Antibody and drug conjugate and application thereof

Abstract

The present invention relates to antibodies and antibody-drug conjugates, and more particularly to antibodies and antibody-drug conjugates (ADCs) targeting TROP2 and / or NECTIN4 as well as compositions containing said antibodies or ADCs and therapeutic applications thereof.

Description

Technical Field

[0001] The present invention relates to antibodies and antibody-drug conjugates, and more particularly to antibodies and antibody-drug conjugates (ADCs) targeting TROP2 and / or NECTIN4, compositions containing said antibodies or ADCs, and their therapeutic applications. Background Art

[0002] Trop2 (trophoblast cell-surface antigen 2, human trophoblast cell surface glycoprotein antigen 2), also known as tumor-associated calcium signal transducer 2 (TACSTD2), epithelial glycoprotein 1 (EGP-1), gastrointestinal antigen 733-1 (GA733-1), membrane surface marker 1 (M1S1), is a type I cell surface glycoprotein encoded by the TACSTD2 gene. Trop2 is highly expressed in human cancers, and the signal pathway mediated by it mainly promotes the growth, proliferation and metastasis of tumor cells by regulating the calcium ion signal pathway, cyclin expression and reducing the adhesion of fibronectin. Therefore, Trop2 has become an attractive therapeutic target for cancer treatment. However, since Trop2 is expressed not only in various cancer tissues such as ovarian cancer, pancreatic cancer, gastric cancer, breast cancer, etc., but also in a large number of normal human tissues such as breast, cervix, prostate, skin, stomach, thymus, etc.; therefore, drugs targeting Trop2 have potential on-target toxicity. For example, when the skin and oral mucosa are greatly affected by the drug, side effects such as stomatitis and rash are likely to occur.

[0003] The only currently marketed Trop2 ADC drug is Gilead (sacituzumab govitecan; SG). In the ASCENT study, the incidence of adverse reactions above grade 3 caused by SG reached 45%, and the two side effects of neutropenia and diarrhea were even marked with a black box warning by the FDA; in addition, 9% of the patients had rashes and 5% of the patients had eye toxicity. Datopotamab Deruxtecan (DS-1062, Dato-DXd) is a Trop2-targeted Dxd antibody-drug conjugate (ADC) jointly developed by Daiichi Sankyo and AstraZeneca. To control toxicity, Dato-DXd uses a monoclonal antibody targeting Trop2 with medium affinity to connect with a less toxic DNA topoisomerase I inhibitor (Dx d), and controls the DAR value at 4. However, in the cynomolgus monkey toxicity experiment, due to the lung toxicity caused by Dxd, the HNSTD (highest non-severe toxicity dose) of Dato-DXd was only 10 mg / kg, which is lower than the HNSTD of 30 mg / kg of DS-8201 with a DAR value of 8 in the monkey toxicity study.

[0004] Nectin4 (Nectin cell adhesion molecule 4) is a type I transmembrane cell adhesion molecule belonging to the Nectin family. This protein was previously known as a homolog of the poliovirus receptor (PVR / CD155) and was also called the poliovirus receptor-related (PRR) protein. During physiological development, Nectin4 is specifically expressed during embryonic and fetal development and is expressed at very low levels in adult tissues. It forms physical connections between adjacent cells and is crucial for enabling intercellular communication, migration, and other important cellular processes. Nectin4 is overexpressed in a variety of tumor cells and is used as a marker for cancer recurrence and metastasis, and is associated with poor prognosis in a variety of cancers, including urothelial carcinoma, breast cancer, ovarian cancer, pancreatic cancer, non-small cell lung cancer, gastric cancer, hepatocellular carcinoma, and bladder cancer, etc.

[0005] Compared with popular targets such as HER2, EGFR, and Trop2, there are currently fewer targeted drugs for Nectin4, and there is only one ADC product globally on the market. (Enfortumab Vedotin; EV) is a "first-in-class" new drug jointly developed by Seagen and Astellas. However, since Nectin4 is expressed to some extent in normal skin tissue, Padcev carries a black box warning for severe skin toxicity.

