aeruginosa.

[0337] In one embodiment, a host cell of the invention comprises a nucleic acid encoding one or more or all chains of an antibody molecule of the invention and a nucleic acid encoding an RMD enzyme. [V. Production and purification of the antibody molecule of the present invention]

[0338] In one embodiment, the present invention provides a method for preparing an antibody molecule of the present invention, wherein the method comprises culturing a host cell under conditions suitable for expressing a nucleic acid encoding an antibody molecule of the present invention, and optionally isolating the antibody. In a certain embodiment, the method further comprises recovering the antibody molecule of the present invention from the host cell.

[0339] In one embodiment, a method for preparing an antibody molecule of the 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 comprising the nucleic acid, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0340] To recombinantly produce an antibody molecule of the invention, nucleic acid encoding an antibody (e.g., an antibody described above, e.g., any 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 acid is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody).

[0341] Antibody molecules prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, and hydrophilicity, and these will be apparent to one of ordinary skill in the art. The purity of the antibody molecules of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like. [VI. Determination method]

[0342] The antibody molecules provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art. In one aspect, the antibodies of the invention are tested for their antigen binding activity, for example, by known methods such as ELISA, Western blotting, etc. Binding to the bound antigen can be determined using methods known in the art, exemplary methods of which are disclosed herein, such as biofilm interferometry and SPR.

[0343] The present invention also provides assays for identifying antibodies with biological activity. Biological activity can include, for example, binding to an antigen, binding to a cell surface antigen, or inhibition or activation of an antigen. Antibodies that exhibit such biological activity in vivo and / or in vitro are also provided.

[0344] In certain embodiments, the antibodies of the invention are tested for such biological activities.

[0345] The present invention also provides methods for identifying properties of antibodies, such as druggability-related properties. The druggability-related properties include, for example, thermal stability, such as long-term thermal stability.

[0346] Cells for use in any of the above in vitro assays include cell lines that naturally express the antigen or are engineered to express the antigen. Such cells also include cell lines that express the antigen and cell lines that are not normally expressing the antigen and are transfected with DNA encoding the antigen.

[0347] It will be appreciated that any of the above assays can be performed using the immunoconjugates of the invention in place of or in addition to the antibody molecules of the invention.

[0348] It will be appreciated that any of the above assays can be performed using the antibody molecules of the invention and additional active agents.

[0349] In some embodiments, the antigen is EGFR (eg, human EGFR) and / or B7-H3 (eg, human B7-H3). VII. Pharmaceutical compositions and pharmaceutical preparations

[0350] In some embodiments, the present invention provides a composition comprising any of the antibody molecules described herein, or fragments thereof (preferably antigen-binding fragments thereof), or immunoconjugates thereof. Preferably, the composition is a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, e.g., a pharmaceutical composition, comprises an antibody molecule, fragment thereof, or immunoconjugate thereof of the present invention in combination with one or more other therapeutic agents.

[0351] In some embodiments, the other therapeutic agent is selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective active agents, small molecule drugs or immunomodulators (such as activators of co-stimulatory molecules or inhibitors of immune checkpoint molecules); preferably, the second therapeutic agent is a small molecule drug, preferably, the small molecule drug is selected from KRAS small molecule inhibitors, such as KRAS G12C inhibitors (such as AMG510 (Sotorasib) or GFH925), KRAS G12D (such as MRTX1133) or KRAS G12S inhibitors.

[0352] In some embodiments, the composition is used to prevent or treat diseases such as acute and chronic inflammatory diseases, infections (e.g., chronic infections), tumors, and the like. For example, the disease is a tumor (e.g., cancer) or an infection. In some embodiments, the tumor is a tumor that has escaped immune function. Preferably, the tumor is an epithelial cancer, such as a gastrointestinal tumor, a lung tumor, or a skin tumor, such as skin cancer (e.g., squamous cell carcinoma of the skin, head and neck cancer, 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, squamous cell lung 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, and the like.

[0353] The present invention also includes compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising an antibody of the present invention or an immunoconjugate thereof and / or compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising a polynucleotide encoding an antibody of the present invention. In certain embodiments, the composition comprises 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.

[0354] These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0355] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic agents, absorption delaying agents, and the like that are physiologically compatible. Pharmaceutical carriers suitable for use in the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions.

[0356] 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, glycol, water, ethanol, etc. For the use of excipients and their applications, see also "Handbook of Pharmaceutical Excipients", Fifth Edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0357] If desired, the composition may also contain a small amount of a wetting agent or emulsifier, or a pH buffer. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may contain standard pharmaceutical carriers and / or excipients, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin.

[0358] The compositions of the present invention may be in a variety of forms. These include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. Commonly preferred compositions are in the form of injectable solutions or infusible solutions. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal (ip), 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.

[0359] Pharmaceutical preparations containing the antibodies described herein can be prepared by mixing the antibodies of the present invention having the desired degree of purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of a lyophilized preparation or an aqueous solution.

[0360] The pharmaceutical composition or formulation of the present invention may also contain more than one active ingredient, which is required for the specific indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents. In some embodiments, the other therapeutic agent is selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, small molecule drugs, or immunomodulators (such as activators of costimulatory molecules or inhibitors of immune checkpoint molecules); preferably, the second therapeutic agent is a small molecule drug, preferably, the small molecule drug is selected from KRAS small molecule inhibitors, such as KRAS G12C inhibitors (such as AMG510 (Sotorasib) or GFH925), KRAS G12D (such as MRTX1133), or KRAS G12S inhibitors.

[0361] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0362] The pharmaceutical compositions of the present invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (eg, by injection or infusion).

[0363] Therapeutic compositions should generally be sterile and stable under the conditions of manufacture and storage. Compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or lyophilized forms. Sterile injectable solutions can be prepared by adding the active compound (i.e., antibody molecule) in the desired amount to a suitable solvent, followed by filtration sterilization. Dispersions are typically prepared by incorporating the active compound into a sterile vehicle containing a base dispersion medium and other ingredients. Coating agents such as lecithin may be used. In the case of dispersions, the proper fluidity of the solution can be maintained by using a surfactant. Prolonged absorption of injectable compositions can be achieved by including substances that delay absorption, such as monostearate and gelatin.

[0364] Kits containing the antibody molecules described herein are also within the scope of the present invention. The kits may contain one or more other elements, including, for example: a package insert; other reagents, such as a label or a reagent for conjugation; a pharmaceutically acceptable carrier; and a device or other materials for administration to a subject. [VIII. Combination Products or Kits]

[0365] In some embodiments, the present invention also provides a combination product comprising an antibody or antigen-binding fragment thereof, or an immunoconjugate 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, other antibodies, a cytotoxic agent, a vaccine, an anti-infective agent, a small molecule drug, or an immunomodulator (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.

[0366] In some embodiments, the combination product is used to prevent or treat diseases such as acute and chronic inflammatory diseases, infections (e.g., chronic infections), tumors, and the like. For example, the disease is a tumor (e.g., cancer) or an infection. In some embodiments, the tumor is a tumor that has escaped immune function. Preferably, the tumor is an epithelial cancer, such as a gastrointestinal tumor, a lung tumor, or a skin tumor, such as skin cancer (e.g., squamous cell carcinoma of the skin, head and neck cancer, 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, squamous cell lung 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, and the like.

[0367] In some embodiments, two or more components of the combination product may be co-administered to a subject sequentially, separately or simultaneously.

[0368] In some embodiments, the present invention also provides a kit comprising an antibody, pharmaceutical composition, immunoconjugate or combination product of the present invention, and optionally a package insert directing administration.

[0369] In some embodiments, the present invention also provides a pharmaceutical product comprising the antibody, pharmaceutical composition, immunoconjugate, or combination product of the present invention, and optionally, the pharmaceutical product further comprises a package insert for guiding administration. [IX. Uses of the Antibody Molecule of the Present Invention]

[0370] In another aspect, the present invention relates to a method for preventing or treating a tumor (e.g., cancer) in a subject, comprising administering to the subject an effective amount of an antibody molecule, pharmaceutical composition, immunoconjugate, combination product, or kit disclosed herein. In some embodiments, the tumor in the subject of the present invention includes solid tumors and hematological tumors. In some embodiments, the tumor is an immune evasion tumor. In some embodiments, the tumor is cancer.

[0371] 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 or adjacent to normal tissue of the same subject, or compared to normal cells of the same tissue of a healthy subject: (i) overexpressing wild-type EGFR (e.g., wild-type EGFR with elevated nucleic acid or protein levels) and / or expressing mutated EGFR, compared to normal cells in adjacent tissues or normal cells in the same tissue in healthy subjects, such as EGFR comprising a mutation 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) overexpressing wild-type KRAS (e.g., having elevated nucleic acid or protein levels of wild-type KRAS) compared to normal cells in adjacent tissues or compared to normal cells in the same tissue in healthy subjects, or expressing mutated KRAS, preferably, the mutated KRAS comprises a G12 or G13 mutation, such as G12D or G12C; (iii) having elevated nucleic acid or protein levels of B7-H3 compared to normal cells in adjacent tissue or compared to normal cells in the same tissue in healthy subjects; and / or (iv) The tumor cells are resistant to tyrosine kinase inhibitors, such as first-generation (erlotinib) and third-generation (osimertinib), for example, resistant to osimertinib.

[0372] In another aspect, the present invention relates to a method for preventing or treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of an antibody molecule, pharmaceutical composition, immunoconjugate, combination product, or kit disclosed herein. In one embodiment, the infectious disease is a chronic infection.

[0373] The subject can be a mammal, for example, a primate, preferably a higher primate, for example, 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 or is at risk of suffering from a disease described herein (e.g., a tumor, infection, or autoimmune disease as described herein). In certain embodiments, the subject receives or has received other treatments, such as chemotherapy and / or radiation therapy.

[0374] In other aspects, the present invention provides the use of an antibody molecule or fragment thereof or an immunoconjugate or composition or combination product or kit in the production or preparation of a drug for treating the relevant diseases or conditions mentioned herein.

[0375] In some embodiments, the antibodies or antibody fragments or immunoconjugates or compositions or combination products or kits of the invention delay the onset of a disorder and / or symptoms associated with the disorder.

[0376] In some embodiments, the antibodies or pharmaceutical compositions or immunoconjugates or combination products or kits of the present invention can also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the prevention and / or treatment described herein.

[0377] In some embodiments, treatment modalities include surgery (eg, tumor resection) or radiation therapy.

