Bispecific antibodies that bind egfr and b7-h3

Abstract

Provided is an artificially designed bispecific antibody molecule, in particular an anti-B7-H3 / EGFR bispecific antibody molecule, which can simultaneously bind to B7-H3 and EGFR.

Description

Invention Field

[0001] This invention generally relates to the fields of immunology and antibody engineering. Specifically, it relates to novel, artificially designed bispecific antibody molecules, particularly bispecific antibodies that simultaneously bind to EGFR and B7-H3, polynucleotides encoding said antibody molecules or their strands, vectors containing said polynucleotides, host cells containing said polynucleotides or vectors, immunoconjugates and pharmaceutical compositions containing said antibody molecules, and the use of said antibody molecules in the immunotherapy, prevention, and / or diagnosis of diseases. Background of the Invention

[0003] Globally, lung cancer is one of the most common types of cancer, accounting for approximately 80%-85% of all lung cancer cases. NSCLC can be classified into common types such as lung adenocarcinoma, lung squamous cell carcinoma, and large cell lung cancer. Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor, a large transmembrane glycoprotein with a molecular weight of approximately 170 kDa. It belongs to the ErbB receptor family and is the most common oncogenic driver gene in non-small-cell lung cancer (NSCLC). Approximately 40% of NSCLC cases in Asian patients and about 15% in Caucasian populations are caused by EGFR mutations (Gower A, Wang Y, Giaccone G. Oncogenic drivers, targeted therapies, and acquired resistance in non-small-cell lung cancer. J. Mol. Med. 2014; 92:697-707.). EGFR overexpression and / or aberrant mutation activation are also observed in other cancers, such as epithelial-derived cancers like kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, and head and neck cancer.

[0004] For the abnormal activation and amplification of EGFR in NSCLC, there are currently two main targeted therapies: EGFR-TKI small molecule inhibitors (such as gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, and ametinib) and large monoclonal antibodies (cetuximab, panitumumab, nexituzumab, and nimotuzumab). Although EGFR-TKI small molecule inhibitors remain the standard treatment for NSCLC, they primarily target patients with tyrosine kinase activity mutations. Resistance to these inhibitors often results from mutations in the target gene, necessitating continuous development of new targeted drugs targeting novel mutation sites. This significantly limits the clinical application of these drugs and represents a severe challenge for the entire EGFR-TKI small molecule industry.

[0005] The EGFR activation mutation region mainly occurs in the tyrosine kinase domain of EGFR exons 18-21 (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 resistance 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. Thus, they can exert antitumor killing effects through multiple mechanisms of action.

[0006] B7-H3 (also known as CD276) is a type I transmembrane protein (Picarda E, Ohaegbulam KC, Zang X. Molecular pathways: targeting B7–H3(CD276) for human cancer immunotherapy. Clin Cancer Res. 2016; 22:3425–31. and Yang S, Wei W, Zhao Q. B7-H3, a checkpoint molecule, as a target for cancer immunotherapy. Int J Biol Sci. 202016:1767–73), which is structurally very similar to PD-L1 and belongs to the B7 / CD28 superfamily. While expressed at low levels in most normal human tissues, it 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 can participate in the regulation of the immune function of toxic 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 distinctions between normal and tumor-derived circulating endothelial cells. Br J Cancer. 2014; 111:149–56). There is evidence that B7-H3 expression may be related to EGFR gene expression status 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). Due to the favorable selective expression profile of B7-H3, some biotechnology companies have developed B7-H3 monoclonal monomers or B7-H3-ADC drugs for research and treatment in related tumor diseases.

[0007] Antibody molecules can specifically bind to their corresponding antigens and are increasingly becoming important therapeutic, preventative, and / or diagnostic agents for various diseases (e.g., cancer, autoimmune diseases, inflammatory diseases, infectious diseases, etc.). However, monospecific antibodies targeting only one target have some limitations in clinical application. Patients may develop drug resistance or become unresponsive after receiving monospecific antibody therapy. With research into cancer and many other diseases, it has been recognized that multiple signal transduction pathways are often involved in the occurrence and development of diseases, and single-target immunotherapy is often insufficient to treat many diseases.

[0008] Because multispecific antibodies (e.g., bispecific antibodies) can specifically bind to different antigens, they can be designed to act on two or more different signal transduction pathways simultaneously. These advantageous properties open up broad application prospects for multispecific antibodies (e.g., bispecific antibodies).

[0009] 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, maintaining binding activity at each antigen-binding site to its corresponding epitope, and other properties. Simultaneously, there is a need to obtain physically and biologically stable bispecific antibody profiles with improved productivity and scalability, allowing for better productivity and scalability. Invention Overview

[0011] A first aspect of the 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.

[0012] The antibody or antigen-binding fragment of the present invention, which binds to EGFR and B7-H3, improves the efficacy, safety, and selectivity of the antibody molecule by reducing EGFR affinity and increasing B7-H3 affinity. Simultaneously, the use of GlymaxX low-fucose technology enhances antibody-dependent cell-mediated cytotoxicity (ADCC). Compared to emivantamab (JNJ-372) for NSCLC-EGFR exon 20 insertion mutations, B7-H3 has a broader expression profile than cMET, making it applicable to a wider range of tumor treatments. Furthermore, the introduction of the B7-H3 parent not only enhances the EGFR blocking activity of the bispecific antibody but also improves the overall ADCC effect of the bispecific antibody.

[0013] The bispecific antibody or antigen-binding fragment thereof that binds EGFR and B7-H3 provided by this invention has one or more of the following characteristics:

[0014] (a) It binds specifically to one or two antigens with high affinity;

[0015] (b) It is easy to express in cultured cells in vitro, and the chains of the antibody molecule can be correctly coupled or paired.

[0016] (c) It has good physical stability, especially good long-term thermal stability; and it can maintain biological activity for a long time.

[0017] (d) After specifically binding to one or two antigens, exert biological functions by modulating (e.g., inhibiting or activating) the signal transduction pathways involved by each antigen;

[0018] (e) To leverage the effectsor function;

[0019] (f) It has better anti-tumor activity.

[0020] In one implementation, different antigen binding sites bind to the same epitope on the same antigen, or different epitopes.

[0021] In one implementation, the first antigen-binding region or the second antigen-binding region is derived from a human, humanized, or chimeric antibody.

[0022] In some embodiments, the antibodies of the present invention, such as bispecific antibodies, further comprise a heavy chain constant domain. In one embodiment, the heavy chain constant domain is derived from IgG1. It should be understood that the Fc region in the constant domain can be mutated to achieve antibody stabilization or enhance effector function.

[0023] In one embodiment, the first antigen-binding region is specific to the first antigen, and in one embodiment, the first antigen is EGFR.

[0024] In one embodiment, the second antigen binding region is specific to the second antigen, and in one embodiment, the second antigen is B7-H3.

[0025] The antibodies of the present invention may further comprise additional antigen-binding regions that bind to other antigens to constitute multispecific antibodies. The types of other antigens that the antibody molecules of the present invention specifically bind to are not particularly limited; antigens may be, for example, cytokines, growth factors, hormones, signal transduction proteins, inflammatory mediators, ligands, cell surface receptors, or fragments thereof. In one embodiment, the other antigens that the antibody molecules of the present invention specifically bind to 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 ligands and / or receptors of these molecules.

[0026] In one aspect, the present invention provides a nucleic acid encoding any one or more polypeptide chains in the antibody molecule of the present invention, a vector containing the nucleic acid, and a host cell containing the nucleic acid or the vector.

[0027] In one aspect, the present invention provides a vector comprising a polynucleotide encoding any one or more polypeptide chains in the antibody molecule of the present invention, preferably an expression vector, such as pcDNA3.1.

[0028] In one aspect, the present invention provides a method for generating the antibody molecule or a fragment thereof of the present invention.

[0029] In some embodiments, the present invention provides immunoconjugates, pharmaceutical compositions, kits, combination products, or articles comprising the antibodies of the present invention.

[0030] In some embodiments, the antibodies, pharmaceutical compositions, or immunoconjugates or 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, etc. For example, the disease is a tumor (e.g., cancer) or infection. In some embodiments, the tumor is a tumor immune escape. Preferably, the tumor is a gastrointestinal tumor, a lung tumor, or a skin tumor. In some embodiments, the infection is a chronic infection.

[0031] In another aspect, the present invention relates to methods for preventing or treating a disease in a subject or individual, the methods comprising administering to the subject an effective amount of any antibody or fragment thereof described herein, a pharmaceutical composition or immunoconjugate or combination product or kit. For example, the disease is a tumor (e.g., cancer) or an infection. In some embodiments, the tumor is a tumor immune escape. In one embodiment, the tumor is a gastrointestinal tumor, a lung tumor, or a skin tumor. In one embodiment, the infection is a chronic infection.

[0032] In another aspect, the present invention also relates to the use of any antibody or fragment thereof or immunoconjugate described herein in 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 a tumor immune escape. In one embodiment, the tumor is a gastrointestinal tumor, a lung tumor, or a skin tumor. In one embodiment, the infection is a chronic infection.

[0033] The present invention also relates to a method for detecting antigens in a sample.

[0034] In another aspect, the present invention relates to the following specific embodiments:

[0035] 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.

[0036] 2. The bispecific antibody of embodiment 1, wherein the second antigen-binding region comprises HCDR1, 2 and 3 sequences of the heavy chain variable region as shown in SEQ ID NO:3, 5 or 7, and LCDR1, 2 and 3 sequences of the light chain variable region as shown in SEQ ID NO:4, 6 or 8. Preferably, HCDR1 adopts the Abm protocol, and HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 adopt the Kabat protocol.

[0037] 3. The bispecific antibody described in Implementation Scheme 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

[0038] (i) The HCDR1, HCDR2 and HCDR3 are the three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:3; and the LCDR1, LCDR2 and LCDR3 are the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:4.

[0039] (ii) The HCDR1, HCDR2, and HCDR3 are the three complementary determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:5; and the LCDR1, LCDR2, and LCDR3 are the three complementary determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:6; or

[0040] (iii) The HCDR1, HCDR2 and HCDR3 are the three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:7; and the LCDR1, LCDR2 and LCDR3 are the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:8.

[0041] Preferably, HCDR1 adopts the Abm scheme, and HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 adopt the Kabat scheme.

[0042] 4. The bispecific antibody described in Implementation Scheme 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

[0043] (i) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:15, HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:16, and HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:17.

[0044] LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:18; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:19; LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:20; or,

[0045] (ii) HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:21, HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:22, and HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:23.

[0046] LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:24; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:25; LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:26; or...

[0047] (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.

[0048] LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:30, LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:31, and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:32.

[0049] 5. The bispecific antibody according to any one of embodiments 1-4, wherein the second antigen-binding region comprises a heavy chain variable region VH, wherein the VH comprises or consists of an 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.

[0050] 6. The bispecific antibody according to any one of embodiments 1-5, wherein the second antigen-binding region comprises a light chain variable region VL, wherein the VL comprises or consists of an 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.

[0051] 7. A bispecific antibody according to any one of implementation schemes 1-6, wherein the second antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein

[0052] (i) The VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:3, or is composed of the amino acid sequence shown in SEQ ID NO:4, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:4, or is composed of the amino acid sequence shown in SEQ ID NO:4;

[0053] (ii) The VH comprises the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:5, or is composed of said amino acid sequence, and the VL comprises the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:6, or is composed of said amino acid sequence; or

[0054] (iii) The VH comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:7, or is composed of the amino acid sequence shown in SEQ ID NO:8, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:8, or is composed of the amino acid sequence shown in SEQ ID NO:8.

[0055] 8. A bispecific antibody according to any one of embodiments 1-7, wherein the second antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein VH and VL respectively comprise or consist of the amino acid sequences shown below:

[0056] SEQ ID NO:3 and SEQ ID NO:4;

[0057] SEQ ID NO:5 and SEQ ID NO:6; or

[0058] SEQ ID NO:7 and SEQ ID NO:8.

[0059] 9. A bispecific antibody according to any one of embodiments 1-7, 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...

[0060] The HCDR1, HCDR2, and HCDR3 are the three complementary determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:1; and the LCDR1, LCDR2, and LCDR3 are the three complementary determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:2.

[0061] Preferably, the CDR is determined using the Kabat scheme.

[0062] 10. The bispecific antibody of embodiment 9, wherein the first antigen-binding region HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:9; HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:10; HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:11; and the first antigen-binding region LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:12; LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:13; and LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:14.

[0063] 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 shown in SEQ ID NO:1 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:1.

[0064] 12. A bispecific antibody according to any one of embodiments 9-11, wherein the first antigen-binding region comprises a light chain variable region VL, wherein the VL comprises or consists of the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:2.

[0065] 13. A bispecific antibody according to any one of embodiments 9-12, wherein the first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:1, or is composed of said amino acid sequence, and the VL comprises the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:2, or is composed of said amino acid sequence.

[0066] 14. A bispecific antibody according to any one of embodiments 9-13, 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.

[0067] 15. A bispecific antibody according to any one of embodiments 1-14, 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...

[0068] HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:9;

[0069] HCDR2 contains or consists of the amino acid sequence of SEQ ID NO:10;

[0070] HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:11;

[0071] LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:12;

[0072] LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:13; and

[0073] LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:14;

[0074] and

[0075] The second antigen-binding region specifically binds to B7H3, which includes HCDR1, HCDR2, and HCDR3 of the heavy chain variable region VH, and LCDR1, LCDR2, and LCDR3 of the light chain variable region VL.

[0076] (i)HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:15;

[0077] HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:16;

[0078] HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:17;

[0079] LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:18;

[0080] LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:19; and

[0081] LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:20;

[0082] (ii) HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:21;

[0083] HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:22;

[0084] HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:23;

[0085] LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:24;

[0086] LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:25; and

[0087] LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:26; or

[0088] (iii) HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:27;

[0089] HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:28;

[0090] HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:29;

[0091] LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:30;

[0092] LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:31; and

[0093] LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:32.

[0094] 16. The bispecific antibody of embodiment 15, wherein the first antigen-binding region comprises a VH containing or composed of the amino acid sequence shown in SEQ ID NO:1 and a VL containing or composed of the amino acid sequence shown in SEQ ID NO:2, and the second antigen-binding region comprises a VH and a VL containing or composed of the amino acid sequences shown below:

[0095] SEQ ID NO:3 and SEQ ID NO:4;

[0096] SEQ ID NO:5 and SEQ ID NO:6; or

[0097] SEQ ID NO:7 and SEQ ID NO:8.

[0098] 17. A bispecific antibody according to any one of embodiments 1-16, comprising an Fc region, preferably wherein the Fc region has low fucosylation, for example, low fucosylation obtained by treatment with GlymaxX technology.

[0099] 18. The bispecific antibody of implementation scheme 17, which includes a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region are the same or different.

[0100] 19. The bispecific antibody according to embodiment 17 or 18, wherein the first Fc region and the second Fc region are human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, for example, containing or consisting of an amino acid sequence SEQ ID NO:46 or 47 or having at least 90% identity with it, for example, 95%, 96%, 97%, 99% or higher.

[0101] 20. The bispecific antibody according to implementation scheme 18 or 19, 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.

[0102] 21. The bispecific antibody of implementation scheme 20, wherein the mutation is introduced based on Innobody technology.

[0103] 22. The multispecific antibody according to implementation scheme 21, 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.

[0104] 23. The bispecific antibody of implementation scheme 22, wherein...

[0105] a) One Fc region polypeptide contains or is composed of the amino acid sequence shown in SEQ ID NO:49 or 50, while another Fc region polypeptide contains or is composed of the amino acid sequence shown in SEQ ID NO:52 or 53;

[0106] b) One Fc region polypeptide contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:49 or 50, while the other Fc region polypeptide contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:52 or 53; or

[0107] c) One Fc region polypeptide contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:49 or 50 and contains the mutations Y349T, K370S, and K409D, while the other Fc region contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:52 or 53 and contains the mutations S364R and D399K.

[0108] 24. The bispecific antibody of implementation scheme 20, wherein the mutation is introduced based on the Knob-into-Hole technology, wherein corresponding Knob mutations and Hole mutations are introduced in the first Fc region and the second Fc region.

[0109] 25. The bispecific antibody of implementation scheme 24, wherein...

[0110] a) One Fc region polypeptide contains the mutant T366W, while another Fc region polypeptide contains T366S, L368A, and Y407V (numbered according to the EU index), or

[0111] b) One Fc region contains amino acid substitutions S354C and T366W, and another Fc region contains amino acid substitutions Y349C, T366S, L368A and Y407V (numbered according to EU index).

[0112] 26. The bispecific antibody according to any one of embodiments 1-25, wherein the first and / or second antigen-binding regions (e.g., the heavy chain variable regions therein) may also be linked to one or two heavy chain constant regions (e.g., the heavy chain constant regions of human IgG1, human IgG2, human IgG3, or human IgG4), the heavy chain constant regions comprising CH1 and Fc regions, linked via or not via hinge regions, for example, the C-terminus of the heavy chain variable region is linked to the N-terminus of CH1 of the heavy chain constant region.

[0113] 27. The bispecific antibody according to embodiment 26, wherein CH1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:42 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:42.

[0114] 28. The bispecific antibody described in embodiments 1-27, wherein the first and / or second antigen-binding regions (e.g., the light chain variable regions therein) may also be linked to the light chain constant regions, for example, the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region.

[0115] 29. The bispecific antibody described in Implementation Scheme 28, wherein the light chain constant region is the kappa light chain constant region or the lambda light chain constant region.

[0116] 30. The bispecific antibody of embodiment 20, wherein the light chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO:54 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:54.

[0117] 31. The bispecific antibody according to any one of embodiments 1-30, wherein the bispecific antibody is an IgG-like antibody having the following characteristics: Figure 1 The configuration shown.

[0118] 32. The bispecific antibody described in 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

[0119] Heavy chain 1 includes a heavy chain variable region and a first heavy chain constant region of the first antigen-binding region; light chain 1 includes a light chain variable region and a first light chain constant region of the first antigen-binding region; and heavy chain 2 includes a heavy chain variable region and a second heavy chain constant region of the second antigen-binding region; light chain 2 includes a light chain variable region and a second light chain constant region of the second antigen-binding region.

[0120] 33. The bispecific antibody according to embodiment 32, wherein the heavy chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:33.

[0121] 34. The bispecific antibody according to embodiment 32 or 33, wherein the light chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:34.

[0122] 35. The bispecific antibody according to any one of embodiments 32-34, wherein the heavy chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:33, and the light chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:34, and the light chain 1 is composed of the amino acid sequence shown in SEQ ID NO:34.

[0123] 36. The bispecific antibody according to any one of embodiments 32-35, wherein the 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 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:35, 37 or 39.

[0124] 37. The bispecific antibody according to any one of embodiments 32-36, wherein the 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 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:36, 38 or 40.

[0125] 38. The bispecific antibody according to any one of implementation schemes 32-37, wherein...

[0126] (1) The heavy chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:35; and the light chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:36;

[0127] (2) The heavy chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:37; and the light chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:38 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:38;

[0128] (3) The heavy chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO:39 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:39; and the light chain 2 comprises or consists of the amino acid sequence shown in SEQ ID NO:40 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:40.

[0129] 39. The bispecific antibody according to any one of implementation schemes 32-38, wherein...

[0130] Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:33, or is composed of said amino acid sequence; and light chain 1 comprises the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:34, or is composed of said amino acid sequence; and heavy chain 2 and light chain 2 respectively comprise the amino acid sequences shown in SEQ ID NO:, or comprise amino acid sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the shown amino acid sequences, or are composed of the sequences shown in SEQ ID NO:.

[0131] i) SEQ ID NO:35 and SEQ ID NO:36;

[0132] ii) SEQ ID NO:37 and SEQ ID NO:38;

[0133] iii) SEQ ID NO:39 and SEQ ID NO:40.

[0134] 40. The bispecific antibody described in any one of implementation schemes 32-39, wherein,

[0135] (i) Heavy chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:33, and light chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:34.

[0136] Heavy chain 2 contains or is composed of the amino acid sequence shown in SEQ ID NO:35, and light chain 2 contains or is composed of the amino acid sequence shown in SEQ ID NO:36; or,

[0137] (ii) Heavy chain 1 contains or is composed of the amino acid sequence shown in SEQ ID NO:33, and light chain 1 contains or is composed of the amino acid sequence shown in SEQ ID NO:34.

[0138] Heavy chain 2 contains or is composed of the amino acid sequence shown in SEQ ID NO:37, and light chain 2 contains or is composed of the amino acid sequence shown in SEQ ID NO:38; or,

[0139] (iii) Heavy chain 1 contains or is composed of the amino acid sequence shown in SEQ ID NO:33, and light chain 1 contains or is composed of the amino acid sequence shown in SEQ ID NO:34.

[0140] Heavy chain 2 contains or consists of the amino acid sequence shown in SEQ ID NO:39, and light chain 2 contains or consists of the amino acid sequence shown in SEQ ID NO:40.

[0141] 41. The bispecific antibody or antigen-binding fragment thereof that binds EGFR and B7-H3 as described in any one of embodiments 1-40, wherein the antibody or antigen-binding fragment thereof has one or more of the following characteristics:

[0142] (i) The antibody can, on the one hand, block the binding of EGFR ligand to EGFR, inhibit biological signal transduction, and block the corresponding biological activities of tumors; on the other hand, it can stimulate EGFR endocytosis and eventually be degraded by intracellular lysosomes, etc.

[0143] (ii) The antibody uses an EGFR antibody parental 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;

[0144] (iii) The antibody uses the high-affinity B7-H3 antibody parent sequence on the basis of low affinity for EGFR, which greatly improves the EGFR signal blocking activity and improves the pharmacodynamic bioactivity and pharmacodynamic safety window of the bispecific antibody of the present invention.

[0145] (iv) The antibody is a low-fucosylated antibody;

[0146] (ii) The antibody has high pharmacodynamic and biological activity and good safety;

[0147] (v) The antibody has excellent tumor killing and inhibitory effects;

[0148] (vi) The antibody has excellent ADCC in vitro and in vivo efficacy;

[0149] (vii) The antibody described in combination with the KRAS small molecule inhibitor has excellent anti-tumor synergistic effect.

[0150] 42. An isolated nucleic acid encoding any one of the chains of the bispecific antibody binding EGFR and B7-H3 as described in any one of embodiments 1-41.

[0151] 43. A vector comprising the nucleic acid of embodiment 42, preferably an expression vector, preferably pcDNA, such as pcDNA3.1.

[0152] 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 yeast cell or mammalian cell (e.g., 293 cell or CHO cell, such as 293F cell or 293T cell or CHO-S cell).

[0153] 45. The host cell of embodiment 44, which is glycoengineered to express RMD enzyme, preferably, the host cell is a CHO cell.

[0154] 46. ​​The host cell of implementation scheme 45, which contains nucleic acid encoding RMD enzyme.

[0155] 47. The host cell of embodiment 46, wherein the RMD enzyme comprises or is at least 90% identical to the amino acid sequence shown in SEQ ID NO:41, or is composed of the amino acid sequence shown in SEQ ID NO:41, preferably, the RMD enzyme is derived from Pseudomonas aeruginosa.

[0156] 48. A method for preparing a bispecific antibody binding to EGFR and B7-H3, the method comprising culturing a host cell according to any one of embodiments 44-47 under conditions suitable for expressing a nucleic acid encoding a bispecific antibody of any one of embodiments 1-41, optionally isolating the antibody or an antigen-binding fragment thereof, optionally further comprising recovering the antibody or an antigen-binding fragment thereof from the host cell (or host cell culture medium).

[0157] 49. An immunoconjugate comprising a bispecific antibody of any one of the aforementioned embodiments 1-41 conjugated to a therapeutic agent or a diagnostic agent.

[0158] 50. A pharmaceutical composition comprising a bispecific antibody of any one of embodiments 1-41 or an immunoconjugate of embodiment 49, and optionally a pharmaceutical excipient.

[0159] 51. The pharmaceutical composition of embodiment 50, further comprising a second therapeutic agent; 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 (e.g., 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 (e.g., AMG510 (Sotorasib) or GFH925), KRASG12D (e.g., MRTX1133), or KRAS G12S inhibitors.