[0006] Nanobodies are small proteins composed of single-chain antibody molecules, which have high specificity and affinity, and have a smaller volume, higher stability, and deeper tissue penetrability compared with traditional antibodies. This makes them have great potential in tumor treatment. They can precisely treat by recognizing and targeting specific antigens on the surface of tumor cells and can penetrate into deep tumor tissues that conventional antibodies cannot reach. Nanobodies can be designed to deliver drugs or radioactive isotopes into tumor cells to achieve the effect of killing tumor cells. In addition, they can also be used in a variety of other treatment methods such as photodynamic therapy and immunoassay. Therefore, the application of nanobodies in the field of tumor treatment is receiving extensive attention and is expected to become one of the important means of future tumor treatment (Bannas, Hambach, and Koch-Nolte 2017).

[0007] Antibody-based drugs have promoted the progress in the field of cancer treatment, but many antibody-based drugs are still not ideal in terms of safety and efficacy. A major challenge faced by anti-cancer drugs remains the selectivity of the drugs, that is, while ensuring that the drugs effectively attack cancer, minimizing the collateral damage of the drugs to normal cells. To overcome the limitations of single-target drugs in this regard, a strategy of combining multiple monoclonal antibodies targeting different targets has been proposed. However, the published studies on different mAb combination therapies have shown that, depending on factors such as different target combinations, there are significant differences in the combined effects. For example, the simultaneous injection of trastuzumab (anti-HER2) and bevacizumab (anti-VEGF-A) has shown encouraging results in HER2+ metastatic breast cancer; however, the combination of bevacizumab and cetuximab (anti-EGFR) in the treatment of advanced colorectal cancer has not led to 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.

[0008] Therefore, there is still an urgent need in the art for new drug molecules with improved tumor specificity and selectivity to reduce on-target toxicity, increase the dosing window, and expand the therapeutic index. Summary of the Invention

[0010] Through the analysis of immunohistochemistry and bioinformatics data of clinical pathological samples (see, for example, Annals of Oncology 28: 769–776, 2017; doi: 10.1093 / annonc / mdw678; and Front. Oncol. 12: 858865. doi: 10.3389 / fonc.2022.858865), it was found that Trop2 and Nectin4 proteins showed a co-expression pattern in many tumor tissues or cells, such as urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, lung cancer, head and neck cancer, and thyroid cancer, etc., while the co-expression ratio was relatively low in normal tissues. Therefore, the present inventors proposed and designed an antibody molecule that simultaneously targets Trop2 and Nectin4 to reduce the on-target toxicity of the two targets and improve the efficacy. On this basis, the present inventors further proposed an innovative antibody mode based on nanobodies, so that the final antibody molecular weight is only about 60% of the molecular weight of the conventional four-chain antibody, thereby achieving a higher proportion of enrichment of the antibody molecule in tumor tissues, and further improving the efficacy. Based on these designs and discoveries, the present inventors established the multispecific antibodies, their drug conjugate molecules and uses of the present invention, especially their uses in cancer treatment.

[0011] Accordingly, in a first aspect, the present invention provides a multispecific antibody that binds Trop2 and Nectin4, and a pharmaceutical composition and use thereof, wherein the antibody comprises at least one antigen-binding domain that specifically binds Trop2 and at least one antigen-binding domain that specifically binds Nectin4.

[0012] In a second aspect, the present invention provides an anti-Trop2 VHH domain and an anti-Trop2 antibody, as well as an anti-Nectin4 VHH domain and an anti-Nectin4 antibody, and antigen-binding molecules, immunoconjugates, immunofusions, and pharmaceutical compositions and uses thereof that contain the same.

[0013] In a third aspect, the present invention provides an antibody-drug conjugate molecule (ADC) that contains the antibody of the present invention, and a pharmaceutical composition and use thereof.

[0014] In some embodiments, the antibodies and antibody-drug conjugates of the present invention have one or more of the following advantages:

[0015] (1) Binding to target cells that express human Nectin4 and / or Trop2 with high affinity.

