Antigen binding molecule, and drug conjugate thereof and use thereof

Abstract

The present invention relates to an antigen binding molecule and an antibody-drug conjugate (ADC), and in particular to an antibody and an ADC targeting MSLN and / or FOLR1, a composition containing the antibody or ADC, and a therapeutic use thereof.

Description

Antigen-binding molecules and their drug conjugates and uses

[0001] Cross-reference to related applications

[0002] This application is based on and claims priority to PCT International Patent Application No. PCT / CN2024 / 134541, filed on November 26, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to antigen-binding molecules and antibody-drug conjugates, and more particularly to antigen-binding molecules and antibody-drug conjugates (ADCs) targeting FOLR1 and / or MSLN, as well as compositions containing said antigen-binding molecules or ADCs and their therapeutic applications. Background Technology

[0004] FRα, also known as FOLR1 or folate-binding protein, is a glycosylphosphatidylinositol-anchored receptor whose primary physiological function is folic acid transport. It has a high affinity for tetrahydrofolate, but it only participates in approximately 20% of serum folate transport (Fathima Zahra Nawaz and Edward T. Kipreos, Trends in Endocrinology & Metabolism, March 2022, Vol. 33, No. 3). In normal tissues, FRα is expressed only in certain organs, such as the placenta, fallopian tubes, kidneys, lungs, and choroid plexus, and the vast majority of FRα receptors are located on the luminal surface of cells, not directly interacting with folate or folate receptor-targeted molecules in the circulatory system (except in the kidneys). In the kidneys, FRα primarily functions to reabsorb folate, preventing its loss in urine. After reabsorption, folic acid is generally not stored in the kidneys, therefore no significant toxic effects have been observed in animals and patients receiving FRα-targeted drug therapy (P. Varagaanti et al, Life Sciences 326(2023)121802).

[0005] However, FRα is highly expressed in a variety of tumors, especially epithelial tumors, such as ovarian cancer, endometrial cancer, non-small cell lung adenocarcinoma, triple-negative breast cancer, and pleural carcinoma. FRα is highly expressed in approximately 80% of epithelial ovarian cancers (EOCs) and is positively correlated with the histological grade and progression of the disease. Furthermore, FRα expression has been detected in approximately 70% of triple-negative breast cancers, 50% of endometrial cancers, as well as pancreatic cancer and colorectal cancer. It not only mediates the uptake of folic acid by tumor cells but also promotes tumor cell growth and proliferation through signal transduction and transcription factors. This makes it an ideal tumor target for the development of targeted antibody drugs (Heather J. Bax et al., British Journal of Cancer (2023) 128:342–353).

[0006] Currently, the only FRα-targeted ADC on the market is ImmunoGen's Elahere (mirvetuximab soravtansine, IMGN853, MIRV). In the Phase III MIRASOL clinical trial, Elahere significantly improved overall survival and progression-free survival compared to chemotherapy, and resulted in significant tumor reduction or disappearance in more patients. Regarding toxicity, common adverse events include abdominal pain, diarrhea, constipation, nausea, vomiting, blurred vision, corneal lesions, fatigue, elevated aspartate aminotransferase (AST), elevated alanine aminotransferase (ALT), peripheral neuropathy, decreased albumin, leukopenia, neutropenia, decreased hemoglobin, and decreased appetite. Elahere's prescribing information includes a boxed warning about ocular toxicity, including visual impairment, corneal lesions, dry eye, photophobia, ocular pain, and uveitis. In addition, several FRα-targeted ADCs are in clinical trials, including Eisai's MORAb-202 / farletuzumab-ecteribulin, which uses the small molecule ecteribulin as a small molecule payload. Although it has a good ORR, significant pulmonary toxicity appeared at lower doses during dose escalation in the Phase I clinical trial, which is presumably related to the small molecule payload. STRO-002 / luveltamab tazevibulin is an FRα-targeted ADC developed by Super, which uses site-directed conjugation technology. Similar to the previous two drugs, it uses microtubule inhibitors as small molecule payloads. In clinical data, its efficacy and toxicity are not significantly better than Elahere (mirvetuximab soravtansine). In addition to traditional microtubule inhibitor ADCs, there are several ADCs targeting FRα that use topoisomerase inhibitors as payloads. The most representative one is Rinatabart Sesutecan (PRO1184) developed by Propharm. PRO1184 uses ethitecan as the payload and combines it with a novel hydrophilic payload linker sesutecan. It has a DAR value of 8 and has shown good safety and efficacy in phase I / II clinical trials.

[0007] Since ADC drugs targeting FRα all require FRα expression levels as biomarkers, such as IMGN853 which shows significant efficacy in patients with TPS>75%, and most other ADC drugs also require TPS>25% to achieve good efficacy, there is still considerable room for improvement in covering the patient population with low FRα expression.

[0008] Mesothelin (MSLN) is a tumor-associated antigen found in various cancers. The MSLN gene encodes a 71 kDa precursor protein, which can be hydrolyzed by furin proteases into two parts: one fragment, mesothelin (MSLN), with a molecular weight of 40 kDa, is anchored to the cell membrane surface via glycosylphosphatidylinositol; the other fragment, megakaryocyte colony-stimulating factor (MPF), with a molecular weight of 31 kDa, is released into the bloodstream. The exact role of MSLN under normal physiological conditions remains unclear. MSLN gene knockout mice have been reported to have normal development and reproductive capabilities, suggesting that MSLN function may not be essential for life. In ovarian cancer drug resistance studies, MSLN has been shown to help tumor cells escape paclitaxel-induced apoptosis by activating the PI3K / AKT and MAPK / ERK signaling pathways. Studies have also shown that MSLN expression is closely related to IL-6, and its overexpression can activate IL-6 / sIL-6R signal transduction, thereby promoting the proliferation of tumor cells (Jiang Lv & Peng Li, Biomarker Research (2019) 7:18).

[0009] MSLN is highly expressed in various malignant tumors and is closely related to cancer cell proliferation, local invasion, metastasis, and anti-apoptosis. A tissue microarray study analyzing 12,679 tumor tissues found that MSLN is widely expressed in ovarian cancer, pancreatic cancer, endometrial cancer, lung adenocarcinoma, and mesothelioma. Weidemann et al, Biomedicines 2021, 9, 397) In healthy tissues, MSLNs are limited to normal mesothelial cells of the pleura, peritoneum and pericardium, making them an ideal target for tumor therapy (Tyvette S Hilliard, Cancers 2018, 10, 277; Brendan L. Hagertya & Kazuaki Takabe, World J Oncol. 2023; 14(5): 340-349).

[0010] Currently, there are no approved drug treatments targeting MSLN. Several cell therapy (CAR-T) therapies are under development or actively explored in early clinical trials. ADC drugs are mainly developed based on microtubule inhibitors. The most advanced is Rongchang Bio's RC88, which demonstrated anti-tumor efficacy in a Phase I clinical trial. However, its objective response rate (ORR) of 37.2% in ovarian cancer is slightly lower than that of FRα ADCs. Early-developed MSLN ADC drugs such as SEL-403 and anetumab ravtansine have not yet achieved significant clinical results. Due to its widespread expression in tumors, there is still considerable room for optimization in MSLN-targeted therapy.

[0011] Nanobodies are small antibody molecules composed of single-chain heavy-chain antibody molecules. They possess high specificity and affinity, and compared to traditional antibodies, they exhibit smaller size, higher stability, and deeper tissue penetration. This makes them highly promising for cancer therapy, enabling precise treatment by recognizing and targeting specific antigens on the surface of tumor cells, and allowing them to penetrate deep tumor tissues inaccessible to conventional antibodies. Nanobodies can be designed to deliver drugs or radioisotopes to tumor cells to kill them. Furthermore, nanobodies have a natural advantage in the construction of bispecific and multispecific antibodies, avoiding the problem of light-heavy chain mismatch. Therefore, the application of nanobodies in cancer therapy is receiving widespread attention and is expected to become one of the important means of future cancer treatment (Bannas, Hambach, and Koch-Nolte 2017).

[0012] Antibody-based drugs have advanced the field of cancer treatment, but many still fall short in terms of safety and efficacy. A major challenge for anticancer drugs remains drug selectivity—that is, minimizing collateral damage to normal cells while ensuring effective cancer targeting. To overcome the limitations of single-target drugs in this regard, strategies have been proposed to combine multiple monoclonal antibodies targeting different targets. However, published studies on combination therapies of different mAbs have shown that the effects of combination therapy vary greatly depending on factors such as the different target combinations. For example, the simultaneous injection of trastuzumab (anti-HER2) and bevacizumab (anti-VEGF-A) in HER2+ metastatic breast cancer has shown encouraging results; however, the combination of bevacizumab with cetuximab (anti-EGFR) in advanced colorectal cancer has not yielded favorable results. See, for example, Maruani A et al., Bispecifics and antibody-drug conjugates: A positive synergy. Drug Discov Today Technol. 2018 Dec; 30:55-61.

[0013] Therefore, antibody drug research still urgently needs new drug molecules with improved tumor specificity and selectivity to reduce on-target toxicity, expand the therapeutic window, or cover a wider range of patients.

[0014] Invention Overview

[0015] Through in-depth analysis of databases such as TCGA and GTEx, as well as single-cell sequencing results, we found that FRα and MSLN are highly correlated in the expression of various tumors. Analysis of single-cell sequencing data from nine ovarian cancer patients showed that eight samples co-expressed FRα and MSLN, with all patients expressing at least one of them. Analysis of individual cells showed that the co-expression rate reached 71%, and more than 95% of cells expressed at least one of the two antigens.

[0016] Therefore, the inventors have discovered antigen-binding molecules targeting FOLR1, such as VHH antibodies, antigen-binding molecules targeting MSLN, such as VHH antibodies, and antigen-binding molecules based on these molecules that simultaneously target FRα and MSLN. These molecules can achieve one or more of the following properties: 1. Dual-target antibodies target FRα and MSLN on the same cell to improve the efficacy window; 2. Dual-target antibodies target FRα and MSLN on different cells to cover a wider patient population. Furthermore, the molecular design employs a dual-epitope design for FRα to maximize patient coverage, including patients with low FRα expression. One epitope-targeting antibody uses a fast Kon / fast Koff binding mechanism, while the other epitope-targeting antibody uses VHH, which has pH-dependent binding properties (stronger binding in the slightly acidic environment of tumors than in the neutral pH environment of normal tissues). This design results in better tumor binding specificity and improved tumor penetration. Based on these designs and discoveries, the inventors have established the multispecific antibodies and drug conjugates of this invention and their uses, particularly in cancer treatment.

[0017] The antibody-drug conjugate of the present invention has the following advantages:

[0018] (1) It binds to target cells expressing human FOLR1 and / or MSLN with high affinity;

[0019] (2) It has a significant lateral killing effect;

[0020] (3) It has high anti-tumor efficacy, stronger killing effect on tumor cells, and stronger inhibitory effect on tumor growth, especially tumors with high expression of FOLR1 and / or MSLN; it has significantly enhanced or even unexpected anti-tumor activity.

[0021] (4) It has better product uniformity;

[0022] (5) It has low toxicity;

[0023] (6) It has a larger treatment / safety window;

[0024] (7) Targeting FRα and MSLN on the same cell, the efficacy window is improved through multivalent binding;

[0025] (8) Targeting FRα and MSLN on different cells, covering a wider patient population, and / or applicable to more indications;

[0026] (9) It has good physicochemical stability; and / or

[0027] (10) It has good drug-like properties.

[0028] The invention is further illustrated in the following figures and specific embodiments. However, these figures and specific embodiments should not be considered as limiting the scope of the invention, and modifications readily apparent to those skilled in the art will be included within the spirit of the invention and the scope of protection of the appended claims.

[0029] Brief description of the attached diagram

[0030] Figure 1: Binding of the fully human single-domain antibody FOLR1 to OVCAR3 cells at pH 6.5.

[0031] Figure 2: Binding of the fully human single-domain antibody FOLR1 to OVCAR3 cells at pH 7.4.

[0032] Figure 3: Binding of the fully human single-domain FOLR1 antibody to HEK293 cells at pH 7.4.

[0033] Figure 4: Comparison of cell binding of the fully human single-domain antibody H2B6 to FOLR1 at different pH values.

[0034] Figure 5: Comparison of cell binding of the fully human single-domain antibody H2B5 to FOLR1 at different pH values.

[0035] Figure 6: Relationship between candidate FOLR1 single-domain antibody molecules and antigen-binding epitopes of Farletuzumab.

[0036] Figure 7: Binding of the V-H2B6 PTM-removed variant to OVCAR3 cells at pH 6.5.

[0037] Figure 8: Binding of the V-H2B6 PTM-removed variant to OVCAR3 cells at pH 7.4.

[0038] Figure 9: Binding of the H2B6-modified hydrophilic variant to OVCAR3 cells at pH 6.5.

[0039] Figure 10: Binding of the H2B6-modified hydrophilic variant to OVCAR3 cells at pH 7.4.

[0040] Figure 11: Binding of a combined variant of H2B6 with PTM removal and enhanced hydrophilicity modification to OVCAR3 cells at pH 6.5.

[0041] Figure 12: Binding of a combined variant of H2B6 with PTM removal and enhanced hydrophilicity modification to OVCAR3 cells at pH 7.4.

[0042] Figure 13: Binding of a combined variant of H2B6 with PTM removal and enhanced hydrophilicity modification to cynoFOLR cells at pH 6.5.

[0043] Figure 14: Binding of a combined variant of H2B6 with PTM removal and enhanced hydrophilicity modification to cynoFOLR cells at pH 7.4.

[0044] Figure 15: Binding of anti-MSLN VHH antibody to CHOS-cynoMSLN cells.

[0045] Figure 16: Binding of anti-MSLN VHH antibody to OVCAR3 cells.

[0046] Figure 17: Binding of anti-MSLN VHH antibody to NCI-H2110 cells.

[0047] Figure 18: Binding activity of the 4-C2 sequence-optimized variant in NCI-H2110 cells.

[0048] Figure 19: Binding activity of NCI-H2110 cells with the optimized 4-C2 sequence variant.

[0049] Figure 20: Binding activity of AsPC-1 cells with the first round of immunogenic variant of 4-C2.

[0050] Figure 21 (A and B): OVCAR3 cell binding of the second round of deimmunogenic variant of 4-C2.

[0051] Figure 22: OVCAR3 cell binding of the deimmunogenic variant of Farletuzumab.

[0052] Figure 23: OVCAR3 cell binding of the symmetrically structured FOLR1xMSLN bispecific antibody.

[0053] Figure 24: EFE-184 cells bind to the symmetrically structured FOLR1xMSLN bispecific antibody.

[0054] Figure 25: Endocytosis of OVCAR3 cells with symmetrical FOLR1xMSLN double antibody.

[0055] Figure 26: Binding of FOLR1 dual epitope antibody to HT1197 cells at pH 6.5.

[0056] Figure 27: Binding of FOLR1 dual epitope antibody to OVCAR3 cells at pH 6.5.

[0057] Figure 28: Binding of FOLR1 dual epitope antibody to HEK293 cells at pH 7.4.

[0058] Figure 29: Binding of the FOLR1 dual epitope antibody to EFO27 cells at pH 7.4.

[0059] Figure 30: Binding of the FOLR1xMSLN dual-target triepitope polyclonal antibody to OVCAR3 cells at pH 6.5.

[0060] Figure 31: Binding of FOLR1xMSLN dual-target tri-epitope polyclonal antibody to NCI-H2110 cells at pH 6.5.

[0061] Figure 32: Binding of the FOLR1xMSLN dual-target triepitope polyclonal antibody to EFE-184 cells at pH 6.5.

[0062] Figure 33: Binding of the FOLR1xMSLN dual-target triepitope polyclonal antibody to H292 cells at pH 6.5.

[0063] Figure 34: Binding of the FOLR1xMSLN dual-target triepitope polyclonal antibody to CAOV3 cells at pH 6.5.

[0064] Figure 35: Binding of the FOLR1xMSLN dual-target tri-epitope polyclonal antibody to EFO27 cells at pH 7.4.

[0065] Figure 36: Binding of the FOLR1xMSLN dual-target triepitope polyclonal antibody to MDA-MB-453 cells at pH 7.4.

[0066] Figure 37: Binding of FOLR1xMSLN dual-target triepitope polyclonal antibodies V-F21 and V-F8 to H292 cells at pH 6.5.

[0067] Figure 38: Binding of FOLR1xMSLN dual-target triepitope polyclonal antibodies V-F21 and V-F8 to H1781 cells at pH 6.5.

[0068] Figure 39: Binding of FOLR1xMSLN dual-target triepitope polyantibodies V-F21 and V-F8 to CAOV3 cells at pH 6.5.

[0069] Figure 40: Internalization of the FOLR1xMSLN dual-target triepitope polyclonal antibody V-F21 in OVCAR3 cells at pH 6.5.

[0070] Figure 41: Detection of the killing activity of the candidate FOLR1xMSLN bispecific antibody-VC-MMAE ADC in OVCAR3 cells in vitro.

[0071] Figure 42: Detection of the killing activity of the candidate FOLR1xMSLN bispecific antibody-VC-MMAE ADC in HT29 cells in vitro.

[0072] Figure 43: Detection of the killing activity of the candidate FOLR1xMSLN bispecific antibody VA-Exd ADC in OVCAR3 cells in vitro.

[0073] Figure 44: In vitro killing activity of candidate FOLR1xMSLN bispecific antibody VA-Exd ADC in EFO27 cells.

[0074] Figure 45: Antitumor efficacy of candidate ADC drugs in mouse OVCAR3 CDX subcutaneous transplantation model.

[0075] Figure 46: Mouse body weight in the mouse OVCAR3 CDX subcutaneous transplantation model of candidate ADC drugs.

[0076] Figure 47: In vitro killing of OVCAR3 cells by FOLR1 dual epitope dual and polyclonal VA-Exd ADC molecules.

[0077] Figure 48: In vitro killing of OVCAR3 cells by candidate dual and polyclonal antibody VA-Exd ADC molecules.

[0078] Figure 49: Antitumor efficacy of candidate ADC drugs in mouse N87 CDX subcutaneous transplantation model.

[0079] Figure 50: Mouse body weight in the mouse N87 CDX subcutaneous transplantation model of candidate ADC drugs.

[0080] Figure 51: In vitro killing of OVCAR3 cells by candidate dual and polyclonal antibody Gluc-Exd ADC.

[0081] Figure 52: In vitro killing of T-47D cells by candidate dual and polyclonal antibody Gluc-Exd ADC.

[0082] Figure 53: In vitro killing of H441 cells by candidate dual and polyclonal antibody Gluc-Exd ADC.

[0083] Figure 54: Killing of HEC-1-A cells in vitro by candidate dual and polyclonal antibody Gluc-Exd ADC.

[0084] Figure 55: In vitro killing effect of V-F8 and V-F21 Gluc-Exd ADCs on OVCAR3 cells.

[0085] Figure 56: Antitumor efficacy of candidate ADC drugs in mouse H292 CDX subcutaneous transplantation model.

[0086] Figure 57: Changes in mouse body weight in the mouse H292 CDX subcutaneous transplantation model of candidate ADC drugs.

[0087] Figure 58: Antitumor efficacy of candidate ADC drugs in mouse OVCAR3 CDX subcutaneous transplantation model.

[0088] Figure 59: Antitumor efficacy of candidate ADC drugs in mouse HCC1806 CDX subcutaneous transplantation model.

[0089] Figure 60: In vitro killing of OVCAR3 cells by V-F2_d1 Gluc-Exd and RDVT-Exd ADCs.

[0090] Figure 61: In vitro killing of OVCAR3 cells by V-F8 Gluc-Exd and RDVT-Exd ADCs.

[0091] Figure 62: In vitro killing of OVCAR3 cells by V-F21 Gluc-Exd and RDVT-Exd ADCs.

[0092] Figure 63: In vitro killing of MDA-MB-453 cells by V-F21 Gluc-Exd and RDVT-Exd ADCs.

[0093] Figure 64: In vitro paracytosis of V-F21 Gluc-Exd and RDVT-Exd ADCs.

[0094] Figure 65: Antitumor efficacy of candidate ADC drugs in mouse H292 CDX subcutaneous transplantation model.

[0095] Figure 66: Antitumor efficacy of candidate ADC drugs in mouse N87 CDX subcutaneous transplantation model.

[0096] Figure 67: Antitumor efficacy of candidate ADC drugs in mouse ovarian cancer PDX subcutaneous transplantation model.

[0097] Figure 68: Schematic diagram of the “2+2” symmetric dual-target bispecific antibody based on Farletuzumab corresponding to Table 18.

[0098] Figure 69: Schematic diagram of the FOLR1 dual epitope antibody corresponding to Table 21.

[0099] Figure 70: Schematic diagram of the FOLR1xMSLN dual-target three-epitope polyclonal antibody corresponding to Table 25.

[0100] Figure 71: Antitumor efficacy of candidate ADC drugs in mouse SW620 CDX subcutaneous transplantation model.

[0101] Figure 72: Antitumor efficacy of candidate ADC drugs in mouse HEC-1-A CDX subcutaneous transplantation model.

[0102] Figure 73: Antitumor efficacy of candidate ADC drugs in mouse HT-1197CDX subcutaneous transplantation model.

[0103] Figure 74: Antitumor efficacy of candidate ADC drugs in mouse CAPAN-2CDX subcutaneous transplantation model.

[0104] Invention Details

[0105] 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.

[0106] For the purpose of interpreting this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate.

[0107] definition

[0108] 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.

[0109] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0110] In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations where the term comprises the mentioned elements, integers, or steps. For example, when referring to an antibody variable region that “comprising” a specific sequence, it is also intended to cover the antibody variable region that comprises that specific sequence.

[0111] In this document, the term "antigen-binding molecule" refers to a molecule, such as a protein or peptide, or a molecule derived therefrom, that contains an antigen-binding domain or antigen-binding site capable of binding to a target antigen. In this invention, when the target antigen is FOLR1 and / or MSLN, antigen-binding molecules binding FOLR1 and / or MSLN are also referred to as FOLR1-binding molecules, MSLN-binding molecules, or FOLR1 / MSLN-binding molecules. Antigen-binding molecules include, for example, antibodies and their antigen-binding fragments, as well as various fusions constructed based on antibodies or antigen-binding fragments, such as VHH-Fc antibodies, multi / bispecific antibodies, and chimeric antigen receptors (CARs). As will be apparent to those skilled in the art, the antigen-binding site of an antibody typically contains amino acid residues from a "complementarity-determining region" or "CDR".

[0112] In this document, the term "antibody" refers to a polypeptide containing at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen. This term encompasses a wide range of antibody structures, including, but not limited to, monoclonal antibodies, single-chain or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies, heavy chain antibodies, chimeric or humanized antibodies, intact antibodies, and antibody fragments, provided they exhibit the desired antigen-binding activity.

[0113] In this article, "intact antibody" or "full-length antibody" may be used interchangeably, referring to an immunoglobulin molecule containing at least two heavy chains (H) and two light chains (L). Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this article) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated as VL in this article) and a light chain constant region.

[0114] In this document, the terms "antibody fragment" and "antigen-binding fragment" are used interchangeably and refer to a molecule distinct from the intact antibody that contains a portion of the intact antibody and is capable of binding the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies; camelid antibodies (heavy chain antibodies) or fragments thereof (e.g., VHH); and monospecific, bispecific, or multispecific antibodies formed from antibody fragments. Unless otherwise stated herein or explicitly contradicted by the context, the term "antibody" as used herein is equivalent to "antibody or an antibody fragment thereof." In some embodiments of the invention, the antibody fragment includes cysteine ​​residue portions for forming interchain disulfide bonds between heavy chains, such as cysteine ​​residues in the antibody hinge region, to provide amino acid residue sites usable for thiol coupling chemistry. In other embodiments of the invention, the antibody fragment includes cysteine ​​residues introduced into the Fc region to provide amino acid residue sites usable for thiol coupling chemistry.

[0115] In this document, the terms "antigen binding site" and "antigen binding domain" are used interchangeably to refer to the region in an antibody molecule that actually binds to the antigen. The antigen binding site of the antibody molecule used in this invention is preferably provided by a variable domain (i.e., "VHH") from a heavy chain antibody or an antibody fragment such as Fab.

[0116] In this document, the term "multispecific" refers to an antigen-binding molecule (e.g., an antibody) having at least two antigen-binding sites, each of which binds to a different antigen or a different epitope, for example, to different epitopes on different antigens or different epitopes on the same antigen. Correspondingly, "single-specific" refers to the ability to bind to only one epitope. "Dual-specific" refers to the ability to bind to two different antigens or epitopes.

[0117] In this article, the antibody-related terms "valence" or "valence number" refer to the total number of antigen-binding sites in an antibody molecule, or the number of antigen-binding sites with the same antigen-binding specificity. For example, a quadrivalent antibody means that the antibody molecule contains a total of 4 antigen-binding sites; the antibody molecule can be a "2+2" type bispecific antibody, that is, the antibody has two different antigen-binding specificities, wherein for each antigen-binding specificity, there are 2 identical antigen-binding sites.

[0118] In this document, the terms “FLOR1” and “FRα” are used interchangeably and refer to folate receptor α (FRα). Unless otherwise stated, this term includes any variant of human FLOR1, including sequence variants, especially naturally occurring variants, allelic variants, and post-translational modification and conformational variants, and covers its species homologs. Furthermore, it should be understood that this term covers not only FLOR1 expressed naturally or recombinantly in cells or expressed on natural or recombinant cells, but also fusion proteins containing FOLR1 or fragments thereof, such as extracellular fragments. An example of FOLR1 is the human FOLR1 protein containing the amino acid sequence at UniProt:P15328. Another example of FOLR1 is the monkey FOLR1 protein containing the amino acid sequence at NCBI:NP_001181576.1. In this document, unless otherwise specified, the term “FOLR1” refers to FOLR1 derived from humans. In this document, “antigen-binding specificity against FOLR1,” that is, “antigen-binding domain that specifically binds to FOLR1,” is preferably provided by a VHH domain or Fab.

[0119] In this document, the term "FOLR1 positive" cell refers to a cell that expresses FOLR1 positively on its cell surface, such as cancer cells, modified cancer cells, or modified non-tumor cells. The FOLR1 expression level on the cell surface can be determined by any conventional method known in the art for determining cell surface antigen expression levels, such as FACS detection or immunofluorescence staining. FOLR1 is expressed at significantly higher levels on a variety of tumor cells than on normal tissues / cells, for example, OVCAR3 (human ovarian cancer cells). Preferably, in this document, FOLR1 positive cells are FOLR1 positive tumor cells.

[0120] In this document, the terms “mesothelin” and “MSLN” are used interchangeably and refer to mesothelin (MSLN). Unless otherwise stated, this term includes any variant of human MSLN, including sequence variants, especially naturally occurring variants, allelic variants, and post-translational modification variants and conformational variants, and covers its species homologs. Furthermore, it should be understood that this term covers not only MSLN expressed naturally or recombinantly in cells or expressed on natural or recombinant cells, but also recombinantly expressed fusion proteins containing MSLN or fragments thereof. An example of an MSLN is the human MSLN protein containing the amino acid sequence NCBI:AAH09272.1. Another example of an MSLN is the monkey MSLN protein containing the amino acid sequence NCBI:XM_005590816.3. In this document, unless otherwise specified, the term “MSLN” refers to MSLN derived from humans. In the antibody of the present invention, the "antigen binding specificity against MSLN", that is, the "antigen binding domain that specifically binds to MSLN", is provided by the VHH domain.

[0121] In this document, the term "MSLN-positive" cell refers to a cell that expresses MSLN on its cell surface positively, such as cancer cells, modified cancer cells, or modified non-tumor cells. The MSLN expression level on the cell surface can be determined by any conventional method known in the art for determining cell surface antigen expression levels, such as FACS detection or immunofluorescence staining. MSLN expression levels are significantly higher in a variety of tumor cells than in normal tissues / cells, for example, NCI-H2110 (human non-small cell lung cancer cells). Preferably, in this document, MSLN-positive cells are MSLN-positive tumor cells.

[0122] In this paper, the term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigenic epitope). In this context, "binding affinity" reflects the intrinsic binding affinity of a 1:1 interaction between members of a binding pair. Binding affinity is typically expressed as the binding dissociation equilibrium constant (K0). D It can be described by ) and measured by commonly known methods in the art, such as surface plasmon resonance (SPR) techniques.

[0123] In this paper, the term “avidity” or “binding affinity” refers to the combined strength of the interactions between multiple binding sites of a molecule (antibody) and the same target.

[0124] In this paper, the term "immunoglobulin" 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). Immunoglobulin heavy chains can be classified into one of five categories based on the type of their constant domains, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM). Some of these categories can be further subdivided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains can also be classified into one of two types based on the amino acid sequence of their constant domains, called κ and λ.

[0125] In this document, the term "isotype" refers to the antibody type determined by the antibody heavy chain constant region. For example, the antibody according to the invention may be an IgA (e.g., IgA1 or IgA2), IgG1, IgG2 (e.g., IgG2a or IgG2b), IgG3, IgG4, IgE, IgM, and IgD antibody, having a heavy chain constant region of the aforementioned immunoglobulin type. Furthermore, the invention contemplates not only antibodies employing native sequence constant regions but also antibodies containing variant sequence constant regions.

[0126] In this document, the term "variable region" or "variable domain" refers to a domain of the antibody's heavy or light chain involved in antibody-antigen binding. In the case of heavy chain antibodies, such as those derived from camelid heavy chains, a single VH domain (also referred to herein as the VHH domain) may be sufficient to impart antigen-binding specificity. Like the variable regions of the heavy and light chains of conventional IgG antibodies, the VHH domain contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs), arranged in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. According to some aspects of the invention, one or more residues in the variable region of the antibody can be modified, for example, by modifying one or more CDR regions and / or by modifying one or more framework regions, particularly by substituting conserved residues, to obtain antibody variants that still substantially retain at least one biological property (e.g., antigen-binding ability) of the parent antibody. In other aspects, the antibody variable region can be modified by CDR transplantation. Since the CDR sequence is responsible for most antibody-antigen interactions, recombinant antibody variants that mimic the properties of known antibodies can be constructed. In such antibody variants, a CDR sequence from a known antibody is grafted onto a framework region of a different antibody with different properties, and one to several residues can be mutated as needed, such as reverting to a mutation to refine the desired properties of the antibody. The properties of the mutated and / or modified antibody or ADC conjugate containing it can be evaluated in in vitro or in vivo assays, such as target antigen binding properties or other desired functional properties, such as endocytic activity, pharmacokinetics, and in vivo tumor-killing activity. Therefore, variants of any variable regions (e.g., VHH) given herein are also contemplated in this invention.