[0378] In some embodiments, the therapeutic agent is selected from an anti-angiogenic agent, a chemotherapeutic agent, an additional antibody, a cytotoxic agent, a vaccine, an anti-infective agent, a small molecule drug, or an immunomodulatory agent.

[0379] In some embodiments, the small molecule drug is selected from a KRAS small molecule inhibitor, such as a KRAS G12C inhibitor (such as AMG510 (Sotorasib) or GFH925), a KRAS G12D (such as MRTX1133) or a KRAS G12S inhibitor.

[0380] Immunomodulators include immune checkpoint inhibitors and co-stimulatory molecule activators.

[0381] In further embodiments, the antibodies or fragments thereof of the invention are used in combination with a small molecule inhibitor of KRAS, such as a KRAS G12C inhibitor (e.g., AMG510 (Sotorasib) or GFH925), a KRAS G12D (e.g., MRTX1133), or a KRAS G12S inhibitor.

[0382] In some embodiments, the antibodies or fragments thereof of the invention may be administered in combination with a therapy comprising adoptive transfer of T cells (eg, cytotoxic T cells or CTLs) expressing a chimeric antigen receptor (CAR).

[0383] In some embodiments, the antibodies or fragments thereof of the present invention can be administered in combination with an anti-tumor agent.

[0384] In some embodiments, the antibodies or fragments thereof of the present invention may be administered in combination with cytokines. The cytokines may be administered as fusion molecules with the antibody molecules of the present invention, or as separate compositions. In one embodiment, the antibodies of the present invention are administered in combination with one, two, three, or more cytokines (e.g., as fusion molecules or as separate compositions).

[0385] In some embodiments, the antibodies or fragments thereof of the present invention can be combined with conventional cancer therapies in the art, including but not limited to: (i) radiation therapy; (ii) chemotherapy, or the use of cytotoxic drugs, which generally affect rapidly dividing cells; (iii) targeted therapy, or agents that specifically affect the deregulation of cancer cell proteins; (iv) immunotherapy, or enhancing the host immune response (e.g., vaccines); (v) hormone therapy, or blocking hormones (e.g., when the tumor is hormone-sensitive), (vi) angiogenesis inhibitors, or blocking blood vessel formation and growth, and (vii) palliative care.

[0386] In some embodiments, the antibodies or fragments thereof of the present invention can be combined with conventional methods for enhancing host immune function.

[0387] The various combination therapies described above can be further combined for treatment.

[0388] Such combination therapies encompass both combined administration (where two or more therapeutic agents are contained in the same formulation or in separate formulations), and separate administration, in which case administration of the antibodies of the invention can occur before, concurrently with, and / or after administration of the other therapies, e.g., treatment modalities and / or therapeutic agents. The antibody molecule and / or other therapies, e.g., therapeutic agents or treatment modalities, can be administered during active disease or during remission or less active disease. The antibody molecule can be administered before, concurrently with, after, or during remission of the other treatment.

[0389] The antibodies of the present invention (as well as pharmaceutical compositions or immunoconjugates comprising the same, and any additional therapeutic agents) can be administered by any suitable method, including parenteral, intrapulmonary, and intranasal administration, and, if desired for local treatment, intralesional administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, such as by injection, for example, intravenous or subcutaneous, depending in part on whether the administration is short-term or chronic. Various administration schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0390] For the prevention or treatment of disease, the appropriate dosage of the antibodies of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient as a single treatment or over a series of treatments.

[0391] The dosage and treatment regimen of the antibody molecules of the invention can be determined by one of ordinary skill in the art. In some embodiments, the dosage regimen is adjusted to provide the optimal desired response (e.g., therapeutic response).

[0392] In some embodiments, the antibodies of the invention (and pharmaceutical compositions or immunoconjugates comprising the same) (alone or in combination with other therapeutic agents) can be administered twice a week, once a week, or once every two weeks.

[0393] It will be appreciated that any treatment can be performed using the immunoconjugates or compositions or combination products or kits of the invention in place of or in addition to the antibodies of the invention.

[0394] [Example 1. Construction of anti-B7-H3 / EGFR bispecific antibody] The anti-B7-H3 / EGFR bispecific antibody molecule of the present invention is assembled into an IgG1 antibody format (as shown in Figure 1) by utilizing the Innobody technology platform (Application No.: PCT / CN2021 / 143141, Invention Title: Protein Containing Heterodimeric Antibody Fc and Method for Preparing the Same) developed by Innovent Biologics (Suzhou) Co., Ltd., or by conventional methods in the art, by combining anti-B7-H3 and anti-EGFR parent antibodies. This bispecific antibody format contains four polypeptide chains and can bind to two antigens: Antigen A is EGFR and Antigen B is B7-H3.

[0395] Among them, the parent antibodies used to construct the bispecific antibody are the anti-EGFR monoclonal antibody anti-Zalutumumab (hereinafter referred to as Zalu, Publication No.: WO02100348A2, Invention Name: Human monoclonal antibodies to epidermal growth factor receptor (EGFR)) and the anti-B7-H3 monoclonal antibody, which are derived from the Innovent Biologics fusion tumor technology screening platform and the antigen binding region sequences of the anti-B7-H3 monoclonal antibodies Hz20G5.26 and Hz19A2.25 were obtained through antibody humanization (Application No.: PCT / CN2021 / 140449, Invention Title: Anti-B7-H3 Antibodies and Their Uses (Hz20G5, Hz19A2) and the antigen-binding region sequence of the anti-B7-H3 monoclonal antibody Hz5C2.9, derived from Innovent Biologics' fusion tumor technology screening platform and humanized; simultaneously, mutations in the Fc region were performed using Innobody technology to enhance heterodimer formation of specific antibodies (Application Number: PCT / CN2021 / 143141, Invention Title: Proteins Containing Heterodimer Antibody Fc and Methods for Preparing Same). Three bispecific antibodies that simultaneously bind to B7H3 and EGFR were screened and obtained, designated Hz5C2.9 / Zalu bsAb, Hz19A2.25 / Zalu bsAb, and Hz20G5.26 / Zalu bsAb bispecific antibody molecules, respectively. Specific amino acid sequence numbers are shown in Table 1 and Table A - Sequence Information. All three bispecific antibodies adopt an IgG-like structure. Schematic diagrams of their structures are shown in Figure 1. Table 1. Sequence details of B7H3 / EGFR bsAb molecules and positive control Gp120 / Zalu 1 Zhou, T., Xu, L., Dey, B. et al. Structural definition of a conserved neutralization epitope on HIV-1 gp120. Nature

[0445] , 732-737 (2007)

[0396] [Example 2. Preparation of anti-B7-H3 / EGFR bispecific antibody molecules]

[0397] 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 listed in Table 1 were respectively inserted into the vector pcDNA3.1 (Invitrogen, V790-20) to obtain the heavy chain plasmid and light chain plasmid at the anti-EGFR end, and the heavy chain plasmid and light chain plasmid at the anti-B7-H3 end, respectively.

[0398] 2. Bispecific Antibody Preparation Process Utilizing GlymaxX technology (ProBioGen AG, see patent publication number: WO2011035884A1, the entire contents of which are incorporated herein by reference in their entirety for the purposes of the present invention), the RMD enzyme (GDP-6-deoxy-D-lyxo-4-hexylose reductase, sequence: SEQ ID NO: 41) plasmid was transiently transfected into ExpiCHO (Invitrogen, A29133) cells along with the heavy and light chain plasmids of the anti-EGFR parent antibody and the heavy and light chain plasmids of the anti-B7-H3 parent antibody, respectively, via PEI MAX (Polysciences, A804355) to express the low-fucose anti-EGFR and anti-B7-H3 parent antibody proteins. After 7 days, the cell fermentation broth was harvested, filtered and clarified, and captured using Hitrap Mabselect Sure chromatography columns (GE Healthcare, 11-0034-95) to obtain antibodies against EGFR parent and B7H3 parent.

[0399] After concentration determination using the A280 method, the parent antibodies were mixed in a 1:1 molar ratio and an appropriate amount of the reducing agent, GSH, was added. The reaction was allowed to react overnight at room temperature. The reducing agent was removed by ultrafiltration, and the reaction was terminated. Fine purification was performed using a MonoS cation exchange chromatography column (GE Healthcare, 17-5168-01). The anti-B7-H3 parent solution was in 20 mM sodium phosphate buffer (pH 6.6), and the anti-EGFR parent solution was in 20 mM sodium phosphate buffer (pH 6.6) containing 1 M sodium chloride. The elution gradient was 0-50% (30 column volumes). The resulting protein solution was ultrafiltered and exchanged into PBS (Gibco, 70011-044). Purity was determined by SEC-HPLC.

[0400] Example 3. Affinity Detection of Hz20G5.26 / Zalu bsAb Bispecific Antibody Molecules The binding affinity (KD) of the bispecific antibody of the present invention to B7H3 and EGFR was determined using biofilm thin layer interferometry (BLI).

[0401] Half an hour before the experiment, depending on the number of samples, an appropriate number of AHC sensors (18-5060, Sartorius) were soaked in SD buffer (1x PBS, 0.1% BSA, 0.05% Tween-20). The above-prepared antibodies, human B7H3 (B73-H52E2, Acro Biosystem), and human EGFR (EGR-H5222, Acro Biosystem) were diluted to 100 nM, respectively.

[0402] SD buffer, antibody solution, human B7H3, and human EGFR were added to a 96-well black polystyrene microplate (Greiner, 655209). Detection was performed using a ForteBio Octet Red96e. The plate was arranged according to the sample position and the sensor position was selected. Instrument parameters were set as follows: Run steps: 120 s baseline equilibration, 100 s addition of immobilized antibody, 120 s baseline equilibration, 100 s antigen binding, and 120 s dissociation. The speed was 1000 rpm and the temperature was 30°C. After completion of the experiment, KD values ​​were analyzed using ForteBio Octet analysis software. The results are shown in Table 2 below. Table 2: Affinity detection of B7H3 / EGFR bs bispecific antibody molecules

[0403] [Example 4. Screening of anti-B7-H3 / EGFR bispecific antibody molecules] Using the Innobody technology platform, parental sequences of anti-B7-H3 and anti-EGFR antibodies were expressed and assembled into bispecific antibodies (Hz5C2.9 / Zalu bsAb, Hz19A2.25 / Zalu bsAb, and Hz20G5.26 / Zalu bsAb). The B7H3 / Zalu bsAb bispecific antibodies were tested for in vitro activity in non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) using growth inhibition assays and antibody-dependent cell-mediated cytotoxicity (ADCC) cell reports. Safety was also validated in a human skin squamous cell carcinoma cell line (A431).