[0160] 52. A pharmaceutical combination product comprising a bispecific antibody of any one of embodiments 1-41, an immunoconjugate of embodiment 49, or a pharmaceutical composition of embodiment 50, and one or more second therapeutic agents, preferably selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, small molecule drugs, or immunomodulators (e.g., 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 (e.g., AMG510 (Sotorasib) or GFH925), KRAS G12D (e.g., MRTX1133), or KRAS G12S inhibitors.

[0161] 53. A method for preventing or treating tumors or infectious diseases in a subject, the method comprising administering to the subject an effective amount of any one of embodiments 1-41, or the immunoconjugate of embodiment 49, or the pharmaceutical composition of embodiment 50.

[0162] 54. The method of embodiment 53, further comprising administering one or more other therapies to the subject in combination, the therapies including, for example, treatment modalities and / or other therapeutic agents, preferably, the treatment modalities including surgical treatment and / or radiotherapy, or the therapeutic agents selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, small molecule drugs, or immunomodulators (e.g., 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 (e.g., AMG510 (Sotorasib) or GFH925), KRAS G12D (e.g., MRTX1133), or KRAS G12S inhibitors.

[0163] 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 a pharmaceutical composition of embodiment 51 or a pharmaceutical combination product of embodiment 52.

[0164] 56. The method of any one of embodiments 53-55, wherein the tumor is cancer, such as a solid tumor or a hematologic malignancy, including epithelial cancers 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 squamous cell carcinoma of the head and neck), esophageal cancer (e.g., squamous cell carcinoma of the esophagus), intestinal cancer (e.g., colon cancer, rectal cancer, colorectal cancer) or lung cancer (e.g., non-small cell lung cancer, squamous cell carcinoma of the lung, adenocarcinoma of the lung).

[0165] 57. The method of any one of embodiments 53-56, wherein the tumor cells of the tumor are...

[0166] (i) Overexpression of wild-type EGFR (e.g., wild-type EGFR with elevated nucleic acid or protein levels) compared to normal cells in adjacent tissues or normal cells in the same tissue in healthy subjects, and / or expression of mutated EGFR, preferably, the mutated EGFR comprising one or more mutations selected from R521K, L858R, T790M, G719X, C797S, Y1069C, Exon19 deletion (Del19), Exon20ins (e.g., S768_D770dup), preferably, the mutated EGFR comprising R521K / Y1069C, R521K, L858R / T790M / C797S, Del19 / T790M / C797S or S768_D770dup;

[0167] (ii) Overexpressing wild-type KRAS (e.g., wild-type KRAS with elevated nucleic acid or protein levels) compared to normal cells in adjacent tissues or normal cells in the same tissue in healthy subjects, or expressing mutated KRAS, preferably, said mutated KRAS containing a G12 or G13 mutation, such as G12D or G12C.

[0168] (iii) B7-H3 with elevated nucleic acid or protein levels compared to normal cells in adjacent tissues or compared to normal cells in the same tissue in healthy subjects; and / or

[0169] (iv) The tumor cells are resistant to tyrosine kinase inhibitors, such as first-generation (erlotinib) and third-generation (osimertinib), for example, resistant to osimertinib.

[0170] 58. The method of embodiment 57, wherein the tumor cells of the tumor express mutated EGFR and mutated KRAS, for example, EGFR with a mutation of R521K and KRAS with a mutation of G120D.

[0171] 59. A method for detecting antigens EGFR and / or B7-H3 in a sample, the method comprising:

[0172] (a) Contacting the sample with the bispecific antibody as described in any one of embodiments 1-41; and

[0173] (b) Detecting the formation of a complex between an antibody or its antigen-binding fragment and EGFR and / or B7-H3, optionally, the antibody is labeled with a detectable tag.

[0174] 60. Use of the antibody or antigen-binding fragment thereof described in any one of embodiments 1-41, and / or the isolated nucleic acid described in embodiment 42, and / or the vector described in embodiment 43, and / or the host cell described in any one of embodiments 44-47, and / or the immune conjugate described in embodiment 49, and / or the pharmaceutical composition described in embodiment 50 or 51 or the pharmaceutical combination product of embodiment 52, in the preparation of a medicament for the prevention and / or treatment of a disease in a subject.

[0175] This invention also covers any combination of any embodiments described herein. Any embodiment described herein, or any combination thereof, is applicable to any and all antibodies, fragments thereof, or immunoconjugates, or pharmaceutical compositions or combinations thereof, products, kits, methods, and uses of the invention described herein. Brief description of the attached diagram

[0177] Combined with the following appendix Figure 1 By reading this, one will better understand the preferred embodiments of the invention described in the following detailed description. For illustrative purposes, the figures show presently preferred embodiments. However, it should be understood that the invention is not limited to the embodiments shown in the figures.

[0178] Figure 1 A schematic diagram of the structure of the anti-B7-H3 / EGFR bispecific antibody of the present invention is shown.

[0179] Figure 2 The diagram shows the screening and proliferation inhibition experiment of the anti-B7-H3 / EGFR bispecific antibody of the present invention.

[0180] Figure 3 The ADCC experimental diagram of the anti-B7-H3 / EGFR bispecific antibody of the present invention is shown.

[0181] Figure 4 The graph shows the expression of B7H3 and EGFR in Hz20G5.26 / Zalu bsAb of the present invention in different cell lines.

[0182] Figure 5 The figure shows the experimental results of the proliferation inhibition of Hz20G5.26 / Zalu bsAb in CRC tumor cell lines CCK-81, NCI-H508, HT-55 and LS180, with antibody dilutions as follows:

[0183] CCK-81, NCI-H508, HT-55: The highest final concentration of the antibody was 125 nM, 4-fold dilution;

[0184] LS180: The highest final concentration of the antibody is 300 nM, diluted 4 times.

[0185] Figure 6 The figure shows the experimental results of the inhibition of proliferation of the Hz20G5.26 / Zalu bsAb of the present invention in HNSCC tumor cell lines TE-1 and Colo680, wherein the antibody dilutions are as follows: TE-1, Colo680: the highest final antibody concentration is 300 nM, 3.16-fold dilution.

[0186] Figure 7.1 The Hz20G5.26 / Zalu bsAb of the present invention was demonstrated in different NSCLC-EGFR WT Figure showing the experimental results of proliferation inhibition in tumor cell lines;

[0187] Figure 7.2 The figure shows the experimental results of the inhibition of proliferation of the Hz20G5.26 / Zalu bsAb of the present invention in the NSCLC-EGFR classical mutant tumor cell line;

[0188] Figure 7.3 The figure shows the experimental results of the inhibition of proliferation of the Hz20G5.26 / Zalu bsAb of the present invention in NSCLC-EGFR abnormally expanded tumor cell lines;

[0189] Figure 7.4 The figure shows the experimental results of the proliferation inhibition of the Hz20G5.26 / Zalu bsAb of the present invention in rare NSCLC-EGFR mutant tumor cell lines;

[0190] Figure 7.5 The diagram shows the results of a comparative experiment on the efficacy of the present invention in combination with Hz20G5.26 / Zalu bsAb and gp120 / Zalu and gp120 / hz20G5.26;

[0191] The antibody or small molecule TKI (Osimertinim or Erlotinib) is diluted as follows:

[0192] Figure 7.1 NCI-292, NCI-H322: The highest final concentration of the antibody is 300 nM, 3.16-fold dilution;

[0193] Figure 7.2 NCI-H1650 and NCI-H1975: The highest final concentration of the antibody is 300 nM, diluted 3.16 times.

[0194] Figure 7.3 NCI-H1703 and SK-MES-1 are antibody molecules with a maximum final concentration of 300 nM and a 3.16-fold dilution. Small molecule TKIs (Osimertinim and Erlotinib) have a maximum final concentration of 1000 nM and a 3.16-fold dilution.

[0195] Figure 7.4 H1975 (EGFR) L858R / T790M / C797S The highest final concentration among various antibody molecules and Osimertinib was 300 nM, diluted 3.16 times; H322 (EGFR S768-D77dup The highest final concentration among various antibody molecules and Osimertinib is 300 nM, diluted 4-fold; PC9+B7H3 (EGFR Del19 / T790M / C79S The highest final concentration among various antibody molecules and Osimertinib is 100 nM, diluted 4-fold.

[0196] Figure 7.5 LS180 and H292: The highest final concentration of the antibody is 300 nM, 4-fold dilution; SK-MES-1: The final concentration is 300 nM, 3.16-fold dilution.

[0197] Figure 8 The figure shows the results of a comparative experiment on the synergistic effect of the present invention's Hz20G5.26 / Zalu bsAb combined with the AMG510 small molecule inhibitor in the H358 NSCLC cell line.

[0198] Figure 8 (A): H358 in vitro proliferation inhibition experiment: The highest concentration of each antibody molecule was 300 nM, 4-fold dilution;

[0199] Figure 8 (B): Combined drug trial of Hz20G5.26 / Zalu bsAb with KRAS G12C inhibitor AMG510 (MedChem Expresses, HY-114277): AMG510+bsAb@10nm means adding a fixed 10nM Hz20G5.26 / Zalu bsAb to each well, while AMG510 is serially diluted in each well (final concentration, 1000nM, 4-fold dilution); AMG510 alone: ​​final concentration up to 1000nM, 4-fold dilution; Hz20G5.26 / Zalu bsAb alone: ​​final concentration up to 10nM, 5-fold dilution; a total of 5 dilutions.

[0200] Figure 9 The figure shows the results of a comparative experiment on the synergistic effect of the present invention's Hz20G5.26 / Zalu bsAb combined with the KRASG12D small molecule inhibitor MRTX1133 in the LS180 CRC cell line.

[0201] Figure 9(A): Combined drug trial of Hz20G5.26 / Zalu bsAb with G12D inhibitor MRTX1133 (MCE, cat: HY-134813A-10mg): MRTX1133+bsAb@100nm means adding a fixed 100nM Hz20G5.26 / Zalu bsAb to each well, while serially diluting MRTX1133 in each well (final concentration, 1000nM, 4-fold dilution); MRTX1133 alone: ​​final concentration up to 1000nM, 4-fold dilution; Hz20G5.26 / Zalu bsAb alone: ​​final concentration up to 100nM, 4-fold dilution; a total of 6 dilutions.

[0202] Figure 10 The figure shows the experimental results of the blocking effect of the Hz20G5.26 / Zalu bsAb of the present invention on EGFR phosphorylation signaling in the H358 cell line.

[0203] Figure 11 The figure shows the experimental results of the blocking effect of the Hz20G5.26 / Zalu bsAb of the present invention on EGFR phosphorylation signaling induced by EGFR ligands (EGF or TGF-α) in the H358 cell line.

[0204] Figure 12.1 The present invention demonstrates Hz20G5.26 / Zalu bsAb in NSCLC-EGFR WT Figure 1 shows the ADCC reporter assay results in tumor cell lines; among the three cell lines, the highest final concentration of antibody molecule was 6.25 nM after a 4-fold dilution.

[0205] Figure 12.2 The diagram shows the ADCC reporter assay results of Hz20G5.26 / Zalu bsAb of the present invention in NSCLC-EGFR mutant tumor cell lines; in the H1975 cell line, the highest final concentration of antibody molecule was 10 nM, 4-fold dilution; and in the H1975 cell line, the highest final concentration of antibody molecule was 6.25 nM, 4-fold dilution.

[0206] Figure 12.3 The figure shows the ADCC reporter assay results of Hz20G5.26 / Zalu bsAb of the present invention in NSCLC-EGFR aberrantly amplified tumor cell lines; in the SK-MES-1 cell line, the highest final concentration of antibody molecule was 6.25 nM, 4-fold dilution; and in the H1703 cell line, the highest final concentration of antibody molecule was 100 nM, 4-fold dilution.

[0207] Figure 12.4The figure shows the ADCC reporter assay results of Hz20G5.26 / Zalu bsAb in NSCLC-KRAS mutant tumor cell lines of the present invention; in the H358 cell line, the highest final concentration of antibody molecule was 25 nM, and the 4-fold dilution ( Figure 12.4 (A)); and in the H358 cell line, the highest final concentration of the antibody molecule was 1.56 nM, 4-fold dilution ( Figure 12.4 (B));

[0208] Figure 13.1 The figure shows the ADCC reporter assay results of Hz20G5.26 / Zalu bsAb in the NSCLC-EGFR wild-type tumor cell line huPBMC; wherein,

[0209] Figure 13.1 (A) In the H292 cell line, the highest final concentration of antibody molecule was 2.5 nM after a 4-fold dilution;

[0210] Figure 13.1 (B): In the H322 cell line, the highest final concentration of antibody molecule was 100 nM, after a 4-fold dilution;

[0211] Figure 13.1 (C): In the H292 cell line, the highest final concentration of antibody molecule was 100 nM, after a 4-fold dilution;

[0212] Figure 13.2 The figure shows the ADCC reporter assay results of Hz20G5.26 / Zalu bsAb of the present invention in the NSCLC-EGFR mutant tumor cell line huPBMC; among which, in the H1975 cell line, the highest final concentration of antibody molecule was 35 nM, 4-fold dilution; in the H1650 cell line, the highest final concentration of antibody molecule was 100 nM, 4-fold dilution.

[0213] Figure 13.3 The figure shows the ADCC reporter assay results of Hz20G5.26 / Zalu bsAb in the NSCLC-EGFR aberrantly amplified tumor cell line huPBMC.

[0214] Figure 13.3 (A) In the SK-MES-1 cell line, the highest final concentration of antibody molecules was 35 nM after a 4-fold dilution;

[0215] Figure 13.3 (B): In the H1703 cell line, the highest final concentration of antibody molecule was 25 nM, after a 4-fold dilution;

[0216] Figure 13.3(C): In the SK-MES-1 cell line, the highest final concentration of antibody molecules was 25 nM, after a 4-fold dilution;

[0217] Figure 13.4 The figure shows the results of the ADCC reporter assay of Hz20G5.26 / Zalu bsAb of the present invention in the NSCLC-KRAS mutant tumor cell line huPBMC. The highest final concentration of antibody molecule was 35 nM in both cell lines, with a 4-fold dilution.

[0218] Figure 14 The figure shows the results of an in vitro study of the skin toxicity of the Hz20G5.26 / Zalu bsAb drug of the present invention. The antibody molecules were diluted with cell culture medium in two different skin cell lines. The final concentration of the antibody molecules was 300 nM, a 3.16-fold dilution.

[0219] Figure 15 Figure A shows the experimental results of the effect of the Hz20G5.26 / Zalu bsAb of the present invention on the antitumor effect of NCl-H292 tumor-bearing mice; Figure 15 B shows the experimental results of the effect of the Hz20G5.26 / Zalu bsAb of the present invention on the antitumor effect of NCI-H292 tumor-bearing mice - survival curve; Figure 15 Figure C shows the experimental results of the effect of the Hz20G5.26 / Zalu bsAb of the present invention on the body weight of NCI-H292 tumor-bearing mice.

[0220] Figure 16 Figure A shows the experimental results of the effect of the present invention Hz20G5.26 / Zalu bsAb on the antitumor effect of SK-MES-1 tumor-bearing mice; Figure 16 Figure B shows the experimental results of the effect of the present invention's Hz20G5.26 / Zalu bsAb on the body weight of SK-MES-1 tumor-bearing mice.

[0221] Figure 17 Figure A shows the experimental results of the effect of the present invention Hz20G5.26 / Zalu bsAb on the antitumor effect of SK-MES-1 tumor-bearing mice; Figure 17 Figure B shows the experimental results of the effect of the present invention's Hz20G5.26 / Zalu bsAb on the body weight of SK-MES-1 tumor-bearing mice.

[0222] Figure 18 Figure A shows the experimental results of the effect of the combination of Hz20G5.26 / Zalu bsAb and AMG510 on the antitumor effect of NCI-H358 tumor-bearing mice. Figure 18Figure B shows the experimental results of the effect of the combination of Hz20G5.26 / Zalu bsAb and AMG510 on the body weight of NCI-H358 tumor-bearing mice. Invention Details

[0224] Unless otherwise specified, 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 pertains. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims.

[0225] I. Definition

[0226] It should be understood that the present invention is not limited to the specific methodologies, schemes, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only 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 pertains.

[0227] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0228] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0229] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0230] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, they also cover situations consisting of the stated elements, integers, or steps, unless otherwise specified. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0231] When “first” and “second” are mentioned in this article, it is only to distinguish between two structural domains or two chains, and does not indicate the location of the two structural domains in any way.

[0232] As used herein, the amino acid positions of all variable regions of the heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), and referred to herein as the “Kabat numbering”.

[0233] As used herein, when referring to amino acid positions in domains other than the variable region of an antibody (e.g., constant regions, such as the Fc region), the numbers are assigned according to the EU numbering system described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) and are referred to herein as “EU numbers”. When a position number and / or amino acid residue is assigned to a particular antibody isotype, it is intended to be applicable to the corresponding position and / or amino acid residue in any other antibody isotype, as is known to those skilled in the art.

[0234] General information about the nucleotide sequences of the light and heavy chains of human immunoglobulins is given in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0235] The term “antibody” is used in the broadest sense herein to refer to a protein that contains an antigen-binding site, encompassing 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.

[0236] The terms “full-length antibody,” “complete antibody,” and “intact antibody” are used interchangeably herein to refer to naturally occurring glycoproteins comprising at least two heavy chains (H) and two light chains (L) linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions (complementarity-determining regions (CDRs) interspersed with more conserved regions (framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions do not directly participate in antibody-antigen binding but exhibit various effector functions.

[0237] A "half-antibody" or "half-polymer" refers to a monovalent antigen-binding polypeptide. In some embodiments, the half-antibody or half-polymer comprises a VH / VL unit and optionally at least a portion of an immunoglobulin constant domain. In some embodiments, the half-antibody or half-polymer comprises an immunoglobulin heavy chain associated with an immunoglobulin light chain, or an antigen-binding fragment thereof. In some embodiments, the half-antibody or half-polymer is monospecific, i.e., binds to a single antigen or epitope. In some specific embodiments, the half-antibody binds to EGFR and not to B7-H3. In some specific embodiments, the half-antibody binds to B7-H3 and not to EGFR. Those skilled in the art will readily understand that a half-antibody may have an antigen-binding domain consisting of a single variable domain (e.g., derived from the genus *Camelidae*).

[0238] The term "antigen-binding fragment" in antibody refers to a molecule distinct from a full-length antibody. It comprises a portion of the full-length antibody but can bind to the antigen of the full-length antibody or compete with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment originates) for antigen binding. Antigen-binding fragments can be prepared using recombinant DNA technology 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, diabody antibodies, single-domain antibodies (sdAb), and nanobodies.

[0239] The terms "Fab fragment" or "Fab" are used interchangeably herein to refer to an immunoglobulin fragment consisting of two polypeptide chains containing the immunoglobulin heavy chain variable region VH, the heavy chain constant domain CH1, the light chain variable region VL, and the light chain constant domain CL. One polypeptide chain contains VH and a constant domain selected from CH1 and CL from the N-terminus to the C-terminus, and the other polypeptide chain contains VL and another constant domain selected from CL and CH1 from the N-terminus to the C-terminus, wherein the VH and VL domains pair to form an antigen-binding site. A Fab' fragment differs from a Fab fragment by the addition of several residues (including one or more cysteine ​​residues from the antibody hinge region) at the carboxyl terminus of the heavy chain CH1 domain. Fab'-SH is the designation for a Fab' in which the cysteine ​​residues of the constant domain carry a free thiol group. The F(ab')2 antibody fragment was originally generated as a pair of Fab' fragments with a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known.

[0240] In this document, the term "bispecific antibody" comprises an antigen-binding domain that specifically binds to two antigens or two epitopes. Unless otherwise stated, the order of the antigens bound to 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" may be used interchangeably. In some embodiments, the bispecific antibody comprises two halves, wherein each halves comprises at least a portion of a single heavy chain variable region and optionally a heavy chain constant region, and at least a portion of a single light chain variable region and optionally a light chain constant region. In some embodiments, the bispecific antibody comprises two halves, wherein each halves 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 halves, each halves comprising a single heavy chain variable region and a single light chain variable region, and wherein the first halves bind to a first antigen / epitope but not to a second antigen, and the second halves bind to a second antigen / epitope but not to the first antigen.

[0241] The multispecific antibodies of the present invention may include linkers. As used herein, the term "linker" refers to any molecule that enables direct linkage between different portions of a multispecific antibody. Examples of linkers that establish covalent links between different portions of a multispecific antibody include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyethylene, or copolymers of PEG and polypropylene glycol. In some embodiments, the term "peptide linker" according to the present invention refers to a sequence of amino acids that links the amino acid sequences of the various portions of the multispecific antibody together. Preferably, the peptide linker has a length sufficient to link two entities in such a way that they maintain their conformation relative to each other without impeding the desired activity. The peptide linker may primarily comprise or may not primarily comprise the following amino acid residues: Gly, Ser, Ala, or Thr. Useful connectors 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 connectors also include glycine-alanine polymers, alanine-serine polymers, and other flexible connectors.

[0242] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of its constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains: a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in native antibodies, an immunoglobulin Fc domain contains the second and third constant domains (CH2 and CH3 domains) of two heavy chains derived from IgG, IgA, and IgD antibodies; or it contains the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) of two heavy chains derived from IgM and IgE antibodies. Unless otherwise stated herein, amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also known 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) of the Fc region may or may not be present. Two Fc regions can dimerize to form a dimer Fc, and two distinct Fc regions can heterodimerize to form a heterodimeric Fc. In this document, 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 immunoglobulins, nor the heavy chain constant region CH1 and light chain constant region CL, but in some cases may include the hinge region at the N-terminus of the heavy chain constant region. In one embodiment, the human IgG heavy chain Fc region extends from Asp221, Cys226, or Asp231 to the carboxyl terminus of the heavy chain. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region belongs to the human IgG4 subclass. In one embodiment, the Fc region belongs to the human IgG1 subclass.

[0243] In this article, "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 Fc scaffolds in bispecific or multispecific antibodies. Therefore, a "heterodimeric Fc scaffold" refers to a scaffold containing two different Fc regions or formed by the dimerization of two different Fc regions, which can be linked to an antigen-binding domain (e.g., a variable region of the heavy and / or light chain of an antibody that can bind to a target molecule, or an antigen-binding fragment of the antibody, or a soluble portion of a ligand or receptor that can bind to a target molecule) at its N-terminus or C-terminus, for the purpose of constituting a multispecific antibody such as a bispecific antibody.

[0244] Amino acid mutations are represented as (original amino acid, amino acid position, mutant amino acid). For example, when the mutation site is located in the Fc region, "T366W" means that the T at EU position 366 is replaced by W. When referring to a combination of mutations, the mutations in the combination are connected by "and" or " / ". "R521K / Y1069C" indicates that it contains both the mutations R521K and Y1069C. It should be noted that when describing a mutation, the specific position also includes its corresponding amino acid position on other polypeptide chains. For example, if C220 is mentioned, it covers the 220th amino acid of the IgG1 heavy chain under the EU number, as well as the corresponding amino acid in other heavy chains, such as the 131st amino acid in IgG2, IgG3, or IgG4. When describing a mutation, the original amino acid at a specific position can be the described amino acid or another amino acid at the corresponding position.

[0245] The complementarity-determining region (CDR) or CDR is a region within the variable domain of an antibody that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the variable region of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, while those located within the variable region of the antibody light chain are called 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 or a combination of many known antibody CDR assignment systems, including, for example: Chothia (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)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDapartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The 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.

[0246] For example, depending on the different CDR determination schemes, the residues of each CDR are as follows.

[0247]

[0248] CDRs can also be determined based on having the same Kabat number position as a reference CDR sequence (e.g., any of the exemplary CDRs of this invention).

[0249] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the foregoing manner.

[0250] Unless otherwise stated, in this invention, when referring to the position of a residue in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it means the position numbered 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)).

[0251] In one embodiment, the CDR of the antibody of the present invention is determined by the Kabat rule, or by the AbM rule, or by a combination thereof.