[0127] In this document, the term "complementarity-determining region" or "CDR region" or "hypervariant region" refers to 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. In the VHH domain of the antibody of this invention, CDRs are sequentially numbered starting from the N-terminus and are commonly referred to as CDR1, CDR2, and CDR3. A specific CDR sequence within a VHH domain can be determined using methods known in the art, such as the Kabat, AbM, Chothia, Contact, and IMGT schemes, to define the regional extent of the CDR and combinations thereof. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the aforementioned methods and combinations thereof. Furthermore, it is understood in the art that although CDRs differ between antibodies, only a limited number of amino acid positions within a CDR directly participate in antigen binding. Using at least two of the Kabat, Chothia, AbM, and Contact methods, a minimal overlapping region can be determined, thereby providing a “minimum binding unit” for antigen binding. Such a minimum binding unit can be a sub-part of a CDR. The remaining residues of the CDR sequence, as will be apparent to those skilled in the art, can be determined by the antibody’s structure and protein folding. Therefore, the invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues may be substituted.

[0128] Unless otherwise stated, in this invention, when referring to the position of residues in the antibody variable region and CDR (including heavy chain variable region residues), it means the numbering position according to the Kabat numbering system.

[0129] In this document, the terms "VHH" and "VHH domain" are used interchangeably to refer to a heavy chain variable domain derived from a heavy chain antibody lacking a light chain, sometimes also called a single variable domain fragment (sVD). Therefore, a VHH differs from the conventional VH of a four-chain immunoglobulin in that it does not require pairing with a light chain variable domain to form an antigen-binding site. Such VHH molecules can be derived from antibodies produced in camelid species (e.g., camels, alpacas, dromedaries, llamas, and guanacos). Other species besides camelids may also produce naturally occurring heavy chain antibodies lacking a light chain, and these VHHs are also within the scope of this invention. In some cases, for the therapeutic application of antibodies or their derivatives, it is desirable to reduce their immunogenicity.

[0130] Heavy chain antibodies (HcAbs) are novel antibody molecules found in camels and sharks. These antibodies are characterized by the natural absence of light chains, consisting only of heavy chains. Despite the lack of light chains, heavy chain antibodies retain the ability to bind antigens. Other species besides camels can also produce naturally occurring heavy chain antibodies lacking light chains. When heavy chain antibodies are mentioned herein, they generally refer to single-chain antibodies containing VHH and a heavy chain constant region or Fc region. The heavy chain antibodies mentioned herein can also dimerize to form dimerized heavy chain antibodies, which are also included within the scope of "heavy chain antibodies" in this invention.

[0131] In this document, the term "immunoglobulin Fc region," used interchangeably with "Fc region" and "Fc domain," defines the C-terminal region of the immunoglobulin heavy chain, which comprises at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions. Fc regions that can be used in the antibodies of this invention include, but are not limited to, Fc regions of IgG1, IgG2, IgG3, or IgG4 having native or variant sequences. 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.

[0132] In this document, the term "Fc region" or "Fc domain" excludes the heavy chain variable region (VH) and light chain variable region (VL) of immunoglobulins, as well as the heavy chain constant region (CH1) and light chain constant region (CL); but may include the CH2 and CH3 domains, and may or may not contain an immunoglobulin hinge region, or may contain a complete or partial hinge region. For example, in some instances, the Fc region may consist of or be composed of the CH2 and CH3 domains from the N-terminus to the C-terminus. In other instances, the Fc region may consist of or be composed of a complete or partial immunoglobulin hinge region, the CH2 and CH3 domains, or a combination of the immunoglobulin hinge region, the CH2 and CH3 domains, from the N-terminus to the C-terminus. When "Fc region" is mentioned herein, it encompasses both the native Fc region and variant Fc regions.

[0133] In this document, the term "natural Fc region" encompasses the Fc regions of various naturally occurring immunoglobulins, 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 human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or Asn231 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. In some embodiments, the human IgG heavy chain Fc region carries at its N-terminus the complete or partial hinge region sequence of the natural immunoglobulin, for example, the sequence E216 to P230 or the sequence D221 to P230 according to EU designations.

[0134] In this document, the term "variant Fc region" refers to a polypeptide containing a modified Fc region relative to the native Fc region sequence. The modification can be the addition, deletion, or substitution of amino acid residues. Substitution can include both naturally occurring and non-natural amino acids. The purpose of the modification can be to alter the binding of the Fc region to its receptor and the resulting effector function, or to prevent undesirable heavy chain mismatches, or to site-directedly introduce amino acid modifications that can be used to conjugate other active molecules.

[0135] In this paper, 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 Fc receptor binding, C1q binding and complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity (ADCC). Depending on the intended use of the antibody molecule, the Fc region of the antibody can be modified to give it altered effector functions relative to antibody molecules with a wild-type Fc region, such as reduced or eliminated Fcγ receptor binding.

[0136] In this document, the terms “flexible linker” or “connector” or “linker peptide” are used interchangeably to refer to a short amino acid sequence consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination, or from the hinge region of an immunoglobulin.

[0137] In this document, the “percentage of identity (%)” for an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to those in the specific amino acid sequence shown in this specification, after comparing the candidate sequence with the specific amino acid sequence shown herein and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of sequence identity. In some embodiments, the invention contemplates variants of the antibody molecules of the invention that have a considerable degree of identity with respect to the antibody molecules and their sequences specifically disclosed herein, for example, an identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or higher. These variants may contain conserved modifications.

[0138] For polypeptide sequences, "conservative modification" includes substitutions, deletions, or additions to the polypeptide sequence that result in the replacement of a certain amino acid with a chemically similar amino acid. Tables providing conserved substitutions of functionally similar amino acids are well known in the art. The following eight groups contain amino acids that are conservedly substituted for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modification" is used in particular to refer to amino acid modifications that do not significantly affect or alter the desired properties (e.g., binding characteristics and / or internalization characteristics) of the antibody containing the amino acid sequence.

[0139] In this document, the terms "binding" or "specific binding" mean that the binding interaction 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 detecting the binding ability of an antibody to an antigen using the ELISA assay described in the examples, detecting the binding ability of an antibody to cells expressing an antigen using the FACS assay described in the examples, or detecting the affinity constant K using the SPR technique described in the examples. D .

[0140] In this document, the term "epitope" refers to the antigenic moiety that an antibody specifically binds to. An epitope may consist of continuous and / or discontinuous amino acids forming a conformational spatial unit. Different antibodies binding to the same antigen can be grouped by epitope grouping using a competitive binding assay. An antibody and a reference antibody are considered "competitively binding antibodies" when the test antibody blocks the binding of a reference antibody to an antigen (e.g., FLOR1 or MSLN) by 50% or more in a competitive binding assay; and conversely, when the reference antibody blocks the binding of the test antibody to the antigen (e.g., FLOR1 or MSLN) by 50% or more in a competitive binding assay. Competitively binding antibodies may bind to the same epitope region as the reference antibody, such as identical epitopes, adjacent epitopes, or overlapping epitopes. The competitive binding assay can be performed by methods known in the art, such as solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay, or the methods described in the examples herein.

[0141] In this document, a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from nonhuman CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs (e.g., CDRs) in a humanized antibody correspond to those in nonhuman antibodies, and all or substantially all of the FRs correspond to those in human antibodies. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. The “humanized form” of an antibody (e.g., a nonhuman antibody) refers to an antibody that has been humanized. In some embodiments, the humanized antibody of the present invention has a framework region sequence “derived” from a specific human lineage sequence. Here, “derived” means that the amino acid sequence of the antibody framework region has at least 85% or 90% identity with the corresponding framework region amino acid sequence encoded by the human lineage immunoglobulin gene, and that the antibody retains antigen-binding activity.

[0142] In this document, if an amino acid sequence (e.g., VHH) is specific for two different antigens or antigenic determinants (e.g., FLOR1 or MSLN from different mammalian species, such as human FLOR1 or human MSLN, cynomolgus monkey FLOR1 or cynomolgus monkey MSLN), then it is said to be "cross-reactive" to these two different antigens or antigenic determinants. Antibodies exhibiting human-monkey species cross-reactivity, particularly having similar binding affinity for human-monkey antigens, is advantageous, as this property can facilitate preclinical drug development of antibodies, such as toxicological assays of ADC molecules composed of antibodies. In some embodiments, the antibodies of the present invention preferably exhibit human-monkey species cross-reactivity.

[0143] In this document, the terms "endocytosis" and "internalization" are used interchangeably, referring to the process by which a ligand / receptor complex is internalized and delivered into the cytosol or translocated to a suitable intracellular compartment, triggered by the binding of a ligand to a corresponding receptor on the cell surface. In some embodiments, the antibodies of the present invention initiate endocytosis mediated by FLOR1 and / or MSLN receptors upon binding to FLOR1 and / or MSLN expressed on the cell surface. In this document, endocytosis and endocytosis rate can be determined, for example, by the methods described in the examples, to characterize the endocytic activity of the antibody. In some embodiments, the antibodies of the present invention having endocytic activity can be used as a tool for delivering antitumor drugs into cancer cells in the ADCs of the present invention.

[0144] In this document, the term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including progeny cells of this type. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny. Host cells can be 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.

[0145] In this document, the term "expression vector" refers to a vector containing a recombinant polynucleotide and an expression control sequence that effectively links the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including clomids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0146] In this document, the terms "immunoconjugate," "immunofusion," or "immunocoupler" are used interchangeably and generally refer to a molecule formed by conjugating or fusing one or more immunoglobulin-associated molecules or fragments thereof (e.g., antibodies or fragments thereof) with one or more other molecules. In some cases, the other molecules may be protein-like molecules, such as peptides, polypeptides, or proteins. In some cases, the other molecules may also be non-protein-like molecules, such as chemical toxins or drugs like small molecule drugs or antitumor compounds. In some cases, the other molecules may be the same as immunoglobulin-associated molecules or fragments thereof. In some cases, the other molecules may be different from immunoglobulin-associated molecules or fragments thereof. The one or more other molecules may be the same or different from each other. For example, the other molecules may be target-binding elements and / or effector elements, such as chemotherapeutic agents, toxins, drugs (e.g., immunotherapeutic agents), radioactive elements, probes, or signaling molecules, etc.

[0147] In this document, the terms “individual” or “subject” are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, an individual is a human.

[0148] In this article, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual receiving treatment. 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 cases involving tumor or cancer treatment, "treatment" encompasses antitumor biological effects that can be induced by artificial intervention (e.g., through the administration of drugs), including but not limited to, reductions in tumor volume, number of tumor cells, proliferation, or survival.

[0149] In this document, the terms “cancer” and “tumor” are used interchangeably to refer to or describe a physiological disorder in mammals characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas, solid tumors, and non-solid tumors. In some embodiments, cancers suitable for treatment by the antibodies or immunoconjugates or immunofusions of the present invention include MSLN-positive and / or FOLR1-positive tumors / cancers, including their metastatic forms. Examples of cancer include the specific cancers mentioned in the embodiments section herein.

[0150] As used herein, "antibody-drug conjugate (ADC)" refers to a compound in which an antigen-binding molecule is linked to a (small molecule) drug, such as an antitumor compound, via a linker. In this context, it may also be referred to as an antigen-binding molecule-drug conjugate.

[0151] "Linker-payload" is a term well known to those skilled in the art, referring to a compound formed by linking a linker to a payload (e.g., a drug).

[0152] The term "linker" refers to a structural segment that connects a drug to an antigen-binding molecule. It should be understood that a linker has functional groups that can form bonds with the functional groups of the antigen-binding molecule before linking to it. Non-limiting examples of linkers include those involved in embodiments and examples of the present invention.

[0153] As used herein, the term "alkyl" refers to a fully saturated branched or unbranched hydrocarbon group. Alkyl groups preferably contain 1-16 carbon atoms, i.e., C16-C26. 1-16 Alkyl, more preferably C10 1-12 Alkyl, C 1-10 Alkyl, C 1-8 Alkyl, C 1-6 Alkyl, C 1-4 Alkyl or C 1- 2. Alkyl groups. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc.

[0154] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group containing 2-16 carbon atoms and at least one double bond but no triple bonds. The alkenyl group preferably contains 2-12 carbon atoms, i.e., C64. 2-12 Alkenyl, more preferably C 2-10 alkenyl, C 2-8 alkenyl, C 2-6 alkenyl or C 2-4 Alkenyl groups. Representative examples of alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, and hexenyl.

[0155] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group containing 2-16 carbon atoms and at least one triple bond. The alkynyl group preferably contains 2-12 carbon atoms, i.e., C64. 2-12 The alkynyl group, more preferably C, is a alkynyl group. 2-10 alkynyl group, C 2-8 alkynyl group, C 2-6 alkynyl or C 2-4. Alynyl group. Representative examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, penynyl, and hexynyl.

[0156] The term "haloalkyl" refers to an alkyl group as defined above, which is substituted with one or more (e.g., up to 12, such as 1, 2, 3, 4, 5, 6, 7, or 8) halogen groups. Halogenated alkyl groups include perhalogenated alkyl groups. A perhalogenated alkyl group is an alkyl group in which all hydrogen atoms are replaced by halogen atoms. When a haloalkyl group is a polyhalogenated alkyl group, i.e., containing two or more halogens, the halogens may be the same or different. Non-limiting examples of haloalkyl groups include monofluoromethyl, difluoromethyl, trifluoromethyl, monochloromethyl, dichloromethyl, trichloromethyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, trifluoroethyl, dichloroethyl, trichloroethyl, difluoropropyl, perfluoroethyl, and perfluoropropyl.

[0157] The term "haloalkenyl" refers to an alkenyl group as defined above, which is substituted by one or more (e.g., up to 12, such as 1, 2, 3, 4, 5, 6, 7, or 8) halogen groups. When the haloalkenyl is a polyhalogenated alkenyl, i.e., containing more than two halogens, the halogens may be the same or different.

[0158] The term "haloalkynyl" refers to an alkynyl group as defined above, which is substituted by one or more (e.g., up to 12, such as 1, 2, 3, 4, 5, 6, 7, or 8) halogen groups. When the haloalkynyl group is a polyhaloalkynyl group, i.e., containing more than two halogens, the halogens may be the same or different.

[0159] The term “halogen” or “halogenated” refers to fluorine (-F), chlorine (-Cl), bromine (-Br), or iodine (-I).

[0160] The term "hexuronic acid" refers to compounds obtained by oxidizing the primary hydroxyl group of a hexose to a carboxyl group. Hexuronic acids include common uronic acids obtained by oxidizing hexoses, including but not limited to glucuronic acid, mannuronic acid, and galacturonic acid.

[0161] The term "amino acid" as used herein has the meaning conventionally understood in the art. Amino acids can be L or D isomers. The notation of amino acids follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. For ease of understanding, the names of some common amino acids and their corresponding abbreviations are listed in the table below:

[0162] The amino acids in this invention also include other amino acids such as citrulline (Cit; C). It should be understood that citrulline (Cit; C) is frequently used in the linker portion of an ADC. Therefore, unless otherwise specified and consistent with the context, the single-letter abbreviation "C" representing an amino acid in a linker or linker fragment involving an ADC represents Cit; and in a linker or linker fragment not involving an ADC, the single-letter abbreviation "C" representing an amino acid represents Cys.

[0163] Unless otherwise specified, the amino acids in this invention refer to L-amino acids.

[0164] The term "optional" or "optionally" means that the event or condition described below either occurs or does not occur, and the description includes instances where the event or condition occurs as well as instances where the event or condition does not occur. For example, when a group or structure is "optionally substituted," the group or structure may or may not be substituted.

[0165] In this article, "pharmaceutically acceptable" means that it can be administered to an individual or subject without producing biologically or otherwise undesirable side effects, such as serious and intolerable side effects.

[0166] Where there is no contradiction in the context, "pharmaceutical acceptable" and "medicinal" are used interchangeably in this article.

[0167] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the molecules of the present invention (e.g., antibody-drug conjugates), and that such salt is not biologically or otherwise undesirable. The ADC conjugates of the present invention can exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable, non-toxic acid addition salt refers to a salt formed by the ADC conjugate of the present invention with an organic or inorganic acid, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, etc. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the ADC conjugates of the present invention with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed by reacting with an organic base containing an N group.

[0168] The term "solvent" refers to an association formed by one or more solvent molecules with the antibody-drug conjugate of this invention. Solvents that form solvates include solvents commonly used in the pharmaceutical industry, such as water, ethanol, diethyl ether, isopropanol, ethyl acetate, and dimethyl sulfoxide.

[0169] The term "drug:antibody ratio" or "DAR" refers to the ratio of the drug portion (D) to the antibody portion in the antibody-drug conjugate molecule (e.g., the compound of formula I) described herein. It should be understood that for antibody-drug conjugate molecules, DAR is an integer; for example, the DAR of the antibody-drug conjugate molecule of the present invention can be an integer from 1 to 16, such as 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, or, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The prepared antibody-drug conjugate typically contains one or more antibody-drug conjugate molecules with different DARs, which are usually characterized by the average DAR, i.e., the overall ratio of the drug fraction (D) conjugated to the Ab fraction described herein to the Ab fraction in the product, as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA, electrophoresis and / or HPLC). This DAR is referred to as the average DAR in this document and may be a decimal. In some embodiments, the average DAR value of the conjugates of the present invention is 1 to 16, for example 2-16, 4-12, 3-5, 5-7, 7-9, 3-8, 2-6, 4-6, for example 3.0-8.0, 3.5-4.5, 5.5-6.5 or 7.5-8.5, for example 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4 6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, a range with two of these values ​​as endpoints. As stated above, it should be understood that when referring to the average DAR value, the ADC of the present invention refers to a population of ADC molecules or a mixture of ADC molecules containing ADC molecules having the same and / or different DAR values.

[0170] The term “therapeutic agent” as used herein encompasses any substance that is effective in preventing or treating diseases such as cancer, including chemotherapeutic agents, cytotoxic agents, immunomodulators (such as immunosuppressants), other antibodies, small molecule drugs, angiogenesis inhibitors, or cytokines.

[0171] "Chemotherapy agents" include chemical compounds that are useful in treating cancer or immune system diseases.

[0172] The term "cytotoxic agent" is used in this invention to refer to substances that inhibit or prevent cell function and / or cause cell death or damage.

[0173] As used herein, the term "immunomodulator" refers to a natural or synthetic active agent or drug that inhibits or modulates (e.g., activates) an immune response. An immune response can be humoral or cellular. Immunomodulators include immunosuppressants or immune agonists, such as immune checkpoint inhibitors or immune checkpoint agonists.

[0174] The term "drug" refers to a compound that can regulate biological processes, particularly altering or preventing pathological processes. In this article, "drug" preferably refers to antitumor compounds.

[0175] The term "small molecule drug" refers to a low molecular weight drug that can regulate biological processes, particularly altering or preventing pathological processes. "Small molecule" is defined as a molecule with a molecular weight less than 10 kDa, typically less than 2 kDa, and preferably less than 1 kDa, more preferably less than 500 kDa. Small molecule drugs include, but are not limited to, organic molecules having the molecular weights defined above, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can penetrate cells more readily, are less susceptible to degradation, and are less likely to elicit an immune response than large molecules.

[0176] "Antitumor compounds" are pharmaceutically active compounds that have an effect on tumors, including but not limited to cytotoxic agents or chemotherapeutic agents, such as the cytotoxic agents disclosed in WO2021 / 173773 and US5658920, such as camptothecin compounds eczetidine (a topoisomerase I inhibitor Exatecan), Dxd (a novel topoisomerase I inhibitor Exatecan derivative), and auristatin compounds such as monomethyl auristatin E (MMAE) and MMAF.

[0177] The term "effective amount" refers to such an amount or dose of the antigen-binding molecule or ADC molecule or 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. Depending on the intended effect, it may include "therapeutic effective amount" and "preventive effective amount".

[0178] "Therapeutic effective dose" refers to the amount that effectively achieves the desired therapeutic outcome at the required dose and for the required duration. Therapeutic effective dose can vary depending on various factors such as disease state, individual age, sex, and weight. Therapeutic effective dose is the amount at which any toxic or harmful effects are less than the beneficial therapeutic effect. Relative to untreated subjects, "therapeutic effective dose" 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 60%, and still more preferably at least about 80%. The ability of the antibody of the present invention to inhibit measurable parameters (e.g., tumor volume) can be evaluated in animal model systems that predict efficacy in human tumors.

[0179] "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 in subjects before or at an early stage of the disease, the prophylactic effective dose is less than the therapeutic effective dose.

[0180] 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.

[0181] The term "pharmaceutical excipient" refers to diluents, adjuvants (such as Freund's adjuvants (complete and incomplete)), carriers, or stabilizers that are applied together with the active substance.

[0182] 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.

[0183] The terms “drug combination,” “combination product,” “drug conjugate,” or “combination product” refer to non-fixed or fixed combinations, including but not limited to pillboxes and pharmaceutical compositions. The term “non-fixed combination” means that the active ingredients (e.g., (i) the antigen-binding molecule or ADC molecule of the present invention, including its pharmaceutically acceptable salt, 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 of two or more active agents. In some embodiments, the antigen-binding molecule or ADC molecule of the present invention and other therapeutic agents used in the drug combination are administered at levels not exceeding those achieved when used alone. 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. The components may each be in separate formulations, and their formulations may be the same or different.

[0184] The terms "combination therapy" or "treatment in combination" refer to the administration of two or more therapeutic agents or modalities of treatment (e.g., radiation therapy or surgery) to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of 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. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0185] 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.

[0186] When used herein, the terms “molecule of the invention” or “molecule according to the invention” or “molecule described herein” are used interchangeably to include antigen-binding molecules (covering VHH antibodies, heavy chain antibodies, multispecific antibodies and other antigen-binding molecules), immunoconjugates, immunofusions, antibody-drug conjugates, or pharmaceutically acceptable salts or solvates thereof as defined herein (e.g., in the Embodiments and Examples sections).

[0187] I. The multispecific antibody of the present invention

[0188] I. Antigen-binding molecules, nucleic acids, and their preparation methods

[0189] In some embodiments, the present invention relates to antigen-binding molecules that specifically bind to MSLN and / or FLOR1, such as VHH antibodies, heavy chain antibodies, full-length antibodies, or multispecific antibodies. The antigen-binding molecules of the present invention also comprise TCR molecules or CAR molecules that specifically bind to MSLN and / or FLOR1.

[0190] I.MSLN single-domain antibodies and their heavy chain antibodies

[0191] In a first aspect, this invention relates to an antigen-binding molecule that specifically binds to MSLNs, preferably, said antigen-binding molecule is a single-domain antibody that specifically binds to MSLNs or a heavy-chain antibody comprising the single-domain antibody VHH. In some embodiments, the antibody of this invention or its antigen-binding fragment binds to mammalian MSLNs, such as human MSLNs or cynomolgus monkey MSLNs.

[0192] In some embodiments, the anti-MSLN single-domain antibody of the present invention is a VHH antibody comprising or composed of a heavy chain variable region, wherein the heavy chain variable region typically has the following structure: FR1-VHH CDR1-FR2-VHH CDR2-FR3-VHH CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; and VHH CDR1 to VHH CDR3 refer to complementarity-determining regions 1 to 3. The CDR sequence in the VHH variable region can be determined according to any CDR definition scheme, for example, according to AbM, Chothia, Kabat, IMGT, or any combination thereof; more preferably, the CDR is defined according to Kabat or AbM, or a combination thereof; and more preferably, the CDR is defined according to AbM. In this document, the anti-MSLN single-domain antibody or VHH antibody is also referred to as VHH. MSLN .

[0193] In some embodiments, the anti-MSLN VHH antibody of the present invention comprises three complementarity-determining regions (CDRs) contained in the VH shown in any one of SEQ ID NO: 29, 33, 37, 42-45, 47, 49, 51-72; preferably, the CDR sequence is defined according to ABM.

[0194] In some embodiments, the anti-MSLN VHH antibody of the present invention comprises or is composed of a heavy chain variable region, said heavy chain variable region comprising the three complementarity-determining regions (CDRs) contained in the VH shown in any one of SEQ ID NO: 29, 33, 37, 42-45, 47, 49, 51-72; preferably, said CDR sequence is defined according to ABM.

[0195] In some embodiments, the anti-MSLN VHH antibody of the present invention comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or the anti-MSLN VHH of the present invention comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein

[0196] The VHH CDR1 comprises, or is composed of, an amino acid sequence selected from SEQ ID NO:30, 34, or 38, or is composed of said amino acid sequence, or the VHH CDR1 comprises an amino acid sequence having one, two, or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence selected from SEQ ID NO:30, 34, or 38; or

[0197] The VHH CDR2 comprises, or is composed of, the amino acid sequence of SEQ ID NO:31, 35, or 39, 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:31, 35, or 39; or

[0198] The VHH CDR3 comprises or is composed of an amino acid sequence selected from SEQ ID NO:32, 36, 40, 46, 48 or 50, or the VHH CDR3 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO:32, 36, 40, 46, 48 or 50.

[0199] In some embodiments, the anti-MSLN VHH antibody of the present invention comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or the anti-MSLN VHH of the present invention comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein

[0200] (i) The VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:30, the VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:31, and the VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:46, 32, 48 or 50.

[0201] (ii) The VHH CDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:34, the VHH CDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:35, and the VHH CDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:36; or

[0202] (iii) The VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:38, the VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:39, and the VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:40.

[0203] In some embodiments, the anti-MSLN VHH antibody of the present invention comprises or is composed of a heavy chain variable region, said heavy chain variable region

[0204] (i) comprising or consisting of 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 any one of SEQ ID NO: 29, 33, 37, 42-45, 47, 49, 51-72; or

[0205] (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO: 29, 33, 37, 42-45, 47, 49, 51-72; or

[0206] (iii) An amino acid sequence having 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 selected from any one of SEQ ID NO:29, 33, 37, 42-45, 47, 49, 51-72, preferably, the amino acid changes do not occur in the CDR region.

[0207] In some embodiments, the anti-MSLN VHH antibody of the present invention comprises or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO:29, 33, 37, 42-45, 47, 49, 51-72.

[0208] In some embodiments, the VHH antibody against MSLN of the present invention comprises a CDR amino acid sequence and / or a framework (FR) amino acid sequence derived from a camelid heavy chain antibody produced by immunizing a camelid animal (e.g., an alpaca). In some embodiments, the VHH monoclonal antibody of the present invention derived from a camelid heavy chain antibody can be engineered, for example, to comprise a framework region sequence derived from a human amino acid sequence (i.e., a human antibody) or other non-camelid mammal species. Therefore, in one embodiment, the VHH antibody of the present invention is a chimeric antibody.

[0209] In one embodiment, the anti-MSLN VHH antibody of the present invention is a humanized antibody. For example, the original VHH sequence is humanized using a "best-matching" method, which includes...

[0210] (i) The amino acid sequence of the VHH framework region was compared and analyzed using the human pedigree V gene database to select the best pedigree sequence;

[0211] (ii) Replace the best-matching human CDR sequence with the VHH CDR sequence to generate a humanized VHH sequence;

[0212] (iii) Mutations that restore multiple residues in the mutant framework region and optionally remove them by post-translational modification (PTM);

[0213] (iv) Optional sequencing of VHH antibodies.

[0214] Typically, humanization is performed in a manner that preserves the favorable binding properties of single-domain antibodies. Assays for determining the biological properties of humanized single-domain antibodies, such as binding affinity, are well known in the art, in order to identify and select suitable mutations or combinations of humanized residues.

[0215] In another aspect of the invention, the invention also provides a heavy chain antibody comprising the heavy chain variable region of the anti-MSLN VHH antibody of the invention.

[0216] In some embodiments, the anti-MSLN single-domain antibody or VHH (e.g., camel-derived VHH or its humanized form) of the present invention can be linked to a constant region or a portion thereof, such as the Fc region, of a human antibody to produce a heavy chain antibody comprising a VHH-constant region, VHH-CH1-Fc, or VHH-Fc. In one embodiment, the heavy chain antibody comprises the VHH antibody of the present invention and an Fc region located at its C-terminus.

[0217] In some embodiments, the anti-MSLN heavy chain antibody of the present invention comprises VHH as defined herein. MSLN Or the heavy chain variable region therein, and the heavy chain constant region or the Fc region of the heavy chain constant region. In some embodiments, in VHH MSLN Or its heavy chain variable region is directly or via a joint connected to its heavy chain constant region or Fc region, such as an Fc region containing a complete or partial hinge region. Preferably, the Fc region contains a complete hinge region and contains a 220S transition. In some embodiments, when the Fc region contains a complete hinge region, such as the complete hinge region shown in SEQ ID NO:139 or the complete hinge region shown in SEQ ID NO:141, the VHH MSLN It is directly connected to the Fc area.

[0218] In one embodiment, the anti-MSLN heavy chain antibody comprises the Fc portion derived from a camel (e.g., an alpaca). In one embodiment, the heavy chain antibody is generated and isolated by immunizing the camel, such as an alpaca. Various methods are known in the art for immunizing camel animals and isolating VHH antibodies or heavy chain antibodies against the target antigen.

[0219] In some embodiments, the anti-MSLN heavy chain antibody contains a constant region derived from human or non-human primate (e.g., cynomolgus monkey) antibodies, such as a constant region derived from human IgG1, human IgG2, human IgG3, or human IgG4.

[0220] In some embodiments, the anti-MSLN heavy chain antibody comprises an Fc region derived from a human or non-human primate (e.g., a cynomolgus monkey). In yet another embodiment, the heavy chain antibody comprises a human IgG Fc region, such as a human IgG1, human IgG2, human IgG3, or human IgG4 Fc region, preferably a human IgG1 or human IgG4 Fc region.