[0404] Cell line source and culture medium: PC9 (NSCLC): Shanghai Yubo Biotechnology, YB-H3210D; Culture medium: MEM + 10% FBS + 1% Pen / strep TE-1 (HNSCC): CoBioer, CBP60655; Culture medium: RPMI 1640 + 10% FBS + 1% Pen / strep SK-MES-1 (NSCLC): CoBioer, CBP60152; Culture medium: MEM + 1% NEAA + 1 mM sodium pyruvate + 10% FBS + 1% Pen / strep A431: skin squamous cell carcinoma cells, ATCC, CRL-1555; culture medium: DMEM + 10% FBS + 1% Pen / strep Construction of PC9+hB7H3 and CHO-S+hB7H3 cell lines: Lentvirus+hB7H3 lentivirus (hB7H3 (UniProt, Q5ZPR3-1), Lentvirus (PPL plasmid and protein shared library, BC000141)) was constructed and packaged, and PC9 (NSCLC, Shanghai Yubo Bio, YB-H3210) and CHO-S (Thermo) were infected with Lentvirus+hB7H3 respectively. PC9+hB7H3 and CHO-S+hB7H3 stable cell lines were obtained by pressure screening and sorting.

[0405] Experimental methods:

[0406] 1. Growth inhibition assay (1) NSCLC: 1500-2500 cells / 100ul were plated on a 96-well low-adsorption plate (Corning, CLS7007-24EA) for 3D cell culture. HNSCC: 1500-2000 cells / 100ul were plated in 96-well white-bottom plates (NUNC, 136101) for 2D cell culture. (2) Add the antibody molecules prepared in advance (maximum concentration 300 nM, 3.16-fold dilution) to the corresponding cell wells, mix well, and culture at 37°C 5% CO2 for 5 days. (3) After 5 days of continuous culture for proliferation inhibition experiment, the Cell-Titer reagent (Promega, G7572) prepared in advance was added to the corresponding cell wells and kept in the dark at room temperature for 15-25 minutes. HNSCC cells were directly detected by a multifunctional microplate reader (Molecular Devices, Spectra MAXi3). (4) NSCLC: Transfer the Cell-Titer and cell mixture into a 96-well white-bottom plate (NUNC, 136101) and detect using a multifunctional microplate reader (Molecular Devices, Spectra MAXi3).

[0407] The results are shown in Figure 2. The percentage of surviving cells (Figures 2A, 2B, 2C, 2D, and 2F) represents the relative survival rate of the tested cells. The corresponding cell labels and categories are shown in Figure 2 and the accompanying legends. For example, PC9 represents a type of NSCLC, and TE-1 represents a type of HNSCC. A431 is a skin squamous cell carcinoma cell that does not respond to antibody treatment and is used to measure drug side effects.

[0408] The growth inhibition percentage (%) in Figure 2E was obtained after analysis and statistics based on the results in Figure 2 (% of surviving cells), as follows: at the highest concentration of 300 nM of the antibody, the maximum cell inhibition rate of the drug in each cell type was calculated as: 300 nM, (1-% of surviving cells) × 100%.

[0409] In HNSCC and NSCLC cell lines, the results of the proliferation inhibition experiments of three bispecific antibody molecules, Hz5C2.9 / Zalu bsAb, Hz19A2.25 / Zalu bsAb, and Hz20G5.26 / Zalu bsAb (Figures 2A, B, and C), showed that the three specific bispecific antibody molecules had obvious tumor killing effects in HNSCC and NSCLC. Among them, the bispecific antibody molecule Hz20G5.26 / Zalu bsAb not only had excellent efficacy, but also had poor in vitro efficacy in A431 (Figure 2D), indicating that Hz20G5.26 / Zalu bsAb can reduce or lower the side effects such as skin toxicity of the clinical use of EGFR antibody series drugs.

[0410] 2. ADCC cell report assay (1) According to the effector-target ratio (10:1), target cells (CHO-S+hB7H3 or NCI-H522 (CoBioer, CBP60140) constructed above) and ADCC effector cells (Promega, G7102) were evenly mixed in a 96-well white bottom plate (NUNC, 136101). (2) Add the antibody molecules prepared by dilution in advance (for the detection of CHO-S+HB7H3: the final concentration of the antibody after dilution is up to 6.25nM, 4-fold serial dilution; for the detection of NCI-H522: the final concentration of the antibody after dilution is up to 100nM, 4-fold serial dilution) to the corresponding cell wells, mix well, and culture at 37℃ 5% CO2 for 20 hours. (3) Add the Bio-glo reagent (Promega, G755B) prepared in advance into the cell well plate, keep it at room temperature away from light for 10-15 minutes, and detect it using a multi-function microplate reader (Molecular Devices, Spectra MAXi3).

[0411] The experimental results are shown in Figure 3, where the vertical axis is the multiple of IgG1 obtained with IgG1 as the control. In NSCLC and HNSCC tumor cell lines, experimental results, including cell proliferation inhibition (Figure 2) and ADCC reporter assays (Figure 3), showed that the Hz20G5.26 / Zalu bsAb exhibited the best overall in vitro efficacy. In the A431 skin squamous cell carcinoma cell line, the Hz20G5.26 / Zalu bsAb exhibited the lowest in vitro toxicity (a measure of the toxicity of EGFR inhibitors to human skin). Therefore, the bispecific antibodies of the present invention, particularly the Hz20G5.26 / Zalu bsAb, exhibit minimal toxicity to human skin and, while eliciting minimal or no toxic side effects, offer a wider effective and safe dosage, resulting in a wider efficacy window and improved safety.

[0412] Example 5. In vitro pharmacodynamic activity of the Hz20G5.26 / Zalu bsAb bispecific antibody molecule in inhibiting proliferation After the EGFR ligand binds to the EGFR on the surface of tumor cells, EGFR will form a homodimer and transmit the signal into the cell. Through various cascade reactions, the tumor cells will produce biological activities such as proliferation, invasion, metastasis and anti-apoptosis.

[0413] The anti-EGFR antigen-binding portion of the Hz20G5.26 / Zalu bsAb binds to EGFR on the surface of tumor cells. This not only blocks the binding of EGFR ligands to EGFR, inhibiting signaling and blocking the corresponding tumor bioactivity, but also stimulates EGFR endocytosis, ultimately leading to its degradation by intracellular lysosomes. EGFR is an epithelial-derived broad-spectrum tyrosine kinase receptor. The Hz20G5.26 / Zalu bsAb utilizes the parental sequence of an EGFR antibody with low affinity for EGFR, significantly reducing the toxic side effects of EGFR monoclonal antibodies on normal epithelial tissues such as the skin. B7-H3 is highly expressed in a variety of tumor cell lines and lowly expressed in normal tissues and organs. It is a TAA with extremely high selectivity and a wide expression spectrum. Hz20G5.26 / Zalu bs Ab uses the high-affinity Hz20G5.26 parent of B7-H3 on the basis of its low affinity for EGFR, which greatly improves the EGFR signal blocking activity and enhances the pharmacodynamic bioactivity and pharmacodynamic safety window of Hz20G5.26 / Zalu bs Ab.

[0414] [Example 5.1. Detection of B7-H3 and EGFR Expression in Tumor Cell Lines] The drug sensitivity of the Hz20G5.26 / Zalu bsAb in tumor cell lines is positively correlated with the abundance of receptor expression on the tumor cell surface. A selection of cell lines from NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), CRC (colon cancer), and normal cell lines were screened for relative expression of B7-H3 and EGFR. In vitro pharmacodynamic activity of the Hz20G5.26 / Zalu bsAb was tested and validated based on the abundance of B7-H3 and EGFR expression on the cell surface. The results are shown in Figure 4.

[0415] Experimental methods: 1. Resuspend various cells in FACS buffer, take 100,000-200,000 cells and spread them on a 96-well plate (Corning, CLS3799-50EA), add 10ug / ml EGFR antibody (Cetuximab), 10ug / ml B7-H3 antibody (Hz20G5.26mAb) and 10ug / ml IgG to the cell plate, and incubate at 4 degrees for 1 hour. 2. Wash twice with PBS, add the prepared APC-anti human Fc antibody (Biolegend, 410712) to the corresponding cell wells, and incubate at 4°C for 30-40 minutes. 3. Wash twice with PBS and perform FACS analysis (BD, Celesta). The results are shown in Figure 4.

[0416] Figure 4 shows the expression of B7H3 and EGFR in different cell lines. Cell lines with high B7H3 expression and dependence on the EGFR signaling pathway (such as cell lines containing EGFR mutations obtained by subsequent sequencing analysis (results not shown) or cell lines with EGFR overexpression) can be selected for activity testing of bispecific antibodies.

[0417] Example 5.2. Inhibition of proliferation of various tumor cell lines using the Hz20G5.26 / Zalu bsAb bispecific antibody molecule 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%), and esophageal cancer (43%-89%). It is a broad-spectrum anti-tumor tyrosine kinase target protein. As a broad-spectrum anti-tumor target protein, EGFR has the highest mutation rate among non-small cell lung cancer patients (the global average mutation rate is approximately 35%, and it reaches 40% in China). Among NSCLC patients with EGFR mutations, classic mutations (L858R, T790M, or Exon 19 deletion) account for 85-90%. Uncommon mutations, such as EGFR exon 20ins, T790M primary point mutations and compound mutations, and other point mutations located between exons 18 and 21, represented by G719X, and sequence duplication mutations, account for approximately 10% of EGFR mutations.

[0418] H1975-EGFRL858R / T790M / C797S, PC9+B7H3-EGFRDel19 / T790M / C797S and H322-EGFRS768_D770dup cell lines were constructed and packaged respectively. Lentvirus+EGFRDel19 / T790M / C797S and Lentvirus+EGFRS768_D770dup viruses were used to infect NCI-H1975 (ATCC, CRL-5908), PC9+hB7H3 (constructed as described above) and H322, respectively. By pressure screening, H1975-EGFRL858R / T790M / C797S, PC9+B7H3-EGFRDel19 / T790M / C797S and H322-EGFRS768_D770dup stable cell lines were obtained.