[0252] In one embodiment of the present invention, the HCDR1 of VH in the antigen-binding region that binds to B7-H3 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.

[0253] In one embodiment of the present invention, the CDRs of VH and VL in the antigen-binding region that binds to EGFR are determined by the Kabat rule.

[0254] The term "hinge region" refers to a portion of an antibody heavy chain polypeptide that connects the CH1 and CH2 regions in the wild-type antibody heavy chain, such as the IgG1 hinge region, for example, the sequence D221 to P230 according to EU numbering. Hinge regions of other IgG subclasses can be identified by aligning with cysteine ​​residues in the hinge region of the IgG1 subclass sequence.

[0255] As used herein, the terms “binding site” or “antigen binding site” or “antigen binding region” refer to any portion of an antibody molecule, such as a multispecific antibody or a bispecific antibody, that binds to a specific target or antigen. An antigen-binding region can be, for example, the antibody or immunoglobulin itself or an antibody fragment. Such an antigen-binding region may or may not have a tertiary structure independent of the remaining portion of BsAB and may bind or not bind its antigen / epitope as a standalone entity. In some embodiments, the antigen-binding region includes a VH / VL pair consisting of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), a heavy chain variable region derived from a camelid heavy chain antibody, a VH-like single domain (v-NAR) from a shark-like IgNAR, a camelidized human VH domain, or a humanized camelid antibody heavy chain variable region.

[0256] When "first antigen-binding region" is mentioned in the context of multispecific or bispecific antibodies, 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 or bispecific antibody may contain one or more first antigen-binding regions. Similarly, a bispecific antibody may contain both a first antigen-binding region and a second antigen-binding region, but may contain one or more first antigen-binding regions and one or more second antigen-binding regions.

[0257] When it is mentioned that "the antigen-binding region is derived from the 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 region's specific antigen-binding fragment, such as Fab, is or is derived from the corresponding fragment of the antibody, such as Fab; 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 the antibody.

[0258] 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 a different epitope of a different antigen. The antibodies provided herein are typically multispecific antibodies, such as bispecific antibodies. A multispecific antibody is an antibody that has binding specificity to at least two different antigenic epitopes. In one embodiment, such a bispecific antibody is provided herein, which has binding specificity against a first antigen and a second antigen.

[0259] The term "immunoglobulin molecule" refers to a protein with a structure that contains naturally occurring antibodies. For example, IgG immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. Each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, from the N-terminus to the C-terminus, followed by three heavy chain constant domains (CH1, CH2, and CH3). Similarly, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, from the N-terminus to the C-terminus, followed by a light chain constant domain (CL). The heavy chains of immunoglobulins can be classified into one of five categories, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further subdivided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can be classified into one of two types based on the amino acid sequence of their constant domains, called κ and λ. Immunoglobulins are essentially composed of two Fab molecules linked by an immunoglobulin hinge region and an Fc domain.

[0260] The term "effective function" refers to those biological activities attributable to the Fc region of immunoglobulins that vary with 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 phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen-presenting cell uptake of antigens, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0261] The term "valent" antibody refers to the number of antigen-binding sites present in the antibody molecule. "Bivalent," "trivalent," and "quadrivalent" antibodies refer to antibodies with 2, 3, and 4 antigen-binding sites, respectively.

[0262] As used herein, the terms “binding” or “specific binding” mean that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific 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 by radioimmunoassay (RIA), thin-layer interferometry, MSD assay, or surface plasmon resonance (SPR).

[0263] "Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can usually be determined by the dissociation constant (K).D This indicates that the dissociation constant is the dissociation rate constant and the association rate constant (Kdissociation and Kassociation, respectively). dis and K on The ratio of affinity to kinetic binding affinity. Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio kinetic binding assay described in this paper.

[0264] The term "knob-in-hole" mutation, or "knob-in-hole" mutation, is used herein to refer to the introduction of mutations into a first Fc polypeptide and a second Fc polypeptide, respectively, using a "knob-in-hole" technique to form a protrusion ("knob") and a complementary hole ("hole") at the interface between the first and second Fc polypeptides. Knob-in-hole techniques are known in the art to modify interfaces between different chains of an antibody molecule to facilitate proper association between the chains. Typically, this technique involves introducing a "protrusion / knob" at the interface of one chain and a corresponding "hole / hole" at the interface of the other chain to which it is to pair, allowing the protrusion to be placed within the hole. 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 chain to which it is to pair. The protrusion can be constructed by replacing a small amino acid side chain from the CH3 domain interface of the heavy chain constant domain of one chain with a larger side chain (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller side chains (e.g., alanine or threonine), compensating holes of the same or similar size as the protrusions are constructed at the interface of the CH3 domain of the heavy chain constant domain of the other chain to be paired. Another alternative interface is the CL domain of the Fab fragment described above, containing the light chain and the CH1 domain of the heavy chain, which promotes proper heterodimerization between the two chains of the Fab fragment by constructing protrusion-hole interactions.

[0265] In this document, antibody constant regions or antibody constant domains, including CH1, CL, and Fc domains, as well as CH2, CH3, and optionally CH4 domains constituting the Fc domain, can be selected according to the intended function of the antibody molecule. For example, the constant region can be an IgA, IgD, IgE, IgG, or IgM region, particularly the immunoglobulin constant domain of human IgG, such as the constant domain of human IgG1, IgG2, IgG3, or IgG4, preferably the constant domain of human IgG1. As another example, the Fab fragment of the antibody can contain the CH and CL constant regions derived from IgG1. As yet another example, the Fc region of the antibody can contain the CH2 and CH3 domains derived from IgG1. Immunoglobulin constant regions can have native or variant sequences.

[0266] The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve antibody production or activation of specific immune cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can be used as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. In some embodiments described herein, a first antigen and a second antigen are two distinct antigens.

[0267] The terms “tumor-associated antigen” or “cancer antigen” can be used interchangeably to refer to molecules (typically proteins, carbohydrates, or lipids) that are preferably expressed fully or as fragments (e.g., MHC / peptides) on the surface of cancer cells compared to normal cells, and said molecules can be used in the preferential targeting of drugs to cancer cells.

[0268] The term "immune checkpoint molecule" refers to a class of inhibitory signaling molecules 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.

[0269] The term "cytokine" is a general term for proteins released by one group of cells that act as intercellular mediators to another cell.

[0270] "Immune conjugates" are antibodies conjugated to one or more other substances (including but not limited to cytotoxic agents or markers).

[0271] As used in this article, the term "EGFR" refers to the epidermal growth factor receptor, a tyrosine kinase receptor, a large transmembrane glycoprotein with a molecular weight of approximately 170 kDa. It belongs to the ErbB receptor family and is the most common oncogenic driver gene in NSCLC. EGFR activation mutations mainly occur in the tyrosine kinase domains of exons 18-21 of EGFR. The binding region of EGFR antibodies is mainly located in the extracellular ligand domain of EGFR, which can avoid the occurrence of drug resistance 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 specific ADCC (antibody-dependent cell-mediated cytotoxicity) to work with immune cells to kill tumors. In this way, they can exert anti-tumor killing effects through multiple mechanisms of action.

[0272] The terms “B7-H3”, “B7H3”, and “CD276” are used interchangeably in this document. B7-H3 is a type I transmembrane glycoprotein belonging to the B7 / CD28 superfamily, sequence-similar to the extracellular domain of PD-L1. B7-H3 has 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 replication, the extracellular structure of B7-H3 can be an IgV-IgC-like domain (2Ig-B7-H3) or an IgV-IgC-IgV-IgC-like domain (4Ig-B7-H3). The sequence of cynomolgus monkey B7-H3 shares approximately 90% homology with its human counterpart.

[0273] "Effective functions" refer to biological activities that can be attributed to the Fc region of an antibody and vary with antibody isotypes. 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 receptors); and B cell activation.

[0274] The term "GlymaxX technology" refers to a technology platform developed by ProBioGen to enhance the ADCC effect of dual antibodies. It is a CHO host cell that stably overexpresses bacterial RMD protein. By interfering with the formation of the substrate GDP-fucose, it ultimately blocks the fucose modification in the Fc region, thereby enhancing the ADCC effect (WO2011035884A1).

[0275] The term "effective amount" refers to such an amount or dose of the antibody, fragment, conjugate, or composition of the present invention, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention. The effective amount can be readily determined by a physician skilled in the art by considering a variety of factors, such as: the species of the mammal; its size, age, and general health; the specific disease involved; the degree or severity of the disease; the individual patient's response; the specific antibody administered; the administration method; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0276] "Therapeutic effective amount" refers to the amount that effectively achieves the desired therapeutic outcome at the required dose and for the required duration. Therapeutic effective amount of an antibody or antibody fragment, or its conjugate or composition, can vary depending on various factors such as disease state, individual age, sex, weight, and the ability of the antibody or antibody fraction to elicit the desired response in the individual. Therapeutic effective amount is also a amount in which any toxic or harmful effects of the antibody or antibody fragment, or its conjugate or composition, are less than the therapeutically beneficial effects. Relative to an untreated subject, "therapeutic 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%. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that predicts efficacy in human tumors. Alternatively, this property of the composition can be evaluated by testing the inhibitory ability of the compound, said inhibition in vitro by a assay known to a skilled craftsman.

[0277] "Prophylactic effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because prophylactic doses are administered to individuals before or at an early stage of the disease, the prophylactic effective dose will be less than the therapeutic effective dose.

[0278] The term "variable region" or "variable domain" refers to a domain of the heavy or light chain of an antibody that participates in antibody-antigen binding. The variable domains of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al., Kuby Immunology, 6) th (ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to provide antigen binding specificity. Furthermore, antibodies binding to a specific antigen can be isolated using VH or VL domains derived from antibodies that bind to that antigen, to screen libraries of complementary VL or VH domains, respectively.

[0279] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny, regardless of the number of passages. Progeny cells may not be identical to parental cells in their nucleic acid content and may contain mutations. Mutant progeny cells with the same function or biological activity screened or selected from the initially transformed cells are included herein. Host cells are any type of cell system that can be used to produce the antibody molecules of this 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.

[0280] The term "antitumor effect" refers to biological effects that can be demonstrated through a variety of means, including but not limited to, for example, reduction in tumor volume, reduction in the number of tumor cells, reduction in tumor cell proliferation, or reduction in tumor cell survival.

[0281] The terms “tumor” and “cancer” are used interchangeably in this article to cover both solid tumors and liquid tumors.

[0282] The terms “cancer” and “cancerous” refer to or describe a physiological disorder in mammals that is typically characterized by unregulated cell growth. In some embodiments, cancers suitable for treatment with the antibodies of the present invention include epithelial-derived cancers, such as skin cancer (e.g., squamous cell carcinoma of the skin, head and neck cancer such as squamous cell carcinoma of the head and neck), esophageal cancer (e.g., squamous cell carcinoma of the esophagus), intestinal cancer (e.g., colon cancer, rectal cancer, colorectal cancer), or lung cancer (e.g., non-small cell lung cancer, squamous cell carcinoma of the lung, adenocarcinoma of the lung), including metastatic forms of those cancers.

[0283] The term "tumor" refers to all neoplasmic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when used in this article.

[0284] The term "infectious disease" refers to a disease caused by a pathogen, including, for example, viral infections, bacterial infections, fungal infections, or protozoan infections such as parasitic infections.

[0285] The term "chronic infection" refers to an infection in which the infectious agent (e.g., pathogens such as viruses, bacteria, protozoa such as parasites, fungi, or the like) has induced an immune response in the infected host but has not yet been cleared or eliminated from the host as in the case of an acute infection.

[0286] As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and facilitates the detection of the conjugated or fused reagent. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling) or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition. The term is intended to cover both direct labeling of probes or antibodies by conjugating (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of probes or antibodies by reacting with another directly labeled reagent. Examples of indirect labeling include the detection of primary antibodies using fluorescently labeled secondary antibodies and the end labeling of biotinylated DNA probes so that they can be detected using fluorescently labeled streptomycin proteins.

[0287] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human 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.

[0288] "Isolated" antibodies are antibodies that have been separated from components of their native environment. In some embodiments, the antibody is purified to a purity exceeding 95% or 99%, 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 used to assess antibody purity, see, for example, Flatman et al., J. Chromatogr. B848:79-87 (2007).

[0289] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain them, but which are located outside the chromosome or at a chromosomal location different from their natural chromosomal location.

[0290] The sequence identity between sequences is calculated as follows. To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position.

[0291] Mathematical algorithms can be used to compare sequences and calculate the percentage of identity between two sequences. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http: / / www.gcg.com) is used in the GAP program integrated into the GCG software package, employing a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two amino acid sequences. In yet another preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used, employing an NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two nucleotide sequences. A particularly preferred set of parameters (and, unless otherwise specified, a set of parameters to be used) is a Blossum 62 scoring matrix employing a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5. Alternatively, the percentage of identity between two amino acid or nucleotide sequences can be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17), which has been incorporated into the ALIGN program (version 2.0). Additionally or alternatively, the nucleic acid and protein sequences described herein can be further used as “query sequences” to perform searches against public databases to, for example, identify other family member sequences or related sequences.

[0292] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0293] The term "pharmaceutical excipients" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), carriers, excipients, or stabilizers that are applied together with the active substance.

[0294] When used herein, "treatment" means to slow, interrupt, block, alleviate, stop, reduce, or reverse the progression or severity of existing symptoms, conditions, illnesses, or diseases. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In some embodiments, the antibody molecules of the present invention are used to delay disease development or to slow disease progression.

[0295] When used herein, “prevention” includes the suppression of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some implementations, subjects with a family history of cancer are candidates for preventative protocols. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

[0296] The term "effective amount" refers to such an amount or dose of the antibody, fragment, composition, or combination of the present invention, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention.

[0297] "Therapeutic effective amount" refers to the amount that, at the required dose and for the required duration, effectively achieves the desired therapeutic outcome. A therapeutic effective amount is also a amount in which any toxic or harmful effects of the antibody, antibody fragment, composition, or combination are less than the beneficial therapeutic effect. Relative to an untreated subject, the "therapeutic effective amount" preferably inhibits or improves a measurable parameter by at least about 40%, and more preferably at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%.

[0298] "Prophylactic effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because prophylactic doses are administered to individuals before or at an early stage of the disease, the prophylactic effective dose will be less than the therapeutic effective dose.

[0299] The term "therapeutic agent" as used herein encompasses any substance that is effective in preventing or treating diseases, such as tumors (e.g., cancer) and infections (e.g., chronic infections), including anti-angiogenic agents, chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs, or immunomodulators.

[0300] "Chemotherapy agents" include chemical compounds that are useful in the treatment of cancer, including but not limited to antitumor agents, including alkylating agents; antimetabolites; natural products; antibiotics; enzymes; miscellaneous reagents; hormones and antagonists; antiestrogens; antiandrogens; and nonsteroidal antiandrogens, etc.

[0301] As used in this article, the term "immunomodulator" refers to natural or synthetic active agents or drugs that inhibit or modulate immune responses. Immune responses can be humoral or cellular. Immunomodulators include inhibitors of immune checkpoint molecules and activators of co-stimulatory molecules.

[0302] The term "small molecule drug" refers to low molecular weight organic compounds that can regulate biological processes.

[0303] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes; chemotherapeutic agents or drugs; growth inhibitors; enzymes and fragments thereof such as lysozymes; antibiotics; and toxins such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof.

[0304] "Tumor immune escape" refers to the process by which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when this evasion weakens, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0305] "Immunogenicity" refers to the ability of a specific substance to trigger an immune response. Tumors are immunogenic, and enhancing tumor immunogenicity helps to eliminate tumor cells through an immune response.

[0306] As used in this article, “antibody agonist activity” refers to the biological activity of an antibody in activating the antigen it binds to.

[0307] "Anti-angiogenic agents" refer to compounds that block or interfere with blood vessel development to some extent. Anti-angiogenic agents can be, for example, small molecules or antibodies that bind to growth factors or growth factor receptors involved in promoting angiogenesis.

[0308] The term "pharmaceutical combination or combination product" refers to non-fixed combination products or fixed combination products, including but not limited to pillboxes and pharmaceutical compositions. The term "non-fixed combination" means that active ingredients (e.g., (i) the immunoconjugates of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of the components produces an effect greater than that achieved by using any one component alone in treating a disease or condition. Each component may be in a separate formulation, and the formulations may be the same or different.

[0309] The terms "combination therapy" or "combination therapy" refer to the administration of two or more therapeutic agents to treat cancer or infection as described in this disclosure. Such administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed proportion of active ingredients. Alternatively, such administration includes co-administration, separate administration, or sequential administration of the individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. In some embodiments, administration also includes using each type of therapeutic agent at substantially the same time, or at different times in a sequential manner. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0310] The term "vector," as used herein, refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell that has already been introduced therein. Some vectors are capable of directing the expression of the nucleic acid to which they are effectively linked. Such vectors are referred to herein as "expression vectors."

[0311] "Subject / Patient Sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of tissue or cell samples can be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture 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 any stage of pregnancy or development in the subject. Tissue samples may contain compounds that are naturally occurring and do not contaminate with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. Examples of tumor samples in this document include, but are not limited to, tumor biopsies, fine-needle aspirations, bronchoalveolar 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 characteristics, and preserved tumor samples, such as formalin-fixed, paraffin-embedded, or frozen tumor samples.

[0312] II. The antibody molecule of the present invention

[0313] This invention provides a novel antibody molecule that can be used for the immunotherapy, prevention, and / or diagnosis of various diseases. The antibody molecule of this invention comprises at least two, three, or four antigen-binding regions and can function as a bispecific or multispecific antibody; preferably, it functions as a bispecific antibody.

[0314] In some embodiments, the bispecific or multispecific antibodies of the present invention comprise a first binding specificity against EGFR and a second binding specificity against B7H3, as well as optionally other binding specificities.

[0315] Therefore, one aspect of the present invention relates to a bispecific antibody comprising

[0316] The first antigen-binding region and the second antigen-binding region specifically bind EGFR, and / or the second antigen-binding region specifically binds B7-H3.

[0317] The bispecific antibody of the present invention can be prepared using bispecific antibody forms or techniques known in the art. Specific exemplary bispecific forms that can be used in the context of the present invention can be found, for example, 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 form comprises an IgG-like antibody (Fan et al. (2015) Journal of Hematology & Oncology. 8: 130). The most common type of IgG-like antibody comprises two Fab regions and two Fc regions, the heavy and light chains of each Fab potentially 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.

[0318] Specifically binds to the antigen-binding region of EGFR

[0319] In some implementations, 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.

[0320] In some embodiments, the antigen-binding region that specifically binds to EGFR contains known EGFR-specific antibodies, such as the EGFR antibodies disclosed in WO02100348A2, such as CDRs 1, 2, 3, 4, 5, or 6 of the Zalutumumab monoclonal antibody.

[0321] In some embodiments, the antigen-binding region that specifically binds to EGFR includes known EGFR-specific antibodies such as the EGFR antibody disclosed in WO02100348A2, such as the 1st, 2nd, and 3rd heavy chain variable regions (CDRs) of Zalututumumab monoclonal antibody, namely HCDR1, HCDR2, and HCDR3.

[0322] In some embodiments, the antigen-binding region that specifically binds to EGFR includes known EGFR-specific antibodies such as the EGFR antibody disclosed in WO02100348A2, such as the 1st, 2nd, and 3rd light chain variable regions (CDRs) of Zalutumumab monoclonal antibody, namely LCDR1, LCDR2, and LCDR3.

[0323] In some embodiments, the antigen-binding region that specifically binds to EGFR includes known EGFR-specific antibodies such as the EGFR antibody disclosed in WO02100348A2, such as the three heavy chain variable regions (CDRs) and three light chain variable regions (CDRs) of Zalutumumab monoclonal antibody.

[0324] In some embodiments, the antigen-binding region that specifically binds to EGFR includes known EGFR-specific antibodies such as the EGFR antibody disclosed in WO02100348A2, such as the heavy chain variable region and light chain variable region of Zalutumumab monoclonal antibody.

[0325] In some embodiments, the antigen-binding region that specifically binds to EGFR contains known antibodies that specifically bind to EGFR, such as the EGFR antibody disclosed in WO02100348A2, such as the Fab of Zalutumumab monoclonal antibody.

[0326] In some embodiments, the antigen-binding region that specifically binds to EGFR includes 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 includes 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 includes three HCDRs from the heavy chain variable region and three LCDRs from the light chain variable region.

[0327] In some aspects, the antigen-binding region that specifically binds to EGFR includes a heavy chain variable region (VH). In some aspects, the antigen-binding region that specifically binds to EGFR includes a light chain variable region (VH). In some aspects, the antigen-binding region that specifically binds to EGFR includes both a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region includes three complementarity-determining regions (CDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region includes three complementarity-determining regions (CDRs) from the light chain variable region: LCDR1, LCDR2, and LCDR3.

[0328] In some implementations, the heavy chain variable region specifically binds to the antigen-binding region of EGFR.

[0329] (i) comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:1; or

[0330] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:1; or

[0331] (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, preferably, the amino acid changes do not occur in the CDR region.

[0332] In some implementations, the light chain variable region specifically binds to the antigen-binding region of EGFR.

[0333] (i) comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:2; or

[0334] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:2; or

[0335] (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, preferably, the amino acid changes do not occur in the CDR region.

[0336] In some implementations, the three complementarity-determining regions (HCDRs) from the heavy chain variable region that specifically bind to the antigen-binding region of EGFR, HCDR1, HCDR2, and HCDR3, are selected from...

[0337] (i) the three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:1, or

[0338] (ii) A sequence that, relative to any one of (i), contains at least one and no more than 5, 4, 3, 2, or 1 amino acid alteration (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions.

[0339] For example, the CDR is determined using the Kabat scheme.

[0340] In some implementations, the three complementarity-determining regions (LCDRs) from the light chain variable region that specifically bind to the antigen-binding region of EGFR, LCDR1, LCDR2, and LCDR3, are selected from...

[0341] (i) the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:2, or

[0342] (ii) A sequence that, relative to any one of (i), contains at least one and no more than 5, 4, 3, 2, or 1 amino acid alteration (preferably an amino acid substitution, preferably a conservative substitution) in the three LCDR regions.

[0343] For example, the CDR is determined using the Kabat scheme.

[0344] In some embodiments, the antigen-binding region that specifically binds to EGFR includes a complementarity-determining region (HCDR) contained in the heavy chain variable region consisting of three amino acid sequences of SEQ ID NO:1 and a complementarity-determining region (LCDR) contained in the light chain variable region consisting of three amino acid sequences of SEQ ID NO:2.

[0345] In some embodiments, HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:9, or HCDR1 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:9.

[0346] In some embodiments, HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:10, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:10.

[0347] In some embodiments, HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:11, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:11.

[0348] In some embodiments, LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:12, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:12.

[0349] In some embodiments, LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:13, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:13.

[0350] In some embodiments, LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:14, or comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:14.

[0351] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to EGFR includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 as described above.

[0352] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to EGFR includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as described above.

[0353] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to EGFR includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein...

[0354] HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:9;

[0355] HCDR2 contains or consists of the amino acid sequence of SEQ ID NO:10;

[0356] HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:11;

[0357] LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:12;

[0358] LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:13; and

[0359] LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:14.

[0360] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to EGFR includes 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.

[0361] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to EGFR includes VH and VL, wherein

[0362] The VH contains the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0363] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to EGFR includes VH and VL, wherein VH and VL respectively contain or consist of the amino acid sequences shown below: SEQ ID NO:1 and SEQ ID NO:2.

[0364] Specifically binds to the antigen-binding region of B7-H3

[0365] 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, for example, the monoclonal antibody numbered Hz20G5 (e.g., Hz20G5.26 mentioned in the embodiments of the present invention).

[0366] In some embodiments, the antigen-binding region that specifically binds to B7-H3 contains known antibodies that specifically bind to B7-H3, such as the B7-H3 antibody described in PCT / CN2021 / 140449, for example, CDRs 1, 2, 3, 4, 5, or 6 of the monoclonal antibody numbered Hz20G5.