[0221] In one embodiment, the heavy chain antibody that specifically binds to MSLN according to the present invention can dimerize with another polypeptide chain (e.g., another heavy chain antibody, the same or different) containing the Fc region via the Fc region. Therefore, in one embodiment, the present invention also provides homologous or heterologous multimeric proteins comprising the heavy chain antibody of the present invention. In a preferred embodiment, the protein preferably comprises a heavy chain antibody formed by pairing two identical heavy chain antibody chains.

[0222] The Fc region or its mutations described in this context are applicable to the heavy chain antibodies of this invention that specifically bind to MSLN.

[0223] In some embodiments, the Fc region is derived from IgG1, which comprises or consists of an amino acid sequence shown in any one of SEQ ID NO: 122, 123, 124, 142-145, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 122, 123, 124, 142-145.

[0224] In some embodiments, the Fc region is derived from IgG4, which comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:128.

[0225] In some embodiments, the single-domain antibody VHH that specifically binds to MSLN, or a heavy-chain antibody containing it, such as VHH-Fc, binds to MSLN-positive cells, for example, superior to known antibodies such as mirvetuximab.

[0226] II. FOLR1 single-domain antibodies and their heavy chain antibodies

[0227] In a second aspect, the present invention relates to an antigen-binding molecule that specifically binds to FOLR1, preferably, said antigen-binding molecule is a single-domain antibody that specifically binds to FOLR1 or a heavy-chain antibody comprising the single-domain antibody VHH. In some embodiments, the antibody of the present invention or its antigen-binding fragment binds to mammalian FOLR1, such as human FOLR1 or cynomolgus monkey FOLR1.

[0228] In some embodiments, the anti-FOLR1 single-domain antibody of the present invention is a VHH antibody comprising or composed of a heavy chain variable region, wherein the heavy chain variable region typically has the following structure: FR1-VHH CDR1-FR2-VHH CDR2-FR3-VHH CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; and VHH CDR1 to VHH CDR3 refer to complementarity-determining regions 1 to 3. The CDR sequence in the VHH variable region can be determined according to any CDR definition scheme, for example, according to AbM, Chothia, Kabat, IMGT, or any combination thereof; more preferably, the CDR is defined according to Kabat or AbM, or a combination thereof; and more preferably, the CDR is defined according to AbM. However, it should be understood that the CDR can also be defined in any other manner known in the art. In this document, the anti-FOLR1 single-domain antibody or VHH antibody will also be referred to as VHH. FOLR1 .

[0229] In some embodiments, the anti-FOLR1 VHH antibody of the present invention comprises three complementarity-determining regions (CDRs) contained in the VH shown in any one of SEQ ID NO: 1, 5, 7, 9, 13, 16, 20, 22-28, 129, 130, 131; preferably, the CDR sequence is defined according to ABM.

[0230] In some embodiments, the anti-FOLR1 VHH antibody of the present invention comprises or is composed of a heavy chain variable region, said heavy chain variable region comprising the three complementarity-determining regions (CDRs) contained in the VH shown in any one of SEQ ID NO: 1, 5, 7, 9, 13, 16, 20, 22-28, 129, 130, 131; preferably, said CDR sequence is defined according to AbM.

[0231] In some embodiments, the anti-FOLR1 VHH antibody of the present invention comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or the anti-FOLR1 VHH of the present invention comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein

[0232] The VHH CDR1 comprises, or is composed of, an amino acid sequence selected from SEQ ID NO:2, 10, or 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 selected from SEQ ID NO:2, 10, or 14; or

[0233] The VHH CDR2 comprises, or is composed of, the amino acid sequence of SEQ ID NO:3, 11, 15, 17, 18, 19, 21, or 41, or is composed of said amino acid sequence, or the VHH CDR2 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:3, 11, 15, 17, 18, 19, 21, or 41; or

[0234] The VHH CDR3 comprises or is composed of an amino acid sequence selected from SEQ ID NO:4, 6, 8 or 12, or the VHH CDR3 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence selected from SEQ ID NO:4, 6, 8 or 12.

[0235] In some embodiments, the anti-FOLR1 VHH antibody of the present invention comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or the anti-FOLR1 VHH of the present invention comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein

[0236] (i) The VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:2, the VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:3, and the VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:4, 6 or 8;

[0237] (ii) The VHH CDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:10, the VHH CDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:11, 17, 18, 19, 21 or 41, and the VHH CDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:12; or

[0238] (iii) The VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:14, the VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:15, and the VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:12.

[0239] In some embodiments, the anti-FOLR1 VHH antibody of the present invention comprises or is composed of a heavy chain variable region, wherein the heavy chain variable region

[0240] (i) comprising or consisting of 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 any one of SEQ ID NO: 1, 5, 7, 9, 13, 16, 20, 22-28, 129, 130, 131; or

[0241] (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO: 1, 5, 7, 9, 13, 16, 20, 22-28, 129, 130, 131; or

[0242] (iii) An amino acid sequence having 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 shown in any one of SEQ ID NO: 1, 5, 7, 9, 13, 16, 20, 22-28, 129, 130, or 131, preferably, the amino acid alterations do not occur in the CDR region.

[0243] In some embodiments, the anti-FOLR1 VHH antibody of the present invention comprises or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO: 1, 5, 7, 9, 13, 16, 20, 22-28, 129, 130, 131.

[0244] In some embodiments, the anti-FOLR1 VHH antibody of the present invention is a fully human antibody.

[0245] In some embodiments, the anti-FOLR1 VHH antibody of the present invention can be modified, for example by performing a post-translational modification (PTM) removal mutation; or by performing a hydrophilic modification, the hydrophilic modification including, for example, the following steps

[0246] (i) Analyze the hydrophilicity and hydrophobicity of the amino acids on the antibody molecular backbone.

[0247] (ii) Select hydrophilic amino acids to replace hydrophobic amino acids for mutant combination design, analyze affinity stability, and then select a reasonable combination;

[0248] (iii) Optional detection of the hydrophilicity of the modified antibody; and

[0249] (iv) Optional sequencing of VHH antibodies.

[0250] In some embodiments, the antiFOLR1 VHH antibody of the present invention may simultaneously contain mutations for PTM removal and hydrophilicity modification.

[0251] In another aspect of the invention, the invention also provides a heavy chain antibody comprising the heavy chain variable region of the anti-FOLR1 VHH antibody of the invention.

[0252] In some embodiments, the anti-FOLR1 single-domain antibody or VHH (e.g., camel-derived VHH or its humanized form) of the present invention can be linked to a constant region or a portion thereof, such as the Fc region, of a human antibody to produce a heavy chain antibody comprising a VHH-constant region, VHH-CH1-Fc, or VHH-Fc. In one embodiment, the heavy chain antibody comprises the VHH antibody of the present invention and an Fc region located at its C-terminus.

[0253] In some embodiments, the anti-FOLR1 heavy chain antibody of the present invention comprises VHH as defined herein. FOLR1 Or the heavy chain variable region therein, and the heavy chain constant region or the Fc region of the heavy chain constant region. In some embodiments, in VHH FOLR1 Or its heavy chain variable region is directly or via a joint connected to its heavy chain constant region or Fc region, such as an Fc region containing a complete or partial hinge region. Preferably, the Fc region contains a complete hinge region and contains a 220S transition. In some embodiments, when the Fc region contains a complete hinge region, such as the complete hinge region shown in SEQ ID NO:139 or the complete hinge region shown in SEQ ID NO:141, the VHH FOLR1 It is directly connected to the Fc area.

[0254] In one embodiment, the heavy chain antibody comprises the Fc portion from a camel (e.g., an alpaca). In one embodiment, the heavy chain antibody is generated and isolated by immunizing the camel, such as an alpaca. Various methods are known in the art for immunizing camel animals and isolating VHH antibodies or heavy chain antibodies against the target antigen.

[0255] In some embodiments, the heavy chain antibody includes a constant region derived from human or non-human primate (e.g., cynomolgus monkey) antibodies, such as a constant region derived from human IgG1, human IgG2, human IgG3, or human IgG4.

[0256] In some embodiments, the heavy chain antibody comprises an Fc region derived from a human or non-human primate (e.g., a cynomolgus monkey). In yet another embodiment, the heavy chain antibody comprises a human IgG Fc region, such as a human IgG1, human IgG2, human IgG3, or human IgG4 Fc region, preferably a human IgG1 or human IgG4 Fc region.

[0257] In one embodiment, the heavy chain antibody that specifically binds to FOLR1 according to the present invention can dimerize with another polypeptide chain (e.g., another heavy chain antibody, the same or different) containing the Fc region via the Fc region. Therefore, in one embodiment, the present invention also provides homologous or heterologous multimeric proteins comprising the heavy chain antibody of the present invention. In a preferred embodiment, the protein preferably comprises a heavy chain antibody formed by pairing two identical heavy chain antibody chains.

[0258] The Fc region or its mutations described in this context are applicable to the heavy chain antibodies of this invention that specifically bind to FOLR1.

[0259] In some embodiments, the Fc region is derived from IgG1, which comprises or consists of an amino acid sequence shown in any one of SEQ ID NO: 122, 123, 124, 142-145, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences shown in SEQ ID NO: 122, 123, 124, 142-145.

[0260] In some embodiments, the Fc region is derived from IgG4, which comprises or consists of the amino acid sequence shown in SEQ ID NO:128 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:128.

[0261] In some embodiments, the single-domain antibody VHH that specifically binds to FOLR1 of the present invention, or a heavy-chain antibody containing it, such as VHH-Fc, has one or more of the following properties:

[0262] (i) The antibody binds to FOLR1-positive cells, for example, exhibiting pH-dependent binding activity, such as stronger binding at acidic pH (≤pH7, ≤pH6.9, ≤pH6.8, ≤pH6.7, ≤pH6.6 or ≤pH6.5, for example, approximately pH6.5) than at neutral or alkaline pH (≥pH7, ≥pH7.1, ≥pH7.2, ≥pH7.3 or ≥pH7.4, for example, approximately pH7.4); preferably, the FOLR1 antibody exhibits stronger binding activity to FOLR1-positive cells in the tumor microenvironment than in the normal tissue environment.

[0263] (ii) does not bind to the same epitope as Farletuzumab or belongs to the same epitope group; and / or

[0264] (iii) It has significant hydrophilicity, for example, variants containing hydrophilic modification mutations have improved hydrophilicity.

[0265] III. Other anti-FOLR1 antibodies

[0266] In a third aspect, the present invention relates to an antigen-binding molecule, preferably an antibody or antigen-binding fragment thereof that specifically binds to FOLR1, which has a reduced risk of immunogenicity compared to Farletuzumab antibody.

[0267] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to FOLR1 is an antibody or antigen-binding fragment thereof with an immunogenicity-reducing modification on the light chain variable region of a Farletuzumab antibody while maintaining the affinity of the Farletuzumab antibody. In some embodiments, the immunogenicity-reducing modification on the light chain variable region is on LCDR1, LCDR2, and / or LCDR3, for example, the immunogenicity-reducing modification on the light chain variable region is on LCDR2.

[0268] In some embodiments, the antibody that specifically binds to FOLR1 or its antigen-binding fragment comprises HCDR1, HCDR2, and HCDR3, as well as LCDR1, LCDR2, and LCDR3, wherein

[0269] The HCDR1, HCDR2, and HCDR3 are those contained in VH as shown in SEQ ID NO:73, and the LCDR1, LCDR2, and LCDR3 are those contained in VL as shown in SEQ ID NO:77, 81, 83, 85, 86, 88, 90, or 91; or

[0270] The HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:74; the HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:75; the HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:76; the LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:78; the LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:79, 82, 84, 87, 89, 92, 107, or 132; and the LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:80.

[0271] In some embodiments, the antibody that specifically binds to FOLR1 or its antigen-binding fragment comprises a light chain variable region (VL), wherein the light chain variable region comprises 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: 77, 81, 83, 85, 86, 88, 90, or 91, or an amino acid sequence selected from SEQ ID NO: 77, 81, 83, 85, 86, 88, 90, or 91, or is composed of said sequences.

[0272] In some embodiments, the antibody that specifically binds to FOLR1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:73 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence. The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:77, 81, 83, 85, 86, 88, 90, or 91 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0273] In some embodiments, the antibody that specifically binds to FOLR1 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein

[0274] The light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:77, 81, 83, 85, 86, 88, 90 or 91, and the heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:73.

[0275] In some embodiments, the antibody or antigen-binding fragment that specifically binds to FOLR1 includes an Fc region. The Fc region or mutations thereof, as defined in this context, apply to the antibody or antigen-binding fragment that specifically binds to FOLR1. In some embodiments, the antibody or antigen-binding fragment that specifically binds to FOLR1 includes a heavy chain constant region derived from the constant regions of (human) IgG1, IgG2, IgG3, or IgG4, such as the constant regions of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the heavy chain constant region or Fc region includes a mutation that reduces binding to the Fcγ receptor, such as the L234A / L235A mutation.

[0276] In some embodiments, the antibody that specifically binds to FOLR1 or its antigen-binding fragment comprises a light chain constant region, which is a lambda or kappa light chain constant region, such as a human lambda or kappa light chain constant region. Preferably, the light chain constant region is a kappa light chain constant region, which comprises the amino acid sequence shown in SEQ ID NO:127, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:127, or an amino acid sequence having 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 conserved amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:127, or is composed of said amino acid sequence.

[0277] In some embodiments, the FOLR1-specific antibody or its antigen-binding fragment comprises a heavy chain, which includes, or is composed of, the heavy chain variable region and heavy chain constant region of the FOLR1 antibody or its antigen-binding fragment described herein or in this section. In some embodiments, the FOLR1-specific antibody or its antigen-binding fragment comprises a light chain, which includes, or is composed of, the light chain variable region and light chain constant region of the FOLR1 antibody or its antigen-binding fragment described herein. In some embodiments, the FOLR1-specific antibody or its antigen-binding fragment comprises a heavy chain and a light chain, for example, two heavy chains and two light chains, such as two heavy chains and two light chains, wherein the heavy chain includes, or is composed of, the heavy chain variable region and heavy chain constant region of the FOLR1 antibody or its antigen-binding fragment described herein, and the light chain includes, or is composed of, the light chain variable region and light chain constant region of the FOLR1 antibody or its antigen-binding fragment described herein.

[0278] In some embodiments, the antibody that specifically binds to FOLR1 is a monoclonal antibody. In some embodiments, the antibody that specifically binds to FOLR1 is a humanized antibody or a chimeric antibody. In some embodiments, the antigen-binding fragment is an antibody fragment selected from: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, dAb (domain antibody), bivalent antibody, or linear antibody.

[0279] In some implementations, the antibody that specifically binds to FOLR1 is a full-length antibody.

[0280] In some implementations, the antibody that specifically binds to FOLR1 is a multispecific antibody, such as a bispecific antibody.

[0281] In some embodiments, the antigen-binding fragment that specifically binds to FOLR1 is Fab (also referred to herein as Fab). FOLR1 ).

[0282] In some embodiments, the Fab comprises two polypeptide chains containing antibody VH, CH1, VL, and CL domains, wherein VH pairs with VL and CH1 pairs with CL to form an antigen-binding region. In some embodiments, in the Fab, one chain contains VH and CH1 from the N-terminus to the C-terminus (i.e., VH-CH1) or consists of VH and CH1, and the other chain contains VL and CL from the N-terminus to the C-terminus (i.e., VL-CL) or consists of VL and CL. In some embodiments, in the multispecific antibody of the present invention, the Fab can be linked to the N-terminus of the Fc region via the C-terminus of the chain containing VH. Preferably, the Fab comprises a VH-CH1 chain and a VL-CL chain, and can be linked to the Fc region via the C-terminus of the CH1 of the VH-CH1 chain. In some embodiments, the link is a direct link or a linker link. In this document, the Fab chain containing VH-CH1 is also referred to as the Fab heavy chain, and the Fab chain containing VL-CL is also referred to as the Fab light chain. In some embodiments, CH1 is CH1 derived from (human) IgG1, IgG2, IgG3, or IgG4, preferably CH1 derived from IgG1. In some embodiments, CH1

[0283] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:135;

[0284] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:135; or

[0285] (iii) An amino acid sequence comprising or consisting of 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:135.

[0286] In some embodiments, the CL is a Kappa light chain constant region or a Lambda light chain constant region. In some embodiments, the CL is a Kappa light chain constant region. In some embodiments, the CL...

[0287] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:127;

[0288] (ii) Containing or consisting of the amino acid sequence of SEQ ID NO:127; or

[0289] (iii) An amino acid sequence comprising or consisting of 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:127.

[0290] IV. The multispecific antibody of the present invention

[0291] In a fourth aspect, the antigen-binding molecule of the present invention is a multispecific antibody that specifically binds to FOLR1 and MSLN, wherein the antibody comprises at least one antigen-binding domain specifically binding to FOLR1 and at least one antigen-binding domain specifically binding to MSLN. The multispecific antibody of the present invention can also be a multispecific antibody specifically binding to more than one epitope of FOLR1, such as a bispecific antibody, wherein the antibody comprises at least one antigen-binding domain specifically binding to one epitope of FOLR1 and at least one antigen-binding domain specifically binding to another epitope of FOLR1. In some aspects, to increase the half-life of the antibody of the present invention in animal circulation, the antibody of the present invention further comprises other domains, such as immunoglobulin Fc regions, for antibody dimerization and / or half-life extension. The antibody of the present invention can take any suitable form, such as an IgG-like multispecific antibody, which comprises two or more peptide chains, wherein domains located on the same chain are linked by a linker or directly as needed.

[0292] In some embodiments, the present invention relates to a multispecific antibody comprising

[0293] (i) one or more antigen-binding domains that specifically bind to FOLR1; and optionally

[0294] (ii) One or more antigen-binding domains that specifically bind to MSLN.

[0295] In some embodiments, the multispecific antibody comprises two antigen-binding domains that specifically bind to FOLR1, wherein the two antigen-binding domains specifically bind to different epitopes of FOLR1.

[0296] In some embodiments, the valence (i.e., the total number of antigen-binding domains) of the multispecific antibody according to the invention is 2-6.

[0297] In some embodiments, the multispecific antibody comprises two antigen-binding domains that specifically bind to FOLR1, such as Fab, and two antigen-binding domains that specifically bind to MSLN, such as VHH. Preferably, the two antigen-binding domains that specifically bind to FOLR1 are the same, and / or the two antigen-binding domains that specifically bind to MSLN are the same.

[0298] In some embodiments, the multispecific antibody comprises one or two antigen-binding domains, such as Fab, that specifically bind to a first epitope of FOLR1, one or two antigen-binding domains, such as VHH, that specifically bind to a second epitope of FOLR1, and one or two antigen-binding domains, such as VHH, that specifically bind to an MSLN. Preferably, the antigen-binding domains specifically binding to the first epitope of FOLR1 are identical, the antigen-binding domains specifically binding to the second epitope of FOLR1 are identical, and / or the antigen-binding domains specifically binding to an MSLN are identical. In some embodiments, the multispecific antibody comprises two antigen-binding domains, such as Fab, that specifically bind to a first epitope of FOLR1, two antigen-binding domains, such as VHH, and two antigen-binding domains, such as VHH, that specifically bind to an MSLN. Preferably, the antigen-binding domains specifically binding to the first epitope of FOLR1 are identical, the antigen-binding domains specifically binding to the second epitope of FOLR1 are identical, and / or the antigen-binding domains specifically binding to an MSLN are identical. In some embodiments, the multispecific antibody comprises one antigen-binding domain, such as Fab, that specifically binds to a first epitope of FOLR1, one antigen-binding domain, such as VHH, that specifically binds to a second epitope of FOLR1, and one antigen-binding domain, such as VHH, that specifically binds to an MSLN. In some embodiments, the multispecific antibody comprises two antigen-binding domains, such as Fab, that specifically bind to a first epitope of FOLR1, one antigen-binding domain, such as VHH, that specifically binds to a second epitope of FOLR1, and one antigen-binding domain, such as VHH, that specifically binds to an MSLN. Preferably, the antigen-binding domains that specifically bind to the first epitope of FOLR1 are identical.

[0299] In some embodiments, the multispecific antibody comprises one or two antigen-binding domains, such as Fab, that specifically bind to a first epitope of FOLR1, and one or two antigen-binding domains, such as VHH, that specifically bind to a second epitope of FOLR1. Preferably, the two antigen-binding domains of the first epitopes of FOLR1 are identical, and / or the antigen-binding structures of the two second epitopes of FOLR1 are identical. In some embodiments, the multispecific antibody comprises one antigen-binding domain, such as Fab, that specifically binds to a first epitope of FOLR1, and two antigen-binding domains, such as VHH, that specifically bind to a second epitope of FOLR1. Preferably, the two antigen-binding domains of the first epitopes of FOLR1 are identical, and / or the antigen-binding structures of the two second epitopes of FOLR1 are identical. In some embodiments, the multispecific antibody comprises one antigen-binding domain, such as Fab, that specifically binds to a first epitope of FOLR1, and one or two antigen-binding domains, such as VHH, that specifically bind to a second epitope of FOLR1. Preferably, the two antigen-binding structures of the second epitopes of FOLR1 are identical.

[0300] In some embodiments, the multispecific antibody comprises one or two antigen-binding domains that specifically bind to FOLR1, such as VHH, and one or two antigen-binding domains that specifically bind to MSLN, such as VHH. Preferably, the two antigen-binding domains that specifically bind to FOLR1 are the same, and / or the two antigen-binding domains that specifically bind to MSLN are the same.

[0301] In some embodiments, the multispecific antibody of the present invention is a bispecific antibody that specifically binds to an epitope of FOLR1 and specifically binds to MSLN.

[0302] In some embodiments, the multispecific antibody of the present invention is a trispecific antibody that specifically binds to two epitopes of FOLR1 and specifically binds to MSLN.

[0303] In some embodiments, the multispecific antibody of the present invention is a bispecific antibody that specifically binds to two different epitopes of FOLR1.

[0304] The components of the multispecific antibody of the present invention are described in detail below. Those skilled in the art will understand that, unless the context clearly indicates otherwise, any combination of any technical features of these components is within the scope of this invention. Furthermore, those skilled in the art will understand that, unless the context clearly indicates otherwise, the antibody of the present invention (including any form of antibody) may contain any such combination of features.

[0305] Specifically binds to the antigen-binding domain of FOLR1

[0306] In some embodiments of the multispecific antibody according to the present invention, preferably, the antigen-binding domain that specifically binds to FOLR1 comprises or is composed of an anti-FOLR1 antibody or its antigen-binding fragment, as long as it can specifically bind to FOLR1, including but not limited to, for example, full-length antibodies that specifically bind to FOLR1, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibodies, VHH or heavy chain antibodies, etc.

[0307] In some implementations, the antigen-binding domain that specifically binds to FOLR1 is the anti-FOLR1 VHH (VHH) as defined herein (e.g., in Section II, “FOLR1 Single-Domain Antibodies and Their Heavy Chain Antibodies”). FOLR1 ).

[0308] In some implementations, the antigen-binding domain that specifically binds to FOLR1 is an antigen-binding fragment of Farletuzumab or an anti-FOLR1 antibody as defined herein (e.g., in Section III, “Other Anti-FOLR1 Single-Domain Antibodies”), such as Fab (Fab FOLR1 ).

[0309] In some implementations, antigen-binding domains of FOLR1, such as Fab, are specifically bound. FOLR1 It contains the heavy chain variable region VH and the light chain variable region VL of Farletuzumab. In some embodiments, it specifically binds to the antigen-binding domain of FOLR1, such as Fab. FOLR1 Six CDRs containing the antigen-binding region of Farletuzumab. In some embodiments, antigen-binding domains of FOLR1, such as Fab, are specifically bound. FOLR1 VH or VL containing Farletuzumab, or VH and VL containing Farletuzumab.

[0310] In some implementations, antigen-binding domains of FOLR1, such as Fab, are specifically bound. FOLR1 It comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region VH contains or is composed of the amino acid sequence shown in SEQ ID NO:73, and the light chain variable region VL contains or is composed of the amino acid sequence shown in SEQ ID NO:140.

[0311] In some implementations, antigen-binding domains of FOLR1, such as Fab, are specifically bound. FOLR1It includes heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, among which

[0312] The HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:74;

[0313] The HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:75;

[0314] The HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:76;

[0315] The LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:78;

[0316] The LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:133; and

[0317] The LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:80.

[0318] Antigen-binding domain that specifically binds to MSLN

[0319] In some embodiments of the multispecific antibody according to the present invention, preferably, the antigen-binding domain that specifically binds to MSLN comprises or is composed of an anti-MSLN antibody or its antigen-binding fragment, as long as it can specifically bind to MSLN, including but not limited to, for example, full-length antibodies that specifically bind to MSLN, single-chain Fv, Fab, Fab', (Fab)2, single-domain antibodies, VHH or heavy chain antibodies, etc.

[0320] In some implementations, the antigen-binding domain that specifically binds to MSLN is the anti-MSLN VHH (VHH) as defined herein (e.g., in Section I, “MSLN Single-Domain Antibodies and Their Heavy Chain Antibodies”). MSLN ).

[0321] Immunoglobulin Fc region

[0322] In some embodiments, multispecific antibodies may comprise immunoglobulin Fc regions. The immunoglobulin Fc region can be any immunoglobulin Fc region. The Fc region is the C-terminal constant domain of an immunoglobulin that interacts with cell surface Fc receptors and some proteins of the complement system. Immunoglobulin Fc regions typically contain two or three heavy-chain constant domains (designated CH2, CH3, and CH4) and a hinge region, and are typically present in a dimerized form. The two chains in a dimerized Fc region can be linked by disulfide bonds within the hinge region. In some embodiments, Fc regions from immunoglobulin isotypes IgG1, IgG2, and IgG4 are capable of binding to FcRn receptors and undergoing FcRn-mediated recycling to provide a long circulating half-life. The interaction site between IgG and FcRn has been identified in the Fc region of the covered CH2 and CH3 domains.

[0323] The immunoglobulin Fc region used in the multispecific antibody of the present invention can be derived from any immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region is preferably derived from IgG1, IgG2, or IgG4, or their subtypes. Preferably, the immunoglobulin Fc region comprises an Fc region sequence derived from humans. In some embodiments, the immunoglobulin Fc region comprises at least an immunoglobulin CH2 domain and a CH3 domain. In some embodiments, the immunoglobulin Fc region further comprises a complete hinge region or a partial hinge region. In some embodiments, the immunoglobulin Fc region, from the N-terminus to the C-terminus, comprises a complete hinge region or a partial hinge region of immunoglobulin, a CH2 domain, and a CH3 domain, or is composed of the latter.

[0324] Immunoglobulin Fc regions can fuse to other domains (i.e., FOLR1 or MSLN binding domains, such as VHH). FOLR1 or VHH MSLN or Fab FOLR1 Immunoglobulin Fc is located at the C or N terminus of either the heavy chain variable region or the light chain variable region. It can fuse to other domains via linkers or directly to other domains.

[0325] In some cases, immunoglobulin Fc regions containing hinge region sequences are preferred, which can, for example, promote the dimerization of antibody polypeptide chains and / or provide cysteine ​​residues for coupling with other active molecules. Such hinge sequences may substantially or partially correspond to the hinge regions of IgG1, IgG2, IgG3, or IgG4. For example, the hinge region sequence may comprise all or part of a core hinge region and all or part of a lower hinge region. The core hinge region has the amino acid sequence CPPC in IgG1, IgG2, and IgG3, and the CPSC sequence in IgG4. Preferably, the hinge region contains at least one disulfide bond connecting two Fc chains. In some embodiments, the hinge region sequence comprises the hinge region sequence E216 to P230 from IgG1 or the hinge region sequence D221 to P230 (according to EU numbering), or a corresponding hinge region sequence from another immunoglobulin isotype. In this document, when referring to a complete hinge region, it generally refers to the hinge region sequence corresponding to E216 to P230 of IgG1 (according to EU designations), for example, the complete hinge region of the IgG1 Fc region is the amino acid sequence shown in SEQ ID NO: 139. In this document, when referring to a partial hinge region, it generally refers to the hinge region sequence corresponding to D221 to P230 of IgG1 (according to EU designations), for example, the partial hinge region of the IgG1 Fc region is the amino acid sequence shown in SEQ ID NO: 138. In some embodiments, the complete hinge region may contain a C220S mutation. In some embodiments, the amino acid sequence of the complete hinge region containing the C220S mutation is as shown in SEQ ID NO: 141. In some embodiments, the Fc region contains the hinge region shown in SEQ ID NO: 138, 139, or 141.

[0326] 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 is an Fc region containing a complete hinge region. In some embodiments, the Fc region is an Fc region containing a complete hinge region with a C220S mutation. In some embodiments, the Fc region is an Fc region containing a partial hinge region.

[0327] It should be understood that the immunoglobulin Fc region of the antigen-binding molecule of the present invention may, as needed, include a complete hinge region, a partial hinge region, or no hinge region at its N-terminus. In some embodiments, when the Fc region is attached to the C-terminus of the Fab heavy chain at its N-terminus, since the Fab heavy chain already includes the portion of the hinge region corresponding to E216-C220 (EU number) of the IgG1 Fc region at its C-terminus of CH1, such as the amino acid sequence shown in SEQ ID NO:137, the Fc region does not need to include the aforementioned portion. Therefore, in some embodiments, when the Fc region is attached to the C-terminus of the Fab heavy chain at its N-terminus, the Fc region generally includes a partial hinge region, that is, the hinge region included at its N-terminus corresponds to the hinge region of the IgG1 Fc region D221 to P230 (EU number), such as the amino acid sequence shown in SEQ ID NO:138. In some embodiments, when the Fc region is connected at its N-terminus to the C-terminus or N-terminus of VHH, it may generally contain a complete hinge region, i.e., the hinge region contained at its N-terminus corresponds to the hinge region of IgG1 Fc region E216-P230 (EU number), such as the amino acid sequence shown in SEQ ID NO:139; particularly, a complete hinge region containing the C220S mutation, such as the amino acid sequence shown in SEQ ID NO:141.

[0328] In some embodiments, the Fc region comprises an amino acid sequence shown in any one of SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:128 or 142-145, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with said amino acid sequence, or is composed of said amino acid sequence.