[0419] Experimental methods:

[0420] Proliferation inhibition assay (1) NSCLC: 1500-2500 cells / 100 μl were plated on a 96-well low-adhesion plate (Corning, CLS7007-24EA) for 3D cell culture. The following cell lines and specific amounts were used: NSCLC: 2000 cells / 100 μl / well: NCI-H292: Chinese Academy of Sciences Cell Bank, 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 above

[0421] CRC and HNSCC: 1500-2000 cells / 100 μL were plated in 96-well white-bottom plates (NUNC, 136101) for 2D cell culture. The following cell lines and specific amounts were used: CRC: 1500 cells / 100ul / well CCK-81:CoBioer,CBP60581 HT-55:CoBioer,CBP60012 H508:CoBioer,CBP60795 LS180:CoBioer,CBP60034 HNSCC: 1500 cells / 100ul / well TE-1:CoBioer,CBP60655 Colo680: CoBioer, CBP60452. (2) Add the antibody molecules prepared by dilution in advance to the corresponding Place the cells in a well plate, mix well, and culture in a 37°C 5% CO2 incubator for 5 days.

[0422] (3) After 5 days of continuous culture for proliferation inhibition experiment, the Cell-Titer reagent (Promega, G7572) prepared in advance was added to the cell wells and kept in the dark at room temperature for 15-25 minutes. CRC and HNSCC cells were directly detected by a multifunctional microplate reader (Molecular Devices, Spectra MAXi3).

[0423] (4) NSCLC: Transfer the Cell-Titer and cell mixture into a 96-well white-bottom plate (NUNC, 136101) and detect using a multifunctional microplate reader (Molecular Devices, Spectra MAXi3).

[0424] EGFR is a broad-spectrum anti-tumor target protein. In vitro proliferation inhibition and anti-tumor efficacy revealed that the Hz20G5.26 / Zalu bsAb exhibited anti-tumor effects in esophageal cancer cell lines (as shown in Figure 6). In the TE-1 esophageal cancer cell line, the Hz20G5.26 / Zalu bsAb exhibited superior anti-tumor efficacy compared to JNJ372. In other tumor cell lines, such as colon cancer (as shown in Figure 5), the Hz20G5.26 / Zalu bsAb achieved the same anti-tumor effects as an EGFR monoclonal antibody, particularly in the LS180 colon cancer cell line, where its in vitro anti-tumor efficacy was superior to that of an EGFR monoclonal antibody. In the NCI-H508 colon cancer cell line, the maximum anti-tumor killing rate reached 84%.

[0425] In lung cancer, not all cancers develop due to primary EGFR mutations. The average EGFR mutation rate in patients with non-small cell lung cancer is approximately 35%. Standard-of-care EGFR-TKI small molecules, such as erlotinib (Selleck Chemicals, cat: S7786) or osimertinib (Selleck Chemicals, cat: S7297), are primarily targeted at patients with sensitive gene mutations. However, these mutations often lead to drug resistance, necessitating the continuous development of new targeted agents targeting novel mutation sites. In lung cancer, the Hz20G5.26 / Zalu bsAb exhibits in vitro antitumor efficacy not only against NSCLC cell lines harboring EGFR mutations but also against wild-type EGFR and amplified EGFR NSCLC cell lines. Compared to the in vitro antitumor efficacy of JNJ-372, the Hz20G5.26 / Zalu bsAb exhibits significantly superior overall in vitro antitumor efficacy in NSCLC cell lines.

[0426] In NSCLC-EGFRWT tumor cell lines (Figure 7.1), the maximum anti-tumor killing rate of NCI-H292 in vitro was 74.2±2.8%; in NSCLC-EGFR classic mutations (L858R, T790M or Exon19 In the NSCLC-EGFR abnormal amplification tumor cell line (Figure 7.2), the maximum in vitro anti-tumor inhibition and killing rate against NCI-H1975 (EGFRL858R / T790M) (Figure 7.2B) was 51.2±1.4%, and the IC50 was 0.99nM, which was superior to JNJ-372 and EGFR monoclonal antibody in vitro; in the NSCLC-EGFR abnormal amplification tumor cell line (Figure 7.3), the maximum in vitro anti-tumor killing rate against SK-MES-1 was 60.7±15.9%, and the IC50 was 0.40nM, which was superior to JNJ-372 and EGFR monoclonal antibody in overall in vitro efficacy. In addition, SK-MES-1 and NCI-H1703, tumor cell lines with abnormal amplification of NSCLC-EGFR, were non-sensitive and resistant to first-generation (erlotinib) and third-generation (osimertinib) drugs. Osimertinib, a third-generation EGFR-TKI small molecule inhibitor that has been marketed and used to treat T790M mutations, has poor efficacy against rare EGFR exon20ins mutations. To explore the efficacy of Hz20G5.26 / Zalu bsAb against EGFR exon20ins tumors, we constructed the H322-EGFRS768_D770dup stable cell line based on NCI-H322-EGFRWT cells. The in vitro proliferation inhibition and anti-tumor efficacy showed (Figure 7.4) that Hz20G5.26 / Zalu bsAb has in vitro pharmacodynamic activity against EGFR exon20ins. With the use and treatment of EGFR-TKI small molecule inhibitors, NSCLC patients continue to develop double (such as Del19 / T790M, L858R / T790M) or triple (such as Del19 / T790M / C797S, L858R / T790M / C797S) drug-resistant mutations, which is also an important research and development direction of the next generation of EGFR-TKI. To verify the pharmacodynamic activity of Hz20G5.26 / Zalu bsAb against EGFR-TKI triple-resistant mutations, we constructed H1975-EGFRL858R / T790M / C797S and PC9+B7H3-EGFRDel19 / T790M / C797S stable cell lines. The in vitro proliferation and anti-tumor efficacy showed (Figure 7.4) that Hz20G5.26 / Zalu bsAb had certain in vitro pharmacodynamic activity against EGFRL858R / T790M / C797S and EGFRDel19 / T790M / C797S.

[0427] To further validate the in vitro pharmacodynamic activity of the Hz20G5.26 / Zalu bsAb, we compared its in vitro proliferation inhibition efficacy with that of gp120 / Zalu and gp120 / Hz20G5.26 in combination (Figure 7.5). The results showed that the in vitro efficacy of the Hz20G5.26 / Zalu bsAb was significantly superior to that of the gp120 / Zalu and gp120 / Hz20G5.26 combinations in NCI-H292 (Lung adeno, EGFRWT) and SK-MES-1 (sqNSCLC, EGFRAmp) NSCLC cell lines, as well as the LS180 CRC cell line. The results of the Hz20G5.26 / Zalu bsAb proliferation inhibition and tumor killing experiments are shown in Table 3.

[0428] Table 3. Results of Hz20G5.26 / Zalu bsAb proliferation inhibition and tumor killing experiments

[0429] Example 5.3. Synergistic Effect of the Hz20G5.26 / Zalu bsAb Bispecific Antibody Molecule Combined with a KRAS Small Molecule Inhibitor In cancer patients, besides abnormal EGFR activation, which can lead to cancer development, KRAS gene mutation activation is also a common driver of cancer. KRAS mutations are found in approximately 25% of human cancers and are significantly associated with cancer prognosis and treatment. KRAS-G12D and KRAS-G12C are common abnormal mutations in CRC and NSCLC tumors. With the current use of KRAS small molecule inhibitors, the development of acquired KRAS resistance is anticipated. Combining KRAS small molecule inhibitors with other drugs (such as immunotherapies or targeted therapies) may be a new area of ​​research and development.

[0430] Experimental methods:

[0431] Proliferation inhibition assay (1) NSCLC (H358) (Cobioer, CBP60136) and CRC (LS180) (Cobioer, CBP60034): 1500-2500 cells / 100ul were plated on 96-well low-adhesion plates (Corning, CLS7007-24EA) for 3D cell culture. (2) Add the pre-diluted antibody molecules and small molecule inhibitors to the corresponding cell wells, mix them evenly, and culture them at 37°C in 5% CO2 for 5 days. (3) After 5 days of continuous culture for proliferation inhibition experiment, the prepared Cell-Titer reagent (Promega, G7572) was added to the cell wells and kept in the dark at room temperature for 15-25 minutes. (4) Transfer Cell-Titer and the above cell mixture into a 96-well white-bottom plate (NUNC, 136101) and detect using a multifunctional microplate reader (Molecular Devices, Spectra MAXi3).

[0432] from [NCI-H358(EGFR)] [WT] [,KRAS] [G12C] The results of [)NSCLC] tumor cell lines showed that (Figure 8A), the maximum anti-tumor killing rate of Hz20G5.26 / Zalu bsAb in H358 was 66.9±1.9% in vitro, with an IC50 of 0.48 nM. The anti-tumor efficacy was far superior to that of the EGFR monoclonal antibody Zalu and JNJ-372, and to the combination of Gp120 / Zalu and Gp120 / Hz20G5.26. By combining with AMG510 (a small molecule inhibitor of KRAS-G12C) (MedChem Expresses, HY-114277), it was found that Hz20G5.26 / Zalu and AMG510 had a synergistic effect. 0.98 nM AMG510 was significantly associated with 10 nM Hz20G5.26 / Zalu. Under the action of bsAb, the killing rate of H358 cells increased from 22.51% to 67.12%, and the anti-tumor effect in H358 cells was enhanced (Figure 8B). To further demonstrate the synergistic effect, based on Figure 8B, the drug efficacy synergy score was calculated in SynergyFinder (https: / / synergyfinder.fimm.fi / synergy / 20210817124619827928 / ), and the results are shown in Figures 8C and 8D. Figure 8D shows the sum of the scores for different dimensions, with a total score of 11.895, which is greater than 10, indicating synergistic effect between the two drugs.

[0433] Results from the LS180 (EGFR R521K, KRAS G12D) CRC tumor cell line revealed that the in vitro antitumor efficacy of the Hz20G5.26 / Zalu bsAb not only outperformed the EGFR monoclonal antibodies Zalu and JNJ372, as well as the combination of Gp120 / Zalu and gp120 / Hz20G5.26 (Figure 7.5 ), but also demonstrated a significant synergistic effect when combined with MRTX1133 (a small molecule inhibitor of KRAS-G12D). At 1.56 nM Hz20G5.26 / Zalu bsAb, the anti-LS180 efficacy of 15.62 nM MRTX1133 increased from 36.45% to 61.56%, significantly enhancing the antitumor effect (Figure 9A). Synergy scores were further calculated using SynergyFinder in Figures 9B and 9C, with a total score of 37.714, indicating synergistic interaction between the two drugs.

[0434] Example 5.4. Study on the in vitro efficacy mechanism of the Hz20G5.26 / Zalu bsAb bispecific antibody for proliferation inhibition In proliferation inhibition experiments, the Hz20G5.26 / Zalu bsAb, with the help of the B7H3 antibody, further enhanced EGFR signaling blockade, resulting in a stronger anti-tumor effect. Using Western blotting on NCI-H358 cells, we investigated both EGFR signaling inhibition and EGFR ligand blockade to explore the reasons why the Hz20G5.26 / Zalu bsAb exhibits superior in vitro anti-tumor efficacy compared to EGFR monoclonal antibodies and the combined use of Gp120 / Zalu and Gp120 / Hz20G5.26.