[0367] In some embodiments, the antigen-binding region that specifically binds to B7-H3 includes known antibodies that specifically bind to B7-H3, such as the B7-H3 antibody described in PCT / CN2021 / 140449, for example, the 1st, 2nd, and 3rd heavy chain variable regions CDR of the monoclonal antibody numbered Hz20G5, namely HCDR1, HCDR2, and HCDR3.

[0368] In some embodiments, the antigen-binding region that specifically binds to B7-H3 includes known antibodies that specifically bind to B7-H3, such as the B7-H3 antibody described in PCT / CN2021 / 140449, for example, the 1st, 2nd, and 3rd light chain variable regions CDRs of the monoclonal antibody numbered Hz20G5, namely LCDR1, LCDR2, and LCDR3.

[0369] In some embodiments, the antigen-binding region that specifically binds to B7-H3 includes known antibodies that specifically bind to B7-H3, such as the B7-H3 antibody described in PCT / CN2021 / 140449, for example, the three heavy chain variable regions (CDRs) and three light chain variable regions (CDRs) of the monoclonal antibody numbered Hz20G5.

[0370] In some embodiments, the antigen-binding region that specifically binds to B7-H3 includes known antibodies that specifically bind to B7-H3, such as the B7-H3 antibody described in PCT / CN2021 / 140449, for example, the heavy chain variable region and light chain variable region of the monoclonal antibody numbered Hz20G5.

[0371] In some embodiments, the antigen-binding region that specifically binds to B7-H3 contains known antibodies that specifically bind to B7-H3, such as the B7-H3 antibody described in PCT / CN2021 / 140449, for example, the Fab of the monoclonal antibody numbered Hz20G5.

[0372] In some embodiments, the antigen-binding region that specifically binds to B7-H3 includes 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 B7-H3 includes 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 B7-H3 includes three HCDRs from the heavy chain variable region and three LCDRs from the light chain variable region.

[0373] In some aspects, the antigen-binding region that specifically binds to B7-H3 includes a heavy chain variable region (VH). In some aspects, the antigen-binding region that specifically binds to B7-H3 includes a light chain variable region (VH). In some aspects, the antigen-binding region that specifically binds to B7-H3 includes both a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region includes three complementarity-determining regions (CDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region includes three complementarity-determining regions (CDRs) from the light chain variable region: LCDR1, LCDR2, and LCDR3.

[0374] In some implementations, the heavy chain variable region specifically binds to the antigen-binding region of B7-H3.

[0375] (i) comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 3, 5, or 7; or

[0376] (ii) Containing or consisting of an amino acid sequence of SEQ ID NO: 3, 5 or 7; or

[0377] (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, preferably, the amino acid changes do not occur in the CDR region.

[0378] In some implementations, the light chain variable region specifically binds to the antigen-binding region of B7-H3.

[0379] (i) comprising or consisting of an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:4, 6, or 8; or

[0380] (ii) Containing or consisting of an amino acid sequence of SEQ ID NO:4, 6, or 8; or

[0381] (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 alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:4, 6, or 8, preferably, the amino acid alterations do not occur in the CDR region.

[0382] In some implementations, the three complementarity-determining regions (HCDRs) from the heavy chain variable region that specifically bind to the B7-H3 antigen-binding region, HCDR1, HCDR2, and HCDR3, are selected from...

[0383] (i) the three complementary determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:3, 5 or 7, or

[0384] (ii) A sequence that, relative to any one of (i), contains at least one and no more than 5, 4, 3, 2, or 1 amino acid alteration (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions.

[0385] For example, HCDR1 adopts the Abm scheme, while HCDR2 and HCDR3 are determined by the Kabat scheme.

[0386] 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...

[0387] (i) the three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO:4, 6 or 8, or

[0388] (ii) A sequence that, relative to any one of (i), contains at least one and no more than 5, 4, 3, 2, or 1 amino acid alteration (preferably an amino acid substitution, preferably a conservative substitution) in the three LCDR regions.

[0389] For example, the CDR is determined using the Kabat scheme.

[0390] In some embodiments, the antigen-binding region that specifically binds to B7-H3 includes a complementarity-determining region (HCDR) contained in a heavy chain variable region consisting of three amino acid sequences of SEQ ID NO: 3, 5, or 7, and a complementarity-determining region (LCDR) contained in a light chain variable region consisting of three amino acid sequences of SEQ ID NO: 4, 6, or 8.

[0391] In some embodiments, HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:15, 21 or 27, or comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:15, 21 or 27.

[0392] In some embodiments, HCDR2 comprises, or is composed of, the amino acid sequence of SEQ ID NO:16, 22 or 28, or comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:16, 22 or 28.

[0393] In some embodiments, HCDR3 comprises, or is composed of, the amino acid sequence of SEQ ID NO:17, 23 or 29, or comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:17, 23 or 29.

[0394] In some embodiments, LCDR1 comprises, or is composed of, the amino acid sequence of SEQ ID NO:18, 24 or 30, or the amino acid sequence thereof, or LCDR1 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:18, 24 or 30.

[0395] In some embodiments, LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:19, 25 or 31, or comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:19, 25 or 31.

[0396] In some embodiments, LCDR3 comprises, or is composed of, the amino acid sequence of SEQ ID NO:20, 26 or 32, or comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:20, 26 or 32.

[0397] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to B7-H3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 as described above.

[0398] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to B7-H3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as described above.

[0399] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to B7-H3 includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein...

[0400] (i) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:15, HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:16, and HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:17.

[0401] LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:18; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:19; LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:20; or,

[0402] (ii) HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:21, HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:22, and HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:23.

[0403] LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:24; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:25; LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:26; or...

[0404] (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.

[0405] LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:30, LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:31, and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:32.

[0406] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to B7-H3 includes

[0407] (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;

[0408] (ii) HCDR1 as shown in SEQ ID NO:21, HCDR2 as shown in SEQ ID NO:22, HCDR3 as shown in SEQ ID NO:23; LCDR1 as shown in SEQ ID NO:24, LCDR2 as shown in SEQ ID NO:25, and LCDR3 as shown in SEQ ID NO:26; or

[0409] (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.

[0410] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to B7-H3 includes VH and VL, wherein

[0411] (i) The VH comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:3, or is composed of the amino acid sequence shown in SEQ ID NO:4, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:4, or is composed of the amino acid sequence shown in SEQ ID NO:4;

[0412] (ii) The VH comprises the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:5, or is composed of said amino acid sequence, and the VL comprises the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:6, or is composed of said amino acid sequence; or

[0413] (iii) The VH comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:7, or is composed of the amino acid sequence shown in SEQ ID NO:8, or an amino acid sequence having at least 80%, 85%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:8, or is composed of the amino acid sequence shown in SEQ ID NO:8.

[0414] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to B7-H3 comprises VH and VL, wherein VH and VL respectively comprise or consist of the amino acid sequences shown below:

[0415] SEQ ID NO:3 and SEQ ID NO:4;

[0416] SEQ ID NO:5 and SEQ ID NO:6; or

[0417] SEQ ID NO:7 and SEQ ID NO:8.

[0418] The present invention also relates to antibodies that specifically bind B7-H3, including the antigen-binding region that specifically binds B7-H3 as described above, such as antibodies that include HCDR1, HCDR2 and HCDR3 as defined in the present invention, as well as LCDR1, LCDR2 and LCDR3; or antibodies that include VH or VL as defined in the present invention, or antibodies that include VH and VL as defined in the present invention.

[0419] Fc area

[0420] In some embodiments, the antibody molecules of the present invention, such as multispecific antibodies (e.g., bispecific antibodies), further include an Fc region, wherein the included Fc regions may be the same or different.

[0421] In some implementations, the Fc region has low fucosylation, for example, low fucosylation obtained by treatment with GlymaxX technology.

[0422] In some embodiments, the antibody molecule of the present invention comprises a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region may be the same or different.

[0423] In some implementations, the first Fc region and the second Fc region are different, and they are capable of dimerizing to form a heterodimeric Fc scaffold.

[0424] In this document, the Fc region refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region, and may include native sequence Fc regions and variant Fc regions. Native sequence Fc regions encompass various naturally occurring immunoglobulin Fc sequences, such as the Fc regions of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520). In some embodiments, the Fc region of the present invention comprises antibodies CH2 and CH3. In some embodiments, the antibody Fc region may also have an IgG hinge region or a portion of an IgG hinge region at its N-terminus, for example, an IgG1 hinge region or a portion of an IgG1 hinge region, such as sequences D221 to P230 according to EU numbers. Mutations may be contained in said hinge region.

[0425] Unless otherwise noted herein, the amino acid residues in the Fc region are numbered 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), NIHP Publication 91-3242.

[0426] In some embodiments, the Fc region is a human IgG Fc, such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. In one embodiment, the Fc region comprises an amino acid sequence SEQ ID NO:46 or 47 or an amino acid sequence having at least 90% identity with it, such as 95%, 96%, 97%, 99%, or higher, or is composed of said amino acid sequence.

[0427] As those skilled in the art will understand, in order to promote the formation of heterodimers in the multispecific antibodies of the present invention, the Fc regions comprising the multispecific antibodies of the present invention may contain mutations that facilitate heterodimerization of the first Fc region and the second Fc region. In one embodiment, mutations are introduced in the CH3 regions of both Fc regions.

[0428] Methods for promoting heterodimerization of Fc regions are known in the art. For example, the CH3 regions of the first Fc region and the CH3 regions of the second Fc region are engineered in a complementary manner such that each CH3 region (or the heavy chain containing it) can no longer homodimerize with itself but is forced to heterodimerize with other complementary engineered CH3 regions (so that the CH3 regions of the first and second Fc regions heterodimerize and no homodimer is formed between the two first CH3 regions or the two second CH3 regions).

[0429] Preferably, based on the Knob-in-Hole technique, corresponding Knob mutations and Hole mutations are introduced into the first Fc region and the second Fc region. This technique is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9,617-621 (1996) and Carter, J Immunol Meth 248,7-15 (2001).

[0430] In one particular embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) (knob mutation); while in the CH3 region of another Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (hole mutation), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (numbering according to EU index).

[0431] In another embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and the serine residue at position 354 is replaced with a cysteine ​​residue (S354C) or the glutamate residue at position 356 is replaced with a cysteine ​​residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine ​​residue); while in the CH3 region of another Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (hole mutation), optionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbered according to EU index), optionally the tyrosine residue at position 349 is replaced with a cysteine ​​residue (Y349C) (numbered according to EU index).

[0432] In one specific implementation, one Fc region contains amino acid substitution T366W, and another Fc region contains amino acid substitutions T366S, L368A, and Y407V (numbered according to the EU index).

[0433] In one specific implementation, one Fc region contains amino acid substitutions S354C and T366W, and another Fc region contains amino acid substitutions Y349C, T366S, L368A, and Y407V (numbered according to the EU index).

[0434] Alternatively, based on Innobody technology, corresponding mutations can be introduced into the first and second Fc regions. For example, see PCT / CN2021 / 143141 for details on this technique.

[0435] In a particular implementation scheme

[0436] The first CH3 region contains the S364R / K mutation (preferably S364R), and optionally one or more other mutations. In some embodiments, the second CH3 region contains the K370S / T / A / V mutation (preferably K370S), and optionally one or more other mutations. In some embodiments, the first CH3 region contains the S364R mutation, and the second CH3 region contains the K370S mutation. In some embodiments, the first CH3 region contains the S364R mutation, and the second CH3 region contains the K370S mutation.

[0437] In some embodiments, the first CH3 region contains the S364R / K (preferably S364R) and D399K / R (preferably D399K) mutations. In some embodiments, the second CH3 region contains the K370S / T / A / V (preferably K370S) mutation and the K409D / E (preferably K409D) mutation. In some embodiments, the first CH3 region contains S364R / K+D399K / R, and the second CH3 region contains K370S / T / A / V+Y349T / S / A / V. In some embodiments, the first CH3 region contains S364R+D399K, and the second CH3 region contains K370S+Y349T. In some embodiments, the first CH3 region further contains E375N / Q (preferably E375N) and / or T350V / A (preferably T350V). In some embodiments, the second CH3 region further includes K409D / E (preferably K409D), Q347D / E (preferably Q347D) and / or T350V / A (preferably T350V).

[0438] In some embodiments, the first CH3 region includes S364R+D399K, and the second CH3 region includes K370S+Y349T+K409D. In some embodiments, the first CH3 region further includes E357N. In some embodiments, the second CH3 region further includes Q347D. In some embodiments, the first CH3 region further includes E357N, and the second CH3 region further includes Q347D. In some embodiments, the first CH3 region and the second CH3 region further each include T350V, or both include T350V.

[0439] Therefore, in some embodiments, the first CH3 region includes S364R+D399K, and the second CH3 region includes K370S+Y349T+K409D+Q347D. In some embodiments, the first CH3 region includes S364R+D399K+E357N, and the second CH3 region includes K370S+Y349T+K409D+Q347D. In some embodiments, the first CH3 region includes S364R+D399K+E357N+T350V, and the second CH3 region includes K370S+Y349T+K409D+Q347D+T350V.

[0440] 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 implementations, the CH3 region includes K409E+T411R, and the second CH3 region includes D399K+K370T.

[0441] In some specific implementations, the first and second CH3 regions have the following combinations of mutations:

[0442]

[0443] In one implementation, CH3 in one Fc region contains S364R and D399K mutations, and CH3 in another Fc region contains Y349T, K370S, and K409D mutations.

[0444] Therefore, in one specific embodiment, the multispecific antibody of the present invention comprises heterodimerized two Fc regions, wherein

[0445] a) One Fc region polypeptide contains the mutant T366W, while the other Fc region polypeptide contains T366S, L368A, and Y407V, or

[0446] b) One Fc-region polypeptide contains the mutants S354C and T366W, while the other Fc-region polypeptide contains the mutants Y349C, T366S, L368A, and Y407V, or

[0447] c) One Fc-region polypeptide contains the mutations S364R and D399K, while the other Fc-region polypeptide contains the mutations Y349T, K370S, and K409D.

[0448] Therefore, in one 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.

[0449] Therefore, in one specific embodiment, the multispecific antibody of the present invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:49 or 50, and the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:52 or 53.

[0450] Therefore, in one specific embodiment, the multispecific antibody of the present invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:49 or 50 and comprises the mutations Y349T, K370S, and K409D, and the other Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:52 or 53 and comprises the mutations S364R and D399K.

[0451] In some implementations, the Fc region also contains other mutations that facilitate heterodimer purification.

[0452] Antibody modification

[0453] In one embodiment of the invention, the amino acid alteration described herein includes amino acid substitution, insertion, or deletion. Preferably, the amino acid alteration described herein is an amino acid substitution, and more preferably a conservative substitution.

[0454] In a preferred embodiment, the amino acid alteration described in this invention occurs in a region outside the CDR (e.g., in the FR). More preferably, the amino acid alteration described in this invention occurs in a region outside the heavy chain variable region and / or outside the light chain variable region.

[0455] In some implementations, the substitution is a conservative substitution. A conservative substitution refers to the substitution of one amino acid with another amino acid of the same class, such as the substitution of one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. Exemplary substitutions are shown in Table A below:

[0456] Table A

[0457]

[0458]

[0459] In some implementations, the antibodies provided herein are modified to increase or decrease the degree of antibody glycosylation.

[0460] Antibodies with altered types of glycosylation can be prepared, such as low-fucosylated antibodies with reduced amounts of fucosylated residues or antibodies with increased fractions of GlcNac structure. For example, fucosylation in constant regions (e.g., Fc regions) can be eliminated to obtain, for instance, low or no fucosylation.

[0461] These altered glycosylation patterns have been shown to increase the ADCC (antibody-dependent glycosylation) capacity of antibodies. Such glycosylation modifications can be achieved, for example, by expressing antibodies in host cells with altered glycosylation systems. For instance, antibodies without fucosylation are expressed in host cells with α-1,6-fucosyltransferase 8 (Fut8) knockout (WO2000061739). For example, low- or no-fucosylated antibodies can be obtained by introducing host cells encoding RMDs of enzymes associated with glycan modification (GlymaxX technology, ProBioGen AG, see patent publication number: WO2011035884A1). Alternatively, fucosidases can be used to cleave fucosylate residues from antibodies; for example, α-L-fucosidase removes fucosylate residues from antibodies (Tarentino et al. (1975) Biochem. 14:5516-23).

[0462] Exemplary bispecific antibody molecules

[0463] In some embodiments, the anti-B7-H3 / EGFR bispecific antibody of the present invention has one or more of the following properties:

[0464] (1) The bispecific antibody of the present invention can, on the one hand, block the binding of EGFR ligand to EGFR, inhibit biological signal transduction, and block the corresponding biological activity of tumors; on the other hand, it can stimulate EGFR endocytosis and eventually be degraded by intracellular lysosomes, etc.

[0465] (2) The bispecific antibody of the present invention adopts the parental sequence of EGFR antibody with low affinity for EGFR, which greatly reduces the toxic side effects of a series of EGFR monoclonal antibodies on normal epithelial tissues such as skin;

[0466] (3) The bispecific antibody of the present invention uses the high-affinity antibody parent sequence B7-H3 on the basis of low affinity for EGFR, which greatly improves the EGFR signal blocking activity and improves the pharmacodynamic bioactivity and pharmacodynamic safety window of the bispecific antibody of the present invention.

[0467] (4) The bispecific antibody of the present invention is a low-fucosylation antibody;

[0468] (5) The bispecific antibody of the present invention has high pharmacodynamic and biological activity and safety;

[0469] (6) The bispecific antibody of the present invention has excellent tumor killing and inhibition effects;

[0470] (7) The bispecific antibody of the present invention has excellent ADCC in vitro and in vivo pharmacological activity;

[0471] (8) The combination of the bispecific antibody of the present invention with the KRAS small molecule inhibitor has excellent anti-tumor effect, especially synergistic effect.

[0472] In some embodiments, in the antibody molecule of the present invention, the antigen-binding region that specifically binds to EGFR is linked to the heavy chain constant region CH, for example, the heavy chain variable region is linked to the heavy chain constant region CH, and 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 the antibody molecule of the present invention, the antigen-binding region that specifically binds to EGFR is linked to the light chain constant region, for example, the light chain variable region is linked to the light chain constant region CL, and 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 the antibody molecule of the present invention, in the antigen-binding region that specifically binds to EGFR, the heavy chain variable region is linked to the heavy chain constant region CH and the light chain variable region is linked to the light chain constant region CL.

[0473] In some embodiments, in the antibody molecule of the present invention, the antigen-binding region specifically binding 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, and 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 the antibody molecule of the present invention, the antigen-binding region specifically binding 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, and 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 the antibody molecule of the present invention, in the antigen-binding region specifically binding 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.

[0474] In some implementations, the heavy chain constant region comprises CH1 and Fc regions, connected via or not via the hinge region.

[0475] In some embodiments, the heavy chain constant region is the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, such as the heavy chain constant region of human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO:42 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:42.

[0476] 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 shown in SEQ ID NO:54 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:54.

[0477] 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 an EGFR-specific half antibody with an Fc, and the Fab fragment that specifically binds to B7-H3 forms a B7-H3-specific half antibody with an Fc.

[0478] In some embodiments, the bispecific antibody is an IgG-like antibody, which has the following properties: Figure 1 The configuration shown.

[0479] In some possible embodiments, the bispecific antibody comprises a heavy chain 1 and a light chain 1, as well as a heavy chain 2 and a light chain 2, wherein the heavy chain 1 and light chain 1 constitute a first half-antibody, and the heavy chain 2 and light chain 2 constitute a second half-antibody; wherein the heavy chain 1 comprises a heavy chain variable region and a first heavy chain constant region of a first antigen-binding region; the light chain 1 comprises a light chain variable region and a first light chain constant region of the first antigen-binding region; and the heavy chain 2 comprises a heavy chain variable region and a second heavy chain constant region of a second antigen-binding region; the light chain 2 comprises a light chain variable region and a second light chain constant region of the second antigen-binding region.

[0480] In some possible embodiments, in the bispecific antibody, heavy chain 1 comprises or consists of an amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:33.

[0481] In some possible embodiments, the light chain 1 of the bispecific antibody comprises or consists of the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:34.

[0482] In some possible embodiments, the bispecific antibody, the heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:33, or is composed of said amino acid sequence, and the light chain 1 comprises the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:34, or is composed of said amino acid sequence.

[0483] In some possible embodiments, the heavy chain 2 of the bispecific antibody comprises or consists of the amino acid sequence shown in SEQ ID NO:35, 37 or 39, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:35, 37 or 39.

[0484] In some possible embodiments, the light chain 2 of the bispecific antibody comprises or consists of the amino acid sequence shown in SEQ ID NO:36, 38 or 40, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:36, 38 or 40.

[0485] In some possible implementations, the bispecific antibody,

[0486] (1) The heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:35, or is composed of said amino acid sequence; and the light chain 2 comprises the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:36, or is composed of said amino acid sequence;

[0487] (2) The heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:37, or is composed of said amino acid sequence; and the light chain 2 comprises the amino acid sequence shown in SEQ ID NO:38 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:38, or is composed of said amino acid sequence;

[0488] (3) The heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:39 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:39 or is composed of said amino acid sequence; and the light chain 2 comprises the amino acid sequence shown in SEQ ID NO:40 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:40 or is composed of said amino acid sequence.

[0489] In some possible implementations, the bispecific antibody,

[0490] Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:33, or is composed of said amino acid sequence; and light chain 1 comprises the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:34, or is composed of said amino acid sequence; and heavy chain 2 and light chain 2 respectively comprise the amino acid sequences shown in SEQ ID NO:, or comprise amino acid sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the shown amino acid sequences, or are composed of the sequences shown in SEQ ID NO:.

[0491] i) SEQ ID NO:35 and SEQ ID NO:36;

[0492] ii) SEQ ID NO:37 and SEQ ID NO:38;

[0493] iii) SEQ ID NO:39 and SEQ ID NO:40.

[0494] In some possible implementations, the bispecific antibody,

[0495] (i) Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:33, and light chain 1 comprises the amino acid sequence shown in SEQ ID NO:34.

[0496] Heavy chain 2 contains the amino acid sequence shown in SEQ ID NO:35, and light chain 2 contains the amino acid sequence shown in SEQ ID NO:36; or,

[0497] (ii) Heavy chain 1 contains the amino acid sequence shown in SEQ ID NO:33, and light chain 1 contains the amino acid sequence shown in SEQ ID NO:34.

[0498] Heavy chain 2 contains the amino acid sequence shown in SEQ ID NO:37, and light chain 2 contains the amino acid sequence shown in SEQ ID NO:38; or,

[0499] (iii) Heavy chain 1 contains the amino acid sequence shown in SEQ ID NO:33, and light chain 1 contains the amino acid sequence shown in SEQ ID NO:34.

[0500] Heavy chain 2 contains the amino acid sequence shown in SEQ ID NO:39, and light chain 2 contains the amino acid sequence shown in SEQ ID NO:40.

[0501] III. Immunoconjugates

[0502] In some embodiments, the invention also covers antibodies conjugated with other substances. Therefore, the present invention relates to immunoconjugates comprising antibodies conjugated with other substances (“immunoconjugates”). In some embodiments, the other substances are, for example, therapeutic agents or markers, such as cytotoxic agents, immunomodulators (e.g., immune agonists), or chemotherapeutic agents. Cytotoxic agents include any agent that is harmful 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 antitumor or anticancer agents.

[0503] In addition, the antibody molecules of the present invention can be conjugated with labeled sequences (such as peptides) to facilitate purification.

[0504] 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 diagnostics and detections can be achieved by conjugating antibodies to detectable substances, including but not limited to various enzymes; prosthetic groups; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metal and non-radioactive paramagnetic metal ions used in various positron emission tomography (PET) imaging techniques.

[0505] Furthermore, the antibody molecules of the present invention can be conjugated to a therapeutic or pharmaceutical portion that regulates a given biological response. The therapeutic or pharmaceutical portion includes, but is not limited to, classic chemotherapeutic agents. For example, the pharmaceutical portion can be a protein, peptide, or polypeptide possessing the desired biological activity.

[0506] In addition, the antibody molecules of the present invention can be conjugated to therapeutic components such as radioactive metal ions.