[0329] The immunoglobulin Fc region of the multispecific antibody used in this invention can be the natural Fc region sequence. Alternatively, the Fc region can contain mutations relative to the natural Fc sequence. Mutations include substitutions, insertions, and / or deletions. Such mutations can be made for the purpose of introducing desired therapeutic properties.

[0330] For example, to promote heterodimerization, a Knob-into-Hole (KiH) mutation can be introduced into the CH3 domain. 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). In this case, one Fc strand is designed to contain a large protruding residue (i.e., Knob), while the other Fc strand is designed to contain a complementary pocket (i.e., Hole). Suitable locations for the KiH mutation are known in the art. Exemplary KiH mutations include, but are not limited to, combinations of Knob mutation T366W and Hole mutations T366S, L368A, and Y407V; or combinations of Knob mutation T366Y and Hole mutation Y407T. In some embodiments, the Fc regions may also contain cysteine ​​residue substitutions to obtain non-natural disulfide bond linkages. In some embodiments, one Fc region contains S354C or E356C, and the other Fc region contains Y349C.

[0331] In one specific implementation, the Fc region containing the Knob mutation contains the amino acid substitution T366W, and the Fc region containing the hole mutation contains the amino acid substitutions T366S, L368A, and Y407V (numbered according to the EU index).

[0332] In one specific implementation, the Fc region containing the Knob mutation contains amino acid substitutions S354C and T366W, and the Fc region containing the hole mutation contains amino acid substitutions Y349C, T366S, L368A and Y407V (numbered according to the EU index).

[0333] When the multispecific antibody of the present invention contains an asymmetric double-stranded structure, the Fc region preferably contains a KiH mutation that promotes the correct heterodimerization of the antibody polypeptide chain.

[0334] Furthermore, depending on the specific application of the antibody or antibody-based molecule, the Fc region may also contain mutations that alter effector function. For example, where effector function is not required, the Fc region may contain mutations that reduce or eliminate effector function. In some cases (e.g., when the antibody of the present invention is used as an ADC carrier), preferably, the Fc region contains mutations that reduce or eliminate the interaction between the Fc region and the Fcγ receptor, such as the LALA mutation (L234A / L235A) where lysine (L) at positions 234 and 235 of the Fc region is replaced with alanine (A), to reduce Fcγ receptor-mediated off-target cytotoxicity. Alternatively or additionally, mutations may be introduced into the Fc region to increase binding to FcRn and / or remove protease sites, and / or introduce amino acid modifications that can be used for conjugation to active molecules. Alternatively or additionally, the Fc region may be mutated for antibody production purposes, for example, by removing or replacing amino acids that may undergo post-translational modifications (e.g., glycosylation), to provide improved drugability and developability of the therapeutic antibody.

[0335] Therefore, in one specific embodiment, the multispecific antibody of the present invention comprises an Fc region containing a mutation that reduces or eliminates the Fc region and the Fcγ receptor, such as the LALA mutation. For example, the Fc region containing the LALA mutation 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:124, for example, containing or consisting of the amino acid sequence shown in SEQ ID NO:124. In some embodiments, the Fc region containing the LALA mutation contains a complete hinge region at its N-terminus or contains a complete hinge region having C220S. For example, the Fc region from the N-terminus to the C-terminus contains the amino acid sequence shown in SEQ ID NO:136 or SEQ ID NO:137 and the amino acid sequence shown in SEQ ID NO:124, and is composed of the amino acid sequence shown in SEQ ID NO:136 or SEQ ID NO:137 and the amino acid sequence shown in SEQ ID NO:124.

[0336] Therefore, in one specific embodiment, the multispecific antibody of the present invention comprises two Fc regions heterodimerized, one Fc-region polypeptide comprising the mutations S354C and T366W, and optionally the LALA mutation, and the other Fc-region polypeptide comprising the mutations Y349C, T366S, L368A and Y407V, and optionally the LALA mutation.

[0337] Therefore, in one specific embodiment, the multispecific antibody of the present invention comprises two heterodimerized Fc regions, wherein the Fc regions respectively contain KiH mutations and mutations such as LALA mutations that reduce or eliminate the Fc region and the Fcγ receptor.

[0338] 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%, and 99% identity with the amino acid sequence shown in SEQ ID NO:125 and comprises the mutations S354C and T366W, and optionally the L234A / L235A mutation, and the other Fc region comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with the amino acid sequence shown in SEQ ID NO:126 and comprises the mutations Y349C, T366S, L368A, and Y407V, and optionally the L234A / L235A mutation; optionally, the Fc region further comprises the amino acid sequence shown in SEQ ID NO:136 or SEQ ID NO:137 at its N-terminus.

[0339] Therefore, in one specific embodiment, the multispecific antibody of the present invention comprises two heterodimerized Fc regions, wherein one Fc region polypeptide comprises or is composed of the amino acid sequence shown in SEQ ID NO:125, and the other Fc region comprises or is composed of the amino acid sequence shown in SEQ ID NO:126; optionally, the Fc region further comprises the amino acid sequence shown in SEQ ID NO:136 or SEQ ID NO:137 at its N-terminus.

[0340] connector

[0341] In the multispecific antibody according to the invention, antibody components (e.g., antigen-binding domain and Fc region) can be connected by adapters.

[0342] There are no specific limitations on the linkers that can be used in the antibodies of this invention. Linker sequences are generally flexible. They can consist primarily of amino acids with large side chains, such as glycine, alanine, and serine, which do not have the large side chains that might limit flexibility. Alternatively, they can consist of sequences derived from the hinge region of immunoglobulins. Depending on the attachment site and the component to be attached, those skilled in the art can readily determine the available linker sequence or optimal length.

[0343] Suitable linker lengths can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, or longer. In some cases, the linker sequence length can be shorter, for example less than about 20 or 15 amino acids, such as 2–15 amino acids or 5–10 amino acids.

[0344] Suitable connector sequences include, but are not limited to, G4S; (G4S)2; (G4S)3; GGGSG; GGSGG; GSGGG; GSGGGP; GGEPS; GGEGGGP and GGEGGGSEGGGS; and (G4S)n, where n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5; TS(G4S)n, where n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5; G(G4S)n, where n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5; (G4)n, where n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5; (GRPGS)n, where n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5. The linkers that can be used for the antibody molecules of this invention can also be, for example, but not limited to, the following amino acid sequences: (G3S)2, (G4S)2, (G3S)3, (G4S)3, (G3S)4, (G4S)4, (G3S)5, (G4S)5, (G3S)6, (G4S), GGG, DGGGS, TGEKP, GGRR, EGKSSGSGSESKVD, KESGSVSSEQLAQFRSLD, GGRRGGGS, LRQRDGERP, LRQKDGGGSERP, and GSTGSGSGKPGSGEGSTKG. Alternatively, suitable flexible linker peptides can be rationally designed by simulating the three-dimensional structure of proteins and peptides using computer programs or by using phage display methods.

[0345] In some embodiments, the linker used in the antibody of the present invention is a flexible linker peptide of 5-50 amino acids, preferably comprising a linker peptide with glycine (G) and / or serine (S) and / or threonine residues (T). In one embodiment, the linker has a length of 5-50 amino acids, for example, 5, 10, 15, 20, 25, or 30 amino acids, or an amino acid length falling between any two integers. In some embodiments, the linker comprises an amino acid sequence (G4S). n , where n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6 or 7.

[0346] In some preferred embodiments, in the multispecific antibody according to the invention, the linker for connecting the antigen-binding domain and the half-life extension domain (if present) comprises the amino acid sequence G4S or (G4S)2 or (G4S)3.

[0347] Structure and examples of multispecific antibodies

[0348] The multispecific antibody according to the present invention can take any suitable form, for example, it may contain two, three, or four chains. A bispecific antibody with a "2+2" symmetrical structure.

[0349] In some embodiments, the multispecific antibody is a bispecific antibody with a "2+2" symmetrical structure, such as any of the structures shown in the Format schematic diagram in Figure 68. In some embodiments, the bispecific antibody comprises two Fab atoms as described herein. FOLR1 And 2 VHHs described in this article MSLN In some embodiments, the bispecific antibody is a quadrivalent bispecific antibody.

[0350] In some embodiments, the multispecific antibody is a bispecific antibody that specifically binds to FOLR1 and MSLN, and contains

[0351] The first polypeptide chain, which comprises or is composed of the following: Fab FOLR1 Light chain;

[0352] The second polypeptide chain, from the N-terminus to the C-terminus, comprises or consists of the following: Fab FOLR1 Heavy chain -Fc-VHH MSLN or VHH MSLN -Fab FOLR1 Heavy chain -Fc;

[0353] Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 and the light chain constant region CL, or composed of it,

[0354] The symbol "-" indicates a linker or direct link, preferably a linker of 5-15 amino acids in length, such as a linker peptide; preferably, the Fc region includes a partial hinge region and is directly linked to the Fab heavy chain. In some preferred embodiments, the bispecific antibody structure is shown in Figure 68A or Figure 68B, where the various domains or linkers are as defined in this context.

[0355] In some embodiments, the multispecific antibody is a bispecific antibody that specifically binds to FOLR1 and MSLN, and contains

[0356] The first polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of the following: Fab FOLR1 Light Chain - VHH MSLN or VHH MSLN -Fab FOLR1 Light chain;

[0357] The second polypeptide chain, from the N-terminus to the C-terminus, comprises or consists of the following: Fab FOLR1Heavy chain -Fc;

[0358] Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 and the light chain constant region CL, or composed of it,

[0359] The symbol "-" indicates a linker or direct link, preferably a linker of 5-15 amino acids in length, such as a linker peptide; preferably, the Fc region includes a partial hinge region and is directly linked to the Fab heavy chain. In some preferred embodiments, the bispecific antibody structure is shown in Figure 68C or Figure 68D, where the various domains or linkers are as defined in this context.

[0360] In some embodiments, the multispecific antibody comprises two first polypeptide chains and two second polypeptide chains or is composed of two first polypeptide chains and two second polypeptide chains. Preferably, the two first polypeptide chains are identical and the two second polypeptide chains are identical.

[0361] In the above-mentioned multispecific antibodies, preferably, each antigen-binding domain on the polypeptide chain (e.g., VHH) MSLN Structural domains and Fc regions or VHH MSLN Domains and Fab FOLR1 Heavy chain or Fab FOLR1 The light chains are connected by a linker. Preferably, the linker has a length of 5-15 amino acids. More preferably, when Fab... FOLR1 When the C-end of the heavy chain connects to the N-end of the Fc region, the Fc region includes a portion of the hinge region and is connected to the Fab. FOLR1 Heavy chains are directly connected, and / or when VHH MSLN Domains and Fab FOLR1 N-terminus or Fab of heavy chain FOLR1 When the N-terminus, C-terminus, or C-terminus of the Fc region of the light chain is connected, the linker preferably contains a G4S amino acid sequence, a (G4S)2 amino acid sequence, or a (G4S)3 amino acid sequence.

[0362] Two immunoglobulin Fc regions can associate to form a homodimer through dimerization. In some embodiments, the Fc region contains an amino acid sequence derived from human IgG1 or IgG4. In some embodiments, the Fc region contains a mutation that reduces or eliminates Fcγ receptor binding, such as the LALA mutation.

[0363] In some embodiments, the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, for example, two identical first polypeptide chains and two identical second polypeptide chains, or composed of two identical first polypeptide chains and two identical second polypeptide chains, wherein the first polypeptide chain and the second polypeptide chain are selected from the group consisting of:

[0364] (i) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93 and 94, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences,

[0365] (ii) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93 and 95, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences,

[0366] (iii) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 96 and 97, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences.

[0367] (iv) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 98 and 97, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences,

[0368] (v) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93 and 99, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences, or

[0369] (vi) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93 and 100, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences.

[0370] Bispecific antibodies that specifically bind to the FOLR1 biepitaxes

[0371] In some embodiments, the multispecific antibody is a bispecific antibody that specifically binds to two epitopes of FOLR1, such as any of the structures shown in the Format schematic diagram in Figure 69.

[0372] In some embodiments, the bispecific antibody comprises one or two Fab atoms described herein. FOLR1 and one or two VHHs described herein FOLR1 In some embodiments, the bispecific antibody is a bivalent, trivalent, or quadrivalent bispecific antibody. In some embodiments, the bispecific antibody comprises two Fab groups described herein. FOLR1 And 2 VHHs described in this article FOLR1 Furthermore, the bispecific antibody is a bispecific antibody with a "2+2" symmetrical structure. In some embodiments, the bispecific antibody comprises one Fab described herein. FOLR1 And 2 VHHs described in this article FOLR1 Furthermore, the bispecific antibody is an asymmetric bispecific antibody. In some embodiments, the bispecific antibody comprises one Fab described herein. FOLR1 And 1 VHH as described in this article FOLR1 Furthermore, the bispecific antibody is an asymmetric bispecific antibody.

[0373] In some embodiments, the multispecific antibody is a bispecific antibody that specifically binds to two different epitopes of FOLR1, comprising...

[0374] The first polypeptide chain, which comprises or is composed of the following: Fab FOLR1 Light chain;

[0375] The second polypeptide chain, from the N-terminus to the C-terminus, comprises or consists of the following: Fab FOLR1 Heavy chain -Fc-VHH FOLR1 or VHH FOLR1 -Fab FOLR1 Heavy chain -Fc;

[0376] Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 And the light chain constant region CL, or composed of it;

[0377] Fab FOLR1 With VHH FOLR1 Combining different epitopes on FOLR1;

[0378] The symbol "-" indicates a linker or direct link, preferably a linker of 5-15 amino acids in length, such as a linker peptide; preferably, the Fc region includes a partial hinge region and is directly linked to the Fab heavy chain. In some preferred embodiments, the bispecific antibody structure is shown in Figure 69A or Figure 69B, where the various domains or linkers are as defined in this context.

[0379] In some embodiments, the multispecific antibody comprises two first polypeptide chains and two second polypeptide chains; preferably, the two first polypeptide chains are identical and the two second polypeptide chains are identical. The two immunoglobulin Fc regions can associate to form a homodimer through dimerization. In some embodiments, the Fc region comprises an amino acid sequence derived from human IgG1 or IgG4. In some embodiments, the Fc region contains a mutation that reduces or eliminates Fcγ receptor binding, such as the LALA mutation.

[0380] In some embodiments, the multispecific antibody is a bispecific antibody that specifically binds to two epitopes of FOLR1, comprising...

[0381] The first polypeptide chain, which comprises or is composed of the following: Fab FOLR1 Light chain;

[0382] The second polypeptide chain: from the N-terminus to the C-terminus, it comprises or consists of the following: Fab FOLR1 Heavy chain - first Fc;

[0383] The third polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of the following: VHH FOLR1 -Second Fc or VHH FOLR1 -VHH FOLR1 -Second Fc;

[0384] Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 And the light chain constant region CL, or composed of it;

[0385] Fab FOLR1 With VHH FOLR1 Combining different epitopes on FOLR1;

[0386] The symbol "-" indicates a linker or direct link, preferably a linker of 5-15 amino acids in length, such as a linker peptide; preferably, the first Fc region includes a partial hinge region and is directly linked to the Fab heavy chain, and the second Fc region includes a complete hinge region (preferably the complete hinge region includes a C220S mutation) and is linked to VHH FOLR1 Direct connection. In some preferred embodiments, the bispecific antibody structure is shown in Figure 69C or Figure 69D, where the various domains or linkers are as defined in this context.

[0387] Due to the dimerization effect of the Fc region of immunoglobulins, the second and third polypeptide chains of the aforementioned multispecific antibodies can associate to form heterodimers. Preferably, to promote heterodimerization of the second and third polypeptide chains, Knob-into-hole mutations can be introduced into the first and second Fc regions of the second and third polypeptide chains, for example, introducing Knob mutations such as T366W / T366S, L368A, Y407V mutations into the first Fc region, or introducing Hole mutations such as T366Y / Y407T mutations, and optionally S354C / Y349C, into the second Fc region. Preferably, the Fc region contains an amino acid sequence from human IgG1 or IgG4, and preferably, the Fc region also contains mutations that reduce or eliminate Fcγ receptor binding, such as the LALA mutation.

[0388] In the aforementioned multispecific antibodies, preferably, the antigen-binding domains on the polypeptide chain are connected by linkers, for example, in the VHH... FOLR1 Between structural domains. Preferably, the linker has a length of 5-15 amino acids. Preferably,

[0389] When VHH FOLR1 Domains and Fab FOLR1 When the N-terminus of the heavy chain or the C-terminus of the Fc region are connected, or when two VHHs are connected... FOLR1 When connected, the connector preferably contains a G4S amino acid sequence or a (G4S)2 amino acid sequence or a (G4S)3 amino acid sequence;

[0390] When Fab FOLR1 When the C-end of the heavy chain connects to the N-end of the Fc region, the Fc region includes a portion of the hinge region and is connected to the Fab. FOLR1 Heavy chain direct connection, and / or

[0391] When VHH FOLR1 When the C-terminus of the Fc region is connected to the N-terminus of the Fc region, the Fc region contains a complete hinge region and is connected to the VHH. FOLR1The heavy chain is directly linked, and more preferably, the Fc region contains a C220S mutation. In some embodiments, the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, for example, two identical first polypeptide chains and two identical second polypeptide chains, or composed of two identical first polypeptide chains and two identical second polypeptide chains, wherein the first polypeptide chain and the second polypeptide chain are selected from the group consisting of:

[0392] (i) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93 and 134, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences, or

[0393] (ii) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93 and 101, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences.

[0394] In some embodiments, the bispecific antibody comprises, or is composed of, a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain are selected from the group consisting of:

[0395] (i) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93, 102, and 103, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first, second, and third polypeptide chains composed of said amino acid sequences, or

[0396] (ii) Each of the amino acid sequences represented by SEQ ID NOs: 93, 102 and 104, or the amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with them, or the first, second and third polypeptide chains composed of said amino acid sequences.

[0397] Trispecific antibodies

[0398] In some embodiments, the multispecific antibody is a trispecific antibody that specifically binds to two epitopes of FOLR1 and specifically binds to MSLN, such as any of the structures shown in the Format schematic diagram in Figure 70.

[0399] In some embodiments, the trispecific antibody comprises one or two Fab atoms described herein. FOLR1 and one or two VHHs described herein FOLR1And one or two VHHs described in this article MSLN In some embodiments, the trispecific antibody is a trivalent, quadrivalent, or hexavalent trispecific antibody. In some embodiments, the trispecific antibody comprises two Fab atoms described herein. FOLR1 And 2 VHHs described in this article FOLR1 And 2 VHHs described in this article MSLN Furthermore, the trispecific antibody is a trispecific antibody with a "2+2+2" symmetrical structure. In some embodiments, the trispecific antibody comprises one Fab described herein. FOLR1 1 VHH described in this article FOLR1 And 1 VHH as described in this article MSLN Furthermore, the trispecific antibody is an asymmetric trispecific antibody. In some embodiments, the trispecific antibody comprises two Fab atoms described herein. FOLR1 1 VHH described in this article FOLR1 And 1 VHH as described in this article MSLN Furthermore, the trispecific antibody is an asymmetric trispecific antibody.

[0400] In some embodiments, the multispecific antibody is a trispecific antibody that specifically binds to two different epitopes of FOLR1 and the MSLN, and that contains

[0401] The first polypeptide chain, which comprises or is composed of the following: Fab FOLR1 Light chain;

[0402] The second polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of the following: VHH FOLR1 -Fab FOLR1 Heavy chain -Fc-VHH MSLN Or VHH MSLN -Fab FOLR1 Heavy chain -Fc-VHH FOLR1 ;

[0403] Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 And the light chain constant region CL, or composed of it;

[0404] Fab FOLR1 With VHH FOLR1 Combining different epitopes on FOLR1;

[0405] The symbol "-" indicates a linker or direct link, preferably a linker of 5-15 amino acids in length, such as a linker peptide; preferably, the Fc region includes a partial hinge region and is directly linked to the Fab heavy chain. In some preferred embodiments, the trispecific antibody structure is shown in Figure 70A, where the various domains or linkers are as defined in this context.

[0406] In some embodiments, the multispecific antibody comprises two first polypeptide chains and two second polypeptide chains, or is composed of two first polypeptide chains and two second polypeptide chains; preferably, the two first polypeptide chains are identical and the two second polypeptide chains are identical. The two immunoglobulin Fc regions can associate to form a homodimer through dimerization. In some embodiments, the Fc region contains an amino acid sequence derived from human IgG1 or IgG4. In some embodiments, the Fc region contains a mutation that reduces or eliminates Fcγ receptor binding, such as the LALA mutation.

[0407] In some embodiments, the multispecific antibody is a trispecific antibody that specifically binds to two epitopes of FOLR1 and the MSLN, and comprises or consists of the following chains:

[0408] The first polypeptide chain, which comprises or is composed of the following: Fab FOLR1 Light chain;

[0409] The second polypeptide chain: from the N-terminus to the C-terminus, it comprises or consists of the following: Fab FOLR1 Heavy chain - first Fc;

[0410] The third polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of the following: VHH FOLR1 -VHH MSLN -Second Fc or VHH MSLN -VHH FOLR1 -Second Fc;

[0411] Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 And the light chain constant region CL, or composed of it;

[0412] Fab FOLR1 With VHH FOLR1 Combining different epitopes on FOLR1;

[0413] The symbol "-" indicates a linker or direct link, preferably a linker of 5-15 amino acids in length, such as a linker peptide; preferably, the first Fc region includes a partial hinge region and is directly linked to the Fab heavy chain, and the second Fc region includes a complete hinge region (preferably the complete hinge region includes a C220S mutation) and is linked to VHH FOLR1 or VHH MSLN Direct connection. In some preferred embodiments, the bispecific antibody structure is shown in Figure 70B or Figure 70C, where the various domains or linkers are as defined in this context.

[0414] In some embodiments, the multispecific antibody is a trispecific antibody that specifically binds to two epitopes of FOLR1 and the MSLN, and comprises or consists of the following chains:

[0415] The first polypeptide chain, which comprises or is composed of the following: Fab FOLR1 Light chain;

[0416] The second polypeptide chain: from its N-terminus to its C-terminus, it comprises or consists of the following: VHH FOLR1 -Fab FOLR1 Heavy chain - first Fc;

[0417] The third polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of the following: VHH MSLN -Fab FOLR1 Heavy chain - second Fc; and

[0418] The fourth polypeptide chain, which comprises or is composed of the following: Fab FOLR1 Light chain;

[0419] Preferably, the first polypeptide chain and the fourth polypeptide chain are identical;

[0420] Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 And the light chain constant region CL, or composed of it;

[0421] Fab FOLR1 With VHH FOLR1 Combining different epitopes on FOLR1;

[0422] The symbol "-" indicates a linker or direct link, preferably a linker of 5-15 amino acids in length, such as a linker peptide; preferably, the first and second Fc regions each contain a partial hinge region and are directly linked to the Fab heavy chain. In some preferred embodiments, the trispecific antibody structure is shown in Figure 70D, where the various domains or linkers are as defined in this context.

[0423] Due to the dimerization effect of the Fc region of immunoglobulins, the second and third polypeptide chains of the aforementioned multispecific antibodies can associate to form heterodimers. Preferably, to promote heterodimerization of the second and third polypeptide chains, Knob-into-hole mutations can be introduced into the first and second Fc regions of the second and third polypeptide chains, for example, introducing Knob mutations such as T366W / T366S, L368A, Y407V mutations into the first Fc region, or introducing Hole mutations such as T366Y / Y407T mutations, and optionally S354C / Y349C, into the second Fc region. Preferably, the Fc region contains an amino acid sequence from human IgG1 or IgG4, and preferably, the Fc region also contains mutations that reduce or eliminate Fcγ receptor binding, such as the LALA mutation.

[0424] In the aforementioned multispecific antibodies, preferably, the antigen-binding domains on the polypeptide chain are connected by linkers, for example, between the VHH domains, between the VHH domain and the Fab heavy chain, or between the VHH domain and the C-terminus of the Fc region. Preferably, the linker has a length of 5-15 amino acids.

[0425] When VHH FOLR1 Domains and Fab FOLR1 When the N-terminus of the heavy chain or the C-terminus of the Fc region is connected, or when VHH MSLN Domains and Fab FOLR1 When the N-terminus of the heavy chain or the C-terminus of the Fc region is connected, or VHH FOLR1 With VHH MSLN When connected, the connector preferably contains a G4S amino acid sequence or a (G4S)2 amino acid sequence or a (G4S)3 amino acid sequence;

[0426] When Fab FOLR1 When the C-end of the heavy chain connects to the N-end of the Fc region, the Fc region includes a portion of the hinge region and is connected to the Fab. FOLR1 Heavy chain direct connection, and / or

[0427] When VHH FOLR1 or VHH MSLN When the C-terminus of the Fc region is connected to the N-terminus of the Fc region, the Fc region contains a complete hinge region and is connected to the VHH. FOLR1 or VHHMSLN More preferably, the Fc region contains a C220S mutation. In some embodiments, the multispecific antibody comprises a first polypeptide chain and a second polypeptide chain, for example, two identical first polypeptide chains and two identical second polypeptide chains, or consists of two identical first polypeptide chains and two identical second polypeptide chains, wherein the first polypeptide chain and the second polypeptide chain respectively comprise, or consist of, the amino acid sequences shown in SEQ ID NOs: 93 and 105 having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them.

[0428] In some embodiments, the multispecific antibody comprises, or is composed of, a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain are selected from the group consisting of:

[0429] (i) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93, 102, and 106, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first, second, and third polypeptide chains composed of said amino acid sequences, or

[0430] (ii) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93, 102, and 108, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first, second, and third polypeptide chains composed of said amino acid sequences; or

[0431] (iii) Each of the amino acid sequences represented by SEQ ID NOs: 111, 102 and 112, or the amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with them, or the first, second and third polypeptide chains composed of said amino acid sequences.

[0432] In some embodiments, the multispecific antibody comprises, or is composed of, a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain, wherein the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain each comprise the amino acid sequences shown in SEQ ID NOs: 93, 109, 110, and 93, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or the first, second, third, and fourth polypeptide chains composed of said amino acid sequences.

[0433] Characteristics of the multispecific antibody of this invention

[0434] In some embodiments, the multispecific antibody of the present invention may have one or more of the following properties:

[0435] (i) It binds to tumor cells that co-express FOLR1 and MSLN with high affinity, especially with a synergistic effect to specifically bind to tumor cells that co-express FOLR1 and MSLN;

[0436] (ii) It exhibits species cross-reactivity with cynomolgus monkeys FOLR1 and MSLN;

[0437] (iii) It has FOLR1 and / or MSLN receptor-mediated endocytic activity, especially FOLR1 and MSLN receptor-mediated co-endocytic activity.

[0438] (iv) Specific binding to FOLR1-positive tumor cells (including tumor cells with low or high expression) and / or MSLN-positive tumor cells, especially synergistic specific binding to tumor cells that simultaneously express or overexpress FOLR1 and MSLN.

[0439] (v) It exhibits pH-dependent binding to FOLR1-positive tumor cells, for example, its binding is stronger in the microacidic environment of tumors than in normal tissues.

[0440] In some respects, the multispecific antibodies of the present invention exhibit high binding affinity to tumor cells co-expressing FOLR1 and MSLN. The cell-binding affinity of the antibodies to FOLR1 and / or MSLN-positive tumor cells can be reflected by FACS or ELSA assays (e.g., the assays described in the examples) and optionally compared with a reference antibody.

[0441] In some embodiments, the multispecific antibody of the present invention exhibits cross-reactivity with human and monkey FOLR1 and MSLN.

[0442] In some embodiments, the multispecific antibody of the present invention specifically binds to FOLR1 and MSLN positive tumor cells with high affinity (synergistic effect).

[0443] In some embodiments, the bispecific antibody of the present invention that specifically binds to the FOLR1 biepitaxes has a synergistic effect with the binding of the biepitaxes, i.e., it binds more strongly to FOLR1-positive tumor cells.

[0444] In some embodiments, the trispecific antibody of the present invention, which specifically binds to the FOLR1 dual epitope and the MSLN epitope, has a synergistic effect with the binding of the dual epitope to the MSLN target, i.e., it binds more strongly to FOLR1-positive and MSLN-positive tumor cells.

[0445] In some embodiments, the bispecific antibody of the present invention that specifically binds to FOLR1 and MSLN has a synergistic effect with the binding of FOLR1 and MSLN targets, i.e., higher binding to FOLR1-positive and MSLN-positive tumor cells.

[0446] In some embodiments, the multispecific antibodies of the present invention possess endocytic activity mediated by FOLR1 and / or MSLN receptors, particularly synergistic endocytic activity mediated by FOLR1 and MSLN receptors. The endocytic activity of the antibodies can be evaluated in cell-based assays, such as those described in the examples.

[0447] In some further embodiments, the multispecific antibody of the present invention also has one or more of the following characteristics: (vi) good developability; (vii) good stability; and (viii) favorable pharmacokinetic properties.

[0448] In some embodiments, after one-step purification of the antibody product generated from recombinant mammalian cells using protein A affinity chromatography, the multispecific antibody of the present invention can achieve a purity of 90% or 95% or higher as determined by SEC-HPLC.

[0449] V. Other antigen-binding molecules

[0450] The antigen-binding molecule of the present invention can also be a TCR molecule or a CAR molecule for use in TCR therapy or CAR therapy. Methods for constructing TCR or CAR molecules using antigen-binding domains are known in the art.

[0451] VI. Production and purification of the antibodies of the present invention

[0452] In another aspect, the present invention provides a method for producing the antigen-binding molecules of the present invention, such as antibodies. To produce the antigen-binding molecules of the present invention, such as antibodies, polypeptide chains of the antigen-binding molecules of the present invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production, and then assembled under suitable conditions.