[0435] Experimental methods:

[0436] 1. EGFR signal blocking experiment

[0437] (1) Cell treatment <1> , take NCI-H358 (Cobioer, CBP60136), 7.0E5 cells / well and plate into 6-well plates (NEST, 703011) for overnight culture. <2> , remove the culture medium (RPMI1640 complete medium (RPMI1640+10% FBS)), add serum-free medium RPMI1640 (assay medium) and starve the cells overnight. <3> , remove <2> The serum-free culture medium (assay medium) containing 200 nM Hz20G5.26 / Zalu bsAb, 200 nM Gp120 / Zalu, 200 nM Gp120 / Hz20G5.26, 200 nM Zalu mAb, and 200 nM Gp120 / Zalu + 200 nM Gp120 / Hz20G5.26 were added to the corresponding cell well plates, and the Blank group (culture medium), bsAb group (200 nM Hz20G5.26 / Zalu bsAb), Gp120 / Zalu group (200 nM Gp120 / Zalu), Gp120 / B7H3 group (200 nM Gp120 / Hz20G5.26), and Zalu group (200 nM Zalu) were set up. mAb), Gp120 / Zalu+Gp120 / B7H3 group (200nM Gp120 / Zalu+200nM Gp120 / Hz20G5.26), and treated at 37℃ 5% CO2 for 2 hours.

[0438] (2) Protein extraction and total protein quantification <1> , prepare cell lysis buffer in advance. The composition of cell lysis buffer is: 100-200ul / tube RIPA (Thermo, 89900) + 1:100 phosphatase inhibitor (abcam, ab201112) + 1:20 protease inhibitor (Roche, 11836170001). <2> , Add the cell lysis solution prepared in advance to the cells obtained in (1) above to digest the cells and lyse them on ice for 20 minutes. <3> , Pre-cool the centrifuge at 4℃, centrifuge at 12000rpm / min, 4℃ for 15min, and take the supernatant. <4> , BCA reagent (Beyotime, P0012) was used to calibrate the concentration of the extracted total protein. <5> 2. Dilute the corresponding total protein with LDS Sample buffer (Invitrogen, 2201446), denature at 70℃ for 10 minutes, and store at -40℃ for later use.

[0439] (3) Western blotting <1> , Add the protein sample obtained in (3) and protein marker (Prestained Protein Ladder (Thermo, 26620)) to the precast gel (Thermo, NP0321BOX); run the gel for about 50 minutes using an electrophoresis apparatus (BIO-RAD, TRANS SD CELL) at a voltage of 200V. <2> , take out the film and use a transfer apparatus (Invitrogen, IBCOT2) to transfer the membrane. <3> , Use the prepared blocking solution (containing 5% skim milk powder TBST) to slowly block for 1-2 hours; add EGF Receptor Rabbit mAb (CST, 4267), pEGF Receptor Rabbit mAb (CST, 3777) and GAPDH Rabbit mAb (CST, 2118) antibodies and incubate at 4 degrees overnight. <4> , wash three times with TBST, 8-10 minutes each time, and control the shaking speed at 80-100 rpm / min. <5> , incubate with labeled HRP-Goat Anti-Rabbit IgG (abcam, Ab205718) at room temperature for 1-2 hours. <6> , wash three times with TBST, 8-10 minutes each time, and control the shaking speed at 80-100 rpm / min. <7> , developed with ECL luminescent liquid (Beyotime, P0018AM) and exposed with developer (BIO-RAD, chemi Doc MP).

[0440] The results are shown in Figure 10.

[0441] 2. EGFR ligand blocking experiment (Signal blocking)

[0442] (1) Cell treatment <1> , take NCI-H358 (H358), 7.0E5 cells / well and plate them in 6-well plates (NEST, 703011) for overnight culture. <2> , remove the culture medium (RPMI1640 complete culture medium (RPMI1640+10% FBS)), add serum-free culture medium RPMI1640 and starve overnight. <3> , remove the culture medium, and add culture medium (2 cell wells), culture medium containing 200nM Hz20G5.26 / Zalu bsAb, 200nM Gp120 / Zalu, 200nM Gp120 / Hz20G5.26, 200nM Zalu mAb, and 200nM Gp120 / Zalu+200nM Gp120 / Hz20G5.26 to the corresponding cell well plates, and set up Blank group (culture medium), Control group (culture medium), bsAb group (200nM Hz20G5.26 / Zalu bsAb), Gp120 / Zalu group (200nM Gp120 / Zalu), Gp120 / B7H3 group (200nM Gp120 / Hz20G5.26), Zalu group (200nM Zalu mAb), Gp120 / Zalu+Gp120 / B7H3 group (200nM Gp120 / Zalu+200nM Gp120 / Hz20G5.26), and placed at 37℃ 5% CO2 for 1 hour. <4> One hour later, 30 nM EGF (ACRO, EGF-H52b) (Figure 11, upper panel) and 30 nM TGF-α (R&D, 239-A-100) (Figure 11, lower panel) were added to the Blank group (culture medium), bsAb group (200 nM Hz20G5.26 / Zalu bsAb), Gp120 / Zalu group (200 nM Gp120 / Zalu), Gp120 / B7H3 group (200 nM Zalu mAb), and Gp120 / Zalu+Gp120 / B7H3 group (200 nM Gp120 / Zalu+200 nM Gp120 / Hz20G5.26), respectively, and the cells were incubated at 37°C with 5% CO2 for 1 hour.

[0443] (2) Protein extraction and total protein quantification <1> , prepare cell lysis buffer in advance. The composition of cell lysis buffer is: 100-200ul / tube RIPA (Thermo, 89900) + 1:100 phosphatase inhibitor (abcam, ab201112) + 1:20 protease inhibitor (Roche, 11836170001). <2> , Add the cell lysis solution prepared in advance to the cells obtained in (1) above to digest the cells and lyse them on ice for 20 minutes. <3> , Pre-cool the centrifuge at 4℃, centrifuge at 12000rpm / min, 4℃ for 15min, and take the supernatant. <4> , BCA reagent (Beyotime, P0012) was used to calibrate the concentration of the extracted total protein. <5> 2. Dilute the corresponding total protein with LDS Sample buffer (Invitrogen, 2201446), denature at 70℃ for 10 minutes, and store at -40℃ for later use.

[0444] (3) Western blotting <1> , add the protein sample and protein marker (Prestained Protein Ladder (Thermo, 26620)) to the precast gel (Thermo, NP0321BOX); run the gel for about 50 minutes at a voltage of 200V using an electrophoresis apparatus (BIO-RAD, TRANS SD CELL). <2> , take out the film and use a transfer apparatus (Invitrogen, IBCOT2) to transfer the membrane. <3> , Use the prepared blocking solution (containing 5% skim milk powder TBST) to slowly block for 1-2 hours; add EGF Receptor Rabbit mAb (CST, 4267), pEGF Receptor Rabbit mAb (CST, 3777) and GAPDH Rabbit mAb (CST, 2118) antibodies and incubate at 4 degrees overnight. <4> , wash three times with TBST, 8-10 minutes each time, and control the shaking speed at 80-100 rpm / min. <5> , incubate with labeled HRP-Goat Anti-Rabbit IgG (abcam, Ab205718) at room temperature for 1-2 hours. <6> , wash three times with TBST, 8-10 minutes each time, and control the shaking speed at 80-100 rpm / min. <7> , developed with ECL luminescent liquid (Beyotime, P0018AM) and exposed with developer (BIO-RAD, chemi Doc MP).

[0445] The results are shown in Figure 11.

[0446] The results of the signal blocking mechanism study (Figures 10 and 11) showed that Hz20G5.26 / Zalu bsAb was stronger in both signal blocking and ligand blocking than the EGFR monoclonal antibody Zalu and the combined effects of Gp120 / Zalu and Gp120 / Hz20G5.26; this result also revealed why Hz20G5.26 / Zalu bsAb was more effective than the EGFR monoclonal antibody, Gp120 / Zalu and Gp120 / Hz20G5.26 in anti-CRC, NSCLC and HNSCC tumors in vitro.

[0447] Example 6. In vitro ADCC activity of the Hz20G5.26 / Zalu bsAb bispecific antibody molecule Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism by which anti-tumor antibodies exert their anti-tumor effects. Its principle is to use the antibody Fab segment to bind to the antigen epitope on the surface of tumor cells, and its Fc segment to bind to the FcR on the surface of cytotoxic immune cells (NK cells, macrophages, neutrophils, etc.), directly killing tumor cells through immune cell mediation. The ADCC effect is mainly achieved through the antibody Fc and the FcR Ⅲ a receptor on the NK surface.

[0448] Due to the introduction of the Hz20G5.26 parent, Hz20G5.26 / Zalu bsAb not only improved the EGFR antibody blocking activity in Hz20G5.26 / Zalu bsAb, but also improved the ADCC effect of Hz20G5.26 / Zalu bsAb as a whole (presumably because Hz20G5.26 binds to a specific epitope near the membrane end of B7H3, thus triggering a strong ADCC function); at the same time, the use of GlymaxX low-fucose technology further enhanced the ADCC effect of Hz20G5.26 / Zalu bsAb.

[0449] Therefore, the Hz20G5.26 / Zalu bsAb bispecific antibody molecule can exert multiple anti-tumor effects through two mechanisms of action: EGFR signaling blockade and ADCC. The specific experimental steps are as follows:

[0450] [Example 6.1.] ADCC reporter experiment In the ADCC reporting experiment, ADCC effector cells (Promega, G7102) were engineered to overexpress the FcR III a (V158) receptor in Jurkat T cells. These engineered cells can rapidly respond to the strength of ADCC through the NFAT-RE driver element within Jurkat T cells.

[0451] Experimental methods: (1) According to the effector-target ratio (10:1), tumor cells (cell lines listed in Table 3) and ADCC effector cells (Promega, G7102) were mixed evenly (1.5E4 cells:1.5E5 cells / well / 100ul (1.5E5:1.5E6 cells / ml)) in a 96-well white bottom plate (NUNC, 136101). (2) Add the pre-diluted antibody molecules to the corresponding cell wells, mix evenly, and culture at 37°C in 5% CO2 for 20 hours. (3) Add the Bio-glo reagent (Promega, G755B) prepared in advance into the cell well plate, keep it at room temperature away from light for 10-15 minutes, and detect it using a multi-function microplate reader (Molecular Devices, Spectra MAXi3).