[0507] Antibodies can also be attached to a solid support, which is particularly useful for immunoassays or the purification of target antigens.

[0508] In some embodiments, the immune conjugate is used to prevent or treat diseases such as acute and chronic inflammatory diseases, infections (e.g., chronic infections), tumors, etc. For example, the disease is a tumor (e.g., cancer) or an infection. In some embodiments, the tumor is a tumor immune escape. Preferably, the tumor is an epithelial cancer, such as a gastrointestinal tumor or 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 squamous cell carcinoma of the head and neck), esophageal cancer (e.g., squamous cell carcinoma of the esophagus), intestinal cancer (e.g., colon cancer, rectal cancer, colorectal cancer), or lung cancer (e.g., non-small cell lung cancer, squamous cell carcinoma of the lung, adenocarcinoma of the lung). 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 protozoan infection, etc.

[0509] IV. The nucleic acid of the present invention and the host cell containing it.

[0510] In one aspect, the present invention provides a nucleic acid encoding any of the above-mentioned antibodies or fragments thereof, or any strand thereof. In one embodiment, a vector comprising said nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising said nucleic acid or said 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 preparing antibodies or antigen-binding fragments thereof. In another embodiment, the host cell is prokaryotic.

[0511] For example, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO:3-8 and 33-40, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO:3-8 and 33-40.

[0512] As will be 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. The nucleic acid sequence encoding the molecule of the present invention can be generated using methods well known in the art, such as de novo solid-phase DNA synthesis or by PCR amplification.

[0513] In one aspect, the present invention provides a nucleic acid encoding any of the above-mentioned antibodies or any antibody chains. When expressed from a suitable expression vector, the polypeptide encoded by said nucleic acid is capable of exhibiting binding ability to human EGFR / or B7-H3 antigens.

[0514] In another aspect, the present invention provides nucleic acids encoding any of the above-mentioned bispecific antibodies. When expressed from a suitable expression vector, the polypeptide encoded by said nucleic acid is capable of binding to human EGFR / or B7-H3 antigens. In one embodiment, the nucleic acids encoding the respective chains of the bispecific antibody may be in the same vector or in different vectors. In another embodiment, the nucleic acids encoding the respective chains of the bispecific antibody may be introduced into the same or different host cells for expression. Therefore, in some embodiments, a method for producing the bispecific antibody of the present invention includes the step of culturing host cells containing nucleic acids encoding the respective chains of the molecule under conditions suitable for expression of the respective chains of the molecule to produce the bispecific antibody of the present invention.

[0515] In one embodiment, one or more vectors containing 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, kinases, λ phages, or yeast artificial chromosomes (YAC). In one embodiment, the vector is, for example, a pcDNA vector, such as pcDNA3.1.

[0516] Once the expression vector or DNA sequence for expression has been prepared, it can be transfected or introduced into suitable host cells. Various techniques can be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, cells are cultured in a medium and screened for suitable activity. The methods and conditions used to culture the resulting transfected cells and to recover the generated antibody molecules are known to those skilled in the art and can be varied or optimized based on methods known in this specification and the prior art, depending on the specific expression vector used and the mammalian host cells.

[0517] Additionally, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells.

[0518] In one embodiment, a host cell comprising one or more of the polynucleotides of the present invention is provided. In some embodiments, a host cell comprising the expression vector of the present invention is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells 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 preparing antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, such as bacteria, such as Escherichia coli.

[0519] 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 suit suspension growth can be used. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (HEK 293 or 293F cells), 293 cells, young hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer 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, and myeloma cell lines such as Y0, NSO, P3X63, and Sp2 / 0. For a review of suitable mammalian host cell lines for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKCLo, Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cell is a CHO cell, such as CHOS cells, CHOK1SV cells, or CHOK1SV GS-KO, or the host cell is a 293 cell, such as HEK293 cells. 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 contains 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 with it, preferably, the RMD enzyme is derived from Pseudomonas aeruginosa.

[0520] In one embodiment, the host cell of the present invention comprises nucleic acid encoding one or more or all of the chains of the antibody molecule of the present invention and nucleic acid encoding the RMD enzyme.

[0521] V. Production and purification of the antibody molecules of the present invention

[0522] In one embodiment, the present invention provides a method for preparing the antibody molecule of the present invention, wherein the method includes culturing the host cell under conditions suitable for expressing a nucleic acid encoding the antibody molecule of the present invention, and optionally isolating the antibody. In another embodiment, the method further includes recovering the antibody molecule of the present invention from the host cell.

[0523] In one embodiment, a method for preparing the antibody molecule of the present invention is provided, wherein the method includes culturing a host cell containing a nucleic acid encoding the antibody (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector containing the nucleic acid, as provided above, under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0524] To recombinantly generate the antibody molecules of the present invention, nucleic acids encoding antibodies (such as those described above, such as any one or more polypeptide chains) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids are easily isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0525] The antibody molecules prepared as described herein can be purified using known existing techniques such as high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. The actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art. The purity of the antibody molecules of this invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, and HPLC.

[0526] VI. Determination Method

[0527] The antibody molecules provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity using a variety of assays known in the art. On one hand, the antigen-binding activity of the antibodies of the present invention can be tested, 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 disclosed herein include, for example, biofilm interferometry and SPR.

[0528] The present invention also provides an assay for identifying antibodies with biological activity. Biological activity may include, for example, binding to antigens, binding to cell surface antigens, inhibition or activation of antigens, etc. Antibodies exhibiting such biological activity in vivo and / or in vitro are also provided.

[0529] In some embodiments, the antibodies of the present invention are tested for such biological activity.

[0530] The present invention also provides a method for identifying properties of antibodies, such as drug-related properties. These drug-related properties include, for example, thermal stability, such as long-term thermal stability.

[0531] Cells used in any of the above in vitro assays include cell lines that naturally express antigens or are engineered to express antigens. Such cells also include cell lines that express antigens and cell lines transfected with antigen-encoding DNA that do not normally express antigens.

[0532] It is understood that the immunoconjugates of the present invention can be used to replace or supplement the antibody molecules of the present invention to perform any of the above-described assays.

[0533] It is understood that the antibody molecules of the present invention and other active agents can be used to perform any of the above-described assays.

[0534] In some embodiments, the antigen is EGFR (e.g., human EGFR) and / or B7-H3 (e.g., human B7-H3).

[0535] VII. Pharmaceutical compositions and pharmaceutical preparations

[0536] In some embodiments, the present invention provides compositions comprising any antibody molecule or fragment thereof (preferably an antigen-binding fragment thereof) or an immunoconjugate thereof, preferably pharmaceutical compositions. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, for example a pharmaceutical composition, comprises an antibody molecule or fragment thereof or an immunoconjugate thereof of the present invention, and a combination of one or more other therapeutic agents.

[0537] In some embodiments, other therapeutic agents are selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, small molecule drugs, or immunomodulators (e.g., activators of co-stimulatory molecules or inhibitors of immune checkpoint molecules); preferably, the second therapeutic agent is a small molecule drug, preferably selected from KRAS small molecule inhibitors, such as KRAS G12C inhibitors (e.g., AMG510 (Sotorasib) or GFH925), KRAS G12D (e.g., MRTX1133), or KRAS G12S inhibitors.

[0538] 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, etc. For example, the disease is a tumor (e.g., cancer) or an infection. In some embodiments, the tumor is a tumor immune escape. Preferably, the tumor is an epithelial cancer, such as a gastrointestinal tumor or 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 squamous cell carcinoma of the head and neck), esophageal cancer (e.g., squamous cell carcinoma of the esophagus), intestinal cancer (e.g., colon cancer, rectal cancer, colorectal cancer), or lung cancer (e.g., non-small cell lung cancer, squamous cell carcinoma of the lung, adenocarcinoma of the lung). 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 protozoan infection, etc.

[0539] The present invention also includes compositions comprising antibodies of the present invention or their immunoconjugates (including pharmaceutical compositions or pharmaceutical formulations) and / or compositions comprising polynucleotides encoding antibodies of the present invention (including pharmaceutical compositions or pharmaceutical formulations). In some 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.

[0540] These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.

[0541] As used herein, "pharmaceutical carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. Pharmaceutical carriers suitable for this 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, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextran, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions.

[0542] Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol. For further information on the use and applications of excipients, see "Handbook of Pharmaceutical Excipients," 5th Edition, R.C. Rowe, P.J. Seskey, and S.O. Wen, Pharmaceutical Press, London, Chicago.

[0543] If desired, the compositions may also contain small amounts of wetting agents or emulsifiers, or pH buffers. These compositions may be in 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.

[0544] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), dispersions or suspensions, liposomes, and suppositories. Preferred forms depend on the intended mode of administration and therapeutic use. Commonly preferred compositions are in the form of injectable or infusionable solutions. Preferred modes of administration are 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.

[0545] Pharmaceutical formulations containing the antibodies described herein can be prepared by mixing the antibodies of the present invention, having the desired purity, with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of lyophilized formulations or aqueous solutions.

[0546] The pharmaceutical compositions or formulations of the present invention may also comprise more than one active ingredient, said active ingredient being required for a specific indication to be treated, preferably those active ingredients having 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 agents are selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, small molecule drugs, or immunomodulators (e.g., activators of co-stimulatory molecules or inhibitors of immune checkpoint molecules); preferably, the second therapeutic agent is a small molecule drug, preferably selected from KRAS small molecule inhibitors, such as KRAS G12C inhibitors (e.g., AMG510 (Sotorasib) or GFH925), KRAS G12D (e.g., MRTX1133), or KRAS G12S inhibitors.

[0547] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being a shaped article, such as a film or microcapsule.

[0548] The pharmaceutical compositions of the present invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0549] Therapeutic compositions should generally be sterile and stable under manufacturing and storage conditions. 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 molecules) in a required amount to a suitable solvent, followed by filtration and sterilization. Dispersions are typically prepared by incorporating the active compound into a sterile solvent containing a base dispersion medium and other components. Coating agents such as lecithin can be used. In the case of dispersions, suitable flowability of the solution can be maintained by using surfactants. Prolonged absorption of injectable compositions can be achieved by including substances that delay absorption, such as monostearate and gelatin.

[0550] Kits containing the antibody molecules described herein are also within the scope of this invention. Kits may include one or more other elements, such as: packaging inserts; other reagents, such as markers or conjugation reagents; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

[0551] VIII. Combination products or reagent kits

[0552] In some embodiments, the present invention also provides a combination product comprising the antibody of the present invention or its antigen-binding fragment, or its immunoconjugate, and one or more other therapeutic agents. In some embodiments, the other therapeutic agents are selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, small molecule drugs, or immunomodulators (e.g., activators of co-stimulatory molecules or inhibitors of immune checkpoint molecules); preferably, the second therapeutic agent is a small molecule drug, preferably selected from KRAS small molecule inhibitors, such as KRAS G12C inhibitors (e.g., AMG510 (Sotorasib) or GFH925), KRAS G12D (e.g., MRTX1133), or KRAS G12S inhibitors.

[0553] In some embodiments, the combined product is used to prevent or treat diseases such as acute and chronic inflammatory diseases, infections (e.g., chronic infections), tumors, etc. For example, the disease is a tumor (e.g., cancer) or an infection. In some embodiments, the tumor is a tumor immune escape. Preferably, the tumor is an epithelial cancer, such as a gastrointestinal tumor or 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 squamous cell carcinoma of the head and neck), 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 carcinoma of the lung, adenocarcinoma of the lung). 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 protozoan infection, etc.

[0554] In some regimens, two or more components of the combined product may be administered to the subject sequentially, separately, or simultaneously.

[0555] In some embodiments, the present invention also provides a kit comprising the antibody, pharmaceutical composition, immunoconjugate, or combination product of the present invention, and optionally a packaging insert with instructions for administration.

[0556] In some embodiments, the present invention also provides pharmaceutical articles comprising the antibodies, pharmaceutical compositions, immunoconjugates, and combination products of the present invention, optionally including a packaging insert for instructions of administration.

[0557] IX. Uses of the antibody molecules of the present invention

[0558] In another aspect, the present invention relates to a method for preventing or treating a tumor (e.g., cancer) in a subject, said method comprising administering to said subject an effective amount of an antibody molecule or pharmaceutical composition or immunoconjugate or combination product or kit disclosed herein. In some embodiments, the tumor in the subject of the present invention includes solid tumors and hematologic malignancies. In some embodiments, the tumor is a tumor immune escape. In some embodiments, said tumor is cancer.

[0559] In some implementations, compared to normal cells in the same tissue adjacent to or near normal cells in the same subject, or compared to normal cells in the same tissue of a healthy subject, the tumor cells of the tumor have one or more of the following characteristics:

[0560] (i) Overexpression of wild-type EGFR (e.g., wild-type EGFR with elevated nucleic acid or protein levels) compared to normal cells in adjacent tissues or normal cells in the same tissue in healthy subjects, and / or expression of mutated EGFR, such as EGFR containing mutations listed in Passaro A, et al. Nat Cancer. 2021, preferably, said mutated EGFR containing one or more mutations selected from R521K, L858R, T790M, G719X, C797S, Y1069C, Exon19 deletion (Del19), Exon20ins (e.g., S768_D770dup), preferably, said mutated EGFR containing R521K / Y1069C, R521K, L858R / T790M / C797S, Del19 / T790M / C797S or S768_D770dup;

[0561] (ii) Overexpressing wild-type KRAS (e.g., wild-type KRAS with elevated nucleic acid or protein levels) or expressing mutated KRAS, preferably, the mutated KRAS containing a G12 or G13 mutation, such as G12D or G12C, compared to normal cells in adjacent tissues or normal cells in the same tissue in healthy subjects.

[0562] (iii) B7-H3 with elevated nucleic acid or protein levels compared to normal cells in adjacent tissues or compared to normal cells in the same tissue in healthy subjects; and / or

[0563] (iv) The tumor cells are resistant to tyrosine kinase inhibitors, such as first-generation (erlotinib) and third-generation (osimertinib), for example, resistant to osimertinib.

[0564] 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 the antibody molecule or pharmaceutical composition or immunoconjugate or combination product or kit disclosed herein. In one embodiment, the infectious disease is a chronic infection.

[0565] The subject may be a mammal, such as a primate, preferably a higher primate, such as a human (e.g., a patient with or at risk of having the diseases described herein). In one embodiment, the subject has or is at risk of having the diseases described herein (e.g., a tumor, infection, or autoimmune disease as described herein). In some embodiments, the subject has received or has received other treatments, such as chemotherapy and / or radiation therapy.

[0566] In other respects, the present invention provides the use of antibody molecules or fragments thereof or their immunoconjugates or compositions or combination products or kits in the manufacture or preparation of medicaments for the treatment of the related diseases or conditions mentioned herein.

[0567] In some embodiments, the antibodies or antibody fragments or immunoconjugates or compositions or combination products or kits of the present invention may delay the onset of symptoms and / or symptoms associated with the disease.

[0568] In some embodiments, the antibody or pharmaceutical composition or immunoconjugate or combination product or kit of the present invention may 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.

[0569] In some implementations, treatment methods include surgery (such as tumor resection) or radiation therapy.

[0570] In some implementations, the therapeutic agent is selected from anti-angiogenic agents, chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, anti-infective agents, small molecule drugs, or immunomodulators.

[0571] In some embodiments, the small molecule drug is selected from KRAS small molecule inhibitors, such as KRAS G12C inhibitors (e.g., AMG510 (Sotorasib) or GFH925), KRAS G12D (e.g., MRTX1133), or KRAS G12S inhibitors.

[0572] Immunomodulators include immune checkpoint molecule inhibitors and co-stimulatory molecule activators.

[0573] In some further embodiments, the antibody or fragment thereof of the present invention is used in combination with a KRAS small molecule inhibitor, such as a KRAS G12C inhibitor (e.g., AMG510 (Sotorasib) or GFH925), a KRASG12D inhibitor (e.g., MRTX1133), or a KRAS G12S inhibitor.

[0574] In some embodiments, the antibodies or fragments thereof of the present invention may be administered in combination with treatments comprising adoptive transfer T cells expressing chimeric antigen receptors (CARs), such as cytotoxic T cells or CTLs.

[0575] In some embodiments, the antibody or fragment thereof of the present invention may be administered in combination with an antitumor agent.

[0576] In some embodiments, the antibody or fragment thereof of the present invention may be administered in combination with cytokines. Cytokines may be administered as fusion molecules with the antibody molecule of the present invention, or as individual compositions. In one embodiment, the antibody of the present invention is administered in combination with one, two, three, or more cytokines (e.g., as fusion molecules or as individual compositions).

[0577] In some embodiments, the antibodies or fragments thereof of the present invention may be combined with conventional cancer therapies in the art, including but not limited to: (i) radiotherapy; (ii) chemotherapy, or the application of cytotoxic drugs that generally affect rapidly dividing cells; (iii) targeted therapy, or agents that specifically affect cancer cell protein deregulation; (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 angiogenesis and growth; and (vii) palliative care.

[0578] In some embodiments, the antibodies or fragments thereof of the present invention can be combined with conventional methods for enhancing host immune function.

[0579] The various combination therapies described above can be further combined for treatment.

[0580] Such combination therapies encompass combined administration (where two or more therapeutic agents are contained in the same formulation or separate formulations) and separate administration, in which case the antibody of the present invention may be administered before, simultaneously with, and / or after the administration of other therapies, such as modalities and / or therapeutic agents. The antibody molecule and / or other therapies, such as therapeutic agents or modalities, may be administered during active disease or during remission or less active disease. The antibody molecule may be administered before, simultaneously with, after, or during disease remission of other treatments.

[0581] The antibodies of the present invention (and pharmaceutical compositions or immunoconjugates comprising them, and any other therapeutic agents) may be administered by any suitable method, including parenteral administration, intrapulmonary administration, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Depending to some extent on whether the administration is short-term or long-term, it may be administered via any suitable route, such as by injection, for example, intravenous or subcutaneous injection. Various administration schedules are covered herein, including, but not limited to, single-dose or multiple-dose administration at multiple time points, bolus administration, and pulsatile infusion.

[0582] For the prevention or treatment of disease, the appropriate dosage of the antibody of the present 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 progression of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is appropriately administered to the patient as a single treatment or after a series of treatments.

[0583] The dosage and treatment regimen of the antibody molecules of the present invention can be determined by a technician. In some embodiments, the dosage regimen is adjusted to provide the optimal desired response (e.g., therapeutic response).

[0584] In some embodiments, the antibodies of the present invention (and pharmaceutical compositions or immunoconjugates comprising them) (alone or in combination with other therapeutic agents) may be administered twice a week, once a week, or once every two weeks.

[0585] It is understood that any treatment can be performed using the immunoconjugates or compositions or combination products or kits of the present invention, replacing or supplementing the antibodies of the present invention.

[0586] Example 1. Construction of anti-B7-H3 / EGFR bispecific antibody

[0587] The anti-B7-H3 / EGFR bispecific antibody molecule of the present invention is prepared by assembling an anti-B7-H3 antibody parent and an anti-EGFR antibody parent into an IgG1 antibody form (e.g., using the Innobody Protein Science technology platform of Innovent Biologics (Suzhou) Co., Ltd. (application number: PCT / CN2021 / 143141, invention title: protein containing heterodimeric antibody Fc and its preparation method) or by using conventional methods in the art. Figure 1 (As shown). This bispecific antibody contains four polypeptide chains and can bind to two antigens: antigen A is EGFR and antigen B is B7-H3.

[0588] The parental antibody used to construct the bispecific antibody is the anti-EGFR monoclonal antibody anti-Zalutumumab (hereinafter referred to as Zalu, publication number: WO02100348A2, invention name: Human monoclonal antibodies toepidermal growth factor). The study describes the development of an anti-B7-H3 monoclonal antibody and an anti-EGFR receptor. The antigen-binding region sequences of anti-B7-H3 monoclonal antibodies Hz20G5.26 and Hz19A2.25, obtained through antibody humanization from the Innovent Biologics hybridoma technology screening platform (application number: PCT / CN2021 / 140449, invention title: Hz20G5 and Hz19A2 in the context of anti-B7-H3 antibody and its use), and the antigen-binding region sequence of anti-B7-H3 monoclonal antibody Hz5C2.9, obtained through antibody humanization from the Innovent Biologics hybridoma technology screening platform. Simultaneously, innobody technology is used to mutate the Fc region to enhance the formation of specific antibody heterodimers (application number: PCT / CN2021 / 143141, invention title: protein containing heterodimeric antibody Fc and its preparation method). Three bispecific antibodies that simultaneously bind to B7H3 and EGFR were obtained through screening, designated as Hz5C2.9 / ZalubsAb, Hz19A2.25 / ZalubsAb, and Hz20G5.26 / ZalubsAb, respectively. The specific amino acid sequence numbers are shown in Table 1 and Table A - Sequence Information. All three bispecific antibodies adopt an IgG-like structure; see the schematic diagram for details. Figure 1 .

[0589] Table 1. Sequence details of B7H3 / EGFR bsAb molecules and positive control Gp120 / Zalu.

[0590]

[0591] 1 Zhou,T.,Xu,L.,Dey,B.et al.Structural definition of a conservedneutralization epitope on

[0592] HIV-1 gp120.Nature 445,732–737(2007)

[0593] Example 2. Preparation of anti-B7-H3 / EGFR bispecific antibody molecules

[0594] 1. Construction of bispecific antibody plasmids

[0595] The heavy chain sequences of anti-EGFR antibodies, light chain sequences of anti-EGFR antibodies, heavy chain sequences of anti-B7-H3 antibodies, and light chain sequences of anti-B7-H3 antibodies listed in Table 1 were inserted into the vector pcDNA3.1 (Invitrogen, V790-20) to obtain heavy chain plasmids and light chain plasmids at the anti-EGFR and anti-B7-H3 ends, respectively.

[0596] 2. Bispecific antibody preparation process

[0597] Using GlymaxX technology (ProBioGen AG, see patent publication number: WO2011035884A1, all contents of which are incorporated herein by reference for the purposes of this 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 with heavy chain and light chain plasmids of anti-EGFR parental antibody and heavy chain and light chain plasmids of anti-B7-H3 parental antibody, respectively, via PEI MAX (Polysciences, A804355), to express low-fucose-content anti-EGFR and anti-B7-H3 end antibody parents. Seven days later, the cell fermentation broth was harvested, filtered and clarified, and captured using a Hitrap Mabselect Sure chromatography column (GE Healthcare, 11-0034-95) to obtain antibodies against the EGFR parent and the B7H3 parent.

[0598] After concentration determination using the A280 method, the antibody parents were mixed at a 1:1 molar ratio, and an appropriate amount of reducing agent GSH was added. The mixture was reacted overnight at room temperature, and the reducing agent was removed by ultrafiltration to terminate the reaction. Fine purification was performed using a MonoS cation exchange chromatography column (GE Healthcare, 17-5168-01). The anti-B7-H3 parental solution was 20 mM sodium phosphate buffer (pH 6.6), and the anti-EGFR parental solution was 20 mM sodium phosphate buffer containing 1 M sodium chloride (pH 6.6). The elution gradient was 0-50% (30 column volumes). The eluted protein solution was ultrafiltered and the buffer was replaced with PBS (Gibco, 70011-044). Purity was determined by SEC-HPLC.

[0599] Example 3. Affinity detection of Hz20G5.26 / Zalu bsAb bispecific antibody molecules.

[0600] The affinity (KD) of the bispecific antibody of this invention for binding to B7H3 and EGFR was determined using biofilm thin-layer interferometry (BLI).

[0601] Half an hour before the experiment, according to the sample quantity, take an appropriate number of AHC sensors (18-5060, Sartorius) and soak them in SD buffer (1x PBS, 0.1% BSA, 0.05% Tween-20). Dilute the prepared antibody and human B7H3 (B73-H52E2, Acro Biosystem) and human EGFR (EGR-H5222, Acro Biosystem) to 100 nM respectively.