[0453] For recombinant production, polynucleotides encoding any one or more polypeptide chains of the antigen-binding molecule, such as an antibody, can be isolated and inserted into one or more vectors for further cloning and / or expression in host cells. The polynucleotides can be easily isolated and sequenced using conventional methods. In one embodiment, polynucleotides encoding one or more polypeptide chains of the antigen-binding molecule, such as an antibody, of the present invention are provided. In yet another embodiment, the present invention provides vectors comprising one or more polynucleotides of the present invention, preferably expression vectors. Thus, in one embodiment, the present invention provides a method for producing the antigen-binding molecule, such as an antibody, of the present invention, the method comprising: culturing host cells containing a polypeptide chain encoding the polypeptide chain under conditions suitable for expressing the antigen-binding molecule, such as an antibody; and assembling the polypeptide chain to produce the antigen-binding molecule, such as an antibody, under conditions suitable for assembling the polypeptide chain into the antigen-binding molecule, such as an antibody.

[0454] Expression vectors can be constructed using methods well known to those skilled in the art. Expression vectors include, but are not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YACs). Preferably, the expression vector is pCDNA, such as pCDNA3.4.

[0455] In one embodiment, the present invention also provides a host cell comprising one or more of the polynucleotides of the present invention. In some embodiments, a host cell comprising the expression vector of the present invention is provided. Suitable host cells include prokaryotic microorganisms such as *Escherichia coli*, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can be used. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (HEK293 or 293F 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 (HepG2), CHO cells, NSO cells, myeloma cell lines such as YO, NSO, P3X63, and Sp2 / O, etc. In a preferred embodiment, the host cell is a CHO or HEK293 cell.

[0456] Antigen-binding molecules, such as antibodies, prepared by the methods described herein can be purified using known techniques such as high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. After purification, the purity of the antigen-binding molecules, such as antibodies, of the present invention can be determined using any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, and HPLC. The physical / chemical properties and / or biological activity of the antibodies provided herein can be identified, screened, or characterized using a variety of assays known in the art.

[0457] In a preferred embodiment, the antigen-binding molecules of the present invention, such as antibodies, exhibit good production properties when recombinantly produced in mammalian host cells, such as CHO cells, especially good expression yield and a good byproduct profile.

[0458] II. Immunofusions and Immunoconjugates

[0459] In one aspect, the present invention provides immunofusions or immunoconjugates produced by fusing or conjugating the antigen-binding molecule of the present invention to a heterologous molecule.

[0460] In one embodiment, in the immunofusion, the antigen-binding molecule of the present invention, such as an antibody (or an antigen-binding fragment thereof), is linked directly or via an amino acid linker to a heterologous peptide or polypeptide molecule. Heterologous peptides or polypeptides that may be mentioned include, but are not limited to, proteins or polypeptides that impart another functional activity to the fusion, or tagged peptides that facilitate the purification or detection of the immunofusion.

[0461] In one embodiment, in the immunoconjugate, the antigen-binding molecule of the present invention, such as an antibody (or an antigen-binding fragment thereof), is conjugated to a therapeutic agent, diagnostic agent, or detectable agent. In the conjugate, linkers can be used to covalently link different entities of the conjugate. Suitable linkers include chemical linkers or peptide linkers. Advantageously, the linker is a "cleavable linker" that facilitates the release of the polypeptide upon delivery to the target site. For example, acid-instable linkers, peptidase-sensitive linkers, photostable linkers, dimethyl linkers, or disulfide-containing linkers can be used.

[0462] In embodiments where a therapeutic agent is conjugated, the therapeutic agents suitable for the conjugation include, but are not limited to, cytotoxins (e.g., cell growth inhibitors or cell killers), cell agonists, protein degraders, pharmaceuticals, or radioisotopes.

[0463] In embodiments conjugated with diagnostic or detectable agents, such conjugates 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 agents, including but not limited to a variety of enzymes such as horseradish peroxidase; prosthetic groups such as streptavidin / biotin and avidin / biotin; 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.

[0464] In some preferred embodiments, the immunoconjugate according to the invention is an antibody-drug conjugate (ADC).

[0465] In some embodiments, the present invention provides an antibody-drug conjugate having formula (I): Ab-(LD) p (I)

[0466] Or its pharmaceutically acceptable salts or solvates,

[0467] in:

[0468] Ab is an antigen-binding molecule as defined above, such as antibodies or fragments thereof (e.g., antigen-binding fragments) that specifically bind to MSLN and / or FOLR1 (e.g., human MSLN and / or human FOLR1).

[0469] L is the connector;

[0470] D is a drug, such as an anti-tumor compound; and

[0471] p is an integer selected from 1 to 16, such as an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12.

[0472] It should be understood that p refers to the number of -LDs linked to Ab in the antibody-drug conjugate molecule of formula (I), also known as DAR. In some embodiments, D in formula (I) of the present invention can be any antitumor compound, as long as it has antitumor effects and has substituents or partial structures that can be linked to the linker structure, preferably small molecule antitumor compounds. In some embodiments, D is a therapeutic agent suitable for the conjugate as described above.

[0473] In some implementations, the antitumor compound may be, for example, a cytotoxic agent, such as a camptothecin compound such as Exatecan, Dxd, or an auristatin compound such as monomethyl auristatin E (MMAE) or MMAF.

[0474] In some implementations, D has the structure shown in formula (D-1a) or formula (D-1b):

[0475] Where R 1a Selected from H and C1-C6 alkyl groups;

[0476] R 2a Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and -SR 5a ;

[0477] R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ;and

[0478] R 4a and R 5a Independently selected from H and C1-C4 alkyl groups;

[0479] Where R 1b R 2b R 3b R 4b R 5b and R 8b Each was independently selected from C 1-8 Alkyl; preferably C 1-4 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl;

[0480] R 6b and R 7b Each was independently selected from C 1-8 Alkyl groups, such as methoxy, ethoxy, or propoxy;

[0481] R 9b Selected from C 1-8 Alkyl groups and COOH; preferably C 1-4 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; and

[0482] R 10b Selected from OH and H.

[0483] It should be understood that a key with a wavy line in the structure indicates that the key is connected to another structure or segment.

[0484] In some implementation schemes,

[0485] In some implementation schemes, R 1a For H; R 2aIt is a C1-C6 alkyl group; R 3a It is a halogen, preferably -F; R 4a It is a C1-C4 alkyl group, preferably ethyl.

[0486] In some implementation schemes, R 1b R 4b and R 8b Each was independently selected from C 1-2 Alkyl; preferably methyl;

[0487] R 2b R 3b and R 5b Each was independently selected from C 3-4 alkyl;

[0488] R 6b and R 7b Each was independently selected from C 1-2 alkoxy groups; and

[0489] R 9b Selected from C 1-4 Alkyl and R 10b For OH; or R 9b It is COOH and R 10b For H.

[0490] In some implementations, D has the structure shown in formula (D-2a) or formula (D-2b):

[0491] Where R 1a R 2a R 3a and R 4a As defined above; or

[0492] Where R 1b R 2b R 3b R 4b R 5b R 6b R 7b R 8b R 9b and R 10b As defined above.

[0493] In some implementations, D has the structure shown in formula (D-3a) or (D-3b):

[0494] In some implementations, the unspecified chiral neutrals are each independently of the R or S configuration.

[0495] In some implementations, D has the structure shown in formula (D-4a) or (D-4b):

[0496] In some implementations, -L- has the following structure: -Z-L1-L2-L3-

[0497] in

[0498] Z is selected from Where m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7 or 8;

[0499] L1 is selected from non-existent, Where n1 and m1 are independently integers selected from 0 to 20, for example, integers selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16;

[0500] L2 is a single amino acid or a peptide residue selected from 2-8 amino acids; and

[0501] L3 is selected from: Where X is selected from -NH-, -O-, and -S-; R 1c Selected from: C 1-8 Alkyl-, Halogenated C 1-8 Alkyl-, C 1-8 Alkyl group, halogen, nitro group, and cyano group; Su is selected from pentose, penturonic acid, hexose, and hexuronic acid; n2 is 0, 1, 2, 3, or 4; n5 is 0, 1, 2, or 3; and n3 and n4 are independently 1, 2, 3, 4, 5, or 6.

[0502] It should be understood that in the above -Z-L1-L2-L3-, Z is connected to Ab, and L3 is connected to D. Unless otherwise specified, the divalent groups mentioned herein are connected in the directions shown. That is, the left side of the Z group (e.g., the maleimide moiety) is connected to Ab, and the right side (e.g., the carbonyl group) is connected to L1, or connected to L1 when L1 is absent; the left side of L3 (amino group) is connected to L2, and the right side (carbonyl group) is connected to D.

[0503] In some implementation schemes, Z is selected from Where m is 1, 2, 3, 4, 5, 6, 7 or 8.

[0504] In some implementation schemes, Z is selected from

[0505] In some implementations, L1 is selected from non-existent, Where n1 is an integer independently selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7 or 8.

[0506] In some implementations, L1 is selected from non-existent,

[0507] In some implementations, L2 is a single amino acid or a peptide residue consisting of 2, 3, 4, 5, 6, or 7 amino acids.

[0508] The amino acid in L2 is preferably an L-amino acid. In addition to α-amino acids, it can also be an amino acid with structures such as β-alanine, ε-aminohexanoic acid, and γ-aminobutyric acid. Furthermore, it can also be a non-natural amino acid, such as an N-methylated amino acid. It should be understood that the amino acid in L2 exists as an amino acid residue linked to two end groups.

[0509] The amino acids in L2 can be independently selected from the amino acids defined in the user-defined section, such as phenylalanine (Phe), tyrosine (Tyr), leucine (Leu), glycine (Gly), alanine (Ala), valine (Val), lysine (Lys), citrulline (Cit), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), threonine (Thr), and glutamine (Gln).

[0510] In some implementations, L2 is selected from: -Ala-, -Val-, -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, -Arg-Asp-Val-Thr- (i.e., RDVT), -Gly-Gly-Phe-Gly-.

[0511] In some implementations, L2 is selected from -Gly-, -Val-Ala-, -Arg-Asp-Val-Thr-, and -Val-Cit-;

[0512] It should be understood that L2 is connected to L1 or Z through the amino group of the amino acid on the left, and to L3 through the carbonyl group of the amino acid on the right, which is consistent with the explanation below.

[0513] In some implementations, L3 is Among them, Su and R 1c As defined above ； n2 and n5 are independently 0 or 1; preferably 0.

[0514] In some implementations, L3 is Su is defined as above.

[0515] In some embodiments, Su is selected from xylose, arabinose, xyuronic acid, arabinuronic acid, glucose, galactose, mannose, glucuronic acid, galacturonic acid, and mannuronic acid.

[0516] In some implementation schemes, Su is selected from

[0517] In some implementation schemes, Su is selected from

[0518] In some implementations, Su is

[0519] In some implementations, Su is

[0520] In some implementations, L3 is selected from:

[0521] It should be understood that L3 is connected to L2 via the amino group on the left and to D via the carbonyl group on the right, which is consistent with the explanation below.

[0522] In some implementations, -Z-L1-L2-L3- is selected from the following structures

[0523] It should be understood that, unless otherwise specified and without contradiction in the context, for the ADC of the present invention, the left-hand bond of the divalent group shown herein is connected to Ab or a group near the Ab end, and the right-hand bond of the divalent group is connected to D or a group near the D end. For example, when L2 is -Val-Ala-, the amino group on the left side of Val is connected to L1, and the carbonyl group on the right side of Ala is connected to L3;

[0524] In some implementations, Ab is covalently linked to L via a sulfur atom, that is, linked to L via -S-.

[0525] It should be understood that the sulfur (-S-) mentioned above is sulfur derived from Ab.

[0526] In some embodiments, the antibody-drug conjugate is selected from...

[0527] Where Ab is the antigen-binding molecule defined in the text; and p is as defined above.

[0528] In some embodiments, the antibody-drug conjugate has an average DAR of 2-10, 3-5, 5-7, 6-9, 6-10, 4-8, 7-9, or 2-4.

[0529] It should be understood that when the maleimide portion of the linker in the ADC is linked to an Ab, it is linked to the Ab through an S atom, which originates from the Ab. The Ab, under the action of a reducing agent such as TCEP, opens disulfide bonds, especially interchain disulfide bonds, to generate a thiol group (-SH), which then links to the terminal functional group of the linker, such as the maleimide portion.

[0530] In some implementations, the S atom attached to Ab comes from the cysteine ​​residue of Ab.

[0531] III. Pharmaceutical Combinations, Drug Conjugates, and Reagent Kits

[0532] In one aspect, the present invention provides compositions, such as pharmaceutical compositions, comprising an antigen-binding molecule, such as the molecules described herein formulated with a pharmaceutically acceptable excipient, such as the antigen-binding molecule, immunofusion compound, or immunoconjugate described herein. As used herein, "pharmaceutically acceptable excipient" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents, etc. 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). In some embodiments, the molecule of the present invention, such as the antigen-binding molecule of the present invention or the immunoconjugate or immunofusion compound of the present invention, is the sole active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may comprise the molecule of the present invention (encompassing the antigen-binding molecule, immunoconjugate, immunofusion compound, antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof) with one or more therapeutic agents.

[0533] In another aspect, the present invention also provides a pharmaceutical combination comprising the molecules of the present invention with one or more therapeutic agents.

[0534] The therapeutic agents applicable to the pharmaceutical compositions and combinations thereof of the present invention may be therapeutic agents selected from any of the following categories (i)-(iv): (i) drugs that enhance antigen presentation (e.g., tumor antigen presentation); (ii) drugs that enhance effector cell responses (e.g., B cell and / or T cell activation and / or mobilization); (iii) drugs that reduce immunosuppression; and (iv) drugs that have antitumor effects.

[0535] The pharmaceutical compositions of the present invention may contain a "therapeutic effective amount" or a "preventive effective amount" of the molecules of the present invention.

[0536] Kits containing the molecules of this invention are also within the scope of this invention. Kits may include one or more other elements, such as: instructions for use; other reagents, such as markers or agents for conjugation; pharmaceutically acceptable carriers or excipients; and devices or other materials for administration to a subject.

[0537] IV. Uses and Methods

[0538] Based on the excellent targeting properties of the molecules of the present invention (e.g., the antigen-binding molecules, immunoconjugates, immunofusions, antibody-drug conjugates, or pharmaceutically acceptable salts or solvates thereof) to tumor cells expressing MSLN and / or FOLR1, as well as the other superior properties mentioned above, the present invention also provides the molecules of the present invention and their applications and methods in the treatment and prevention of MSLN and / or FOLR1-related diseases.

[0539] MSLN and FOLR1 are overexpressed in cancer tissues from various sources (e.g., on cell surfaces), and are therefore suitable targets for developing cancer immunotherapies. In one aspect, the present invention provides the use of molecules of the invention for the prevention and / or treatment in subjects of MSLN and / or FOLR1-related diseases, such as tumors, such as MSLN and / or FOLR1-positive tumors, particularly MSLN and FOLR1-positive tumors. In said application, the molecules of the invention may be administered to the subject as the sole active agent or may be administered to the subject in combination with other therapies or therapeutic agents. These other therapies and therapeutic agents include, for example, drugs that target antigens on the surface of tumor cells to eliminate tumors by binding to and / or blocking these molecules; and drugs that activate the subject's immune system, prompting it to spontaneously eliminate tumors.

[0540] In another aspect, the present invention also provides a method for preventing or treating MSLN and / or FOLR1-related diseases in subjects, comprising administering the molecules of the present invention to subjects in need. In some embodiments, the MSLN and / or FOLR1-related diseases are tumors such as cancers, such as MSLN and / or FOLR1-positive tumors or cancers, such as MSLN-positive tumors or cancers, or FOLR1-positive tumors or cancers, or MSLN-positive and FOLR1-positive tumors or cancers.

[0541] The tumors suitable for the methods and applications of this invention can be selected from various solid tumors, such as: ovarian cancer, ovarian adenocarcinoma, peritoneal cancer, fallopian tube cancer, cervical cancer, breast cancer, ductal carcinoma of the breast, squamous cell carcinoma of the breast, triple-negative breast cancer, colorectal cancer, lung cancer, lung adenocarcinoma, pancreatic cancer, gastric cancer, gastroesophageal adenocarcinoma, bladder cancer, and endometrial cancer. In some embodiments, the solid tumor is an MSLN and / or FOLR1 positive tumor, particularly an MSLN-positive and FOLR1-positive tumor.

[0542] MSLN and / or FOLR1-positive tumors suitable for the methods and applications of this invention can be early, intermediate, or late-stage or metastatic cancers. Furthermore, MSLN and / or FOLR1-positive tumors suitable for the methods and applications of this invention can be tumors that have previously undergone treatment and have experienced immune escape.

[0543] In some embodiments, MSLN and / or FOLR1 positive tumors suitable for prevention or treatment according to the method of the present invention have overexpression of MSLN and / or FOLR1, for example, overexpression of MSLN and / or FOLR1 in their tumor samples (e.g., tumor cells or tissues), for example, compared to the corresponding tissues or cells of a healthy subject and / or to adjacent healthy tissues or healthy cells of the same subject's tumor tissues or cells.

[0544] In some embodiments, MSLN and / or FOLR1-positive tumors suitable for prevention or treatment according to the method of the present invention have at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% MSLN and / or FOLR1-positive cells. In some embodiments, MSLN-positive tumors have at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% MSLN-positive cells. In some embodiments, FOLR1-positive tumors have at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% FOLR1-positive cells. In some implementations, MSLN-positive and FOLR1-positive tumors have at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, and 100% MSLN and FOLR1 double-positive cells. The expression levels of MSLN and / or FOLR1 on tumor biopsies can be assessed by immunohistochemistry. High percentages of MSLN and / or FOLR1-positive cells have been detected in biopsies from various tumors or cancers, such as those described above.

[0545] Preferably, in some embodiments, the method according to the invention is used for the prevention or treatment of tumors having a high percentage of MSLN and / or FOLR1 positive cells, for example, tumors having at least 25%, 50%, 75%, or 100% MSLN and / or FOLR1 positive cells. In some embodiments, the tumor having a high percentage of MSLN positive cells has at least 25%, 50%, 75%, or 100% MSLN positive cells. In some embodiments, the tumor having a high percentage of FOLR1 positive cells has at least 25%, 50%, 75%, or 100% FOLR1 positive cells. In some embodiments, the tumor having a high percentage of both MSLN and FOLR1 positive cells has at least 25%, 50%, 75%, or 100% MSLN and FOLR1 double positive cells.

[0546] In some embodiments, the method according to the invention can also be used to treat tumors with less than 25% or 20% of MSLN and / or FOLR1 positive cells, preferably tumors with less than 25% or 20% of MSLN positive cells but with a high percentage of FOLR1 positive cells; or tumors with less than 25% or 20% of FOLR1 positive cells but with a high percentage of MSLN positive cells. In some embodiments, the tumor has less than 25% or 20% of both MSLN and FOLR1 positive cells. In some embodiments, the tumor has less than 25% of both MSLN and FOLR1 positive cells.

[0547] In some embodiments, the application of the method of the present invention to said cancer results in tumor growth inhibition. In some embodiments, the application of the method of the present invention to said cancer induces tumor regression.

[0548] In any of the above embodiments of the method of the present invention, the application of the molecules according to the present invention may include 1) a therapeutic measure that cures, slows, alleviates, reduces or stops the progression of a diagnosed pathological condition or disease; or 2) a preventive or preventative measure that prevents and / or slows the development of a pathological condition or disease. Therefore, in the method of the present invention, the subject may be an individual who already suffers from a disease, an individual susceptible to a disease, or an individual wishing to prevent a disease. The individual will benefit from the therapeutic or preventative measures and, compared to an individual who has not received the treatment, exhibit a reduction or improvement in the occurrence, recurrence, or development of the disease, condition, symptom, and / or symptoms. In some embodiments, the present invention relates to the treatment of a disease or condition; in other embodiments, the present invention relates to the prevention of a disease or condition.

[0549] The molecules according to the invention, and optionally other therapeutic agents used in combination therewith, can be administered by any suitable method, including parenteral administration, intratumoral administration, and intranasal administration. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Various dosing schedules are covered herein, including, but not limited to, single-dose or multiple-dose administration at multiple time points, bolus administration, and pulsatile infusion.

[0550] For the prevention or treatment of disease, the appropriate dosage of the molecule according to the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the specific type of drug used, the severity and course of the disease, whether the drug 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.

[0551] On the other hand, the present invention provides molecules of the present invention for therapeutic purposes, such as for the prevention and / or prevention of MSLN and / or FOLR1-related diseases as defined herein.

[0552] On the other hand, the present invention provides molecules of the present invention that are used as medicines, such as medicines for preventive or therapeutic purposes of the present invention, such as medicines for the prevention and / or treatment of MSLN and / or FOLR1-related diseases as defined herein.

[0553] On the other hand, the present invention provides the use of the molecules of the present invention for the preparation of medicaments, wherein the medicaments are used for the preventive or therapeutic purposes of the present invention, such as for the treatment and / or prevention of MSLN and / or FOLR1-related diseases as defined herein.

[0554] V. Preparation of the ADC molecule of the present invention

[0555] Another aspect of the present invention provides a method for preparing the ADC of the present invention (I) using the antigen-binding molecule of the present invention. The method includes conjugating the antigen-binding molecule (Ab) of the present invention to a drug (D) via a linker (L).

[0556] In some implementations, the method includes the following steps:

[0557] (a) The antigen-binding molecule (Ab) of the present invention is placed in a buffer solution, a reducing agent and an optional metal salt are added, and the mixture is incubated.

[0558] (b) The linker-loaded component is added to the reaction solution from step (a) for coupling to obtain the crude product; and

[0559] (c) Optionally purify the crude product to obtain the antibody-drug conjugate of the present invention;

[0560] Ab is defined as above.

[0561] In some embodiments, the metal salt is a hydrochloride or sulfate of Zn, Cd, and Hg, preferably ZnCl2.

[0562] In some implementations, the reducing agent in step a) is TCEP.

[0563] In some embodiments, the buffer solution in step a) is a NaOAc-HAc buffer solution, preferably with a pH of 5.0-9.0, for example 5.0-7.0.

[0564] In some implementations, the connector-payload has the following structure: Z'-L1-L2-L3-D, where L1, L2, L3, and D are as defined herein, and Z' is... m is as defined above.

[0565] In some implementations, for the synthesis of Z is The ADC method of formula I also includes an additional hydrolysis step that opens the ring of maleimide.

[0566] In some implementations, the steps are performed under the specific reaction conditions disclosed in the embodiments of the present invention.

[0567] It should be noted that embodiments obtained by varying the range or specific values ​​of the specific reaction conditions disclosed in the embodiments by 100%, 90%, 80%, 70%, 60%, 40%, 20%, or 10% are also considered in this invention. Embodiments obtained by limiting the embodiments described above to the specific conditions and ranges in the embodiments are also included within the scope of this invention.

[0568] It should be understood that, given that the linker-load (-LD) portion in the compound of Formula I is clearly defined, those skilled in the art can clearly understand the structure of the linker-load used before connection.

[0569] Any or all of the features described above and throughout this application may be combined in various embodiments of the invention. The following examples further illustrate the invention; however, it should be understood that the examples are for illustrative purposes only and should not be construed as constituting any limitation. VI. Specific Implementation Plan

[0570] In some aspects, the present invention relates to the following specific embodiments:

[0571] A1. A VHH antibody that specifically binds to FOLR1, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region comprises the three complementarity-determining regions (CDRs) contained in the VH as shown in any one of SEQ ID NO: 27, 1, 5, 7, 9, 13, 16, 20, 22-26, 28, 129, 130, 131; preferably, the CDR sequence is defined according to ABM.

[0572] A2. The VHH antibody described in embodiment A1, comprising complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or comprising or composed of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein

[0573] (i) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:10, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:17, 11, 18, 19, 21 or 41, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:12.

[0574] (ii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:2, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:3, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:4, 6, or 8; or

[0575] (iii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:14, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:15, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:12.

[0576] A3. The VHH antibody according to embodiment A1 or A2, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region comprises or consists of an amino acid sequence having at least 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: 27, 1, 5, 7, 9, 13, 16, 20, 22-26, 28, 129, 130, 131.

[0577] A4. The VHH antibody according to any one of embodiments A1-A3, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region comprises or consists of an amino acid sequence selected from or consisting of any one of SEQ ID NO: 27, 1, 5, 7, 9, 13, 16, 20, 22-26, 28, 129, 130, 131.

[0578] A5. A heavy chain antibody that specifically binds to FOLR1, comprising the VHH antibody as described in any one of embodiments A1-A4.

[0579] A6. The heavy chain antibody of embodiment A5, comprising the VHH antibody of any one of embodiments A1-A5 linked to the constant region or Fc region of the antibody.

[0580] A7. The heavy chain antibody of embodiment A6, wherein the constant region or Fc region of the antibody is derived from human IgG1, human IgG2, human IgG3 or human IgG4, preferably from the Fc region of human IgG1 or IgG4.

[0581] A8. A heavy chain antibody according to implementation scheme A6 or A7, wherein the Fc region contains a complete hinge region and is directly linked to the VHH antibody.

[0582] A9. The heavy chain antibody according to any one of embodiments A6-A8, wherein the Fc region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 122, 123, 128, or 142-143.

[0583] A10, the heavy chain antibody according to any one of embodiments A6-A9, wherein the Fc region comprises or consists of the amino acid sequence shown in or composed of any one of SEQ ID NO: 122, 123, 128 or 142-143.

[0584] A11, the heavy chain antibody according to any one of embodiments A6-A10, wherein the Fc region contains a C220S mutation.

[0585] A12, the heavy chain antibody according to any one of embodiments A6-A11, wherein the Fc region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 144 or 145.

[0586] A13, the heavy chain antibody according to any one of embodiments A6-A12, wherein the Fc region comprises or is composed of the amino acid sequence shown in SEQ ID NO:144 or 145.

[0587] A14. The heavy chain antibody according to any one of embodiments A6-A13, wherein the Fc region comprises a mutation that reduces or eliminates the Fc region and the Fcγ receptor, such as the LALA mutation.

[0588] A15. The heavy chain antibody according to any one of embodiments A6-A14, wherein the Fc region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:124.

[0589] A16, the heavy chain antibody according to any one of embodiments A6-A15, wherein the Fc region comprises or is composed of the amino acid sequence shown in SEQ ID NO:124.

[0590] A17. A VHH antibody of any one of embodiments A1-A5, or a heavy chain antibody of any one of embodiments A6-A16, wherein the antibody is a fully human antibody.

[0591] A18. An antigen-binding molecule comprising a VHH antibody of any one of embodiments A1-A5 or A17, or a heavy chain antibody of any one of embodiments A6-A16, for example, the antigen-binding molecule being a multispecific antibody such as a bispecific antibody or a trispecific antibody.

[0592] A19. An antigen-binding molecule according to embodiment A18, wherein the antigen-binding molecule specifically binds to two different epitopes of FOLR1, comprising at least one antigen-binding domain of a first epitope of FOLR1 and at least one antigen-binding domain of a second epitope of FOLR1.

[0593] A20, the antigen-binding molecule of embodiment A19, wherein the antigen-binding domain that specifically binds to the first epitope of FOLR1 is a VHH antibody as described in any of embodiments A1-A5 or A17.

[0594] A21. An antigen-binding molecule according to embodiment A19 or A20, wherein the antigen-binding domain of at least one FOLR1-specifically binding second epitope is a Fab fragment specifically binding FOLR1, i.e., Fab FOLR1 Preferably, the Fab fragment comprises a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain comprises or is composed of VH-CH1, and the Fab light chain comprises or is composed of VL-CL.

[0595] A22. The antigen-binding molecule of embodiment A21, wherein the antigen-binding domain that specifically binds to FOLR1 is, for example, Fab. FOLR1 It includes heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, among which

[0596] (i) HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:74; HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:75; HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:76; LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:78; LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:132, 79, 82, 84, 87, 89, 92, or 107; and LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:80; or

[0597] (ii) The HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:74; the HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:75; the HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:76; the LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:78; the LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:133; and the LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:80. A23, an antigen-binding molecule of embodiment A21 or A22, wherein the antigen-binding domain that specifically binds to FOLR1 is, for example, Fab. FOLR1 It includes the heavy chain variable region VH and the light chain variable region VL, in which

[0598] The VH comprises 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:73, and the VL comprises 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:85, 140, 77, 81, 83, 86, 88, 90, or 91.

[0599] A24. An antigen-binding molecule according to any one of embodiments A21-A23, wherein the antigen-binding domain that specifically binds to FOLR1 is, for example, Fab. FOLR1It comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:73, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:85, 140, 77, 81, 83, 86, 88, 90 or 91.

[0600] A25. An antigen-binding molecule according to any one of embodiments A21-A24, wherein the antigen-binding domain that specifically binds to FOLR1 is, for example, Fab. FOLR1 Includes CH1;

[0601] Preferably, CH1 is CH1 derived from (human) IgG1, IgG2, IgG3, or IgG4.

[0602] More preferably, CH1 is CH1 derived from IgG1.

[0603] Optionally, the CH1

[0604] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:135;

[0605] (ii) Contains or consists of the amino acid sequence of SEQ ID NO:135.

[0606] A26. An antigen-binding molecule according to any one of embodiments A21-A25, wherein the antigen-binding domain that specifically binds to FOLR1 is, for example, Fab. FOLR1 Includes the light chain constant region CL;

[0607] Preferably, the CL is a Kappa light chain constant region or a Lambda light chain constant region.

[0608] More preferably, the CL is the constant region of the Kappa light chain.

[0609] Optionally, the CL

[0610] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:127;

[0611] (ii) Contains or consists of the amino acid sequence of SEQ ID NO:127.

[0612] A27. An antigen-binding molecule according to any one of embodiments A18-A26, wherein the antigen-binding molecule further comprises at least one antigen-binding domain that specifically binds to MSLN.

[0613] A28. The antigen-binding molecule of embodiment A27, wherein the antigen-binding domain that specifically binds to MSLN is a VHH antibody as described in any of embodiments B1-B5.

[0614] A29. An antigen-binding molecule according to any one of embodiments A18-A28, wherein the antigen-binding molecule further comprises an Fc region, for example, comprising two identical or different Fc regions.

[0615] A30. The antigen-binding molecule of embodiment A29, wherein the Fc region is a human IgG Fc, such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. For example, the Fc region comprises a complete hinge region or a partial hinge region. For example, the Fc region comprises an amino acid sequence as described in any one of SEQ ID NO: 122, 123, 128, or 142-145, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity with the amino acid sequence, or is composed of the amino acid sequence.