[0452] We selected different EGFR types of NSCLC cell lines (EGFR wild-type, EGFR aberrantly amplified, EGFR mutant, EGFR wild-type and KRAS mutant) and verified the ADCC activity of Hz20G5.26 / Zalu bsAb by ADCC reporter experiments. The ADCC reporter experimental results (Figure 12 and Table 4) showed 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, the experimental results in H292 (NSCLC-EGFR wild-type) and H358 (NSCLC-EGFR wild-type and KRAS mutant) showed 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 4. Results of Hz20G5.26 / Zalu bsAb ADCC reporter gene assay

[0454] [Example 6.2] huPBMC ADCC experiment The above-mentioned ADCC reporting experimental results showed that the ADCC activity of Hz20G5.26 / Zalu bsAb was stronger than that of JNJ372, EGFR monoclonal antibody Zalu, Gp120 / Zalu and Gp120 / Hz20G5.26 combination; in order to more truly and effectively reflect the ADCC activity of Hz20G5.26 / Zalu bsAb, we designed a huPBMC ADCC experiment and used normal human PBMC to verify ADCC activity.

[0455] Experimental methods: (1) Preheat CTS medium (Gibco, A3021002) at 37°C, take huPBMC (Miaotong Biotechnology, PB100C-W) and quickly melt it in a water bath. Slowly add the cells into 8 ml of CTS medium (containing 1% DNase). (2) Centrifuge at 300g for 8min, remove the supernatant, resuspend with 30ml CTS (containing 10uL DNase), transfer to a T75 culture flask, and incubate at 37℃ overnight. (3) Take the suspended cells cultured overnight, centrifuge at 300g / 8min, remove the supernatant, and adjust the cell density with CTS; according to the effector cell (huPBMC) and target cell (tumor cells in Table 5) effector-target ratio of 50:1, spread huPBMC and pre-prepared tumor cells (Target: huPBMC = 1.5E4 / 7.5E5 cells / well / 100ul) on a 96-well low-adsorption plate (Corning, CLS7007-24EA). (4) Add the diluted antibody drug to the corresponding cell well plate and culture at 37℃ 5% CO2 for 8 hours. (5) Centrifuge at 300 g for 5 min, transfer 50 μl of supernatant to a 96-well transparent flat-bottom plate (NUNC, 136101); add 50 μl of pre-prepared LDH reagent (Promega, G1780) to the corresponding wells of the plate, and let it stand at room temperature in the dark for 15-30 min. (6) Take 50ul LDH Stop Solution (Promega, G1780), read at 490nm, and detect with a multi-function microplate reader (Molecular Devices, Spectra MAXi3).

[0456] The results are shown in Figure 13 and Table 5. The huPBMC ADCC assay results of Hz20G5.26 / Zalu bsAb more effectively reflect the actual efficacy results. The huPBMC ADCC results (Figure 13 and Table 5) showed that the huPBMC ADCC activity of Hz20G5.26 / Zalu bsAb was generally consistent with the reported ADCC activity. Across various NSCLC-EGFR tumor cell lines, the huPBMC ADCC activity of Hz20G5.26 / Zalu bsAb was stronger than that of JNJ373 and EGFR monoclonal antibodies, and stronger than the combined ADCC activity of Gp120 / Zalu and Gpl20 / Hz20G5.26. Through huPBMC ADCC, although the anti-tumor killing rate of Hz20G5.26 / Zalu bsAb was the lowest against H1975, the killing rate was still 36.53%. The anti-tumor killing rate of Hz20G5.26 / Zalu bsAb was the highest against H322, reaching 100%.

[0457] Therefore, Hz20G5.26 / Zalu bsAb can further exert its anti-tumor activity in many cancer tumors by enhancing EGFR signal blocking and participating in ADCC through immune cells.

[0458] Table 5. Hz20G5.26 / Zalu bsAb huPBMC ADCC assay results

[0459] Example 6. In vitro safety study of the Hz20G5.26 / Zalu bsAb bispecific antibody molecule EGFR is an epithelial-derived epidermal growth factor. Besides overexpression or abnormal activation, which can lead to cancer, EGFR is also expressed in some epithelial cells and keratinocytes. Compared to the adverse reactions of EGFR-TKI small-molecule inhibitors, which include rash, diarrhea, paronychia, oral mucositis, liver damage, and interstitial lung disease, EGFR monoclonal antibodies also have certain adverse reactions, primarily manifesting as skin toxicity, such as pustular rash, paronychia, and dry and itchy skin. This significantly impacts the quality of life and treatment compliance of cancer patients.

[0460] We selected human skin squamous cell carcinoma cell line (A431, ATCC, CRL-1555) and human skin keratinocytes (HaCat, Cell lines service, 300493) cells as Hz20G5.26 / Zalu bs Ab adverse reaction research models and explored the tolerance of Hz20G5.26 / Zalu bs Ab in vitro experiments.

[0461] Experimental methods (1) A431 and HaCat cells (1500-2000 cells / 100ul) were plated on 96-well low-adsorption plates (Corning, CLS7007-24EA) for 3D cell culture. The culture medium used was DMEM+10% FBS+1% Pen / strep. (2) Add the pre-diluted antibody molecules to the corresponding cell wells, mix evenly, and culture in a 37°C 5% CO2 incubator for 5 days. (3) After 5 days of continuous culture for proliferation inhibition experiment, the prepared Cell-Titer reagent (Promega, G7572) was added to the cell wells and kept in the dark at room temperature for 15-25 minutes. (4) Transfer the Cell-Titer and cell mixture into a 96-well white-bottom plate (NUNC, 136101) and detect using a multifunctional microplate reader (Molecular Devices, Spectra MAXi3).

[0462] The results are shown in Figure 14. The experimental results showed that the in vitro efficacy of Hz20G5.26 / Zalu bsAb on A431 and HaCat cells was much lower than that of EGFR monoclonal antibodies, indicating that the cells have a high tolerance to Hz20G5.26 / Zalu bsAb and have far fewer adverse reactions than EGFR monoclonal antibodies. Furthermore, the cells also have better tolerance to Hz20G5.26 / Zalu bsAb than JNJ372.

[0463] Therefore, on the basis of reducing the affinity for EGER, Hz20G5.26 / Zalu bs Ab applied the Hz20G5.26 parent with high affinity for B7H3, thereby improving the pharmacodynamic bioactivity and pharmacodynamic safety window of Hz20G5.26 / Zalu bs Ab.

[0464] Example 7. In vivo pharmacodynamic activity of the Hz20G5.26 / Zalu bsAb bispecific antibody molecule To demonstrate the in vivo efficacy of the Hz20G5.26 / Zalu bispecific antibody, the antitumor efficacy of the Hz20G5.26 / Zalu bispecific antibody of the present invention was determined using Balb / c nude mice inoculated with the NSCLC-EGFRWT tumor cell line NCI-H292 (ATCC). SPF-rated female Balb / c nude mice (purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were used, with a certification number of 110011211108430747.

[0465] NCI-H292 cells were routinely subcultured for subsequent in vivo experiments. Cells were harvested by centrifugation and resuspended in a mixture of 1× PBS and Matrigel (Corning) in equal proportions to prepare a cell suspension at a concentration of 20 × 106 cells / ml. 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 an NCI-H292 tumor-bearing mouse model.

[0466] Four days after tumor cell inoculation, the tumor volume of each mouse was measured and the mice were divided into groups (5 mice per group). The dosage and method of administration are shown in Table 6.

[0467] Table 6: Grouping, dosage and mode of administration of in vivo experiments

[0468] h-IgG, JNJ372, Gp120 / Zalu, Hz20G5.26 / Zalu, and Zalu mAb were all used at a concentration of 0.5 mg / ml and administered every 3-4 days for a total of four doses (Q3-4D x 4). Dosing was performed on days 4, 8, 12, and 15 after inoculation of NCI-H292 cells. Tumor volume and body weight were monitored twice weekly, as shown in Figure 15A, until day 59. Because some groups experienced early tumor-related mortality, the relative tumor inhibition rate (TGI%) was calculated based on the tumor volume on day 22 after inoculation using the following formula: TGI%=100%*(tumor volume of control group-tumor volume of treatment group) / (tumor volume of control group-tumor volume of control group before drug administration).

[0469] Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated using the following formula: V=L*W2 / 2.

[0470] Body weight was measured using an electronic balance. Mice with tumor volumes exceeding 2000 mm3 or body weight loss exceeding 20% ​​were euthanized.

[0471] Tumor inhibition results are shown in Table 7. On day 22 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. One mouse in each of the Hz20G5.26 / Zalu and Zalu mAb groups achieved complete tumor remission. Mouse survival curves are shown in Figure 15B, demonstrating that Hz20G5.26 / Zalu significantly prolonged mouse survival. In summary, the antitumor efficacy of Hz20G5.26 / Zalu in NCI-H292 tumor-bearing mice is comparable to that of the parental Zalu mAb and superior to that of JNJ372 and the untargeted GP120 / Zalu.

[0472] At the same time, the results of monitoring the weight of mice (Figure 15C) showed that there was no significant decrease in the weight of mice in the Hz20G5.26 / Zalu group within 59 days after inoculation.

[0473] Table 7: Antitumor efficacy statistics

[0474] To demonstrate the in vivo efficacy of the Hz20G5.26 / Zalu bispecific antibody against NSCLC harboring abnormally amplified EGFR, the anti-tumor efficacy of the Hz20G5.26 / Zalu bispecific antibody of the present invention was determined using Balb / c nude mice inoculated with SK-MES-1 cells (Nanjing Kebai). SPF-rated female Balb / c nude mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used, with a certification number of 110011211108966881.

[0475] SK-MES-1 cells were routinely subcultured for subsequent in vivo experiments. Cells were harvested by centrifugation and mixed with PBS (1×) and Matrigel (Corning) in equal proportions to prepare a cell suspension at a concentration of 20 × 106 cells / ml. 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 an SK-MES-1 tumor-bearing mouse model.

[0476] Six days after tumor cell inoculation, the tumor volume of each mouse was measured and the mice were divided into groups (7 mice per group). The dosage and method of administration are shown in Table 8.