[0602] SD buffer, antibody solution, human B7H3, and human EGFR were added to 96-well black polystyrene microplates (Greiner, 655209), respectively. Detection was performed using a Fortebio Octet Red96e. The plate was arranged according to the sample location, and the sensor position was selected. The instrument settings were as follows: baseline equilibration 120s, antibody loading and solidification 100s, baseline equilibration 120s, antigen binding 100s, and dissociation 120s; rotation speed 1000 rpm; temperature 30℃. After the experiment, the KD values ​​were analyzed using ForteBioOctet analysis software. The results are shown in Table 2 below.

[0603] Table 2: Affinity Detection of B7H3 / EGFR bs Bispecific Antibody Molecules

[0604]

[0605]

[0606] Example 4. Screening of anti-B7-H3 / EGFR bispecific antibodies

[0607] Using the Innobody technology platform, parental sequences of anti-B7-H3 and anti-EGFR antibodies were expressed and assembled into bispecific antibody molecules: Hz5C2.9 / Zalu bsAb, Hz19A2.25 / Zalu bsAb, and Hz20G5.26 / Zalu bsAb. The in vitro activity of the B7H3 / Zalu bsAb bispecific antibody molecules in non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) was assessed using a growth inhibition assay and an antibody-dependent cell-mediated cytotoxicity assay (ADCC cell report). Safety was also validated in the human skin squamous cell carcinoma cell line (A431).

[0608] Cell line source and culture medium:

[0609] PC9 (NSCLC): Shanghai Yubo Biotechnology, YB-H3210D; Culture medium: MEM + 10% FBS + 1% Pen / strep

[0610] TE-1(HNSCC): CoBioer, CBP60655; Culture medium: RPMI 1640 + 10% FBS + 1% Pen / strep

[0611] SK-MES-1 (NSCLC): CoBioer, CBP60152; Culture medium: MEM + 1% NEAA + 1mM Sodium Pyruvate + 10% FBS + 1% Pen / strep

[0612] A431: Squamous cell carcinoma cells of the skin, ATCC, CRL-1555; Culture medium: DMEM + 10% FBS + 1% Pen / strep

[0613] Construction of PC9+hB7H3 and CHO-S+hB7H3 cell lines: Lentiviral virus (hB7H3(UniProt, Q5ZPR3-1), Lentiviral virus (PPL plasmid and protein sharing library, BC000141)) was constructed and packaged. PC9 (NSCLC, Shanghai Yubo Biotechnology, YB-H3210) and CHO-S (Thermo) were infected with Lentiviral virus+hB7H3, respectively. Stable PC9+hB7H3 and CHO-S+hB7H3 cell lines were obtained by pressure screening and sorting.

[0614] Experimental methods:

[0615] 1. Growth inhibition assay

[0616] (1) NSCLC: 1500-2500 cells / 100ul were laid in 96-well low-absorption plates (Corning, CLS7007-24EA) for 3D cell culture.

[0617] HNSCC: 1500-2000 cells / 100ul were seeded in 96-well white plates (NUNC, 136101) for 2D cell culture.

[0618] (2) Add the pre-diluted antibody molecules (maximum concentration 300 nM, 3.16 times dilution) to the corresponding cell well plate, mix well, and incubate at 37℃ with 5% CO2 for 5 days.

[0619] (3) After continuous culture for 5 days, the Cell-Titer reagent (Promega, G7572) prepared in advance was added to the corresponding cell wells and kept at room temperature in the dark for 15-25 minutes. The HNSCC cells were then directly detected by a multifunctional microplate reader (Molecular Devices, Spectra MAXi3).

[0620] (4) NSCLC: The Cell-Titer and cell mixture was transferred into a 96-well white plate (NUNC, 136101) and detected by a multi-functional microplate reader (Molecular Devices, Spectra MAXi3).

[0621] See results Figure 2 The percentage of surviving cells (%) Figure 2 A, B, C, D, and F represent the relative cell viability detected. See [link to cell labeling and category information] for the corresponding cell labels. Figure 2 The accompanying diagrams illustrate this, such as PC9 being a type of NSCLC and TE-1 being a type of HNSCC. A431 is a skin squamous cell carcinoma that does not respond to antibody treatment; its purpose is to measure drug side effects.

[0622] Figure 2 The growth inhibition percentage (Growth inhibition%) in E is based on Figure 2 The results (% of surviving cells) were obtained after analysis and statistics, as follows: the maximum cell inhibition rate of the drug in each cell type at the highest antibody concentration of 300 nM, calculated as: 300 nM, (1 - % of surviving cells) × 100%.

[0623] The results of specific molecular proliferation inhibition experiments using three bispecific antibodies—Hz5C2.9 / Zalu bsAb, Hz19A2.25 / Zalu bsAb, and Hz20G5.26 / Zalu bsAb—in HNSCC and NSCLC cell lines ( Figure 2 (A, B, and C) showed that the three specific bispecific antibody molecules all had significant tumor-killing effects in HNSCC and NSCLC. Among them, the Hz20G5.26 / Zalu bsAb bispecific antibody not only had excellent efficacy but also showed poor in vitro efficacy against A431. Figure 2 D) suggests that Hz20G5.26 / Zalu bsAb can reduce or decrease the skin toxicity and other side effects of EGFR antibody series clinical drugs.

[0624] 2. ADCC reporter assay (ADCC cell report assay)

[0625] (1) Mix the target cells (CHO-S+hB7H3 or NCI-H522 (CoBioer, CBP60140) constructed above) and ADCC effector cells (Promega, G7102) evenly into a 96-well white plate (NUNC, 136101) according to the effector-target ratio (10:1).

[0626] (2) Add the pre-diluted antibody molecules (for the detection of CHO-S+HB7H3: the final concentration of the antibody after dilution is up to 6.25 nM, serially diluted 4 times; for the detection of NCI-H522: the final concentration of the antibody after dilution is up to 100 nM, serially diluted 4 times) to the corresponding cell plate, mix well, and incubate at 37℃ with 5% CO2 for 20 hours.

[0627] (3) Add the prepared Bio-glo reagent (Promega, G755B) to the cell plate, let it stand at room temperature in the dark for 10-15 minutes, and then detect it with a multi-functional microplate reader (Molecular Devices, Spectra MAXi3).

[0628] The experimental results are shown in Figure 3 The vertical axis represents the multiple of the effect against IgG1 obtained with IgG1 as the control.

[0629] In NSCLC and HNSCC tumor cell lines, cell proliferation was inhibited ( Figure 2 ) and ADCC report experiment ( Figure 3 Experimental results showed that Hz20G5.26 / Zalu bsAb exhibited the best overall in vitro efficacy. In the A431 skin squamous cell carcinoma line, Hz20G5.26 / Zalu bsAb showed the lowest in vitro toxicity (used to measure the toxicity of EGFR inhibitors to human skin). Therefore, the bispecific antibody of this invention, particularly Hz20G5.26 / Zalu bsAb, exhibits low toxicity to human skin and provides a larger effective and safer drug dose without causing or with minimal adverse reactions, meaning it has a wider efficacy selectivity window and is safer.

[0630] Example 5. Hz20G5.26 / Zalu bsAb bispecific antibody molecule proliferation inhibition in vitro pharmacological activity.

[0631] When EGFR ligands bind to EGFR on the surface of tumor cells, EGFR forms a homodimer, which transmits signals into the cell. Through various cascade reactions, this enables tumor cells to produce biological activities such as proliferation, invasion, metastasis, and anti-apoptosis.

[0632] The antigen-binding moiety of anti-EGFR in Hz20G5.26 / Zalu bsAb binds to EGFR on the surface of tumor cells. This binding blocks the binding of EGFR ligands to EGFR, inhibiting biological signal transduction and blocking corresponding tumor biological activities. Simultaneously, it stimulates EGFR endocytosis, ultimately leading to its degradation by intracellular lysosomes. EGFR is a broad-spectrum epithelial tyrosine kinase receptor. Hz20G5.26 / Zalu bsAb utilizes a parental EGFR antibody sequence with low affinity for EGFR, significantly reducing the toxic side effects of EGFR monoclonal antibodies on normal epithelial tissues such as skin. B7-H3 is highly expressed in various tumor cell lines and lowly expressed in normal tissues and organs. It is a highly selective and broadly expressed TAA. Hz20G5.26 / Zalu bs Ab, based on the low affinity of EGFR, incorporates the high affinity of B7-H3 Hz20G5.26 parent, which greatly enhances EGFR signal blocking activity and improves the pharmacodynamic bioactivity and safety window of Hz20G5.26 / Zalu bs Ab.

[0633] Example 5.1. Detection of B7-H3 and EGFR expression in tumor cell lines

[0634] The drug sensitivity of Hz20G5.26 / Zalu bsAb to tumor cell lines is positively correlated with the abundance of receptor expression on the tumor cell surface. Cell lines were selected from NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), CRC (colon cancer), and normal cell lines for relative expression detection of B7-H3 and EGFR. The in vitro efficacy of Hz20G5.26 / Zalu bsAb was tested and validated based on the expression abundance of B7-H3 and EGFR on the cell surface. The experimental results are as follows: Figure 4 As shown.

[0635] Experimental methods:

[0636] 1. Resuspend all cell lines in FACS buffer. Seed 100,000-200,000 cells into 96-well plates (Corning, CLS3799-50EA). Add 10 μg / ml EGFR antibody (Cetuximab), 10 μg / ml B7-H3 antibody (Hz20G5.26mAb), and 10 μg / ml IgG to the cell wells and incubate at 4°C for 1 hour.

[0637] 2. Wash twice with PBS, add the prepared APC-anti human Fc antibody (Biolegend, 410712) to the corresponding cell plate wells, and incubate at 4°C for 30-40 minutes.

[0638] 3. Wash twice with PBS, and detect by FACS (BD, Celesta). Results are shown below. Figure 4 .

[0639] Figure 4 The expression of B7H3 and EGFR in different cell lines was shown. Cell lines with high B7H3 expression that depend on the EGFR signaling pathway (such as cell lines containing EGFR mutations obtained by subsequent sequencing analysis (results not shown) or cell lines overexpressing EGFR) can be selected for the activity detection of bispecific antibodies.

[0640] Example 5.2. Inhibition of proliferation of Hz20G5.26 / Zalu bsAb bispecific antibody molecules in various tumor cell lines.

[0641] EGFR is highly expressed on the surface of various solid tumor cells, such as lung cancer (30%-80%), head and neck squamous cell carcinoma (36%-100%), colorectal cancer (25%-77%), and esophageal cancer (43%-89%), making it a broad-spectrum anti-tumor tyrosine kinase target protein. As a broad-spectrum anti-tumor target protein, EGFR also has the highest mutation rate among non-small cell lung cancer (NSCLC) patients (the global average mutation rate is approximately 35%, reaching 40% in China). In NSCLC patients carrying EGFR mutations, classic mutations (L858R, T790M, or Exon 19 deletion) account for 85-90%; primary point mutations and compound mutations of EGFR exon 20ins and T790M, as well as other point mutations and sequence duplication mutations located between exons 18-21, represented by G719X, account for approximately 10% of EGFR mutations.

[0642] H1975-EGFR L858R / T790M / C797S PC9+B7H3-EGFR Del19 / T790M / C797S and H322-EGFR S768_D770dup Cell line construction: Construction and packaging of Lentvirus+EGFR separately L858R / T790M / C797S Lentvirus + EGFR Del19 / T790M / C797S and Lentvirus+EGFR S768_D770dup Viruses, respectively using Lentvirus + EGFR L858R / T790M / C797S Lentvirus + EGFR Del19 / T790M / C797S and Lentvirus+EGFR S768_D770du The virus was used to infect NCI-H1975 (ATCC, CRL-5908), PC9+hB7H3 (constructed as described above), and H322. H1975-EGFR was obtained through pressure screening. L858R / T790M / C797SPC9+B7H3-EGFR Del19 / T790M / C797S and H322-EGFR S768_D770dup Stable cell lines.

[0643] Experimental methods:

[0644] Proliferation inhibition experiment

[0645] (1) NSCLC: 1500-2500 cells / 100ul were seeded into 96-well low-absorption plates (Corning, CLS7007-24EA) for 3D cell culture. The following cell lines and specific quantities were used:

[0646] NSCLC:2000cells / 100μl / well:

[0647] NCI-H292: Cell Bank of Chinese Academy of Sciences, SCSP-582

[0648] NCI-H322: Cobioer, CBP60134

[0649] NCI-H1650:ATCC, CRL-5883

[0650] NCI-H1975:ATCC, CRL-5908

[0651] SK-MES-1: CoBioer, CBP60152

[0652] NCI-H1703:ATCC,HTB-43

[0653] PC9+hB7H3: As described above

[0654] CRC and HNSCC: 1500-2000 cells / 100ul were seeded in 96-well white plates (NUNC, 136101) for 2D cell culture, using the following cell lines and specific quantities:

[0655] CRC: 1500 cells / 100ul / well

[0656] CCK-81: CoBioer, CBP60581

[0657] HT-55: CoBioer, CBP60012

[0658] H508: CoBioer, CBP60795

[0659] LS180: CoBioer, CBP60034

[0660] HNSCC: 1500 cells / 100ul / well

[0661] TE-1: CoBioer, CBP60655

[0662] Colo680: CoBioer, CBP60452. (2) Add the pre-diluted antibody molecules to the corresponding cell well plates, mix well, and incubate at 37°C in a 5% CO2 incubator for 5 days.

[0663] (3) After continuous culture for 5 days, the Cell-Titer reagent (Promega, G7572) prepared in advance was added to the cell wells and kept at room temperature in the dark for 15-25 minutes. CRC and HNSCC cells were directly detected by a multifunctional microplate reader (Molecular Devices, Spectra MAXi3).

[0664] (4) NSCLC: The Cell-Titer and cell mixture was transferred into a 96-well white plate (NUNC, 136101) and detected by a multi-functional microplate reader (Molecular Devices, Spectra MAXi3).

[0665] EGFR, as a broad-spectrum anti-tumor target protein, has been shown to have anti-tumor effects in in vitro proliferation inhibition studies. Furthermore, Hz20G5.26 / Zalu bsAb has been found to have anti-tumor effects in esophageal cancer cell lines (e.g., Figure 6 As shown), in the TE-1 esophageal cancer cell line, Hz20G5.26 / Zalu bsAb showed better antitumor effects than JNJ372. In colorectal cancer and other tumor cell lines (such as...), ... Figure 5 As shown in the figure, Hz20G5.26 / Zalu bsAb can achieve the anti-tumor effect of EGFR monoclonal antibodies, especially in the LS180 colorectal cancer cell line, where its in vitro anti-tumor effect is superior to that of EGFR monoclonal antibodies; in the NCI-H508 colorectal cancer cell line, the maximum anti-tumor killing rate reaches 84%.

[0666] In lung cancer, not all cancers develop due to primary EGFR gene mutations; the average EGFR mutation rate in non-small cell lung cancer (NSCLC) patients is approximately 35%. Standard-of-care EGFR-TKIs (such as Erlotinib (Selleck Chemicals, cat: S7786) or Osimertinib (Selleck Chemicals, cat: S7297)) primarily target patients with sensitive gene mutations, but resistance often develops due to these mutations, necessitating the continuous development of new targeted drugs against novel mutation sites. In lung cancer, Hz20G5.26 / Zalu bsAb is effective not only against EGFR-mutated NSCLC cell lines but also against wild-type EGFR and aberrantly amplified EGFR NSCLC cell lines. In NSCLC cell lines, Hz20G5.26 / Zalu bsAb demonstrates significantly superior overall in vitro antitumor efficacy compared to JNJ-372.

[0667] In NSCLC-EGFR WT Tumor cell lines ( Figure 7.1 The maximum in vitro antitumor killing rate of NCI-H292 was 74.2 ± 2.8%; in NSCLC-EGFR classic mutation (L858R, T790M or Exon19 deletion) tumor cell lines ( Figure 7.2 ), for NCI-H1975 (EGFR L858R / T790M ()( Figure 7.2 B) The maximum in vitro antitumor inhibitory killing rate was 51.2±1.4%, and the IC50 was 0.99 nM, showing superior in vitro efficacy compared to JNJ-372 and EGFR monoclonal antibodies; in NSCLC-EGFR aberrantly expanded tumor cell lines ( Figure 7.3 The maximum in vitro antitumor killing rate of SK-MES-1 was 60.7±15.9%, with an IC50 of 0.40 nM. Overall, its in vitro efficacy was superior to JNJ-372 and EGFR monoclonal antibodies. Furthermore, SK-MES-1 and NCI-H1703, abnormally amplified NSCLC-EGFR tumor cell lines, were resistant to both first-generation (erlotinib) and third-generation (osimertinib) drugs. Osimertinib, a marketed third-generation EGFR-TKI small molecule inhibitor used for the treatment of T790M mutations, is an EGFR-TKI inhibitor. exon20ins Rare mutations often result in poor drug efficacy. This study aims to investigate the effect of Hz20G5.26 / Zalu bsAb on EGFR. exon20ins The efficacy of tumor-specific drugs, as observed in NCI-H322-EGFR WT Based on cells, construct H322-EGFR S768_D770dupStable cell lines showed antitumor drug efficacy in vitro by inhibiting cell proliferation. Figure 7.4 ), Hz20G5.26 / Zalu bsAb is a response to EGFR exon20ins It exhibits in vitro pharmacological activity. With the use and treatment of EGFR-TKI small molecule inhibitors, NSCLC patients are increasingly developing dual (e.g., Del19 / T790M, L858R / T790M) or triple (e.g., Del19 / T790M / C797S, L858R / T790M / C797S) resistance mutations, which is also an important research direction for next-generation EGFR-TKIs. To verify the pharmacological activity of Hz20G5.26 / Zalu bsAb against EGFR-TKI triple resistance mutations, we constructed H1975-EGFR... L858R / T790M / C797S and PC9+B7H3-EGFR Del19 / T790M / C797S Stable cell lines showed anti-tumor drug efficacy during in vitro proliferation. Figure 7.4 ), Hz20G5.26 / Zalu bsAb for EGFR L858R / T790M / C797S and EGFR Del19 / T790M / C797S It has certain in vitro pharmacological activity.

[0668] To further verify the in vitro pharmacological activity of Hz20G5.26 / Zalu bsAb, a comparative analysis of the in vitro proliferation inhibition efficacy of Hz20G5.26 / Zalu bsAb combined with gp120 / Zalu and gp120 / Hz20G5.26 was conducted. Figure 7.5 The results showed that at Hz 20G5.26 / Zalu bsAb in NCI-H292 (Lung adeno, EGFR) WT ) and SK-MES-1 (sqNSCLC, EGFR Amp In NSCLC and LS180 CRC tumor cell lines, the in vitro efficacy of Hz20G5.26 / Zalu bsAb was significantly superior to that of the combined use of gp120 / Zalu and gp120 / Hz20G5.26. The results of the proliferation inhibition and tumor killing experiments using Hz20G5.26 / Zalu bsAb are shown in Table 3.

[0669] Table 3. Results of experiments on the proliferation inhibition and tumor killing of Hz20G5.26 / Zalu bsAb.

[0670]

[0671] Example 5.3. Synergistic effect of Hz20G5.26 / Zalu bsAb bispecific antibody molecule combined with KRAS small molecule inhibitor.

[0672] In cancer patients, besides EGFR abnormal activation leading to cancer development, KRAS gene mutation activation is also a common driver of cancer development. Approximately 25% of human cancers are KRAS-mutated, and KRAS mutations are strongly associated with cancer prognosis and treatment. In CRC and NSCLC tumors, KRAS-G12D and KRAS-G12C are common abnormal mutations. With the use of KRAS small molecule inhibitors, acquired KRAS resistance is expected to develop. The combination of KRAS small molecule inhibitors with other drugs (such as immunotherapy or targeted therapy) is a potential area for new drug research and development.

[0673] Experimental methods:

[0674] Proliferation inhibition experiment

[0675] (1) NSCLC(H358)(Cobioer,CBP60136) and CRC(LS180)(Cobioer,CBP60034): 1500-2500 cells / 100ul were seeded in 96-well low-adsorption plates (Corning,CLS7007-24EA) for 3D cell culture.

[0676] (2) Add the pre-diluted antibody molecules and small molecule inhibitors to the corresponding cell well plates, mix well, and incubate at 37°C and 5% CO2 for 5 days.

[0677] (3) After culturing for 5 days in the proliferation inhibition experiment, the prepared Cell-Titer reagent (Promega, G7572) was added to the cell wells and left to stand at room temperature in the dark for 15-25 minutes.

[0678] (4) Transfer the Cell-Titer and the above cell mixture into a 96-well white plate (NUNC, 136101) and detect it using a multi-functional microplate reader (Molecular Devices, Spectra MAXi3).

[0679] From NCI-H358 (EGFR) WT ,KRAS G12C Results of NSCLC tumor cell line studies revealed ( Figure 8 A) The maximum in vitro antitumor killing rate of Hz20G5.26 / ZalubsAb in H358 was 66.9±1.9%, IC50 was 1.9%. 50=0.48nM, the antitumor efficacy is far superior to EGFR monoclonal antibodies Zalu and JNJ-372, and superior to the combination of Gp120 / Zalu and Gp120 / Hz20G5.26; through combination with AMG510 (KRAS-G12C small molecule inhibitor) (MedChem Expresses, HY-114277), the results showed that Hz20G5.26 / Zalu and AMG510 have a synergistic effect. At 0.98nM, AMG510, under the action of 10nM Hz20G5.26 / Zalu bsAb, increased the killing rate of H358 cells from 22.51% to 67.12%, and enhanced the antitumor effect in H358 cells. Figure 8 B). To demonstrate the synergistic effect, further based on Figure 8 B. Calculate the synergistic score of drug efficacy in SynergyFinder (https: / / synergyfinder.fimm.fi / synergy / 20210817124619827928 / ), and see the results below. Figure 8 C and Figure 8 D. Figure 8 D shows the total score across different dimensions, with a total score of 11.895, which is greater than 10, indicating a synergistic effect between the two drugs.

[0680] From LS180 (EGFR) R521K ,KRAS G12D Results from CRC tumor cell lines showed that the in vitro antitumor efficacy of Hz20G5.26 / Zalu bsAb was superior to that of EGFR monoclonal antibodies Zalu, JNJ372, and the combination of Gp120 / Zalu and gp120 / Hz20G5.26. Figure 7.5 Furthermore, it exhibits a significant synergistic effect when used in combination with MRTX1133 (a small molecule inhibitor of KRAS-G12D). Under the conditions of 1.56 nM Hz20G5.26 / Zalu bsAb, the anti-LS180 tumor efficacy of 15.62 nM MRTX1133 increased from 36.45% to 61.56%, demonstrating a significantly enhanced anti-tumor effect. Figure 9 A). Figure 9 B and Figure 9 In C, SynergyFinder was further used to calculate the synergy score, with a total score of 37.714, representing the synergistic effect between the two drugs.

[0681] Example 5.4. Study on the in vitro efficacy mechanism of Hz20G5.26 / Zalu bsAb bispecific antibody molecule in inhibiting proliferation.

[0682] In proliferation inhibition experiments, Hz20G5.26 / Zalu bsAb, with the assistance of B7H3 antibody, further enhanced EGFR signal blocking, thus achieving a stronger anti-tumor killing effect. In NCI-H358 cells, we investigated EGFR signal inhibition and EGFR ligand blockade using Western blotting to explore the reasons why Hz20G5.26 / Zalu bsAb exhibited superior in vitro anti-tumor effects compared to EGFR monoclonal antibodies and the combination of Gp120 / Zalu and Gp120 / Hz20G5.26.

[0683] Experimental methods:

[0684] 1. EGFR signal blocking experiment

[0685] (1) Cell treatment

[0686] <1> Take NCI-H358 (Cobioer, CBP60136), seed 7.0E5 cells / well in 6-well plates (NEST, 703011) and incubate overnight.

[0687] <2> Remove the culture medium (RPMI 1640 complete medium (RPMI 1640 + 10% FBS)) and add serum-free RPMI 1640 (assay medium) for starvation overnight.

[0688] <3> , remove <2> The culture media were prepared by adding serum-free 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 to the corresponding well plates, respectively. The following groups were established: Blank group (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 mAb). The mixture of mAb, Gp120 / Zalu+Gp120 / B7H3 (200nM Gp120 / Zalu+200nM Gp120 / Hz20G5.26) was placed at 37℃ with 5% CO2 for 2 hours.