[0616] A31. The antigen-binding molecule according to embodiment A29 or A30, wherein the Fc region comprises a mutation that reduces or eliminates the interaction between the Fc region and the Fcγ receptor, such as the LALA mutation.

[0617] Preferably, the Fc region containing the LALA mutation contains an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:124, for example, containing or consisting of the amino acid sequence shown in SEQ ID NO:124.

[0618] A32. The antigen-binding molecule according to any one of embodiments A29-A31, wherein the Fc region further contains the amino acid sequence shown in SEQ ID NO:138, SEQ ID NO:139, or SEQ ID NO:141 at its N-terminus.

[0619] A33. An antigen-binding molecule according to any one of embodiments A29-A32, wherein the two Fc regions are different, and wherein the two Fc regions respectively contain a Knob mutation and a Hole mutation, for example, the Knob mutation T366W, and the Hole mutation T366S, L368A, and Y407V combination; or the Knob mutation is T366Y, and the Hole mutation is Y407T; optionally, the Fc region containing the Knob mutation further contains the S354C mutation, and the Fc region containing the Hole mutation further contains the Y349C mutation, for example, the Fc region containing the Knob mutation contains or is composed of the amino acid sequence shown in SEQ ID NO:125, while the Fc region containing the Hole mutation contains or is composed of the amino acid sequence shown in SEQ ID NO:126.

[0620] A34. The antigen-binding molecule according to embodiment A33, comprising the Fc region of the Knob mutation and / or the Fc region comprising the Hole mutation, further comprising the amino acid sequence shown in SEQ ID NO:138, SEQ ID NO:139, or SEQ ID NO:141 at its N-terminus.

[0621] A35. An antigen-binding molecule according to any one of embodiments A18-A34, wherein the antigen-binding domains are connected to each other or to the Fc region via a connector;

[0622] Preferably, the connector comprises (G4S)n, where n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5.

[0623] A36. An antigen-binding molecule according to any one of embodiments A29-A35, wherein when the Fc region is connected at its N-terminus to the C-terminus or N-terminus of the VHH domain, the Fc region contains a complete hinge region, that is, at its N-terminus, a portion of the hinge region corresponding to the E216-P230 (EU number) of the IgG1 Fc region, for example, the amino acid sequence shown in SEQ ID NO:139 or SEQ ID NO:141.

[0624] A37. An antigen-binding molecule according to any one of embodiments A29-A36, wherein when the Fc region is connected at its N-terminus to the C-terminus of the Fab heavy chain, the Fc region includes a partial hinge region, that is, at its N-terminus, a portion of the hinge region corresponding to the IgG1 Fc region E2221-P230 (EU number), for example, the amino acid sequence shown in SEQ ID NO:138.

[0625] B1. A VHH antibody that specifically binds to MSLN, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region comprises the three complementarity-determining regions (CDRs) contained in VH as shown in any one of SEQ ID NO: 70, 29, 33, 37, 42-45, 47, 49, 51-69 and 71-72; preferably, the CDR sequence is defined according to ABM.

[0626] B2. A VHH antibody that specifically binds to MSLN, comprising complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein

[0627] (i) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:30, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:31, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:46, 32, 48 or 50.

[0628] (ii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:34, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:35, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:36; or

[0629] (iii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:38, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:39, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:40.

[0630] B3. The VHH antibody according to embodiment B1 or B2, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region comprises or consists of an amino acid sequence having at least 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: 70, 29, 33, 37, 42-45, 47, 49, 51-69, and 71-72.

[0631] B4. The VHH antibody according to any one of embodiments B1-B3, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region comprises or consists of an amino acid sequence selected from or consisting of any one of SEQ ID NO: 70, 29, 33, 37, 42-45, 47, 49, 51-69 and 71-72.

[0632] B5. A heavy chain antibody that specifically binds to MSLN, comprising the VHH antibody described in any one of embodiments B1-B4.

[0633] B6. The heavy chain antibody of embodiment B5, comprising the VHH antibody of any one of embodiments B1-B5 linked to the constant region or Fc region of the antibody.

[0634] B7. The heavy chain antibody of embodiment B6, wherein the constant region or Fc region of the antibody is derived from human IgG1, human IgG2, human IgG3 or human IgG4, preferably from the Fc region of human IgG1 or IgG4.

[0635] B8. The heavy chain antibody of embodiment B6 or B7, wherein the Fc region contains a complete hinge region and is directly linked to the VHH antibody.

[0636] B9. The heavy chain antibody according to any one of embodiments B6-B8, wherein the Fc region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 122, 123, 128, or 142-143.

[0637] B10, the heavy chain antibody according to any one of embodiments B6-B9, wherein the Fc region comprises or consists of the amino acid sequence shown in or composed of any one of SEQ ID NO: 122, 123, 128 or 142-143.

[0638] B11, the heavy chain antibody according to any one of embodiments B6-B10, wherein the Fc region contains a C220S mutation.

[0639] B12, the heavy chain antibody according to any one of embodiments B6-B11, wherein the Fc region comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 144 or 145.

[0640] B13, the heavy chain antibody according to any one of embodiments B6-B12, wherein the Fc region comprises or is composed of the amino acid sequence shown in SEQ ID NO:144 or 145.

[0641] B14. The heavy chain antibody according to any one of embodiments B6-B13, wherein the Fc region comprises a mutation that reduces or eliminates the Fc region and the Fcγ receptor, such as the LALA mutation.

[0642] B15. The heavy chain antibody according to any one of embodiments B6-B14, wherein the Fc region comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:124.

[0643] B16, the heavy chain antibody according to any one of embodiments B6-B15, wherein the Fc region comprises or is composed of the amino acid sequence shown in SEQ ID NO:124.

[0644] B17. A VHH antibody of any one of embodiments B1-B5, or a heavy chain antibody of any one of embodiments B6-B16, wherein the antibody is a chimeric antibody or a humanized antibody.

[0645] B18. An antigen-binding molecule comprising a VHH antibody of any one of embodiments B1-B5 or B17, or a heavy chain antibody of any one of embodiments B6-B16, for example, the antigen-binding molecule being a multispecific antibody such as a bispecific antibody or a trispecific antibody.

[0646] B19. An antigen-binding molecule according to embodiment B18, wherein the antigen-binding molecule specifically binds to FOLR1 and MSLN, and comprises at least one antigen-binding domain specifically binding to FOLR1 and at least one antigen-binding domain specifically binding to MSLN; for example, it comprises at least one antigen-binding domain specifically binding to a first epitope of FOLR1 and at least one antigen-binding domain specifically binding to a second epitope of FOLR1, and at least one antigen-binding domain specifically binding to MSLN.

[0647] B20, the antigen-binding molecule of embodiment B19, wherein the antigen-binding domain that specifically binds to MSLN is a VHH antibody as described in any of embodiments B1-B5 or B17.

[0648] B21, the antigen-binding molecule of embodiment B19 or B20, wherein the at least one antigen-binding domain that specifically binds to FOLR1, for example, the antigen-binding domain that specifically binds to the first epitope of FOLR1, is a VHH antibody of any one of embodiments A1-A5, or a heavy chain antibody of any one of embodiments A6-A16.

[0649] B22. An antigen-binding molecule according to any one of embodiments B19 to B21, wherein the at least one antigen-binding domain specifically binding to FOLR1, for example, at least one antigen-binding domain specifically binding to a second epitope of FOLR1, is a Fab fragment specifically binding to FOLR1, i.e., Fab FOLR1 Preferably, the Fab fragment comprises a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain comprises or is composed of VH-CH1, and the Fab light chain comprises or is composed of VL-CL.

[0650] B23. The antigen-binding molecule of embodiment B22, wherein the antigen-binding domain that specifically binds to FOLR1 is, for example, Fab. FOLR1 It includes heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, among which

[0651] (i) HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:74; HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:75; HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:76; LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:78; LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:132, 79, 82, 84, 87, 89, 92, or 107; and LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:80; or

[0652] (ii) HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:74; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:75; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:76; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:78; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:133; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:80.

[0653] B24. An antigen-binding molecule according to embodiment B22 or B23, wherein the antigen-binding domain that specifically binds to FOLR1 is, for example, Fab. FOLR1 It includes the heavy chain variable region VH and the light chain variable region VL, in which

[0654] The VH comprises 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:73, and the VL comprises 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:85, 140, 77, 81, 83, 86, 88, 90, or 91.

[0655] B25. The antigen-binding molecule according to any one of embodiments B22-B24, wherein the antigen-binding domain that specifically binds to FOLR1 is, for example, Fab. FOLR1 It comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:73, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:85, 140, 77, 81, 83, 86, 88, 90 or 91.

[0656] B26. The antigen-binding molecule according to any one of embodiments B22-B25, wherein the antigen-binding domain that specifically binds to FOLR1 is, for example, Fab. FOLR1 Includes CH1;

[0657] Preferably, CH1 is CH1 derived from (human) IgG1, IgG2, IgG3, or IgG4.

[0658] More preferably, CH1 is CH1 derived from IgG1.

[0659] Optionally, the CH1

[0660] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:135;

[0661] (ii) Contains or consists of the amino acid sequence of SEQ ID NO:135.

[0662] B27. The antigen-binding molecule according to any one of embodiments B22-B26, wherein the antigen-binding domain that specifically binds to FOLR1 is, for example, Fab. FOLR1 Includes the light chain constant region CL;

[0663] Preferably, the CL is a Kappa light chain constant region or a Lambda light chain constant region.

[0664] More preferably, the CL is the constant region of the Kappa light chain.

[0665] Optionally, the CL

[0666] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:127;

[0667] (ii) Contains or consists of the amino acid sequence of SEQ ID NO:127.

[0668] B28. An antigen-binding molecule according to any one of embodiments B18-B27, wherein the antigen-binding molecule further comprises an Fc region, for example, comprising two identical or different Fc regions.

[0669] B29. The antigen-binding molecule according to embodiment B28, wherein the Fc region is a human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc, for example, the Fc region comprises a complete hinge region or a partial hinge region, for example, the Fc region comprises an amino acid sequence of any one of SEQ ID NO: 122, 123, 128 or 142-145 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher identity with the amino acid sequence or is composed of the amino acid sequence.

[0670] B30, the antigen-binding molecule of embodiment B28 or B29, wherein the Fc region comprises a mutation that reduces or eliminates the interaction between the Fc region and the Fcγ receptor, such as the LALA mutation.

[0671] Preferably, the Fc region containing the LALA mutation contains an amino acid sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:124, for example, containing or consisting of the amino acid sequence shown in SEQ ID NO:124.

[0672] B31, the antigen-binding molecule according to any one of embodiments B28-B30, wherein the Fc region further contains the amino acid sequence shown in SEQ ID NO:138, SEQ ID NO:139, or SEQ ID NO:141 at its N-terminus.

[0673] B32. An antigen-binding molecule according to any one of embodiments B28-B31, wherein the two Fc regions are different, and wherein the two Fc regions respectively contain a Knob mutation and a Hole mutation, for example, the Knob mutation T366W, and the Hole mutation T366S, L368A and Y407V combination; or the Knob mutation is T366Y, and the Hole mutation is Y407T; optionally, the Fc region containing the Knob mutation also contains the S354C mutation, and the Fc region containing the Hole mutation also contains the Y349C mutation, for example, the Fc region containing the Knob mutation contains or is composed of the amino acid sequence shown in SEQ ID NO:125, while the Fc region containing the Hole mutation contains or is composed of the amino acid sequence shown in SEQ ID NO:126.

[0674] B33. The antigen-binding molecule according to embodiment B32, comprising the Fc region of the Knob mutation and / or the Fc region comprising the Hole mutation, further comprising the amino acid sequence shown in SEQ ID NO:138, SEQ ID NO:139, or SEQ ID NO:141 at its N-terminus.

[0675] B34. The antigen-binding molecule according to any one of embodiments B18-B33, wherein the antigen-binding domains are connected to each other or to the Fc region via a connector.

[0676] Preferably, the connector comprises (G4S)n, where n is an integer equal to or greater than 1, such as 1, 2, 3, 4 or 5.

[0677] B35, the antigen-binding molecule of any one of embodiments B28-B34, wherein when the Fc region is connected at its N-terminus to the C-terminus or N-terminus of the VHH domain, the Fc region contains a complete hinge region, that is, at its N-terminus, the portion of the hinge region corresponding to the IgG1 Fc region E216-P230 (EU number), for example, the amino acid sequence shown in SEQ ID NO:139 or SEQ ID NO:141.

[0678] B36, the antigen-binding molecule of any one of embodiments B28-B35, wherein when the Fc region is connected at its N-terminus to the C-terminus of the Fab heavy chain, the Fc region includes a partial hinge region, that is, at its N-terminus, a portion of the hinge region corresponding to the E2221-P230 (EU number) of the IgG1 Fc region, for example, the amino acid sequence shown in SEQ ID NO:138.

[0679] C1, a multispecific antibody, is a bispecific antibody that specifically binds to the first and second epitopes of FOLR1. It contains...

[0680] The first polypeptide chain, which comprises or is composed of the following: Fab FOLR1 Light chain;

[0681] The second polypeptide chain, from the N-terminus to the C-terminus, comprises or consists of the following: Fab FOLR1 Heavy chain -Fc-VHH FOLR1 or VHH FOLR1 -Fab FOLR1 Heavy chain -Fc;

[0682] Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 And the light chain constant region CL, or composed of it;

[0683] Fab FOLR1 With VHH FOLR1 Combining different epitopes on FOLR1;

[0684] The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker of 5-15 amino acids in length, such as a linker peptide.

[0685] C2, the multispecific antibody of implementation plan C1, in which Fab FOLR1 It is a Fab as defined in any of the implementation schemes A21-A26 or B22-B27. FOLR1 .

[0686] C3. The multispecific antibody described in implementation scheme C1 or C2, wherein VHH FOLR1 It is a VHH antibody that specifically binds to FOLR1 as described in any one of the embodiments A1-A5.

[0687] C4. A multispecific antibody according to any one of embodiments C1-C3, wherein the Fc region is the Fc region as described in any one of embodiments A29-A32 or B28-B31.

[0688] C5. The multispecific antibody according to any one of embodiments C1-C4, wherein the Fc region includes a partial hinge region, that is, a portion of the hinge region at its N-terminus corresponding to the E2221-P230 (EU number) of the IgG1 Fc region, for example, the amino acid sequence shown in SEQ ID NO:138; and is directly linked to the Fab heavy chain.

[0689] C6. A multispecific antibody according to any one of embodiments C1-C5, wherein when VHH FOLR1 Domains and Fab FOLR1 When the N-terminus of the heavy chain or the C-terminus of the Fc region is connected, the linker preferably contains a G4S amino acid sequence or a (G4S)2 amino acid sequence or a (G4S)3 amino acid sequence.

[0690] C7. The multispecific antibody according to any one of embodiments C1-C6, wherein VHH FOLR1 The structural domain contains or is composed of heavy chain variable regions, which contain the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NO:27; preferably, the CDR sequence is defined according to ABM.

[0691] C8. A multispecific antibody according to any one of embodiments C1-C7, wherein VHH FOLR1 The domain contains or is composed of a heavy chain variable region, wherein the heavy chain variable region contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or contains or is composed of a heavy chain variable region, wherein the heavy chain variable region contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:10, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:17, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:12.

[0692] C9. The multispecific antibody according to any one of embodiments C1-C8, wherein VHH FOLR1 The domain contains or is composed of a heavy chain variable region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:27.

[0693] C10, the multispecific antibody according to any one of embodiments C1-C9, wherein the Fab FOLR1The device comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:74; HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:75; HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:76; LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:78; LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:132; and LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:80.

[0694] C11. A multispecific antibody according to any one of embodiments C1-C10, wherein the Fab FOLR1 It comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:73, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:85.

[0695] C12, the multispecific antibody according to any one of embodiments C1-C9, wherein the Fab FOLR1 The device comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:74; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:75; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:76; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:78; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:133; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:80.

[0696] C13, the multispecific antibody according to any one of embodiments C1-C9 and C12, wherein the Fab FOLR1 It comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:73, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:140.

[0697] C14. A multispecific antibody according to any one of embodiments C1-C13, wherein the Fab FOLR1 Contains CH1, for example, where CH1 is CH1 of IgG1, preferably containing or consisting of the amino acid sequence of SEQ ID NO:135.

[0698] C15. A multispecific antibody according to any one of embodiments C1-C14, wherein the Fab FOLR1 It contains a light chain constant region CL, for example, wherein the CL is a Kappa light chain constant region, preferably containing or consisting of the amino acid sequence shown in SEQ ID NO:127.

[0699] C16. The multispecific antibody according to any one of embodiments C1-C15 comprises two first polypeptide chains and two second polypeptide chains or is composed of two first polypeptide chains and two second polypeptide chains, preferably, the two first polypeptide chains are the same and the two second polypeptide chains are the same.

[0700] C17. The multispecific antibody according to any one of embodiments C1-C16, wherein the first polypeptide chain and the second polypeptide chain are selected from the group consisting of:

[0701] (i) comprising, respectively, an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences shown in SEQ ID NOs: 93 and 134, or a first or second polypeptide chain composed of said amino acid sequences, or

[0702] (ii) An amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequences shown in SEQ ID NOs: 93 and 101, or a first or second polypeptide chain composed of said amino acid sequences.

[0703] C18. A multispecific antibody according to any one of embodiments C1-C17, wherein the first polypeptide chain and the second polypeptide chain are selected from the group consisting of:

[0704] (i) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93 and 134, or first and second polypeptide chains composed of said amino acid sequences; or

[0705] (ii) comprising the amino acid sequences shown in SEQ ID NOs: 93 and 101, respectively, or the first and second polypeptide chains composed of said amino acid sequences.

[0706] D1. Multispecific antibody, which is a bispecific antibody that specifically binds to the first and second epitopes of FOLR1, and contains or consists of the following:

[0707] The first polypeptide chain, which comprises or is composed of the following: Fab FOLR1 Light chain;

[0708] The second polypeptide chain: from the N-terminus to the C-terminus, it comprises or consists of the following: Fab FOLR1 Heavy chain - first Fc;

[0709] The third polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of the following: VHH FOLR1 -Second Fc or VHH FOLR1 -VHH FOLR1 -Second Fc;

[0710] Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 And the light chain constant region CL, or composed of it;

[0711] Fab FOLR1 With VHH FOLR1 Combining different epitopes on FOLR1;

[0712] The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker of 5-15 amino acids in length, such as a linker peptide.

[0713] D2, the multispecific antibody of implementation plan D1, in which Fab FOLR1 It is a Fab as defined in any of the implementation schemes A21-A26 or B22-B27. FOLR1 .

[0714] D3. The multispecific antibody described in implementation scheme D1 or D2, wherein VHH FOLR1 It is a VHH antibody that specifically binds to FOLR1 as described in any one of the embodiments A1-A5.

[0715] D4. A multispecific antibody according to any one of embodiments D1-D3, wherein the Fc region is the Fc region as described in any one of embodiments A29-A34 or B28-B33.

[0716] D5. A multispecific antibody according to any one of embodiments D1-D4, wherein the first Fc region includes a partial hinge region and is directly linked to the Fab heavy chain, and the second Fc region includes a complete hinge region (preferably the complete hinge region includes a C220S mutation) and is linked to VHH FOLR1 Direct connection;

[0717] Partial hinge regions correspond to portions of the IgG1 Fc region E2221-P230 (EU number), such as the amino acid sequence shown in SEQ ID NO:138; and complete hinge regions correspond to portions of the IgG1 Fc region E216-P230 (EU number), such as the amino acid sequences shown in SEQ ID NO:139 or SEQ ID NO:141.

[0718] D6. A multispecific antibody according to any one of embodiments D1-D6, wherein a Knob mutation such as T366W, L368A, or Y407V mutation is introduced in the first Fc region or a Hole mutation such as T366Y / Y407T mutation is introduced in the second Fc region, and optionally S354C / Y349C.

[0719] D7. A multispecific antibody according to any one of embodiments D1-D6, wherein the Fc region further comprises a mutation that reduces or eliminates Fcγ receptor binding, such as the LALA mutation.

[0720] D8. A multispecific antibody according to any one of embodiments D1-D7, wherein the first Fc region comprises or is composed of the amino acid sequence shown in SEQ ID NO:125, and the second Fc region comprises the amino acid sequence shown in SEQ ID NO:126, and the second Fc region further comprises the amino acid sequence shown in SEQ ID NO:139 or SEQ ID NO:141 at its N-terminus.

[0721] D9. A multispecific antibody according to any one of implementation schemes D1-D8, wherein two VHHs FOLR1 When connected, the connector preferably contains a G4S amino acid sequence or a (G4S)2 amino acid sequence or a (G4S)3 amino acid sequence.

[0722] D10, the multispecific antibody according to any one of the implementation schemes D1-D9, wherein VHH FOLR1 The structural domain contains or is composed of heavy chain variable regions, which contain the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NO:27; preferably, the CDR sequence is defined according to ABM.

[0723] D11. A multispecific antibody according to any one of the implementation schemes D1-D10, wherein VHH FOLR1 The domain contains or is composed of a heavy chain variable region, wherein the heavy chain variable region contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or contains or is composed of a heavy chain variable region, wherein the heavy chain variable region contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:10, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:17, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:12.

[0724] D12, the multispecific antibody according to any one of the implementation schemes D1-D11, wherein VHH FOLR1 The domain contains or is composed of a heavy chain variable region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:27.

[0725] D13. A multispecific antibody according to any one of embodiments D1-D12, wherein the Fab FOLR1 The device comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:74; HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:75; HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:76; LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:78; LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:132; and LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:80.

[0726] D14. A multispecific antibody according to any one of embodiments D1-D13, wherein the Fab FOLR1 It comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:73, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:85.

[0727] D15, the multispecific antibody according to any one of embodiments D1-D12, wherein the FabFOLR1 The device comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:74; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:75; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:76; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:78; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:133; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:80.

[0728] D16, the multispecific antibody according to any one of embodiments D1-D12 and D15, wherein the Fab FOLR1 It comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:73, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:140.

[0729] D17. A multispecific antibody according to any one of embodiments D1-D16, wherein the Fab FOLR1 Contains CH1, for example, where CH1 is CH1 of IgG1, preferably containing or consisting of the amino acid sequence of SEQ ID NO:135.

[0730] D18. A multispecific antibody according to any one of embodiments D1-D17, wherein the Fab FOLR1 It contains a light chain constant region CL, for example, wherein the CL is a Kappa light chain constant region, preferably containing or consisting of the amino acid sequence shown in SEQ ID NO:127.

[0731] D19. A multispecific antibody according to any one of embodiments D1-D18, wherein the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain are selected from the group consisting of:

[0732] (i) comprising, respectively, an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences shown in SEQ ID NOs: 93, 102, and 103, or a first, second, and third polypeptide chain composed of said amino acid sequences, or

[0733] (ii) Each of the following amino acid sequences comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences shown in SEQ ID NOs: 93, 102, and 104, or a first, second, and third polypeptide chains composed of said amino acid sequences.

[0734] D20, the multispecific antibody according to any one of embodiments D1-D19, wherein the first polypeptide chain and the second polypeptide chain are selected from the group consisting of:

[0735] (i) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93, 102, and 103, or the first, second, and third polypeptide chains composed of said amino acid sequences; or

[0736] (ii) Each contains the amino acid sequences shown in SEQ ID NOs: 93, 102 and 104, or the first, second and third polypeptide chains composed of said amino acid sequences.

[0737] E1, a multispecific antibody, preferably a bispecific antibody, specifically binds to FOLR1 and MSLN, and contains...

[0738] The first polypeptide chain, which comprises or is composed of the following: Fab FOLR1 Light chain;

[0739] The second polypeptide chain, from the N-terminus to the C-terminus, comprises or consists of the following: Fab FOLR1 Heavy chain -Fc-VHH MSLN or VHH MSLN -Fab FOLR1 Heavy chain -Fc;

[0740] Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 and the light chain constant region CL, or composed of it,

[0741] The symbol "-" indicates a connection via a connector or a direct connection, preferably a connector with a length of 5-15 amino acids.

[0742] E2, a multispecific antibody, preferably a bispecific antibody, which specifically binds to FOLR1 and MSLN, and contains

[0743] The first polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of the following: Fab FOLR1 Light Chain - VHH MSLN or VHHMSLN -Fab FOLR1 Light chain;

[0744] The second polypeptide chain, from the N-terminus to the C-terminus, comprises or consists of the following: Fab FOLR1 Heavy chain -Fc;

[0745] Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 and the light chain constant region CL, or composed of it,

[0746] The symbol "-" indicates a connection via a connector or a direct connection, preferably a connector with a length of 5-15 amino acids.

[0747] E3, a multispecific antibody based on implementation scheme E1 or E2, wherein Fab FOLR1 It is a Fab as defined in any of the implementation schemes A21-A26 or B22-B27. FOLR1 .

[0748] E4. The multispecific antibody according to any one of implementation schemes E1-E3, wherein VHH MSLN It is a VHH antibody that specifically binds to MSLN as described in any one of embodiments B1-B4.

[0749] E5. A multispecific antibody according to any one of embodiments E1-E4, wherein the Fc region is the Fc region as described in any one of embodiments A29-A32 or B28-B31.

[0750] E6. The multispecific antibody according to any one of embodiments E1-E5, wherein the Fc region includes a partial hinge region, that is, a portion of the hinge region at its N-terminus corresponding to the IgG1 Fc region E2221-P230 (EU number), for example, the amino acid sequence shown in SEQ ID NO:138; and is directly linked to the Fab heavy chain.

[0751] E7. A multispecific antibody according to any one of embodiments E1-E6, wherein when VHH MSLN Domains and Fab FOLR1 When the N-terminus or C-terminus of the Fc region of the heavy or light chain is connected, the linker preferably contains a G4S amino acid sequence or a (G4S)2 amino acid sequence or a (G4S)3 amino acid sequence.

[0752] E8. The multispecific antibody according to any one of the implementation schemes E1-E7, wherein VHH MSLNThe structural domain contains or is composed of heavy chain variable regions, which contain the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NO: 51, 56 or 70; preferably, the CDR sequence is defined according to ABM.

[0753] E9. The multispecific antibody according to any one of the implementation schemes E1-E8, wherein VHH MSLN The domain contains or is composed of a heavy chain variable region, wherein the heavy chain variable region contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or contains or is composed of a heavy chain variable region, wherein the heavy chain variable region contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:30, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:31, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:46.

[0754] E10, the multispecific antibody according to any one of embodiments E1-E9, wherein VHH MSLN The domain contains or is composed of a heavy chain variable region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:51.

[0755] E11, the multispecific antibody according to any one of embodiments E1-E9, wherein VHH MSLN The domain contains or is composed of a heavy chain variable region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:56.

[0756] E12, the multispecific antibody according to any one of implementation schemes E1-E9, wherein VHH MSLN The domain contains or is composed of a heavy chain variable region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:70.

[0757] E13, the multispecific antibody according to any one of embodiments E1-E12, wherein the Fab FOLR1The device comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:74; HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:75; HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:76; LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:78; LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:132; and LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:80.

[0758] E14, the multispecific antibody according to any one of embodiments E1-E13, wherein the Fab FOLR1 It comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:73, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:85.

[0759] E15, the multispecific antibody according to any one of embodiments E1-E12, wherein the Fab FOLR1 The device comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:74; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:75; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:76; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:78; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:133; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:80.

[0760] E16, the multispecific antibody according to any one of embodiments E1-E12 and E15, wherein the Fab FOLR1 It comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:73, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:140.

[0761] E17, the multispecific antibody according to any one of embodiments E1-E16, wherein the Fab FOLR1 Contains CH1, for example, where CH1 is CH1 of IgG1, preferably containing or consisting of the amino acid sequence of SEQ ID NO:135.

[0762] E18, the multispecific antibody according to any one of embodiments E1-E17, wherein the Fab FOLR1 It contains a light chain constant region CL, for example, wherein the CL is a Kappa light chain constant region, preferably containing or consisting of the amino acid sequence shown in SEQ ID NO:127.

[0763] E19, the multispecific antibody according to any one of embodiments E1-E18, comprises two first polypeptide chains and two second polypeptide chains or is composed of two first polypeptide chains and two second polypeptide chains, preferably, the two first polypeptide chains are the same and the two second polypeptide chains are the same.

[0764] E20, the multispecific antibody according to any one of embodiments E1-E19, wherein the first polypeptide chain and the second polypeptide chain are selected from the group consisting of:

[0765] (i) comprising, respectively, an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences shown in SEQ ID NOs: 93 and 95, or a first and second polypeptide chain composed of said amino acid sequences,

[0766] (ii) comprising, respectively, an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences shown in SEQ ID NOs: 93 and 94, or a first and second polypeptide chain composed of said amino acid sequences,

[0767] (iii) A first and second polypeptide chains comprising amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences shown in SEQ ID NOs: 96 and 97, respectively.

[0768] (iv) First and second polypeptide chains comprising amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences shown in SEQ ID NOs: 98 and 97, respectively.

[0769] (v) comprising, respectively, an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences shown in SEQ ID NOs: 93 and 99, or a first or second polypeptide chain composed of said amino acid sequences, or

[0770] (vi) comprising an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequences shown in SEQ ID NOs: 93 and 100, or a first or second polypeptide chain composed of said amino acid sequences.

[0771] E21. A multispecific antibody according to any one of embodiments E1-E20, wherein the first polypeptide chain and the second polypeptide chain are selected from the group consisting of:

[0772] (i) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93 and 95, or the first and second polypeptide chains composed of said amino acid sequences.

[0773] (ii) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93 and 94, or the first and second polypeptide chains composed of said amino acid sequences.

[0774] (iii) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 96 and 97, or the first and second polypeptide chains composed of said amino acid sequences.

[0775] (iv) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 98 and 97, or the first and second polypeptide chains composed of said amino acid sequences.

[0776] (v) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93 and 99, or the first and second polypeptide chains composed of said amino acid sequences, or

[0777] (vi) Each contains the amino acid sequences shown in SEQ ID NOs: 93 and 100, or the first and second polypeptide chains composed of said amino acid sequences.