[0477] Table 8: Grouping, dosage and method of administration of in vivo experiments

[0478] h-IgG, Hz20G5.26 / Zalu, JNJ372, Zalu mAb, and Gp120 / Zalu were all used at a concentration of 0.1 mg / ml. Dosing was performed once on day 6 after SK-MES-1 cell inoculation. Tumor volume and body weight were monitored twice weekly for 24 days, as shown in Figure 16A.

[0479] The relative tumor inhibition rate (TGI%) was calculated on the 24th day after inoculation using the following formula: TGI%=100%*(tumor volume of control group-tumor volume of treatment group) / (tumor volume of control group-tumor volume of control group before drug administration).

[0480] Tumor volume measurement: Vernier calipers were used to measure the maximum longitudinal axis (L) and maximum width axis (W) of the tumor, and tumor volume was calculated using the following formula: V = L * W2 / 2. Body weight was measured using an electronic balance.

[0481] The tumor inhibition rates are shown in Table 9. On day 24 post-inoculation, compared to the h-IgG 1 mg / kg group, the tumor inhibition rates for Hz20G5.26 / Zalu, JNJ372, Zalu mAb, and Gp120 / Zalu were 143%, 122%, 145%, and 113%, respectively. Simultaneous monitoring of mouse body weight (Figure 16B) revealed no significant differences in mouse body weight on day 24 post-inoculation.

[0482] Table 9: Antitumor efficacy statistics on day 24

[0483] To further demonstrate the in vivo efficacy advantages of the Hz20G5.26 / Zalu bispecific antibody, the anti-tumor efficacy of the Hz20G5.26 / Zalu bispecific antibody of the present invention was determined using Balb / c nude mice inoculated with SK-MES-1 cells (Nanjing Kebai). SPF-rated female Balb / c nude mice (purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were used, with a certification number of 110011221103705231.

[0484] SK-MES-1 cells were routinely subcultured for subsequent in vivo experiments. Cells were harvested by centrifugation and mixed with PBS (1×) and Matrigel (Corning) in equal proportions to prepare a cell suspension at a concentration of 25 × 106 cells / ml. 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 an SK-MES-1 tumor-bearing mouse model.

[0485] Fourteen days after tumor cell inoculation, the tumor volume of each mouse was measured and the mice were divided into groups (6 mice per group). The dosage and method of administration are shown in Table 10.

[0486] Table 10: Grouping, dosage and mode of administration of in vivo experiments

[0487] The h-IgG concentration was 0.06 mg / ml, and the concentrations of Hz20G5.26 / Zalu, Gp120 / Zalu, and Gp120 / Hz20G5.26 were all 0.03 mg / ml. Dosing was performed once on day 14 after SK-MES-1 cell inoculation. Tumor volume and body weight of mice were monitored twice weekly until day 35, as shown in Figure 17A.

[0488] The relative tumor inhibition rate (TGI%) was calculated on the 35th day after inoculation using the following formula: TGI%=100%*(tumor volume of control group-tumor volume of treatment group) / (tumor volume of control group-tumor volume of control group before drug administration).

[0489] Tumor volume measurement: Vernier calipers were used to measure the maximum longitudinal axis (L) and maximum width axis (W) of the tumor, and tumor volume was calculated using the following formula: V = L * W2 / 2. Body weight was measured using an electronic balance.

[0490] Tumor inhibition rates are shown in Table 11. On day 35 post-inoculation, compared to the h-IgG 0.6 mg / kg group, the tumor inhibition rates for 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-targeted monoclonal antibody Gp120 / Zalu, Gp120 / Hz20G5.26, or the combination of the two, demonstrating the unique mechanism of action of Hz20G5.26 / Zalu as a bispecific antibody, namely, the synergistic effect of Hz20G5.26 on EGFR signaling blocked by zalu. Simultaneous monitoring of mouse body weight (Figure 17B) showed no significant difference in mouse body weight on day 24 post-inoculation.

[0491] Table 11: Antitumor efficacy statistics on day 35

[0492] Example 8. In vivo pharmacodynamic activity of the Hz20G5.26 / Zalu bsAb bispecific antibody molecule combined with a KRAS small molecule inhibitor To demonstrate the in vivo efficacy of the Hz20G5.26 / Zalu bispecific antibody in combination with a small molecule KRAS inhibitor, NOG mice were inoculated with the KRASG12C mutant NSCLC cell line NCI-H358 (Nanjing Kebai) to determine the antitumor efficacy of the Hz20G5.26 / Zalu bispecific antibody combined with the KRAS small molecule inhibitor AMG510. SPF female NOG mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were used, with a certificate number of 1100112211001153625.

[0493] NCI-H358 cells were routinely subcultured for subsequent in vivo experiments. Cells were harvested by centrifugation and resuspended in a mixture of 1× PBS and Matrigel (Corning) in equal proportions to prepare a cell suspension at a concentration of 25 × 106 cells / ml. On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of NOG mice to establish an NCI-H358 tumor-bearing mouse model.

[0494] Six days after tumor cell inoculation, the tumor volume of each mouse was measured and the mice were divided into groups (7 mice per group). The dosage and method of administration are shown in Table 12.

[0495] Table 12: Grouping, dosage and mode of administration of in vivo experiments

[0496] The h-IgG was used at a concentration of 2 mg / ml, and the Hz20G5.26 / Zalu, JNJ372, and Zalu mAbs were all used at a concentration of 1 mg / ml. Dosing was performed every 3-4 days for a total of 4 doses (Q3-4D x 4). Dosing was performed on days 6, 9, 12, and 15 after inoculation of NCI-H358 cells, and the tumor volume and body weight of the mice were monitored twice weekly. AMG510 was used at a concentration of 1 mg / ml, starting on day 6 after inoculation of NCI-H358 cells and administered daily for a total of 14 doses (QD x 14). Tumor volume and body weight were monitored twice weekly, and body weight was monitored daily during dosing and twice weekly after dosing. Monitoring was continued until day 33, as shown in Figure 18A. The relative tumor inhibition rate (TGI%) was calculated on day 33 after inoculation using the following formula: TGI%=100%*(tumor volume of control group-tumor volume of treatment group) / (tumor volume of control group-tumor volume of control group before drug administration).

[0497] Tumor volume measurement: Vernier calipers were used to measure the maximum longitudinal axis (L) and maximum width axis (W) of the tumor, and tumor volume was calculated using the following formula: V = L * W2 / 2. Body weight was measured using an electronic balance.

[0498] Table 13 shows the tumor inhibition rates. On day 33 after inoculation, compared to the h-IgG 20 mg / kg group, the tumor inhibition rates for AMG510, Hz20G5.26 / Zalu, AMG510 + Hz20G5.26 / Zalu, JNJ372, and Zalu mAbs were 89%, 110%, 130%, 82%, and 111%, respectively. Among all groups, the combination of Hz20G5.26 / Zalu and AMG510 demonstrated the best antitumor efficacy in NCI-H358 tumor-bearing mice, demonstrating a synergistic effect.

[0499] At the same time, the results of monitoring the weight of mice (Figure 18B) showed that the administration of AMG510 caused the weight of mice to gradually decrease, and the weight recovered after the administration was suspended. On the 33rd day, the weight of mice in each group was normal.

[0500] Table 13: Tumor inhibition rate on day 33

[0501] [Sequence Listing: Sequence Information of Anti-B7-H3 / EGFR Bispecific Antibody]

[0502] TW202402801A_112123489_SEQL.xml

Claims

1. A bispecific antibody binding to EGFR and B7-H3, 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 B7-H3, 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, wherein, HCDR1 is composed of the amino acid sequence shown in SEQ ID NO: 15, HCDR2 is composed of the amino acid sequence shown in SEQ ID NO: 16, HCDR3 is composed of the amino acid sequence shown in SEQ ID NO: 17, LCDR1 is composed of the amino acid sequence shown in SEQ ID NO: 18, LCDR2 is composed of the amino acid sequence shown in SEQ ID NO: 19, and LCDR3 is composed of the amino acid sequence shown in SEQ ID NO:

20. The first antigen-binding region comprises HCDR1, HCDR2, and HCDR3 with a heavy chain variable region VH, and LCDR1, LCDR2, and LCDR3 with a light chain variable region VL. Specifically, HCDR1 of the first antigen-binding region is composed of the amino acid sequence shown in SEQ ID NO: 9; HCDR2 is composed of the amino acid sequence shown in SEQ ID NO: 10; HCDR3 is composed of the amino acid sequence shown in SEQ ID NO: 11; and LCDR1 of the first antigen-binding region is composed of the amino acid sequence shown in SEQ ID NO: 12; LCDR2 is composed of the amino acid sequence shown in SEQ ID NO:

15. The amino acid sequence of NO: 13 is composed of the amino acid sequence of SEQ ID NO: 14; and LCDR3 is composed of the amino acid sequence of SEQ ID NO:

14.

2. The bispecific antibody as claimed in claim 1, wherein the second antigen-binding region comprises a heavy chain variable region VH, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:

3.

3. The bispecific antibody as claimed in claim 1 or 2, wherein the second antigen-binding region comprises a light chain variable region VL, wherein the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:

4.

4. The bispecific antibody as claimed in claim 1, wherein the second 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: 3, and the VL comprises the amino acid sequence shown in SEQ ID NO:

4.

5. The bispecific antibody as claimed in claim 1, wherein the second antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL are composed of the amino acid sequences shown below: SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

6. The bispecific antibody as claimed in claim 1, wherein the first antigen-binding region comprises a heavy chain variable region VH, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:

1.

7. The bispecific antibody as claimed in claim 1 or 6, wherein the first antigen-binding region comprises a light chain variable region VL, wherein the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:

2.

8. The bispecific antibody as claimed in claim 1, 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, and the VL comprises the amino acid sequence shown in SEQ ID NO:

2.

9. The bispecific antibody as claimed in claim 1, wherein the first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL are composed of the amino acid sequences shown below: SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

10. The bispecific antibody as claimed in claim 1, wherein the first antigen-binding region comprises a VH containing an amino acid sequence as shown in SEQ ID NO: 1 or composed of such amino acid sequences and a VL containing an amino acid sequence as shown in SEQ ID NO: 2 or composed of such amino acid sequences, and the second antigen-binding region comprises a VH and a VL containing an amino acid sequence as shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

11. The bispecific antibody as claimed in any one of claims 1, 2, 4 to 6 and 8 to 10, comprising an Fc region.

12. The bispecific antibody as claimed in claim 11, wherein, The Fc region has low fucosylation.

13. The bispecific antibody as described in claim 12, wherein, The Fc region has low fucosylation obtained by processing with GlymaxX technology.

14. The bispecific antibody as claimed in claim 11, 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, wherein the first Fc region and the second Fc region are human IgG1 Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc, respectively.