[0689] (2) Protein extraction and total protein quantification

[0690] <1> Prepare cell lysis buffer in advance. The cell lysis buffer consists of: 100-200 μL / tube RIPA (Thermo, 89900) + 1:100 phosphatase inhibitor (abcam, ab201112) + 1:20 protease inhibitor (Roche, 11836170001).

[0691] <2> Add the prepared cell lysis buffer to the cells obtained in (1) above to digest the cells, and lyse on ice for 20 min.

[0692] <3> Pre-cool the centrifuge to 4℃, centrifuge at 12000rpm / min for 15min, and collect the supernatant.

[0693] <4> The concentration of the extracted total protein was determined using BCA reagent (Beyotime, P0012).

[0694] <5> Take the corresponding total protein, dilute it with LDS Sample buffer (Invitrogen, 2201446), denature it at 70℃ for 10 minutes, and store it at -40℃ for later use.

[0695] (3) Western blotting

[0696] <1> Add the protein sample and protein marker (Prestained Protein Ladder (Thermo, 26620)) obtained in (3) to the precast gel (Thermo, NP0321BOX); run the gel at 200V using an electrophoresis apparatus (BIO-RAD, TRANS SD CELL) for about 50 minutes.

[0697] <2> Remove the film and transfer it using a transfer machine (Invitrogen, IBCOT2).

[0698] <3> Block slowly for 1-2 hours using the prepared blocking solution (containing 5% skim milk powder TBST); add EGF Receptor Rabbit mAb (CST, 4267), pEGF Receptor Rabbit mAb (CST, 3777) and GAPDHabbit mAb (CST, 2118) antibodies, and incubate overnight at 4 degrees Celsius.

[0699] <4> Wash three times with TBST, each time for 8-10 minutes, with the shaker speed controlled at 80-100 rpm / min.

[0700] <5> Incubate with labeled HRP-Goat Anti-Rabbit IgG (abcam, Ab205718) at room temperature for 1-2 hours.

[0701] <6> Wash three times with TBST, each time for 8-10 minutes, with the shaker speed controlled at 80-100 rpm / min.

[0702] <7> Developed with ECL luminescent liquid (Beyotime, P0018AM), and exposed using a developer (BIO-RAD, chemi Doc MP).

[0703] The results are as follows Figure 10 .

[0704] 2. EGFR ligand blocking assay (Signal blocking)

[0705] (1) Cell treatment

[0706] <1> Take NCI-H358 (H358), seed 7.0E5 cells / well in a 6-well plate (NEST, 703011) and incubate overnight.

[0707] <2> Remove the culture medium (RPMI 1640 complete medium (RPMI 1640 + 10% FBS)) and add serum-free RPMI 1640 medium for starvation treatment overnight.

[0708] <3> Remove the culture medium and add culture medium (2 wells each) containing 200 nM Hz20G5.26 / ZalubsAb, 200 nM Gp120 / Zalu, 200 nM Gp120 / Hz20G5.26, 200 nM Zalu mAb, 200 nM Gp120 / Zalu + 200 nM Gp120 / Hz20G5.26 to the corresponding well plates. Set up the Blank group (culture medium), Control 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 mAb). mAb), Gp120 / Zalu+Gp120 / B7H3 group (200nM Gp120 / Zalu+200nM Gp120 / Hz20G5.26), were placed at 37℃ with 5% CO2 for 1 hour.

[0709] <4> One hour later, 30 nM EGF (ACRO, EGF-H52b) was 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 Gp120 / Hz20G5.26), Zalu group (200 nM Zalu mAb), and Gp120 / Zalu+Gp120 / B7H3 group (200 nM Gp120 / Zalu+200 nM Gp120 / Hz20G5.26). Figure 11 (See above) and 30 nM TGF-α (R&D, 239-A-100) Figure 11 (See the image below), place at 37℃ with 5% CO2 for 1 hour.

[0710] (2) Protein extraction and total protein quantification

[0711] <1> Prepare cell lysis buffer in advance. The cell lysis buffer consists of: 100-200 μL / tube RIPA (Thermo, 89900) + 1:100 phosphatase inhibitor (abcam, ab201112) + 1:20 protease inhibitor (Roche, 11836170001).

[0712] <2> Add the prepared cell lysis buffer to the cells obtained in (1) above to digest the cells, and lyse on ice for 20 min.

[0713] <3> Pre-cool the centrifuge to 4℃, centrifuge at 12000rpm / min for 15min, and collect the supernatant.

[0714] <4> The concentration of the extracted total protein was determined using BCA reagent (Beyotime, P0012).

[0715] <5> Take the corresponding total protein, dilute it with LDS Sample buffer (Invitrogen, 2201446), denature it at 70℃ for 10 minutes, and store it at -40℃ for later use.

[0716] (3) Western blotting

[0717] <1> Add the protein sample and protein marker (Prestained Protein Ladder (Thermo, 26620)) to the precast gel (Thermo, NP0321BOX); run the gel at 200V using an electrophoresis apparatus (BIO-RAD, TRANS SD CELL) for about 50 minutes.

[0718] <2> Remove the film and transfer it using a transfer machine (Invitrogen, IBCOT2).

[0719] <3> Block slowly for 1-2 hours using the prepared blocking solution (containing 5% skim milk powder TBST); add EGF Receptor Rabbit mAb (CST, 4267), pEGF Receptor Rabbit mAb (CST, 3777) and GAPDHabbit mAb (CST, 2118) antibodies, and incubate overnight at 4 degrees Celsius.

[0720] <4> Wash three times with TBST, each time for 8-10 minutes, with the shaker speed controlled at 80-100 rpm / min.

[0721] <5> Incubate with labeled HRP-Goat Anti-Rabbit IgG (abcam, Ab205718) at room temperature for 1-2 hours.

[0722] <6> Wash three times with TBST, each time for 8-10 minutes, with the shaker speed controlled at 80-100 rpm / min.

[0723] <7> Developed with ECL luminescent liquid (Beyotime, P0018AM), and exposed using a developer (BIO-RAD, chemi Doc MP).

[0724] See results Figure 11 .

[0725] Based on the research results of the signal blocking mechanism ( Figure 10 and Figure 11 The study found that Hz20G5.26 / Zalu bsAb had stronger signal blocking and ligand blocking effects than EGFR monoclonal antibody Zalu and the combined effects of Gp120 / Zalu and Gp120 / Hz20G5.26. This result also explains why Hz20G5.26 / Zalu bsAb is superior to EGFR monoclonal antibody and Gp120 / Zalu and Gp120 / Hz20G5.26 in vitro against CRC, NSCLC and HNSCC tumors.

[0726] Example 6. In vitro pharmacodynamic activity of Hz20G5.26 / Zalu bsAb bispecific antibody molecule ADCC

[0727] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism by which antitumor antibodies exert their antitumor effects. The principle is that the antibody Fab fragment binds to the antigenic epitope on the surface of tumor cells, and its Fc fragment binds to the FcR on the surface of cytotoxic immune cells (NK cells, macrophages, neutrophils, etc.). Through immune cell-mediated direct killing of tumor cells, ADCC is mainly achieved through the binding of antibody Fc to the FcR IIIa receptor on the surface of NK cells.

[0728] The introduction of the Hz20G5.26 parent not only enhanced the EGFR antibody blocking activity of Hz20G5.26 / ZalubsAb, but also improved its overall ADCC activity (presumably due to Hz20G5.26 binding to a specific epitope near the membrane of B7H3, thus triggering strong ADCC function). Furthermore, the use of GlymaxX low-fucose technology further enhanced the ADCC activity of Hz20G5.26 / ZalubsAb.

[0729] Therefore, the Hz20G5.26 / Zalu bsAb dual-characteristic antibody molecule can exert multiple anti-tumor effects through both EGFR signaling blockade and ADCC mechanisms. The specific experimental steps are as follows:

[0730] Example 6.1. ADCC Report Experiment

[0731] In the ADCC reporter assay, ADCC effector cells (Promega, G7102) were engineered to overexpress the FcRⅢa(V158) receptor into Jurkat T cells. These engineered cells were able to rapidly respond to ADCC strength through the NFAT-RE driving element within the Jurkat T cells.

[0732] Experimental methods:

[0733] (1) Mix tumor cells (cell lines listed in Table 3) and ADCC effector cells (Promega, G7102) evenly (1.5E4 cells: 1.5E5 cells / well / 100ul (1.5E5: 1.5E6 cells / ml)) into 96-well white plates (NUNC, 136101) according to the effector-target ratio (10:1).

[0734] (2) Add the pre-diluted antibody molecules to the corresponding cell well plates, mix well, and incubate at 37°C with 5% CO2 for 20 hours.

[0735] (3) Add the prepared Bio-glo reagent (Promega, G755B) to the cell plate, let it stand at room temperature in the dark for 10-15 minutes, and then detect it with a multi-functional microplate reader (Molecular Devices, Spectra MAXi3).

[0736] We selected different EGFR-type tumor cell lines (EGFR wild-type, EGFR amplified, EGFR mutant, EGFR wild-type, and KRAS mutant) in NSCLC tumor cell lines and verified the ADCC activity of Hz20G5.26 / ZalubsAb using ADCC reporter assays. The ADCC reporter assay results (Figure 12 and Table 4) showed that in different types of NSCLC-EGFR tumor cell lines, the ADCC activity of Hz20G5.26 / ZalubsAb was stronger than that of JNJ373 and EGFR monoclonal antibodies. Furthermore, in experiments with H292 (NSCLC-EGFR wild-type) and H358 (NSCLC-EGFR wild-type and KRAS mutant), the ADCC activity of Hz20G5.26 / ZalubsAb was not only stronger than that of JNJ372 and EGFR monoclonal antibodies, but also stronger than that of Gp120 / Zalub and the combined ADCC activity of Gp120 / Hz20G5.26.

[0737] Table 4. Results of the Hz20G5.26 / Zalu bsAb ADCC reporter gene experiment.

[0738]

[0739] Example 6.2 huPBMC ADCC Experiment

[0740] Based on the ADCC report results above, we found that the ADCC activity of Hz20G5.26 / Zalu bsAb was stronger than that of JNJ372, EGFR monoclonal antibody Zalu, Gp120 / Zalu, and the combination of Gp120 / Hz20G5.26. To more accurately and effectively reflect the ADCC activity of Hz20G5.26 / ZalubsAb, we designed a huPBMC ADCC experiment, using normal human PBMCs to verify ADCC activity.

[0741] Experimental methods:

[0742] (1) Preheat CTS medium (Gibco, A3021002) to 37°C, take huPBMC (Miaotong Biotechnology, PB100C-W) and quickly melt it in a water bath, then slowly add the cells to 8ml of CTS medium (containing 1% DNase).

[0743] (2) Centrifuge at 300g for 8min, remove the supernatant, resuspend in 30ml CTS (containing 10uL DNA enzyme), transfer to T75 culture flask, and incubate overnight at 37℃.

[0744] (3) Take the overnight cultured suspension cells, centrifuge at 300g / 8min, remove the supernatant, and adjust the cell density with CTS; according to the effector cell (huPBMC) to target cell (tumor cells in Table 5) effector-target ratio of 50:1, place huPBMC and pre-prepared tumor cells (Target:huPBMC = 1.5E4 / 7.5E5 cells / well / 100ul) into a 96-well low-absorption plate (Corning, CLS7007-24EA).

[0745] (4) Add the diluted antibody drug to the corresponding cell well plate and incubate at 37°C and 5% CO2 for 8 hours.

[0746] (5) Centrifuge at 300g for 5min, take 50ul of supernatant and transfer to a 96-well transparent flat plate ((NUNC,136101); take 50ul of the pre-prepared LDH reagent (Promega,G1780) and add it to the corresponding well plate, and let it stand at room temperature in the dark for 15-30min.

[0747] (6) Take 50 μL of LDH Stop Solution (Promega, G1780), read the value at 490 nm, and detect it using a multi-functional microplate reader (Molecular Devices, Spectra MAXi3).

[0748] The results are shown in Figure 13 and Table 5. The experimental results of Hz20G5.26 / Zalu bsAb in huPBMC ADCC more accurately reflect the actual efficacy. The results of huPBMC ADCC (Figure 13 and Table 5) showed that the ADCC activity of Hz20G5.26 / Zalu bsAb in huPBMCs was basically consistent with the ADCC reporter activity results. In many different types of NSCLC-EGFR tumor cell lines, the ADCC activity of Hz20G5.26 / Zalu bsAb in huPBMCs was stronger than that of JNJ373 and EGFR monoclonal antibodies, and stronger than the ADCC activity of Gp120 / Zalu and Gp120 / Hz20G5.26 combined. Through huPBMC ADCC action, although Hz20G5.26 / Zalu bsAb had the lowest antitumor killing rate in H1975, it still achieved a killing rate of 36.53%. The antitumor killing rate of Hz20G5.26 / Zalu bsAb was the highest in H322, reaching 100%.

[0749] Therefore, Hz20G5.26 / Zalu bsAb, by enhancing EGFR signal blocking and participating in ADCC through immune cells, can further exert the antitumor activity of Hz20G5.26 / Zalu bsAb in a variety of cancer tumors.

[0750] Table 5. Experimental Results of Hz20G5.26 / Zalu bsAb huPBMC ADCC

[0751]

[0752]

[0753] Example 6. In vitro safety study of Hz20G5.26 / Zalu bsAb bispecific antibody molecular drug.

[0754] EGFR is an epidermal growth factor derived from the epidermis. Besides overexpression or abnormal activation leading to cancerous tumors, EGFR is also expressed in some epithelial cells and keratinocytes. Compared to the adverse reactions of EGFR-TKI small molecule inhibitors, such as rash, diarrhea, paronychia, stomatitis, liver damage, and interstitial lung disease, EGFR monoclonal antibodies also have certain adverse reactions, mainly manifested as skin toxicity, such as pustular rashes, paronychia, and dry, itchy skin. This significantly impacts the quality of life and treatment adherence of cancer patients.

[0755] We selected human squamous cell carcinoma cell lines (A431, ATCC, CRL-1555) and human keratinocytes (HaCat, Cell Lines Service, 300493) as adverse reaction study models for Hz20G5.26 / Zalubs Ab to investigate the tolerance of Hz20G5.26 / Zalubs Ab in vitro.

[0756] Experimental methods

[0757] (1) 1500-2000 cells / 100ul of A431 and HaCat cells were seeded into 96-well low-absorption plates (Corning, CLS7007-24EA) for 3D cell culture. The culture medium used was DMEM + 10% FBS + 1% Pen / strep.

[0758] (2) Add the pre-diluted antibody molecules to the corresponding cell well plates, mix well, and incubate at 37°C in a 5% CO2 incubator for 5 days.

[0759] (3) After culturing for 5 days in the proliferation inhibition experiment, the prepared Cell-Titer reagent (Promega, G7572) was added to the cell wells and left to stand at room temperature in the dark for 15-25 minutes.

[0760] (4) Transfer the Cell-Titer and cell mixture into a 96-well white plate (NUNC, 136101) and detect it using a multi-functional microplate reader (Molecular Devices, Spectra MAXi3).

[0761] See results Figure 14 The experimental results showed that the in vitro efficacy of Hz20G5.26 / Zalu bs Ab in A431 and HaCat cells was much lower than that of EGFR monoclonal antibodies, indicating that the cells had high tolerance to Hz20G5.26 / Zalu bs Ab and far fewer adverse reactions than EGFR monoclonal antibodies. At the same time, the cells also showed better tolerance to Hz20G5.26 / Zalu bs Ab than to JNJ372.

[0762] Therefore, Hz20G5.26 / Zalu bs Ab, by using the high-affinity parent Hz20G5.26 B7H3 while reducing EGER affinity, improves the pharmacodynamic bioactivity and pharmacodynamic safety window of Hz20G5.26 / Zalu bs Ab.

[0763] Example 7. In vivo pharmacodynamic activity of Hz20G5.26 / Zalu bsAb bispecific antibody molecule.

[0764] To demonstrate the in vivo efficacy of the Hz20G5.26 / Zalu bispecific antibody molecule, NSCLC-EGFR was used. WT The antitumor efficacy of the Hz20G5.26 / Zalu bispecific antibody of this invention was determined by inoculating Balb / c Nude mice with the tumor cell line NCI-H292 (ATCC). SPF-grade female Balb / c Nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., certificate number NO.110011211108430747) were used in the experiment.

[0765] NCI-H292 cells were passaged using standard methods for subsequent in vivo experiments. Cells were collected by centrifugation and resuspended in a mixture of PBS (1×) and Matrigel Matix (Corning) in equal proportions to prepare a cell concentration of 20×10⁶ cells / mL. 6 Cells / ml suspension. On day 0, 0.2 ml of cell suspension was subcutaneously injected into the right abdominal region of Balb / c Nude mice to establish an NCI-H292 tumor-bearing mouse model.

[0766] 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.

[0767] Table 6: Grouping, Dosage, and Method of Administration in In Vivo Experiments

[0768]

[0769] *: h-IgG is an isotype control antibody, purchased from Equitech-Bio, batch number 210112-0356.

[0770] h-IgG, JNJ372, Gp120 / Zalu, Hz20G5.26 / Zalu, and Zalu mAb were all administered at a concentration of 0.5 mg / ml, once every 3-4 days for a total of 4 doses (Q3-4D x4). The drugs were administered on days 4, 8, 12, and 15 after NCI-H292 cell inoculation. Tumor volume and body weight in mice were monitored twice weekly. Figure 15 As shown in Figure A, monitoring ended after 59 days. Since some groups experienced early mortality due to tumor-related causes, the relative tumor inhibition rate (TGI%) was calculated based on tumor volume on day 22 post-inoculation, using the following formula:

[0771] TGI% = 100% * (Tumor volume in control group – Tumor volume in treatment group) / (Tumor volume in control group – Tumor volume in treatment group)

[0772] Tumor volume before drug administration (group).

[0773] Tumor volume measurement: The maximum long axis (L) and maximum width axis (W) of the tumor were measured using vernier calipers. The tumor volume was calculated using the following formula: V = L * W 2 / 2.

[0774] Weight was measured using an electronic balance. For tumors exceeding 2000 mm²... 3 Mice that have lost more than 20% of their body weight are euthanized.

[0775] The tumor inhibition rate results are shown in Table 7. On day 22 post-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. The mouse survival curves are shown below. Figure 15As shown in Figure B, Hz20G5.26 / Zalu significantly prolonged the survival of mice. In conclusion, the antitumor efficacy of Hz20G5.26 / Zalu in NCI-H292 tumor-bearing mice is comparable to that of the parental ZalumAb, and superior to JNJ372 and the non-targeting GP120 / Zalu.

[0776] The results of monitoring mouse body weight ( Figure 15 C) showed that within 59 days after inoculation, the body weight of mice in the Hz20G5.26 / Zalu group did not decrease significantly.

[0777] Table 7: Statistics on the Efficacy of Antitumor Drugs

[0778]

[0779] *Complete response rate: Complete tumor regression, with a tumor volume of 0.

[0780] To demonstrate the in vivo efficacy of the Hz20G5.26 / Zalu bispecific antibody molecule against NSCLC with abnormal EGFR amplification, the antitumor efficacy of the Hz20G5.26 / Zalu bispecific antibody molecule of this invention was determined by inoculating Balb / c Nude mice with SK-MES-1 cells (Nanjing Kebai). SPF-grade female Balb / c Nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), certificate number NO.110011211108966881, were used in the experiment.

[0781] SK-MES-1 cells were passaged using standard methods for subsequent in vivo experiments. Cells were collected by centrifugation and mixed with PBS (1×) and Matrigel Matix (Corning) in equal proportions to prepare a cell concentration of 20 × 10⁶ cells / mL. 6 Cells / ml of cell suspension. On day 0, 0.2 ml of cell suspension was subcutaneously injected into the right abdominal region of Balb / cNude mice to establish the SK-MES-1 tumor-bearing mouse model.

[0782] 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.

[0783] Table 8: Grouping, Dosage, and Method of Administration in In Vivo Experiments

[0784]

[0785] *: h-IgG is an isotype control antibody, purchased from Equitech-Bio, batch number 210112-0356.

[0786] h-IgG, Hz20G5.26 / Zalu, JNJ372, Zalu mAb, and Gp120 / Zalu were all administered at a concentration of 0.1 mg / ml. The drugs were administered once on day 6 after SK-MES-1 cell inoculation, and tumor volume and body weight were monitored twice weekly. Figure 16 As shown in A, monitoring ended after 24 days.

[0787] The relative tumor inhibition rate (TGI%) was calculated on day 24 after vaccination. The calculation formula is as follows: TGI% = 100% * (Tumor volume of control group – Tumor volume of treatment group) / (Tumor volume of control group – Tumor volume of control group before administration).

[0788] Tumor volume measurement: The maximum long axis (L) and maximum width axis (W) of the tumor were measured using vernier calipers. The tumor volume was calculated using the following formula: V = L * W 2 / 2. Weight was measured using an electronic balance.

[0789] The tumor inhibition rate results are shown in Table 9: On day 24 post-inoculation, compared with the h-IgG, 1 mg / kg group, the tumor inhibition rates of Hz20G5.26 / Zalu, JNJ372, Zalu mAb, and Gp120 / Zalu were 143%, 122%, 145%, and 113%, respectively. Simultaneously, the results of mouse body weight monitoring were also presented. Figure 16 B) shows that there was no significant difference in mouse body weight on day 24 post-inoculation.

[0790] Table 9: Statistics on the efficacy of antitumor drugs on day 24

[0791]

[0792] *Complete response rate: Complete tumor regression, with a tumor volume of 0.

[0793] To further demonstrate the advantages of the Hz20G5.26 / Zalu molecule as a bispecific antibody in vivo, the antitumor efficacy of the Hz20G5.26 / Zalu bispecific antibody molecule of this invention was determined by inoculating Balb / c Nude mice with SK-MES-1 cells (Nanjing Kebai). SPF-grade female Balb / c Nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), certificate number NO.110011221103705231, were used in the experiment.

[0794] SK-MES-1 cells were passaged using standard methods for subsequent in vivo experiments. Cells were collected by centrifugation and mixed with PBS (1×) and Matrigel Matix (Corning) in equal proportions to prepare a cell concentration of 25 × 10⁶ cells / mL. 6Cells / ml of cell suspension. On day 0, 0.2 ml of cell suspension was subcutaneously injected into the right abdominal region of Balb / c Nude mice to establish the SK-MES-1 tumor-bearing mouse model.

[0795] 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.

[0796] Table 10: Grouping, Dosage, and Method of Administration in In Vivo Experiments

[0797]

[0798] *: h-IgG is an isotype control antibody, purchased from Equitech-Bio, batch number 210112-0356.

[0799] The concentration of h-IgG used was 0.06 mg / ml, and the concentrations of Hz20G5.26 / Zalu, Gp120 / Zalu, and Gp120 / Hz20G5.26 were all 0.03 mg / ml. The drugs were administered once on day 14 after SK-MES-1 cell inoculation, and tumor volume and body weight in mice were monitored twice weekly. Figure 17 As shown in A, monitoring ended after 35 days.

[0800] The relative tumor inhibition rate (TGI%) was calculated on day 35 after vaccination. The calculation formula is as follows: 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).

[0801] Tumor volume measurement: The maximum long axis (L) and maximum width axis (W) of the tumor were measured using vernier calipers. The tumor volume was calculated using the following formula: V = L * W 2 / 2. Weight was measured using an electronic balance.

[0802] The tumor inhibition rate results are shown in Table 11: On day 35 post-inoculation, compared with the h-IgG, 0.6 mg / kg group, the tumor inhibition rates of Hz20G5.26 / Zalu, Gp120 / Zalu, Gp120 / Hz20G5.26, and Gp120 / Zalu+Gp120 / Hz20G5.26 were 121%, 31%, 1%, and 16%, respectively. The antitumor efficacy of Hz20G5.26 / Zalu was superior to the control non-targeting monoclonal antibody Gp120 / Zalu, Gp120 / Hz20G5.26, or a combination of both, demonstrating the unique mechanism of Hz20G5.26 / Zalu as a bispecific antibody, namely, the pulling effect of Hz20G5.26 on zalu blocking EGFR signaling. Simultaneously, the results of monitoring mouse body weight (…) Figure 17B) shows that there was no significant difference in mouse body weight on day 24 post-inoculation.