[0778] F1, a multispecific antibody, is a trispecific antibody that specifically binds to two different epitopes of FOLR1 and MSLN, and it contains...

[0779] The first polypeptide chain, which comprises or is composed of the following: Fab FOLR1 Light chain;

[0780] The second polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of the following: VHH FOLR1 -Fab FOLR1Heavy chain -Fc-VHH MSLN ;

[0781] Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 And the light chain constant region CL, or composed of it;

[0782] Fab FOLR1 With VHH FOLR1 Combining different epitopes on FOLR1;

[0783] The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker of 5-15 amino acids in length, such as a linker peptide or a portion of the hinge region.

[0784] F2, a multispecific antibody, is a trispecific antibody that specifically binds to two different epitopes of FOLR1 and MSLN, and it contains...

[0785] The first polypeptide chain, which comprises or is composed of the following: Fab FOLR1 Light chain;

[0786] The second polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of the following: VHH MSLN -Fab FOLR1 Heavy chain -Fc-VHH FOLR1 ;

[0787] Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 And the light chain constant region CL, or composed of it;

[0788] Fab FOLR1 With VHH FOLR1 Combining different epitopes on FOLR1;

[0789] The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker of 5-15 amino acids in length, such as a linker peptide or a portion of the hinge region.

[0790] F3, a multispecific antibody based on implementation scheme F1 or F2, wherein Fab FOLR1 It is a Fab as defined in any of the implementation schemes A21-A26 or B22-B27. FOLR1 .

[0791] F4. A multispecific antibody according to any one of implementation schemes F1-F3, wherein VHH MSLN It is a VHH antibody that specifically binds to MSLN as described in any one of embodiments B1-B4.

[0792] F5. A multispecific antibody according to any one of implementation schemes F1-F4, wherein VHH FOLR1 It is a VHH antibody that specifically binds to FOLR1 as described in any one of embodiments A1-A4.

[0793] F6. A multispecific antibody according to any one of embodiments F1-F5, wherein the Fc region is the Fc region as described in any one of embodiments A29-A34 or B28-B33.

[0794] F7. A multispecific antibody according to any one of embodiments F1-F6, wherein the Fc region includes a partial hinge region, that is, a portion of the hinge region at its N-terminus corresponding to the E2221-P230 (EU number) of the IgG1 Fc region, for example, the amino acid sequence shown in SEQ ID NO:138; and is directly linked to the Fab heavy chain.

[0795] F8. A multispecific antibody according to any one of embodiments F1-F7, wherein when VHH FOLR1 Domains and Fab FOLR1 When the N-terminus of the heavy chain or the C-terminus of the Fc region is connected, the linker preferably contains a G4S amino acid sequence or a (G4S)2 amino acid sequence or a (G4S)3 amino acid sequence.

[0796] F9, the multispecific antibody according to any one of embodiments F1-F8, wherein when VHH MSLN Domains and Fab FOLR1 When the N-terminus of the heavy chain or the C-terminus of the Fc region is connected, the linker preferably contains a G4S amino acid sequence or a (G4S)2 amino acid sequence or a (G4S)3 amino acid sequence.

[0797] F10, the multispecific antibody according to any one of embodiments F1-F9, wherein VHH FOLR1 The structural domain contains or is composed of heavy chain variable regions, which contain the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NO:27; preferably, the CDR sequence is defined according to ABM.

[0798] F11, the multispecific antibody according to any one of embodiments F1-F10, wherein VHH FOLR1The domain contains or is composed of a heavy chain variable region, wherein the heavy chain variable region contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or contains or is composed of a heavy chain variable region, wherein the heavy chain variable region contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:10, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:17, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:12.

[0799] F12, the multispecific antibody according to any one of embodiments F1-F11, wherein VHH FOLR1 The domain contains or is composed of a heavy chain variable region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:27.

[0800] F13, the multispecific antibody according to any one of embodiments F1-F12, wherein VHH MSLN The structural domain contains or is composed of heavy chain variable regions, which contain the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NO: 51, 56 or 70; preferably, the CDR sequence is defined according to ABM.

[0801] F14, the multispecific antibody according to any one of embodiments F1-F13, wherein VHH MSLN The domain contains or is composed of a heavy chain variable region, wherein the heavy chain variable region contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or contains or is composed of a heavy chain variable region, wherein the heavy chain variable region contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:30, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:31, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:46.

[0802] F15, the multispecific antibody according to any one of embodiments F1-F14, wherein VHH MSLN The domain contains or is composed of a heavy chain variable region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:51.

[0803] F16, the multispecific antibody according to any one of embodiments F1-F14, wherein VHH MSLN The domain contains or is composed of a heavy chain variable region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:56.

[0804] F17, the multispecific antibody according to any one of embodiments F1-F14, wherein VHH MSLN The domain contains or is composed of a heavy chain variable region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:70.

[0805] F18, a multispecific antibody according to any one of embodiments F1-F17, wherein the Fab FOLR1 The device comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:74; HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:75; HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:76; LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:78; LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:132; and LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:80.

[0806] F19, the multispecific antibody according to any one of embodiments F1-F18, wherein the Fab FOLR1 It comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:73, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:85.

[0807] F20, the multispecific antibody according to any one of embodiments F1-F17, wherein the Fab FOLR1The device comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:74; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:75; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:76; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:78; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:133; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:80.

[0808] F21, a multispecific antibody according to any one of embodiments F1-F17 and F20, wherein the Fab FOLR1 It comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:73, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:140.

[0809] F22, A multispecific antibody according to any one of embodiments F1-F21, wherein the Fab FOLR1 Contains CH1, for example, where CH1 is CH1 of IgG1, preferably containing or consisting of the amino acid sequence of SEQ ID NO:135.

[0810] F23, the multispecific antibody according to any one of embodiments F1-F22, wherein the Fab FOLR1 It contains a light chain constant region CL, for example, wherein the CL is a Kappa light chain constant region, preferably containing or consisting of the amino acid sequence shown in SEQ ID NO:127.

[0811] F24. The multispecific antibody according to any one of embodiments F1-F23 comprises two first polypeptide chains and two second polypeptide chains or is composed of two first polypeptide chains and two second polypeptide chains, preferably, the two first polypeptide chains are the same and the two second polypeptide chains are the same.

[0812] F25, the multispecific antibody according to any one of embodiments F1-F24, wherein the first polypeptide chain and the second polypeptide chain respectively comprise an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequences shown in SEQ ID NOs: 93 and 105, or composed of said amino acid sequences.

[0813] F26, the multispecific antibody according to any one of embodiments F1-F25, wherein the first polypeptide chain and the second polypeptide chain respectively comprise the amino acid sequences shown in SEQ ID NOs: 93 and 105 or an amino acid sequence composed of said amino acid sequences.

[0814] G1, a multispecific antibody, is a trispecific antibody that specifically binds to two different epitopes of FOLR1 and MSLN, and contains or consists of the following chains:

[0815] The first polypeptide chain, which comprises or is composed of the following: Fab FOLR1 Light chain;

[0816] The second polypeptide chain: from the N-terminus to the C-terminus, it comprises or consists of the following: Fab FOLR1 Heavy chain - first Fc;

[0817] The third polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of the following: VHH FOLR1 -VHH MSLN -Second Fc;

[0818] Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 And the light chain constant region CL, or composed of it;

[0819] Fab FOLR1 With VHH FOLR1 Combining different epitopes on FOLR1;

[0820] The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker of 5-15 amino acids in length, such as a linker peptide.

[0821] G2, a multispecific antibody, is a trispecific antibody that specifically binds to two different epitopes of FOLR1 and MSLN, and contains or consists of the following chains:

[0822] The first polypeptide chain, which comprises or is composed of the following: Fab FOLR1 Light chain;

[0823] The second polypeptide chain: from the N-terminus to the C-terminus, it comprises or consists of the following: Fab FOLR1 Heavy chain - first Fc;

[0824] The third polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of the following: VHH MSLN -VHHFOLR1 -Second Fc;

[0825] Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 And the light chain constant region CL, or composed of it;

[0826] Fab FOLR1 With VHH FOLR1 Combining different epitopes on FOLR1;

[0827] The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker of 5-15 amino acids in length, such as a linker peptide.

[0828] G3, a multispecific antibody based on implementation plan G1 or G2, wherein Fab FOLR1 It is a Fab as defined in any of the implementation schemes A21-A26 or B22-B27. FOLR1 .

[0829] G4, the multispecific antibody described in implementation schemes G1-G3, wherein VHH FOLR1 It is a VHH antibody that specifically binds to FOLR1 as described in any one of embodiments A1-A4.

[0830] G5, the multispecific antibody described in implementation schemes G1-G4, wherein VHH MSLN It is a VHH antibody that specifically binds to MSLN as described in any one of embodiments B1-B4.

[0831] G6. A multispecific antibody according to any one of embodiments G1-G4, wherein the Fc region is the Fc region as described in any one of embodiments A29-A34 or B28-B33.

[0832] G7. A multispecific antibody according to any one of embodiments G1-G6, wherein the first Fc region includes a partial hinge region and is directly linked to the Fab heavy chain, and the second Fc region includes a complete hinge region (preferably the complete hinge region includes a C220S mutation) and is linked to VHH FOLR1 or VHH MSLN Direct connection;

[0833] Partial hinge regions correspond to portions of the IgG1 Fc region E2221-P230 (EU number), such as the amino acid sequence shown in SEQ ID NO:138; and complete hinge regions correspond to portions of the IgG1 Fc region E216-P230 (EU number), such as the amino acid sequences shown in SEQ ID NO:139 or SEQ ID NO:141.

[0834] G8. A multispecific antibody according to any one of embodiments G1-G7, wherein a Knob mutation such as T366W, L368A, or Y407V mutation is introduced in the first Fc region or a Hole mutation such as T366Y / Y407T mutation is introduced in the second Fc region, and optionally S354C / Y349C.

[0835] G9. A multispecific antibody according to any one of embodiments G1-G8, wherein the Fc region further comprises a mutation that reduces or eliminates Fcγ receptor binding, such as the LALA mutation.

[0836] G10, the multispecific antibody according to any one of embodiments G1-G9, wherein the first Fc region comprises or is composed of the amino acid sequence shown in SEQ ID NO:125, and the second Fc region comprises the amino acid sequence shown in SEQ ID NO:126, and the second Fc region further contains the amino acid sequence shown in SEQ ID NO:139 or SEQ ID NO:141 at its N-terminus.

[0837] G11, the multispecific antibody according to any one of embodiments G1-G10, wherein VHH FOLR1 and VHH MSLN When connected, the connector preferably contains a G4S amino acid sequence or a (G4S)2 amino acid sequence or a (G4S)3 amino acid sequence.

[0838] G12, the multispecific antibody according to any one of embodiments G1-G11, wherein VHH MSLN The structural domain contains or is composed of heavy chain variable regions, which contain the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NO: 51, 56 or 70; preferably, the CDR sequence is defined according to ABM.

[0839] G13, the multispecific antibody according to any one of embodiments G1-G12, wherein VHH MSLNThe domain contains or is composed of a heavy chain variable region, wherein the heavy chain variable region contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or contains or is composed of a heavy chain variable region, wherein the heavy chain variable region contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:30, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:31, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:46.

[0840] G14, the multispecific antibody according to any one of embodiments G1-G13, wherein VHH MSLN The domain contains or is composed of a heavy chain variable region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:51.

[0841] G15, the multispecific antibody according to any one of embodiments G1-G13, wherein VHH MSLN The domain contains or is composed of a heavy chain variable region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:56.

[0842] G16, the multispecific antibody according to any one of embodiments G1-G13, wherein VHH MSLN The domain contains or is composed of a heavy chain variable region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:70.

[0843] G17, the multispecific antibody according to any one of embodiments G1-G16, wherein the Fab FOLR1 The device comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:74; HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:75; HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:76; LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:78; LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:132; and LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:80.

[0844] G18, a multispecific antibody according to any one of embodiments G1-G17, wherein the Fab FOLR1 It comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:73, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:85.

[0845] G19, the multispecific antibody according to any one of embodiments G1-G16, wherein the Fab FOLR1 The device comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:74; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:75; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:76; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:78; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:133; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:80.

[0846] G20, a multispecific antibody according to any one of embodiments G1-G16 and G19, wherein the Fab FOLR1 It comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:73, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:140.

[0847] G21, A multispecific antibody according to any one of embodiments G1-G20, wherein the Fab FOLR1 Contains CH1, for example, where CH1 is CH1 of IgG1, preferably containing or consisting of the amino acid sequence of SEQ ID NO:135.

[0848] G22, A multispecific antibody according to any one of embodiments G1-G21, wherein the Fab FOLR1 It contains a light chain constant region CL, for example, wherein the CL is a Kappa light chain constant region, preferably containing or consisting of the amino acid sequence shown in SEQ ID NO:127.

[0849] G23, the multispecific antibody according to any one of implementation schemes G1-G22, wherein VHHFOLR1 The structural domain contains or is composed of heavy chain variable regions, which contain the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NO:27; preferably, the CDR sequence is defined according to ABM.

[0850] G24, the multispecific antibody according to any one of implementation schemes G1-G23, wherein VHH FOLR1 The domain contains or is composed of a heavy chain variable region, wherein the heavy chain variable region contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or contains or is composed of a heavy chain variable region, wherein the heavy chain variable region contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:10, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:17, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:12.

[0851] G25, the multispecific antibody according to any one of embodiments G1-G24, wherein VHH FOLR1 The domain contains or is composed of a heavy chain variable region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:27.

[0852] G26. A multispecific antibody according to any one of embodiments G1-G25, wherein the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain are selected from the group consisting of:

[0853] (i) Each of the following amino acid sequences comprises the amino acid sequences shown in SEQ ID NOs: 111, 102 and 112, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with them, or first, second and third polypeptide chains composed of said amino acid sequences;

[0854] (ii) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93, 102, and 106, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first, second, and third polypeptide chains composed of said amino acid sequences, or

[0855] (iii) Each of the amino acid sequences represented by SEQ ID NOs: 93, 102 and 108, or the amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with them, or the first, second and third polypeptide chains composed of said amino acid sequences.

[0856] G27. A multispecific antibody according to any one of embodiments G1-G26, wherein the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain are selected from the group consisting of:

[0857] (i) Each contains the amino acid sequences shown in SEQ ID NOs: 111, 102 and 112, or the first, second and third polypeptide chains composed of said amino acid sequences;

[0858] (ii) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93, 102, and 106, or the first, second, and third polypeptide chains composed of said amino acid sequences, or

[0859] (iii) Each contains the amino acid sequences shown in SEQ ID NOs: 93, 102 and 108, or the first, second and third polypeptide chains composed of said amino acid sequences.

[0860] H1, a multispecific antibody, is a trispecific antibody that specifically binds to two different epitopes of FOLR1 and MSLN, and contains or consists of the following chains:

[0861] The first polypeptide chain, which comprises or is composed of the following: Fab FOLR1 Light chain;

[0862] The second polypeptide chain: from its N-terminus to its C-terminus, it comprises or consists of the following: VHH FOLR1 -Fab FOLR1 Heavy chain - first Fc;

[0863] The third polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of the following: VHH MSLN -Fab FOLR1 Heavy chain - second Fc; and

[0864] The fourth polypeptide chain, which comprises or is composed of the following: Fab FOLR1 Light chain;

[0865] Preferably, the first polypeptide chain and the fourth polypeptide chain are identical;

[0866] Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 And the light chain constant region CL, or composed of it;

[0867] Fab FOLR1 With VHH FOLR1 Combining different epitopes on FOLR1;

[0868] The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker of 5-15 amino acids in length, such as a linker peptide.

[0869] H2, the multispecific antibody of implementation scheme H1, wherein Fab FOLR1 It is a Fab as defined in any of the implementation schemes A21-A26 or B22-B27. FOLR1 .

[0870] H3, the multispecific antibody described in implementation scheme H1 or H2, wherein VHH MSLN It is a VHH antibody that specifically binds to MSLN as described in any one of embodiments B1-B4.

[0871] H4. The multispecific antibody according to any one of embodiments H1-H3, wherein the VHH FOLR1 It is a VHH antibody that specifically binds to FOLR1 as described in any one of embodiments A1-A4.

[0872] H5, the multispecific antibody according to any one of embodiments H1-H4, wherein the Fc region is the Fc region according to any one of embodiments A29-A34 or B28-B33.

[0873] H6. The multispecific antibody according to any one of embodiments H1-H5, wherein the first Fc region and the second Fc region include a partial hinge region, that is, the portion of the hinge region corresponding to IgG1 Fc region E2221-P230 (EU number) at its N-terminus, for example, the amino acid sequence shown in SEQ ID NO:138; and is directly linked to the Fab heavy chain.

[0874] H7, the multispecific antibody according to any one of embodiments H1-H6, wherein when VHH MSLN Domain or VHH FOLR1 With Fab FOLR1 When the N-terminus of the heavy chain is connected, the linker preferably contains a G4S amino acid sequence or a (G4S)2 amino acid sequence or a (G4S)3 amino acid sequence.

[0875] H8, the multispecific antibody according to any one of the implementation schemes H1-H7, wherein VHH MSLNThe structural domain contains or is composed of heavy chain variable regions, which contain the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NO: 51, 56 or 70; preferably, the CDR sequence is defined according to ABM.

[0876] H9, the multispecific antibody according to any one of the implementation schemes H1-H8, wherein VHH MSLN The domain contains or is composed of a heavy chain variable region, wherein the heavy chain variable region contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or contains or is composed of a heavy chain variable region, wherein the heavy chain variable region contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:30, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:31, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:46.

[0877] H10, the multispecific antibody according to any one of embodiments H1-H9, wherein VHH MSLN The domain contains or is composed of a heavy chain variable region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:51.

[0878] H11, the multispecific antibody according to any one of the implementation schemes H1-H9, wherein VHH MSLN The domain contains or is composed of a heavy chain variable region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:56.

[0879] H12, the multispecific antibody according to any one of the implementation schemes H1-H9, wherein VHH MSLN The domain contains or is composed of a heavy chain variable region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:70.

[0880] H13, the multispecific antibody according to any one of embodiments H1-H12, wherein the Fab FOLR1The device comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:74; HCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:75; HCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:76; LCDR1 comprises or is composed of the amino acid sequence of SEQ ID NO:78; LCDR2 comprises or is composed of the amino acid sequence of SEQ ID NO:132; and LCDR3 comprises or is composed of the amino acid sequence of SEQ ID NO:80.

[0881] H14, the multispecific antibody according to any one of embodiments H1-H13, wherein the Fab FOLR1 It comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:73, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:85.

[0882] H15, the multispecific antibody according to any one of embodiments H1-H12, wherein the Fab FOLR1 The device comprises heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:74; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:75; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:76; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:78; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:133; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:80.

[0883] H16, the multispecific antibody according to any one of embodiments H1-H12 and H15, wherein the Fab FOLR1 It comprises a heavy chain variable region VH and a light chain variable region VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:73, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:140.

[0884] H17, the multispecific antibody according to any one of embodiments H1-H16, wherein the Fab FOLR1 Contains CH1, for example, where CH1 is CH1 of IgG1, preferably containing or consisting of the amino acid sequence of SEQ ID NO:135.

[0885] H18, the multispecific antibody according to any one of embodiments H1-H17, wherein the Fab FOLR1 It contains a light chain constant region CL, for example, wherein the CL is a Kappa light chain constant region, preferably containing or consisting of the amino acid sequence shown in SEQ ID NO:127.

[0886] H19, the multispecific antibody according to any one of embodiments H1-H18, wherein VHH FOLR1 The structural domain contains or is composed of heavy chain variable regions, which contain the three complementary determinant regions (CDRs) contained in the VH shown in SEQ ID NO:27; preferably, the CDR sequence is defined according to ABM.

[0887] H20, the multispecific antibody according to any one of the implementation schemes H1-H19, wherein VHH FOLR1 The domain contains or is composed of a heavy chain variable region, wherein the heavy chain variable region contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or contains or is composed of a heavy chain variable region, wherein the heavy chain variable region contains complementation-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; wherein VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:10, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:17, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:12.

[0888] H21, the multispecific antibody according to any one of embodiments H1-H20, wherein VHH FOLR1 The domain contains or is composed of a heavy chain variable region, which contains or is composed of the amino acid sequence shown in SEQ ID NO:27.

[0889] H22, the multispecific antibody according to any one of embodiments H1-H21, wherein the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain respectively comprise an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences shown in SEQ ID NOs: 93, 109, 110, and 93, or a first, second, third, and fourth polypeptide chain composed of said amino acid sequences.

[0890] H23, the multispecific antibody according to any one of embodiments H1-H22, wherein the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain respectively comprise the amino acid sequences shown in SEQ ID NOs: 93, 109, 110, and 93, or the first, second, third, and fourth polypeptide chains composed of said amino acid sequences.

[0891] J1, an antibody or antigen-binding fragment specifically binding to FOLR1, comprising HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, wherein...

[0892] HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:74; HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:75; HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:76; LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:78; LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:132, 79, 82, 84, 87, 89, 92 or 107; and / or LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:80.

[0893] J2, an antibody or antigen-binding fragment that specifically binds to FOLR1, comprising a heavy chain variable region and a light chain variable region, wherein...

[0894] The light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:77, 81, 83, 85, 86, 88, 90 or 91, and the heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:73.

[0895] J3, the antibody or antigen-binding fragment thereof described in embodiment J1 or J2, further comprising a heavy chain constant region and / or a light chain constant region, for example

[0896] The heavy chain constant region is derived from the constant region of IgG1, IgG2, IgG3, or IgG4, such as the constant region of human IgG1, IgG2, IgG3, or IgG4. For example, the heavy chain constant region or Fc region contains a mutation that reduces binding to the Fcγ receptor, such as the L234A / L235A mutation; or

[0897] The light chain constant region is a lambda or Kappa light chain constant region, such as a human lambda or Kappa light chain constant region. Preferably, the light chain constant region is a Kappa light chain constant region.

[0898] J4. The antibody or antigen-binding fragment thereof as described in any one of embodiments J1-J3, wherein the antigen-binding fragment is an antibody fragment selected from the following: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, dAb (domain antibody), bivalent antibody, or linear antibody.

[0899] K1, a nucleic acid molecule encoding any one of the antibodies described in any one of embodiments A1-A17 and B1-B17, the antigen-binding molecules described in any one of embodiments A18-37 and B18-B36, the multispecific antibodies described in any one of embodiments C1-C18, D1-D20, E1-E21, F1-F26, G1-G27 and H1-H20, or any one of the antibodies described in any one of embodiments J1-J4, or any chain thereof, or encoding any one of the antibodies described in any one of embodiments A1-A17 and B1-B17, the antigen-binding molecules described in any one of embodiments A18-37 and B18-B36, the multispecific antibodies described in any one of embodiments C1-C18, D1-D20, E1-E21, F1-F26, G1-G27 and H1-H20, or any one of the antibodies described in any one of embodiments J1-J4, or any one of the antigen-binding fragments thereof.

[0900] K2, an expression vector comprising the nucleic acid molecule of embodiment K1, preferably pCDNA, such as pCDNA3.4.

[0901] K3, a host cell comprising the nucleic acid molecule described in embodiment K1 or the expression vector described in embodiment K2, preferably, the host cell is prokaryotic or eukaryotic, such as 293 cells or CHO cells.

[0902] K4. A method for preparing an antibody or antigen-binding fragment thereof from any one of embodiments A1-A17 and B1-B17, an antigen-binding molecule from any one of A18-37 and B18-B36, a multispecific antibody from any one of C1-C18, D1-D20, E1-E21, F1-F26, G1-G27 and H1-H23, or any one of J1-J4, the method comprising culturing a host cell containing the nucleic acid molecule from embodiment K1 or the expression vector from embodiment K2 under conditions suitable for chain expression of the antibody or antigen-binding molecule or the multispecific antibody or the antibody or the antigen-binding fragment thereof, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0903] K5, an immunoconjugate or immunofusion comprising any one of the antibodies described in embodiments A1-A17 and B1-B17, any one of the antigen-binding molecules described in embodiments A18-37 and B18-B36, any one of the multispecific antibodies described in embodiments C1-C18, D1-D20, E1-E21, F1-F26, G1-G27 and H1-H23, or any one of the antibodies described in embodiments J1-J4, or an antigen-binding fragment thereof.

[0904] K6, the antibody-drug conjugate shown in formula (I): Ab-(LD) p (I)

[0905] Or its pharmaceutically acceptable salts or solvates,

[0906] in:

[0907] Ab is any one of the antibodies in embodiments A1-A17 and B1-B17, any one of the antigen-binding molecules in embodiments A18-37 and B18-B36, any one of the multispecific antibodies in embodiments C1-C18, D1-D20, E1-E21, F1-F26, G1-G27 and H1-H23, or any one of the antibodies in embodiments J1-J4, or the antigen-binding fragment thereof;

[0908] L is the connector;

[0909] D is a drug, such as an anti-tumor compound; and

[0910] p is an integer selected from 1 to 16, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12.

[0911] K7. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment K6, wherein the antitumor compound is a cytotoxic agent, such as camptothecin or auroretinoin.

[0912] K8. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment K6, wherein D has the structure shown in formula (D-1a) or formula (D-1b):

[0913] Where R 1a Selected from H and C1-C6 alkyl groups;

[0914] R 2a Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and -SR 5a ;

[0915] R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ;and

[0916] R 4a and R 5a Independently selected from H and C1-C4 alkyl groups;

[0917] or

[0918] Where R 1b R 2b R 3b R 4b R 5b and R 8b Each was independently selected from C 1-8 Alkyl groups; such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl;

[0919] R 6b and R 7b Each was independently selected from C 1-8 Alkyl groups, such as methoxy, ethoxy, or propoxy;

[0920] R 9b Selected from C 1-8 Alkyl groups and COOH; for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; and

[0921] R 10b Selected from OH and H.

[0922] K9. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate as described in implementation scheme K8, wherein R 1a For H; R 2a It is a C1-C6 alkyl group; R 3a It is a halogen, preferably -F; R 4a It is a C1-C4 alkyl group, preferably ethyl;

[0923] R 1b R 4b and R 8b Each was independently selected from C 1-2 Alkyl; preferably methyl;

[0924] R 2b R 3b and R 5b Each was independently selected from C 3-4 alkyl;

[0925] R 6b and R 7b Each was independently selected from C 1-2 alkoxy groups; and

[0926] R 9b Selected from C 1-4 Alkyl and R 10b For OH; or R 9b It is COOH and R 10b For H.

[0927] K10. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment K8, wherein D has the structure shown in formula (D-2a) or formula (D-2b):

[0928] Where R 1a R 2a R 3a and R 4a As defined in implementation scheme K8 or M4; or

[0929] Where R 1b R 2b R 3b R 4b R 5b R 6b R 7b R 8b R 9b and R 10b As defined in implementation scheme K8 or K9.

[0930] K11. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvation according to embodiment K8, wherein D has the structure shown in formula (D-3a) or (D-3b):

[0931] K12. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment K8, wherein D has the structure shown in formula (D-4a) or (D-4b):

[0932] K13. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of embodiments K6-K12, wherein...

[0933] -L- has the following structure: -Z-L1-L2-L3-

[0934] in

[0935] Z is selected from Where m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7 or 8;

[0936] L1 is selected from non-existent, Where n1 and m1 are independently selected from 0 to 20 integers, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16;

[0937] L2 is a single amino acid or a peptide residue selected from 2-8 amino acids; and

[0938] L3 is selected from: Where X is selected from -NH-, -O-, and -S-; R 1c Selected from: C 1-8 Alkyl-, Halogenated C 1-8 Alkyl-, C 1-8 Alkyl group, halogen, nitro group, and cyano group; Su is selected from pentose, penturonic acid, hexose, and hexuronic acid; n5 is 0, 1, 2, or 3; n2 is 0, 1, 2, 3, or 4; and n3 and n4 are independently 1, 2, 3, 4, 5, or 6;

[0939] Preferably, L2 is selected from: -Ala-, -Val-, -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit--Arg-Asp-Val-Thr-, -Gly-Gly-Phe-Gly-.

[0940] K14. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment K13, wherein...

[0941] Z is selected from Where m is 1, 2, 3, 4, 5, 6, 7 or 8;

[0942] L1 is selected from non-existent, Where n1 is an integer independently selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0943] L3 is selected from: Where X is selected from -NH-, -O-, and -S-; R 1c Selected from: C 1-8 Alkyl-, Halogenated C 1-8 Alkyl-, C 1-8 Alkyl group, halogen, nitro group, and cyano group; Su is selected from pentose, penturonic acid, hexose, and hexuronic acid; n2 is 0, 1, 2, 3, or 4; n5 is 0, 1, 2, or 3; and n3 and n4 are independently 1, 2, 3, 4, 5, or 6.

[0944] K15. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment K14, wherein...

[0945] Z is selected from

[0946] L1 is selected from non-existent or

[0947] L2 is selected from -Gly-, -Val-Ala-, -Arg-Asp-Val-Thr-, and -Val-Cit-;

[0948] L3 is Among them, Su and R 1c As defined in implementation scheme K14 ； n2 and n5 are independently 0 or 1.

[0949] K16. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate as described in embodiment K15, wherein L3 is Where Su is as defined in implementation scheme K15

[0950] Preferably, Su is selected from

[0951] K17. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate as described in implementation scheme K16, wherein...

[0952] Su selected

[0953] K18. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvation as described in implementation scheme K17, wherein...

[0954] Su

[0955] Preferably, L3 is selected from:

[0956] K19. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate as described in embodiment K18, wherein,

[0957] Su

[0958] K20. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment K13, wherein,

[0959] -Z-L1-L2-L3- is selected from the following structures

[0960] K21. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to embodiment K6, wherein,

[0961] The antibody-drug conjugate is selected from...

[0962] Ab and p are as defined in implementation scheme K6.

[0963] K22. An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of embodiments K6-K21, wherein the antibody-drug conjugate has an average DAR of 2-10, for example 4-8.