15. The bispecific antibody as described in claim 14, wherein, The Fc region contains or consists of an amino acid sequence SEQ ID NO: 46 or 47.

16. The bispecific antibody as claimed in claim 14, wherein a mutation promoting heterodimerization of the first Fc region and the second Fc region is introduced into the first Fc region and the second Fc region.

17. The bispecific antibody as claimed in claim 16, wherein the CH3 of one Fc region contains S364R and D399K mutations, and the CH3 mutation of the other Fc region contains Y349T, K370S and K409D mutations.

18. The bispecific antibody as claimed in claim 17, wherein one Fc region polypeptide comprises or is composed of the amino acid sequence shown in SEQ ID NO: 49 or 50, and the other Fc region polypeptide comprises or is composed of the amino acid sequence shown in SEQ ID NO: 52 or 53.

19. The bispecific antibody as claimed in claim 16, wherein the mutation is introduced based on the Knob-into-Hole technique, wherein the corresponding Knob mutation and Hole mutation are introduced in the first Fc region and the second Fc region.

20. The bispecific antibody as claimed in claim 19, wherein a) one Fc region polypeptide contains the mutant T366W, and the other Fc region polypeptide contains T366S, L368A, and Y407V (numbered in accordance with the EU index), or b) one Fc region contains amino acid substitutions S354C and T366W, and the other Fc region contains amino acid substitutions Y349C, T366S, L368A, and Y407V (numbered in accordance with the EU index).

21. The bispecific antibody as claimed in any one of claims 1, 2, 4 to 6, and 8 to 10, wherein the heavy chain variable region of the first and / or second antigen-binding region may also be linked to one or two heavy chain constant regions comprising CH1 and Fc regions, linked via or not via hinge regions, wherein, The C-terminus of the variable region of the heavy chain is connected to the N-terminus of CH1 of the constant region of the heavy chain, wherein the constant region of the heavy chain is the heavy chain constant region of human IgG1, human IgG2, human IgG3 or human IgG4.

22. The bispecific antibody as claimed in claim 21, wherein the CH1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:

42.

23. The bispecific antibody as claimed in claim 21, wherein the light chain variable region of the first and / or second antigen-binding region may also be linked to the light chain constant region, wherein, The C-terminus of the variable region of the light chain is connected to the N-terminus of the constant region of the light chain.

24. The bispecific antibody as claimed in claim 23, wherein the light chain constant region is a kappa light chain constant region or a lambda light chain constant region.

25. The bispecific antibody as claimed in claim 23, wherein the light chain constant region comprises or is composed of the amino acid sequence shown in SEQ ID NO:

54.

26. A bispecific antibody as claimed in any one of claims 1, 2, 4 to 6, 8 to 10 and 23, comprising 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 a heavy chain variable region and a first heavy chain constant region of a first antigen-binding region; light chain 1 comprises a light chain variable region and a first light chain constant region of a first antigen-binding region; and heavy chain 2 comprises a heavy chain variable region and a second heavy chain constant region of a second antigen-binding region; light chain 2 comprises a light chain variable region and a second light chain constant region of a second antigen-binding region.

27. The bispecific antibody as claimed in claim 26, wherein the heavy chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:

33.

28. The bispecific antibody as claimed in claim 26, wherein the light chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:

34.

29. The bispecific antibody as claimed in claim 26, wherein the heavy chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:

35.

30. The bispecific antibody as claimed in claim 26, wherein the light chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:

36.

31. The bispecific antibody as claimed in claim 30, wherein the heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO: 35; and the light chain 2 comprises the amino acid sequence shown in SEQ ID NO:

36.

32. The bispecific antibody as claimed in claim 26, wherein 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; and the heavy chain 2 and the light chain 2 each comprise the amino acid sequences shown in SEQ ID NO: 35 and SEQ ID NO: 36, respectively.

33. The bispecific antibody as claimed in claim 26, wherein the heavy chain 1 is composed of the amino acid sequence shown in SEQ ID NO: 33, and the light chain 1 is composed of the amino acid sequence shown in SEQ ID NO: 34, the heavy chain 2 is composed of the amino acid sequence shown in SEQ ID NO: 35, and the light chain 2 is composed of the amino acid sequence shown in SEQ ID NO:

36.

34. An isolated nucleic acid encoding a bispecific antibody binding EGFR and B7-H3 as described in any one of claims 1 to 33.

35. An expression vector comprising the nucleic acid as described in claim 34.

36. A host cell comprising a nucleic acid as described in claim 34 or an expression vector as described in claim 35.

37. The host cell as described in claim 36, wherein, The host cell is either a 293 cell or a CHO cell.

38. The host cell as described in claim 36 or 37, which is glycoengineered to express an RMD enzyme, or which contains nucleic acid encoding an RMD enzyme.

39. The host cell of claim 38, wherein the RMD enzyme comprises or is composed of the amino acid sequence shown in SEQ ID NO: 41 or an amino acid sequence having at least 90% identity with it.

40. A method for preparing a bispecific antibody binding to EGFR and B7-H3, the method comprising culturing a host cell as described in any one of claims 36 to 39 under conditions suitable for expressing a nucleic acid encoding the bispecific antibody as described in any one of claims 1 to 33, and isolating the antibody or an antigen-binding fragment thereof.

41. An immunoconjugate comprising a bispecific antibody as described in any one of claims 1 to 33 conjugated to a therapeutic or diagnostic agent.

42. A pharmaceutical composition comprising the bispecific antibody as described in any one of claims 1 to 33 or the immunoconjugate as described in claim 41, and pharmaceutical excipients.

43. The pharmaceutical composition as claimed in claim 42, further comprising a second therapeutic agent; wherein, The second therapeutic agent is a small molecule inhibitor of KRAS.

44. A pharmaceutical combination product comprising a bispecific antibody as described in any one of claims 1 to 33, an immunoconjugate as described in claim 41, or a pharmaceutical composition as described in claim 42, and one or more second therapeutic agents, wherein, The second therapeutic agent is selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, small molecule drugs, or immunomodulators.

45. The pharmaceutical combination product as described in claim 44, wherein, The second therapeutic agent was selected from KRAS small molecule inhibitors.

46. ​​The pharmaceutical combination product as described in claim 45, wherein, The KRAS small molecule inhibitor is selected from KRAS G12C inhibitors, KRAS G12D or KRAS G12S inhibitors.

47. The pharmaceutical combination product as claimed in claim 45, wherein, The KRAS small molecule inhibitor is selected from AMG510 (Sotorasib), GFH925 or MRTX1133.

48. Use of a bispecific antibody as described in any one of claims 1 to 33, or an immunoconjugate as described in claim 41, or a pharmaceutical composition as described in claim 42, in the preparation of a medicament for use in the prevention or treatment of a tumor or infectious disease in a subject.

49. The use as described in claim 48, wherein, The drug is also used in combination with one or more other therapies, which include treatment methods and / or other therapeutic agents.

50. The use as described in claim 49, wherein, The treatment may include surgical treatment and / or radiation therapy, or the treatment agent may be selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, small molecule drugs, or immunomodulators.

51. The use as described in claim 49, wherein, The second therapeutic agent was selected from KRAS small molecule inhibitors.

52. The use as described in claim 51, wherein, The KRAS small molecule inhibitor is selected from KRAS G12C inhibitors, KRAS G12D or KRAS G12S inhibitors.

53. The use as described in claim 51, wherein, The KRAS small molecule inhibitor is selected from AMG510 (Sotorasib), GFH925 or MRTX1133.

54. The use as claimed in any one of claims 48 to 53, wherein the tumor is cancer.

55. The use as described in any one of claims 48 to 53, wherein, The tumor is either a solid tumor or a hematologic malignancy.

56. The use as described in claim 54, wherein, This cancer is an epithelial cancer.

57. The use as described in claim 54, wherein, The tumor could be a gastrointestinal tumor, a lung tumor, or a skin tumor.

58. The use as described in claim 54, wherein, The cancer could be skin cancer, esophageal cancer, colorectal cancer, or lung cancer.

59. The use as described in claim 54, wherein, The cancer could be squamous cell carcinoma of the skin, head and neck cancer, squamous cell carcinoma of the esophagus, colon cancer, rectal cancer, colorectal cancer, non-small cell lung cancer, squamous cell carcinoma of the lung, or adenocarcinoma of the lung.

60. The use as described in claim 59, wherein, The head and neck cancer is a squamous cell carcinoma of the head and neck.

61. The use as claimed in any one of claims 48 to 53, wherein the tumor cells of the tumor (i) overexpress wild-type EGFR and / or express mutant EGFR compared to normal cells in adjacent tissues or normal cells in the same tissue in a healthy subject; (ii) overexpress wild-type KRAS or express mutant KRAS compared to normal cells in adjacent tissues or normal cells in the same tissue in a healthy subject; (iii) have elevated levels of B7-H3 nucleic acid or protein compared to normal cells in adjacent tissues or normal cells in the same tissue in a healthy subject; and / or (iv) the tumor cells are resistant to tyrosine kinase inhibitors.

62. The use as described in claim 61, wherein, (i) Overexpression of wild-type EGFR is wild-type EGFR with elevated nucleic acid or protein levels; and / or the mutated EGFR contains one or more mutations selected from R521K, L858R, T790M, G719X, C797S, Y1069C, Exon19 deletion (Del19), and / or Exon20ins; (ii) Overexpression of wild-type KRAS is wild-type KRAS with elevated nucleic acid or protein levels; (iii) the mutated KRAS contains a mutation at position G12 or G13; and / or (iv) the tyrosine kinase inhibitor is selected from those resistant to first-generation (erlotinib) or third-generation (osimertinib).

63. The use as described in claim 61, wherein, (i) the EGFR mutation includes R521K / Y1069C, R521K, L858R / T790M / C797S, Del19 / T790M / C797S or S768_D770dup mutations; (ii) the KRAS mutation includes G12D or G12C; and / or (iii) the tumor cells are resistant to osimertinib.

64. The use as described in claim 61, wherein the tumor cells of the tumor express mutated EGFR and mutated KRAS.

65. The use as claimed in claim 64, wherein the tumor cells of the tumor contain EGFR with an R521K mutation and KRAS with a G120D mutation.

66. A method for detecting antigens EGFR and / or B7-H3 in a sample, the method comprising (a) contacting the sample with a bispecific antibody as described in any one of claims 1 to 33; and (b) detecting the formation of a complex between the antibody or its antigen-binding fragment and EGFR and / or B7-H3.

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