[0803] Table 11: Statistics on the efficacy of antitumor drugs on day 35

[0804]

[0805] Example 8. In vivo pharmacodynamic activity of Hz20G5.26 / Zalu bsAb bispecific antibody molecule combined with KRAS small molecule inhibitor.

[0806] To demonstrate the in vivo efficacy of the Hz20G5.26 / Zalu bispecific antibody molecule combined with a KRAS small molecule inhibitor, KRAS was used. G12C The antitumor efficacy of the Hz20G5.26 / Zalu bispecific antibody combined with the KRAS small molecule inhibitor AMG510 was determined by inoculating NOG mice with the mutant NSCLC tumor cell line NCI-H358 (Nanjing Kebai). SPF-grade female NOG mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., certificate number NO.1100112211001153625) were used in the experiment.

[0807] NCI-H358 cells were passaged using standard methods for subsequent in vivo experiments. Cells were collected by centrifugation and resuspended in a mixture of PBS (1×) and Matrigel Matix (Corning) in equal proportions to prepare a cell concentration of 25 × 10⁶ cells / year. 6 Cells / ml suspension. On day 0, 0.2 ml of cell suspension was subcutaneously injected into the right abdominal region of NOG mice to establish an NCI-H358 tumor-bearing mouse model.

[0808] 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.

[0809] Table 12: Grouping, Dosage, and Method of Administration in In Vivo Experiments

[0810]

[0811] *: h-IgG is an isotype control antibody, purchased from Equitech-Bio, batch number 210112-0356.

[0812] The h-IgG concentration was 2 mg / ml, and the concentrations of Hz20G5.26 / Zalu, JNJ372, and Zalu mAb were all 1 mg / ml, administered once every 3-4 days for a total of 4 times (Q3-4D x4). Administration was performed on days 6, 9, 12, and 15 after NCI-H358 cell inoculation, with tumor volume and body weight monitored twice weekly. AMG510 was administered at a concentration of 1 mg / ml, starting on day 6 after NCI-H358 cell inoculation, once daily for a total of 14 times (QD x14), with tumor volume monitored twice weekly. Mouse body weight was monitored daily during administration and twice weekly after administration. Figure 18 As shown in Figure A, monitoring ended after 33 days. The relative tumor inhibition rate (TGI%) was calculated on day 33 post-vaccination 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 administration).

[0813] Tumor volume measurement: The maximum long axis (L) and maximum width axis (W) of the tumor were measured using vernier calipers. The tumor volume was calculated using the following formula: V = L * W 2 / 2. Weight was measured using an electronic balance.

[0814] The tumor inhibition rate results are shown in Table 13. On day 33 post-inoculation, compared with the h-IgG, 20 mg / kg group, the tumor inhibition rates of AMG510, Hz20G5.26 / Zalu, AMG510+Hz20G5.26 / Zalu, JNJ372, and Zalu mAb were 89%, 110%, 130%, 82%, and 111%, respectively. Among the groups, Hz20G5.26 / Zalu combined with AMG510 showed the best antitumor efficacy in NCI-H358 tumor-bearing mice, exhibiting a synergistic effect.

[0815] The results of monitoring mouse body weight ( Figure 18 B) shows that AMG510 administration caused a gradual decrease in mouse body weight, which rebounded after administration was discontinued, and the body weight of mice in all groups returned to normal on day 33.

[0816] Table 13: Tumor suppression rate on day 33

[0817]

[0818] Sequence listing: Sequence information of anti-B7-H3 / EGFR bispecific antibody

[0819]

[0820]

[0821]

[0822]

[0823]

[0824]

[0825]

[0826]

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, HCDR2, and HCDR3 are the three complementary determinant regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO: 3; and LCDR1, LCDR2, and LCDR3 are the three complementary determinant regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO: 4; wherein HCDR1 is determined using the AbM protocol, and HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are determined using the Kabat protocol.

2. 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 consists of the amino acid sequence shown in SEQ ID NO:15, HCDR2 consists of the amino acid sequence shown in SEQ ID NO:16, and HCDR3 consists of the amino acid sequence shown in SEQ ID NO:

17. LCDR1 consists of the amino acid sequence shown in SEQ ID NO:18, LCDR2 consists of the amino acid sequence shown in SEQ ID NO:19, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO:

20.

3. The bispecific antibody of claim 1 or 2, wherein the second antigen-binding region comprises a heavy chain variable region VH, wherein the VH comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:

3.

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

3.

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

3.

6. The bispecific antibody according to claim 1 or 2, wherein, The second antigen-binding region includes a light chain variable region VL, wherein the VL contains an amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO:

4.

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

4.

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

4.

9. The bispecific antibody of claim 1 or 2, wherein the second antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 3, and the VL comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

4.

10. The bispecific antibody of claim 1 or 2, 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.

11. The bispecific antibody of claim 1 or 2, wherein the second antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH consists of the amino acid sequence shown in SEQ ID NO: 3, and the VL consists of the amino acid sequence shown in SEQ ID NO:

4.

12. The bispecific antibody of claim 1 or 2, 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 HCDR1, HCDR2, and HCDR3 are the three complementary determinant regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO: 1; and LCDR1, LCDR2, and LCDR3 are the three complementary determinant regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO: 2; wherein the CDRs are determined using the Kabat scheme.

13. The bispecific antibody of claim 1 or 2, 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, and HCDR1 is composed of the amino acid sequence of SEQ ID NO:9; HCDR2 is composed of the amino acid sequence of SEQ ID NO:10; HCDR3 is composed of the amino acid sequence of SEQ ID NO:11; and LCDR1 is composed of the amino acid sequence of SEQ ID NO:12; LCDR2 is composed of the amino acid sequence of SEQ ID NO:13; and LCDR3 is composed of the amino acid sequence of SEQ ID NO:

14.

14. The bispecific antibody of claim 1 or 2, wherein the first antigen-binding region comprises a heavy chain variable region VH, wherein the VH comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

1.

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

1.

16. The bispecific antibody of claim 1 or 2, wherein the first antigen-binding region comprises a heavy chain variable region VH, wherein the VH consists of the amino acid sequence shown in SEQ ID NO:

1.

17. The bispecific antibody of claim 1 or 2, wherein the first antigen-binding region comprises a light chain variable region VL, wherein the VL comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

2.

18. The bispecific antibody of claim 1 or 2, wherein the first antigen-binding region comprises a light chain variable region VL, wherein the VL comprises the amino acid sequence shown in SEQ ID NO:

2.

19. The bispecific antibody of claim 1 or 2, wherein the first antigen-binding region comprises a light chain variable region VL, wherein the VL consists of the amino acid sequence shown in SEQ ID NO:

2.

20. The bispecific antibody of claim 1 or 2, wherein the first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1, and the VL comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

2.

21. The bispecific antibody of claim 1 or 2, 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.

22. The bispecific antibody of claim 1 or 2, wherein the first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH consists of the amino acid sequence shown in SEQ ID NO:1, and the VL consists of the amino acid sequence shown in SEQ ID NO:

2.

23. The bispecific antibody of claim 2, 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 consists of the amino acid sequence of SEQ ID NO:9; HCDR2 consists of the amino acid sequence of SEQ ID NO:10; HCDR3 consists of the amino acid sequence of SEQ ID NO:11; LCDR1 consists of the amino acid sequence of SEQ ID NO:12; LCDR2 consists of the amino acid sequence of SEQ ID NO:13; and LCDR3 consists of the amino acid sequence of SEQ ID NO:14; and The second antigen-binding region specifically binds to B7-H3, which includes HCDR1, HCDR2, and HCDR3 of the heavy chain variable region VH, and LCDR1, LCDR2, and LCDR3 of the light chain variable region VL. HCDR1 consists of the amino acid sequence of SEQ ID NO:15; HCDR2 consists of the amino acid sequence of SEQ ID NO:16; HCDR3 consists of the amino acid sequence of SEQ ID NO:17; LCDR1 consists of the amino acid sequence of SEQ ID NO:18; LCDR2 consists of the amino acid sequence of SEQ ID NO:19; and LCDR3 consists of the amino acid sequence of SEQ ID NO:

20.

24. The bispecific antibody of claim 23, wherein the first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 1, and the VL comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2; and The second antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH contains an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 3, and the VL contains an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

4.

25. The bispecific antibody of claim 23, 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, and 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.

26. The bispecific antibody of claim 23, wherein the first antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH consists of the amino acid sequence shown in SEQ ID NO:1 and the VL consists of the amino acid sequence shown in SEQ ID NO:2; and the second antigen-binding region comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH consists of the amino acid sequence shown in SEQ ID NO:3 and the VL consists of the amino acid sequence shown in SEQ ID NO:

4.

27. The bispecific antibody according to any one of claims 1, 2 and 23-26, comprising an Fc region.

28. The bispecific antibody of claim 27, wherein the Fc region has low fucosylation.

29. The bispecific antibody of claim 28, wherein low fucosylation is obtained after treatment with GlymaxX technology.

30. The bispecific antibody of claim 27, comprising a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region may be the same or different.

31. The bispecific antibody of claim 30, wherein the first Fc region and the second Fc region are human IgG Fc, respectively.

32. The bispecific antibody of claim 31, 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.

33. The bispecific antibody of claim 32, wherein the first Fc region and the second Fc region respectively contain amino acid sequences having at least 90% identity with the amino acid sequences shown in SEQ ID NO: 46 or 47.

34. The bispecific antibody of claim 32, wherein the first Fc region and the second Fc region respectively contain the amino acid sequence shown in SEQ ID NO:46 or 47.

35. The bispecific antibody of claim 32, wherein the first Fc region and the second Fc region are respectively composed of the amino acid sequence shown in SEQ ID NO:46 or 47.

36. The bispecific antibody of any one of claims 30-35, 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.

37. The bispecific antibody of claim 36, wherein the CH3 of one Fc region contains the S364R and D399K mutations, and the CH3 mutation of the other Fc region contains the Y349T, K370S and K409D mutations.

38. The bispecific antibody of claim 37, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 49 or 50 and comprises the mutants Y349T, K370S, and K409D, and the other Fc region comprises an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 52 or 53 and comprises the mutants S364R and D399K.

39. The bispecific antibody of claim 37, wherein one Fc region polypeptide comprises the amino acid sequence shown in SEQ ID NO:49 or 50, and the other Fc region polypeptide comprises the amino acid sequence shown in SEQ ID NO:52 or 53.

40. The bispecific antibody of claim 37, wherein one Fc region polypeptide consists of the amino acid sequence shown in SEQ ID NO:49 or 50, and the other Fc region polypeptide consists of the amino acid sequence shown in SEQ ID NO:52 or 53.

41. The bispecific antibody of claim 36, wherein the mutation is introduced based on Knob-into-Hole technology, wherein corresponding Knob and Hole mutations are introduced in the first Fc region and the second Fc region, wherein... a) One Fc region polypeptide contains amino acid substitution T366W, while the other Fc region polypeptide contains amino acid substitutions T366S, L368A, and Y407V, wherein the amino acid positions are numbered according to the EU index, or b) One Fc region contains amino acid substitutions S354C and T366W, and another Fc region contains amino acid substitutions Y349C, T366S, L368A, and Y407V, wherein the amino acid positions are numbered according to the EU index.

42. The bispecific antibody of any one of claims 1, 2 and 23-26, wherein the first and / or second antigen-binding region is linked to one or two heavy chain constant regions comprising CH1 and Fc regions, linked via or not via hinge regions.

43. The bispecific antibody of claim 42, wherein the heavy chain variable region in the first and / or second antigen-binding region is connected to one or two heavy chain constant regions, wherein the C-terminus of the heavy chain variable region is connected to the N-terminus of CH1 of the heavy chain constant region.

44. The bispecific antibody of claim 42, wherein the heavy chain constant region is the heavy chain constant region of human IgG1, human IgG2, human IgG3 or human IgG4.

45. The bispecific antibody of claim 42, wherein CH1 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

42.

46. ​​The bispecific antibody of claim 42, wherein CH1 comprises the amino acid sequence shown in SEQ ID NO:

42.

47. The bispecific antibody of claim 42, wherein CH1 consists of the amino acid sequence shown in SEQ ID NO:

42.

48. The bispecific antibody of claim 42, wherein the light chain variable region of the first and / or second antigen-binding region is connected to the light chain constant region.

49. The bispecific antibody of claim 42, wherein the light chain variable region of the first and / or second antigen-binding region is connected to the light chain constant region, wherein the C-terminus of the light chain variable region is connected to the N-terminus of the light chain constant region.

50. The bispecific antibody of claim 48 or 49, wherein the light chain constant region is the kappa light chain constant region or the lambda light chain constant region.

51. The bispecific antibody of claim 48 or 49, wherein the light chain constant region comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

54.

52. The bispecific antibody of claim 48 or 49, wherein the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

54.

53. The bispecific antibody of claim 48 or 49, wherein the light chain constant region consists of the amino acid sequence shown in SEQ ID NO:

54.

54. The bispecific antibody according to any one of claims 1, 2, 23-26, and 37-41, wherein the bispecific antibody is an IgG-like antibody 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 includes a heavy chain variable region and a first heavy chain constant region of the first antigen-binding region; light chain 1 includes a light chain variable region and a first light chain constant region of the first antigen-binding region; and heavy chain 2 includes a heavy chain variable region and a second heavy chain constant region of the second antigen-binding region; light chain 2 includes a light chain variable region and a second light chain constant region of the second antigen-binding region.

55. The bispecific antibody of claim 54, wherein the heavy chain 1 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

33.

56. The bispecific antibody of claim 54, wherein heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:

33.

57. The bispecific antibody of claim 54, wherein heavy chain 1 consists of the amino acid sequence shown in SEQ ID NO:

33.

58. The bispecific antibody of any one of claims 54-57, wherein the light chain 1 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

34.

59. The bispecific antibody of claim 58, wherein light chain 1 comprises the amino acid sequence shown in SEQ ID NO:

34.

60. The bispecific antibody of claim 58, wherein the light chain 1 consists of the amino acid sequence shown in SEQ ID NO:

34.

61. The bispecific antibody of claim 54, wherein the heavy chain 1 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 33, and the light chain 1 comprises an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:

34.

62. The bispecific antibody of claim 54, 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.

63. The bispecific antibody of claim 54, wherein the heavy chain 1 consists of the amino acid sequence shown in SEQ ID NO: 33, and the light chain 1 consists of the amino acid sequence shown in SEQ ID NO:

34.

64. The bispecific antibody of any one of claims 54-57 and 59-63, wherein the heavy chain 2 comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:

35.

65. The bispecific antibody of claim 64, wherein heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:

35.

66. The bispecific antibody of claim 64, wherein heavy chain 2 consists of the amino acid sequence shown in SEQ ID NO:

35.

67. The bispecific antibody of any one of claims 54-57, 59-63 and 65-66, wherein the light chain 2 comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:

36.

68. The bispecific antibody of claim 67, wherein the light chain 2 comprises the amino acid sequence shown in SEQ ID NO:

36.

69. The bispecific antibody of claim 67, wherein the light chain 2 consists of the amino acid sequence shown in SEQ ID NO:

36.

70. The bispecific antibody according to any one of claims 54-57 and 59-63, wherein the heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 90% identity with 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 or an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:

36.

71. The bispecific antibody of claim 70, 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.

72. The bispecific antibody of claim 70, wherein the heavy chain 2 consists of the amino acid sequence shown in SEQ ID NO: 35; and the light chain 2 consists of the amino acid sequence shown in SEQ ID NO:

36.

73. The bispecific antibody of claim 54, wherein... Heavy chain 1 contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 33, and light chain 1 contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 34; heavy chain 2 contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 35, and light chain 2 contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

36.

74. The bispecific antibody of claim 54, wherein, Heavy chain 1 contains the amino acid sequence shown in SEQ ID NO:33, and light chain 1 contains the amino acid sequence shown in SEQ ID NO:

34. Heavy chain 2 contains the amino acid sequence shown in SEQ ID NO:35, and light chain 2 contains the amino acid sequence shown in SEQ ID NO:

36.

75. The bispecific antibody of claim 54, wherein, Heavy chain 1 consists of the amino acid sequence shown in SEQ ID NO:33, and light chain 1 consists of the amino acid sequence shown in SEQ ID NO:

34. Heavy chain 2 consists of the amino acid sequence shown in SEQ ID NO:35, and light chain 2 consists of the amino acid sequence shown in SEQ ID NO:

36.

76. An isolated nucleic acid encoding the bispecific antibody binding EGFR and B7-H3 as described in any one of claims 1-75.

77. A vector comprising the nucleic acid of claim 76.

78. The carrier of claim 77, wherein the carrier is an expression carrier.

79. The vector of claim 78, wherein the expression vector is pcDNA.

80. The vector of claim 78, wherein the expression vector is pcDNA3.

1.

81. A host cell comprising the nucleic acid of claim 76 or the vector of any one of claims 77-80, wherein the host cell is a yeast cell or a mammalian cell.

82. The host cell of claim 81, wherein the mammalian cell is a 293 cell or a CHO cell.

83. The host cell of claim 81, wherein the mammalian cell is a 293F cell, a 293T cell, or a CHO-S cell.

84. The host cell of any one of claims 81-83, wherein it is glycoengineered to express RMD enzyme.

85. The host cell of claim 84, comprising nucleic acid encoding RMD enzyme.

86. The host cell of claim 85, wherein the RMD enzyme comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO:

41.

87. The host cell of claim 85, wherein the RMD enzyme comprises the amino acid sequence shown in SEQ ID NO:

41.

88. The host cell of claim 85, wherein the RMD enzyme comprises the amino acid sequence shown in SEQ ID NO:

41.

89. The host cell of claim 84, wherein the RMD enzyme is derived from Pseudomonas aeruginosa.

90. The host cell of any one of claims 85-88, wherein the RMD enzyme is derived from Pseudomonas aeruginosa.

91. A method for preparing a bispecific antibody binding to EGFR and B7-H3, the method comprising culturing a host cell according to any one of claims 81-90 under conditions suitable for expressing a nucleic acid encoding a bispecific antibody according to any one of claims 1-75, optionally isolating the bispecific antibody, and optionally further comprising recovering the bispecific antibody from the host cell or a host cell culture medium.

92. A pharmaceutical composition comprising the bispecific antibody as described in any one of claims 1-75, and optionally a pharmaceutical excipient.

93. The pharmaceutical composition of claim 92, further comprising a second therapeutic agent.

94. The pharmaceutical composition of claim 93, 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.

95. The pharmaceutical composition of claim 94, wherein the immunomodulator is an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule.

96. The pharmaceutical composition of claim 93, wherein the second therapeutic agent is a KRAS small molecule inhibitor.

97. The pharmaceutical composition of claim 96, wherein the KRAS small molecule inhibitor is a KRAS G12C inhibitor, a KRAS G12D inhibitor, or a KRAS G12S inhibitor.

98. The pharmaceutical composition of claim 97, wherein the KRAS G12C inhibitor is AMG510 or GFH925; or the KRAS G12D inhibitor is MRTX1133.

99. A pharmaceutical combination product comprising a bispecific antibody as described in any one of claims 1-75 or a pharmaceutical composition as described in any one of claims 92-98, and one or more second therapeutic agents.

100. The pharmaceutical combination product of claim 99, 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.

101. The pharmaceutical combination product of claim 100, wherein the immunomodulator is an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule.

102. The pharmaceutical combination product of claim 100, wherein the second therapeutic agent is a KRAS small molecule inhibitor.

103. The pharmaceutical combination product of claim 102, wherein the KRAS small molecule inhibitor is a KRAS G12C inhibitor, a KRAS G12D inhibitor, or a KRAS G12S inhibitor.

104. The pharmaceutical combination product of claim 103, wherein the KRAS G12C inhibitor is AMG510 or GFH925; or the KRAS G12D inhibitor is MRTX1133.

105. Use of the bispecific antibody of any one of claims 1-75 or the pharmaceutical composition of claim 92 to prepare a medicament for use in the prevention or treatment of cancer in a subject, wherein the cancer is skin cancer, head and neck cancer, esophageal cancer, intestinal cancer or lung cancer.

106. The use of claim 105, wherein the drug is further administered in combination with one or more other therapies.

107. The use of claim 106, wherein the therapy comprises a treatment method and / or a second therapeutic agent.

108. The use of claim 107, wherein the treatment method includes surgical treatment and / or radiotherapy, or 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.

109. The use of claim 108, wherein the immunomodulator is an activator of a co-stimulatory molecule or an inhibitor of an immune checkpoint molecule.

110. The use of claim 107, wherein the second therapeutic agent is a KRAS small molecule inhibitor.

111. The use according to claim 110, wherein the KRAS small molecule therapeutic agent is a KRAS G12C inhibitor, a KRAS G12D inhibitor, or a KRAS G12S inhibitor.

112. The use according to claim 111, wherein the KRAS G12C inhibitor is AMG510 or GFH925; or the KRAS G12D inhibitor is MRTX1133.

113. Use of the pharmaceutical composition of any one of claims 93-98 or the pharmaceutical combination product of any one of claims 99-104 in the preparation of a medicament for the prevention or treatment of cancer in a subject, wherein the cancer is skin cancer, head and neck cancer, esophageal cancer, intestinal cancer or lung cancer.

114. The use according to any one of claims 105-113, wherein the skin cancer is squamous cell carcinoma of the skin, the head and neck cancer is squamous cell carcinoma of the head and neck, the esophageal cancer is squamous cell carcinoma of the esophagus, the intestinal cancer is colon cancer, rectal cancer or colorectal cancer, or the lung cancer is non-small cell lung cancer, squamous cell carcinoma of the lung or adenocarcinoma of the lung.

115. The use according to any one of claims 105-113, wherein the tumor cells of said tumor (i) Overexpression of wild-type EGFR compared to normal cells in adjacent tissues or normal cells in the same tissues in healthy subjects, and / or expression of mutant EGFR; (ii) Overexpression of wild-type KRAS compared to normal cells in adjacent tissues or compared to normal cells in the same tissues in healthy subjects, and / or expression of mutant KRAS; (iii) B7-H3 with elevated nucleic acid or protein levels compared to normal cells in adjacent tissues or compared to normal cells in the same tissue in healthy subjects; and / or (iv) The tumor cells are resistant to tyrosine kinase inhibitors.

116. The use as described in claim 115, wherein Overexpression of wild-type EGFR is characterized by elevated levels of wild-type EGFR nucleic acids or proteins. The mutated EGFR includes one or more mutations selected from R521K, L858R, T790M, G719X, C797S, Y1069C, Exon19 deletion, and Exon20ins; Overexpression of wild-type KRAS is wild-type KRAS with elevated levels of nucleic acid or protein; The mutated KRAS contains a mutation at position G12 or G13, and / or Tyrosine kinase inhibitors are either first-generation or third-generation tyrosine kinase inhibitors.

117. The use as described in claim 116, wherein The Exon20ins is S768_D770dup; The mutated EGFR includes R521K / Y1069C, R521K, L858R / T790M / C797S, Del19 / T790M / C797S or S768_D770dup; The mutated KRAS includes G12D or G12C; The first-generation tyrosine kinase inhibitor is erlotinib; and / or The third-generation tyrosine kinase inhibitor is osimertinib.

118. The use of claim 115, wherein the tumor cells of said tumor express mutated EGFR and mutated KRAS.

119. The use according to any one of claims 115-118, wherein the tumor cells of said tumor express EGFR with an R521K mutation and KRAS with a G12D mutation.

120. A non-diagnostic or therapeutic method for detecting antigens EGFR and / or B7-H3 in a sample, the method comprising: (a) Contacting the sample with the bispecific antibody according to any one of claims 1-75; and (b) Detecting the formation of a complex between an antibody or its antigen-binding fragment and EGFR and / or B7-H3, optionally, the antibody having a detectable label.

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