[0964] K23. A pharmaceutical composition comprising an antibody according to any one of embodiments A1-A17 and B1-B17, an antigen-binding molecule according to any one of A18-37 and B18-B36, a multispecific antibody according to any one of C1-C18, D1-D20, E1-E21, F1-F26, G1-G27 and H1-H23, or an antibody according to any one of J1-J4 or an antigen-binding fragment thereof, or an immunoconjugate or immunofusion according to embodiment K5, or an antibody-drug conjugate according to any one of embodiments K6-K22 or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient.

[0965] K24. Use of the antibody according to any one of embodiments A1-A17 and B1-B17, the antigen-binding molecule according to any one of A18-37 and B18-B36, the multispecific antibody according to any one of C1-C18, D1-D20, E1-E21, F1-F26, G1-G27 and H1-H23, or the antibody or antigen-binding fragment thereof according to any one of J1-J4, or the immunoconjugate or immunofusion of embodiment K5, or the antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of embodiments K6-K22, or the pharmaceutical composition according to K23 for the preparation of a medicament.

[0966] K25. Use of the implementation scheme K24, wherein the drug is used to treat and / or prevent cancer in an individual.

[0967] K26. Use of implementation scheme K25, wherein the cancer is MSLN-positive cancer.

[0968] K27. Use of implementation scheme K24 or K25, wherein the cancer is a FOLR1 positive cancer.

[0969] K28. Use of any one of the implementation schemes K24-K26, wherein the cancer is MSLN and FOLR1 positive cancer.

[0970] K29. Use of an...

Claims

A VHH antibody that specifically binds to FOLR1, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region comprises the three complementarity-determining regions (CDRs) contained in a VH as shown in any one of SEQ ID NO: 27, 1, 5, 7, 9, 13, 16, 20, 22-26, 28, 129, 130, 131; preferably, the CDR sequence is defined according to ABM. A VHH antibody that specifically binds to FOLR1, comprising complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or comprising or consisting of a heavy chain variable region. The heavy chain variable region includes complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; among which (i) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:10, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:17, 11, 18, 19, 21 or 41, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:

12. (ii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:2, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:3, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:4, 6, or 8; or (iii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:14, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:15, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:

12. The VHH antibody of claim 1 or 2, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region (i) comprising or consisting of 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 any one of SEQ ID NO: 27, 1, 5, 7, 9, 13, 16, 20, 22-26, 28, 129, 130, 131; or (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO: 27, 1, 5, 7, 9, 13, 16, 20, 22-26, 28, 129, 130, 131. A heavy chain antibody that specifically binds to FOLR1, comprising the VHH antibody according to any one of claims 1-3. The heavy chain antibody of claim 4, comprising the VHH antibody of any one of claims 1-3 linked to an antibody constant region or Fc region, preferably, the antibody constant region or Fc region being derived from human IgG1, human IgG2, human IgG3, or human IgG4, optionally, the Fc region comprising a complete hinge region and directly linked to the VHH antibody, preferably, the Fc region comprising a C220S mutation; optionally, the Fc region comprising a mutation that reduces or eliminates the connection between the Fc region and the Fcγ receptor, such as the LALA mutation. The heavy chain antibody of claim 5, comprising the VHH antibody of any one of claims 1-3 linked to an antibody Fc region, wherein the Fc region is an Fc region derived from human IgG1 or IgG4, preferably, the Fc region... (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 122, 123, 124, 128, or 142-145; or (ii) Contains or consists of the amino acid sequence shown in any one of SEQ ID NO: 122, 123, 124, 128 or 142-145. The VHH antibody of any one of claims 1-3, or the heavy chain antibody of any one of claims 4-6, wherein the antibody is a fully human antibody. An antigen-binding molecule comprising the VHH antibody of any one of claims 1-3 and 7 or the heavy chain antibody of any one of claims 4-7, for example, the antigen-binding molecule being a multispecific antibody such as a bispecific antibody. A multispecific antibody that specifically binds to two different epitopes of FOLR1, comprising an antigen-binding domain of at least one first epitope of FOLR1 and an antigen-binding domain of at least one second epitope of FOLR1, and optionally comprising at least one antigen-binding domain of MSLN. Preferably, the antigen-binding domain that specifically binds to the first epitope is a VHH as defined in any one of claims 1-3, i.e., VHH. FOLR1 . The multispecific antibody of claim 9, wherein at least one antigen-binding domain that specifically binds to FOLR1 is a Fab fragment that specifically binds to FOLR1, i.e., Fab FOLR1 Preferably, the Fab fragment comprises a Fab heavy chain and a Fab light chain, wherein the Fab heavy chain comprises or is composed of VH-CH1, and the Fab light chain comprises or is composed of VL-CL. The multispecific antibody of claim 10, wherein the Fab FOLR1 It includes heavy chain variable regions HCDR1, HCDR2, and HCDR3, and light chain variable regions LCDR1, LCDR2, and LCDR3, among which (i) HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:74; HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:75; HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:76; LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:78; LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:132, 79, 82, 84, 87, 89, 92, or 107; and LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:80; or (ii) HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:74; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:75; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:76; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:78; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:133; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:

80. The multispecific antibody of claim 10 or 11, wherein the Fab FOLR1 It includes the heavy chain variable region VH and the light chain variable region VL, in which The VH comprises 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:73, or is composed of the amino acid sequence shown in SEQ ID NO:85, 140, 77, 81, 83, 86, 88, 90, or 91, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:85, 140, 77, 81, 83, 86, 88, 90, or 91, or is composed of the amino acid sequence shown in SEQ ID NO:85, 140, 77, 81, 83, 86, 88, 90, or 91. The multispecific antibody according to any one of claims 10-12, wherein the Fab FOLR1 Containing CH1, for example, wherein said CH1 is CH1 derived from (human) IgG1, IgG2, IgG3 or IgG4, preferably CH1 derived from IgG1, preferably, said CH1 (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:135; (ii) Contains or consists of the amino acid sequence of SEQ ID NO:

135. The multispecific antibody according to any one of claims 10-13, wherein the Fab FOLR1 It includes a light chain constant region CL, for example, wherein the CL is a Kappa light chain constant region or a Lambda light chain constant region, preferably, the CL is a Kappa light chain constant region. (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:127; (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:

127. The multispecific antibody according to any one of claims 9-14, wherein the antigen-binding domain that specifically binds to MSLN is the VHH that specifically binds to MSLN, i.e., VHH MSLN . The multispecific antibody of claim 15, wherein the VHH MSLN It contains or is composed of a heavy chain variable region, the heavy chain variable region comprising the three complementary determinant regions (CDRs) contained in the VH shown in any one of SEQ ID NO: 70, 29, 33, 37, 42-45, 47, 49, 51-69 and 71-72; preferably, the CDR sequence is defined according to ABM. The multispecific antibody of claim 15 or 16, wherein the VHH MSLN The CDRs include complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or they contain or are composed of heavy chain variable regions. The heavy chain variable region includes complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; among which (i) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:30, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:31, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:46, 32, 48 or 50. (ii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:34, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:35, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:36; or (iii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:38, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:39, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:

40. The multispecific antibody according to any one of claims 15-17, wherein the VHH MSLN Contains or is composed of heavy chain variable regions, said heavy chain variable regions (i) comprising or consisting of 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 any one of SEQ ID NO: 70, 29, 33, 37, 42-45, 47, 49, 51-69, and 71-72; or (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO: 70, 29, 33, 37, 42-45, 47, 49, 51-69 and 71-72. The multispecific antibody according to any one of claims 9-18, wherein the multispecific antibody further comprises an Fc region, for example, comprising two identical or different Fc regions. The multispecific antibody of claim 19, wherein the Fc region is human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc or human IgG4 Fc, for example, the Fc region comprises a complete hinge region or a partial hinge region, for example, the Fc region comprises an amino acid sequence as described in any one of SEQ ID NO: 122, 123, 128 or 142-145 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher identity with said amino acid sequence or is composed of said amino acid sequence, optionally, the Fc region comprises a mutation that reduces or eliminates the Fc region and the Fcγ receptor, such as the LALA mutation, for example, the Fc region containing said LALA mutation comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence shown in SEQ ID NO: 124, for example comprising SEQ ID NO: 122, 123, 128 or 142-145, for example, comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with said amino acid sequence, for example, comprising SEQ ID NO: 124, for example, comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with said amino acid sequence, for example, comprising SEQ ID NO: 122, 123, 128 or 142-145, for example, comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% identity with said amino acid sequence, for example, comprising SEQ ID The amino acid sequence shown in NO:124 or composed of it, optionally, the Fc region containing the LALA mutation also contains the amino acid sequence shown in SEQ ID NO:138 or SEQ ID NO:139 at its N-terminus. The multispecific antibody of claim 19 or 20, wherein the two Fc regions are different, and wherein the two Fc regions respectively contain a Knob mutation and a Hole mutation, for example, the Knob mutation T366W, and the Hole mutation T366S, L368A, and Y407V combination; or the Knob mutation is T366Y, and the Hole mutation is Y407T; optionally, the Fc region containing the Knob mutation further contains the S354C mutation, and the Fc region containing the Hole mutation further contains the Y349C mutation, for example, the Fc region containing the Knob mutation contains or is composed of the amino acid sequence shown in SEQ ID NO:125, while the Fc region containing the Hole mutation contains or is composed of the amino acid sequence shown in SEQ ID NO:126; optionally, the Fc region containing the Knob mutation and / or the Fc region containing the Hole mutation also contains the amino acid sequence shown in SEQ ID NO:138 or SEQ ID NO:139 at its N-terminus. The multispecific antibody according to any one of claims 9-21, wherein the antigen-binding domains are connected to each other, or to the Fc region, via a linker, for example, the linker comprising (G4S)n, where n is an integer equal to or greater than 1, such as 1, 2, 3, 4, or 5; when the Fc region is connected at its N-terminus to the C-terminus or N-terminus of the VHH domain, the Fc region comprises a complete hinge region, i.e., at its N-terminus, a hinge region corresponding to the portion of the IgG1 Fc region E216-P230 (EU number), for example, the amino acid sequence shown in SEQ ID NO: 139 or SEQ ID NO: 141; and / or when the Fc region is connected at its N-terminus to the C-terminus of the Fab heavy chain, the Fc region comprises a partial hinge region, i.e., at its N-terminus, a hinge region corresponding to the portion of the IgG1 Fc region E2221-P230 (EU number), for example, the amino acid sequence shown in SEQ ID NO:

138. The multispecific antibody according to any one of claims 9-22, which specifically binds to the first and second epitopes of FOLR1, is preferably a bispecific antibody comprising... The first polypeptide chain comprises or consists of the following: Fab FOLR1 Light chain; The second polypeptide chain, from the N-terminus to the C-terminus, comprises or consists of the following: Fab FOLR1 Heavy chain -Fc-VHH FOLR1 or VHH FOLR1 -Fab FOLR1 Heavy chain -Fc; Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 And the light chain constant region CL, or composed of it; Fab FOLR1 With VHH FOLR1 Combining different epitopes on FOLR1; The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker of 5-15 amino acids in length, such as a linker peptide. The multispecific antibody of claim 23, wherein the first polypeptide chain and the second polypeptide chain are selected from the group consisting of: (i) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93 and 134, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences, or (ii) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93 and 101, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences. The multispecific antibody according to any one of claims 9-22, which specifically binds to the first and second epitopes of FOLR1, is preferably a bispecific antibody, comprising or consisting of the following: The first polypeptide chain comprises or consists of the following: Fab FOLR1 Light chain; The second polypeptide chain: from the N-terminus to the C-terminus, it comprises or consists of the following: Fab FOLR1 Heavy chain - first Fc; The third polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of the following: VHH FOLR1 -Second Fc or VHH FOLR1 -VHH FOLR1 -Second Fc; Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 And the light chain constant region CL, or composed of it; Fab FOLR1 With VHH FOLR1 Combining different epitopes on FOLR1; The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker of 5-15 amino acids in length, such as a linker peptide. The multispecific antibody of claim 25, wherein the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain are selected from the group consisting of: (i) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93, 102, and 103, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first, second, and third polypeptide chains composed of said amino acid sequences, or (ii) Each of the amino acid sequences represented by SEQ ID NOs: 93, 102 and 104, or the amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with them, or the first, second and third polypeptide chains composed of said amino acid sequences. The multispecific antibody according to any one of claims 9-22 is a trispecific antibody that specifically binds to two different epitopes of FOLR1 and the MSLN, and comprises The first polypeptide chain comprises or consists of the following: Fab FOLR1 Light chain; The second polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of the following: VHH FOLR1 -Fab FOLR1 Heavy chain -Fc-VHH MSLN or VHH MSLN -Fab FOLR1 Heavy chain -Fc-VHH FOLR1 ; Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 And the light chain constant region CL, or composed of it; Fab FOLR1 With VHH FOLR1 Combining different epitopes on FOLR1; The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker of 5-15 amino acids in length, such as a linker for peptides or a portion of the hinge region; Preferably, the multispecific antibody comprises two first polypeptide chains and two second polypeptide chains or is composed of two first polypeptide chains and two second polypeptide chains. More preferably, the two first polypeptide chains are identical and the two second polypeptide chains are identical. The multispecific antibody of claim 27, wherein the first polypeptide chain and the second polypeptide chain respectively comprise amino acid sequences shown in SEQ ID NOs: 93 and 105 that have at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with them or amino acid sequences composed of said amino acid sequences. The multispecific antibody according to any one of claims 9-22 is a trispecific antibody that specifically binds to two different epitopes of FOLR1 and the MSLN, and comprises or consists of the following chains: The first polypeptide chain comprises or consists of the following: Fab FOLR1 Light chain; The second polypeptide chain: from the N-terminus to the C-terminus, it comprises or consists of the following: Fab FOLR1 Heavy chain - first Fc; The third polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of the following: VHH FOLR1 -VHH MSLN -Second Fc or VHH MSLN -VHH FOLR1 -Second Fc; Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 And the light chain constant region CL, or composed of it; Fab FOLR1 With VHH FOLR1 Combining different epitopes on FOLR1; The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker of 5-15 amino acids in length, such as a linker peptide. The multispecific antibody of claim 29, wherein the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain are selected from the group consisting of: (i) Each of the following amino acid sequences comprises the amino acid sequences shown in SEQ ID NOs: 111, 102 and 112, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with them, or first, second and third polypeptide chains composed of said amino acid sequences; (ii) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93, 102, and 106, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first, second, and third polypeptide chains composed of said amino acid sequences, or (iii) Each of the amino acid sequences represented by SEQ ID NOs: 93, 102 and 108, or the amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with them, or the first, second and third polypeptide chains composed of said amino acid sequences. The multispecific antibody according to any one of claims 9-22 is a trispecific antibody that specifically binds to two different epitopes of FOLR1 and the MSLN, and comprises or consists of the following chains: The first polypeptide chain comprises or consists of the following: Fab FOLR1 Light chain; The second polypeptide chain: from its N-terminus to its C-terminus, it comprises or consists of the following: VHH FOLR1 -Fab FOLR1 Heavy chain - first Fc; The third polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of the following: VHH MSLN -Fab FOLR1 Heavy chain - second Fc; and The fourth polypeptide chain, which comprises or is composed of the following: Fab FOLR1 Light chain; Preferably, the first polypeptide chain and the fourth polypeptide chain are identical; Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 And the light chain constant region CL, or composed of it; Fab FOLR1 With VHH FOLR1 Combining different epitopes on FOLR1; The symbol "-" indicates a connection via a linker or a direct connection, preferably a linker of 5-15 amino acids in length, such as a linker peptide. The multispecific antibody of claim 31, wherein the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain respectively comprise the amino acid sequences shown in SEQ ID NOs: 93, 109, 110, and 93, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or the first, second, third, and fourth polypeptide chains composed of said amino acid sequences. A VHH antibody that specifically binds to MSLN, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region comprises the three complementarity-determining regions (CDRs) contained in VH as shown in any one of SEQ ID NO: 70, 29, 33, 37, 42-45, 47, 49, 51-69 and 71-72; preferably, the CDR sequence is defined according to ABM. A VHH antibody that specifically binds to MSLN, comprising complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3, or comprising or consisting of a heavy chain variable region. The heavy chain variable region includes complementarity-determining regions (CDRs) VHH CDR1, VHH CDR2, and VHH CDR3; among which (i) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:30, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:31, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:46, 32, 48 or 50. (ii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:34, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:35, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:36; or (iii) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:38, VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:39, and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:

40. The VHH antibody of claim 33 or 34, comprising or consisting of a heavy chain variable region, wherein the heavy chain variable region (i) comprising or consisting of 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 any one of SEQ ID NO: 70, 29, 33, 37, 42-45, 47, 49, 51-69, and 71-72; or (ii) Contains or consists of an amino acid sequence selected from or composed of any one of SEQ ID NO: 70, 29, 33, 37, 42-45, 47, 49, 51-69 and 71-72. A heavy chain antibody that specifically binds to MSLN, comprising the VHH antibody according to any one of claims 33-35. The heavy chain antibody of claim 36, comprising the VHH antibody of any one of claims 33-35 linked to an antibody constant region or Fc region, preferably wherein the antibody constant region or Fc region is derived from human IgG1, human IgG2, human IgG3, or human IgG4, optionally wherein the Fc region comprises a complete hinge region and is directly linked to the VHH antibody, preferably wherein the Fc region comprises a C220S mutation; optionally, the Fc region comprises a mutation that reduces or eliminates the connection between the Fc region and the Fcγ receptor, such as the LALA mutation. The heavy chain antibody of claim 37, comprising the VHH antibody of any one of claims 1-3 linked to an antibody Fc region, wherein the Fc region is an Fc region derived from human IgG1 or IgG4, preferably, the Fc region... (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 122, 123, 124, 128, or 142-145; or (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:122, 123, 124, 128 or 142-145. The VHH antibody of any one of claims 33-35, or the heavy chain antibody of any one of claims 36-38, wherein the antibody is a chimeric antibody or a humanized antibody. An antigen-binding molecule comprising the VHH antibody of any one of claims 33-35 and 39 or the heavy chain antibody of any one of claims 36-39, for example, the antigen-binding molecule being a multispecific antibody such as a bispecific antibody. Multispecific antibodies, preferably bispecific antibodies, It specifically binds to FOLR1 and MSLN, and comprises at least one antigen-binding domain that specifically binds to FOLR1 and at least one antigen-binding domain that specifically binds to MSLN, wherein the antigen-binding domain that specifically binds to MSLN is a VHH as defined in any one of claims 33-35, i.e., VHH. MSLN ; Optionally, the antigen-binding domain that specifically binds to FOLR1 is the Fab domain as defined in any one of claims 10-14. FOLR1 . Optionally, it also includes an Fc region and an optional connector, such as the Fc region as defined in any one of claims 19-21, or the connector as defined in any one of claims 22. The multispecific antibody of claim 41, comprising: The first polypeptide chain comprises or consists of the following: Fab FOLR1 Light chain; The second polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of the following: Fab FOLR1 Heavy chain -Fc-VHH MSLN or VHH MSLN -Fab FOLR1 Heavy chain -Fc; or The first polypeptide chain, from its N-terminus to its C-terminus, comprises or consists of the following: Fab FOLR1 Light Chain - VHH MSLN or VHH MSLN -Fab FOLR1 Light chain; The second polypeptide chain, from the N-terminus to the C-terminus, comprises or consists of the following: Fab FOLR1 Heavy chain -Fc; Fab FOLR1 The heavy chain contains the VH region of the heavy chain variable region, which is an antibody that specifically binds to FOLR1. FOLR1 and CH1 or composed of it, and Fab FOLR1 The light chain contains the VL light chain variable region of an antibody that specifically binds to FOLR1. FOLR1 and the light chain constant region CL, or composed of it, The symbol "-" indicates a connection via a connector or a direct connection, preferably a connector with a length of 5-15 amino acids. The multispecific antibody of claim 42 comprises two first polypeptide chains and two second polypeptide chains or is composed of two first polypeptide chains and two second polypeptide chains, preferably, the two first polypeptide chains are identical and the two second polypeptide chains are identical. The multispecific antibody of claim 42 or 43, wherein the first polypeptide chain and the second polypeptide chain are selected from the group consisting of: (i) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93 and 95, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences, (ii) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93 and 94, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences, (iii) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 96 and 97, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences. (iv) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 98 and 97, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences, (v) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93 and 99, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences, or (vi) comprising, respectively, the amino acid sequences shown in SEQ ID NOs: 93 and 100, or amino acid sequences having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with them, or first and second polypeptide chains composed of said amino acid sequences. An antibody or its antigen-binding fragment that specifically binds to FOLR1, comprising HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, wherein... HCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:74; HCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:75; HCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:76; LCDR1 contains or is composed of the amino acid sequence of SEQ ID NO:78; LCDR2 contains or is composed of the amino acid sequence of SEQ ID NO:132, 79, 82, 84, 87, 89, 92 or 107; and / or LCDR3 contains or is composed of the amino acid sequence of SEQ ID NO:

80. An antibody or its antigen-binding fragment that specifically binds to FOLR1 contains a heavy chain variable region and a light chain variable region, wherein... The light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:77, 81, 83, 85, 86, 88, 90 or 91, and the heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:

73. The antibody or antigen-binding fragment thereof according to claim 45 or 46 further comprises a heavy chain constant region and / or a light chain constant region, for example... The heavy chain constant region is derived from the constant region of IgG1, IgG2, IgG3, or IgG4, such as the constant region of human IgG1, IgG2, IgG3, or IgG4. For example, the heavy chain constant region or Fc region contains a mutation that reduces binding to the Fcγ receptor, such as the L234A / L235A mutation; or The light chain constant region is a lambda or Kappa light chain constant region, such as a human lambda or Kappa light chain constant region. Preferably, the light chain constant region is a Kappa light chain constant region. The antibody or antigen-binding fragment thereof according to any one of claims 45-47, wherein the antigen-binding fragment is an antibody fragment selected from the following: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, dAb (domain antibody), bivalent antibody, or linear antibody. A nucleic acid molecule that encodes any one chain of the antibody according to any one of claims 1-7 and 33-39, or the antigen-binding molecule according to claim 8 or 40, or the multispecific antibody according to any one of claims 9-32 and 41-44, or the antibody according to any one of claims 45-48, or any chain thereof, or encodes any one chain of the antibody according to any one of claims 1-7 and 33-39, or the antigen-binding molecule according to claim 8 or 40, or the multispecific antibody according to any one of claims 9-32 and 41-44, or the antibody according to any one of claims 45-48, or any chain thereof. An expression vector comprising the nucleic acid molecule of claim 49, preferably, the expression vector being pCDNA, such as pCDNA3.

4. A host cell comprising the nucleic acid molecule of claim 49 or the expression vector of claim 50, preferably, the host cell being prokaryotic or eukaryotic, such as 293 cells or CHO cells. A method for preparing the antibody of any one of claims 1-7 and 33-39, or the antigen-binding molecule of claim 8 or 40, or the multispecific antibody of any one of claims 9-32 and 41-44, or the antibody of any one of claims 45-48, or an antigen-binding fragment thereof, the method comprising culturing a host cell containing the nucleic acid molecule of claim 49 or the expression vector of claim 50, under conditions suitable for chain expression of the antibody or antigen-binding molecule or the multispecific antibody or the antibody or the antigen-binding fragment thereof, and optionally recovering the antibody from the host cell (or host cell culture medium). An immunoconjugate or immunofusion comprising any antibody of any one of claims 1-7 and 33-39, or an antigen-binding molecule of any one of claims 8 or 40, or a multispecific antibody of any one of claims 9-32 and 41-44, or an antibody of any one of claims 45-48, or an antigen-binding fragment thereof. The antibody-drug conjugate shown in formula (I): Ab-(L-D) p (I) Or its pharmaceutically acceptable salts or solvates, in: Ab is the antibody according to any one of claims 1-7 and 33-39, or the antigen-binding molecule according to claim 8 or 40, or the multispecific antibody according to any one of claims 9-32 and 41-44, or the antibody or antigen-binding fragment thereof according to any one of claims 45-48; L is the connector; D is a drug, such as an anti-tumor compound; and p is an integer selected from 1 to 16, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12. The antibody-drug conjugate of claim 54 or a pharmaceutically acceptable salt or solvate thereof, wherein the antitumor compound is a cytotoxic agent, such as camptothecin or auratestatin. The antibody-drug conjugate according to claim 54, or a pharmaceutically acceptable salt or solvate thereof, wherein D has the structure shown in formula (D-1a) or formula (D-1b): Where R 1a Selected from H and C1-C6 alkyl groups; R 2a Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and -SR 5a ; R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ;and R 4a and R 5a Independently selected from H and C1-C4 alkyl groups; or Where R 1b R 2b R 3b R 4b R 5b and R 8b Each was independently selected from C 1-8 Alkyl groups; such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; R 6b and R 7b Each was independently selected from C 1-8 Alkyl groups, such as methoxy, ethoxy, or propoxy; R 9b Selected from C 1-8 Alkyl groups and COOH; for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; and R 10b Selected from OH and H. According to claim 56, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate thereof, wherein, R 1a For H; R 2a It is a C1-C6 alkyl group; R 3a It is a halogen, preferably -F; R 4a It is a C1-C4 alkyl group, preferably ethyl; R 1b R 4b and R 8b Each was independently selected from C 1-2 Alkyl; preferably methyl; R 2b R 3b and R 5b Each was independently selected from C 3-4 alkyl; R 6b and R 7b Each was independently selected from C 1-2 alkoxy groups; and R 9b Selected from C 1-4 Alkyl and R 10b For OH; or R 9b It is COOH and R 10b For H. According to claim 56, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate thereof, wherein, D has the structure shown in equation (D-2a) or equation (D-2b): Where R 1a R 2a R 3a and R 4a As defined in claim 56 or 57; or Where R 1b R 2b R 3b R 4b R 5b R 6b R 7b R 8b R 9b and R 10b As defined in claim 56 or 57. According to claim 56, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate thereof, wherein, D has the structure shown in formula (D-3a) or (D-3b): According to claim 56, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate thereof, wherein, D has the structure shown in formula (D-4a) or (D-4b): The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 54-60, wherein -L- has the following structure: -Z-L1-L2-L3- in Z is selected from Where m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7 or 8; L1 is selected from non-existent, Where n1 and m1 are independently selected from 0 to 20 integers, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16; L2 is a single amino acid or a peptide residue selected from 2-8 amino acids; and L3 is selected from: Where X is selected from -NH-, -O-, and -S-; R 1c Selected from: C 1-8 Alkyl-, Halogenated C 1-8 Alkyl-, C 1-8 Alkyl group, halogen, nitro group, and cyano group; Su is selected from pentose, penturonic acid, hexose, and hexuronic acid; n5 is 0, 1, 2, or 3; n2 is 0, 1, 2, 3, or 4; and n3 and n4 are independently 1, 2, 3, 4, 5, or 6; Preferably, L2 is selected from: -Ala-, -Val-, -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit--Arg-Asp-Val-Thr-, -Gly-Gly-Phe-Gly-. The antibody-drug conjugate according to claim 61, or a pharmaceutically acceptable salt or solvate thereof, wherein Z is selected from Where m is 1, 2, 3, 4, 5, 6, 7 or 8; L1 is selected from non-existent, Where n1 is an integer independently selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7 or 8; L3 is selected from: Where X is selected from -NH-, -O-, and -S-; R 1c Selected from: C 1-8 Alkyl-, Halogenated C 1-8 Alkyl-, C 1-8 Alkyl group, halogen, nitro group, and cyano group; Su is selected from pentose, penturonic acid, hexose, and hexuronic acid; n2 is 0, 1, 2, 3, or 4; n5 is 0, 1, 2, or 3; and n3 and n4 are independently 1, 2, 3, 4, 5, or 6. The antibody-drug conjugate according to claim 62, or a pharmaceutically acceptable salt or solvate thereof, wherein Z is selected from L1 is selected from non-existent or L2 is selected from -Gly-, -Val-Ala-, -Arg-Asp-Val-Thr-, and -Val-Cit-; L3 is Among them, Su and R 1c As defined in claim 62 ； n2 and n5 are independently 0 or 1. According to claim 63, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate thereof, wherein, L3 is Wherein Su is as defined in claim 63, Preferably, Su is selected from The antibody-drug conjugate according to claim 64, or a pharmaceutically acceptable salt or solvate thereof, wherein, Su selected The antibody-drug conjugate according to claim 65, or a pharmaceutically acceptable salt or solvate thereof, wherein, Su Preferably, L3 is selected from: The antibody-drug conjugate according to claim 66, or a pharmaceutically acceptable salt or solvate thereof, wherein, Su According to claim 61, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate thereof, wherein, -Z-L1-L2-L3- is selected from the following structures According to claim 54, the antibody-drug conjugate or its pharmaceutically acceptable salt or solvate thereof, wherein, The antibody-drug conjugate is selected from... Wherein Ab and p are as defined in claim 54. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 54-69, wherein, The antibody-drug conjugate has an average DAR of 2-10, for example, 4-8. A pharmaceutical composition comprising an antibody according to any one of claims 1-7 and 33-39, or an antigen-binding molecule according to claim 8 or 40, or a multispecific antibody according to any one of claims 9-32 and 41-44, or an antibody or antigen-binding fragment thereof according to any one of claims 45-48, or an immunoconjugate or immunofusion according to claim 53, or an antibody-drug conjugate according to any one of claims 54-70, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient. Use of the antibody according to any one of claims 1-7 and 33-39, or the antigen-binding molecule according to claim 8 or 40, or the multispecific antibody according to any one of claims 9-32 and 41-44, or the antibody according to any one of claims 45-48 or the antigen-binding fragment thereof, or the immunoconjugate or immunofusion of claim 53, or the antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to any one of claims 54-70, for the preparation of a medicament. The use of claim 72, wherein the drug is used to treat and / or prevent cancer in an individual, preferably the cancer being MSLN and / or FOLR1 positive cancer. The use of claim 73, wherein the cancer is selected from ovarian cancer, ovarian adenocarcinoma, peritoneal cancer, fallopian tube cancer, cervical cancer, breast cancer, mammary ductal carcinoma, mammary squamous cell carcinoma, triple-negative breast cancer, colorectal cancer, lung cancer, lung adenocarcinoma, pancreatic cancer, gastric cancer, gastroesophageal adenocarcinoma, bladder cancer, and endometrial cancer.

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