[0016] (2) Co-binding to target cells that express human Nectin4 and Trop2.

[0017] (3) Co-endocytosis on target cells that express human Nectin4 and Trop2.

[0018] (4) Co-killing effect on target cells that express human Nectin4 and Trop2.

[0019] (5) Having a significant bystander effect.

[0020] (6) Having high anti-tumor efficacy, having a stronger killing effect on tumor cells, having a stronger inhibitory effect on tumor growth, especially tumors with high expression of Nectin4 and / or Trop2; having significantly improved or even unexpected anti-tumor activity.

[0021] In some embodiments, the antibodies and antibody-drug conjugates of the present invention also have one or more of the following advantages:

[0022] (7) Enhanced ability to resist the emergence of tumor drug resistance compared to Nectin4 and Trop2 single-target drugs.

[0023] (8) Resisting tumor drug resistance caused by mutations in the Nectin4 or Trop2 antigen epitopes.

[0024] (9) It has antitumor activity against tumor cells resistant to single-target drugs of Nectin4 and Trop2 (for example, tumor cells expressing high levels of the multidrug resistance protein MDR1); and

[0025] (10) Improved tumor-targeted distribution compared to single-target drugs of Nectin4 or Trop2.

[0026] In some embodiments, the single-target drug is the reference antibody Datopotamab, the reference antibody Enfortumab, or their corresponding ADC drugs.

[0027] In some embodiments, the antibody-drug conjugate of the present invention also has one or more of the following advantages:

[0028] (11) It has better product uniformity.

[0029] (12) It has low toxicity;

[0030] (13) It has good physicochemical stability; and

[0031] (14) It has good drug-likeness.

[0032] The present invention is further illustrated in the following drawings and specific embodiments. However, these drawings and specific embodiments should not be considered as limiting the scope of the present invention, and changes that are easily conceivable by those skilled in the art will be included within the spirit of the present invention and the scope of the appended claims. Brief Description of the Drawings

[0034] Figure 1A Showing the endocytosis detection of an exemplary anti-Trop2 VHH-Fc on BT-474 cells.

[0035] Figure 1B Showing the endocytosis detection of an exemplary anti-Trop2 VHH-Fc on NCI-N87 cells.

[0036] Figure 2A Showing the endocytosis detection of an exemplary anti-Nectin4 VHH-Fc on BT474 cells.

[0037] Figure 2B Showing the endocytosis detection of an exemplary anti-Nectin4 VHH-Fc on NCI-N87 cells.

[0038] Figure 3 Showing the binding of an exemplary anti-Trop2 VHH-Fc to NCI-N87 cells.

[0039] Figure 4Panel A shows the binding of an exemplary anti-Nectin4 VHH-Fc antibody to NCI-N87 cells.

[0040] Figure 4 Panel B shows the binding of an exemplary anti-Nectin4 VHH-Fc antibody to BT474 cells.

[0041] Figure 5 Panel A shows the binding of an exemplary anti-Nectin4 VHH-His antibody to CHO-K1-Human Nectin4 cells.

[0042] Figure 5 Panel B shows the binding of an exemplary anti-Nectin4 VHH-His antibody to CHO-K1-Cyno Nectin4 cells.

[0043] Figure 5 Panel C shows the non-specific binding of an exemplary anti-Nectin4 VHH-His antibody to CHO-K1 cells.

[0044] Figure 6 Shows the expression of Trop2 antigen and Nectin4 antigen on various target cells.

[0045] Figure 7 Panel A shows that the exemplary antibody V-hu21-Fc has no specific binding to the paralogous Nectin1, 2, 3, and Trop1 antigens.

[0046] Figure 7 Panel B shows that the exemplary antibody V-hu23-Fc has no specific binding to the paralogous Nectin1, 2, and 3 antigens and has specific binding to the target antigen Nectin4.

[0047] Figure 7 Panel C shows that the exemplary antibody V-hu23-Fc has no specific binding to the paralogous Trop1 antigen and has specific binding to the target antigen Trop2.

[0048] Figure 8 Panel A shows the binding of an exemplary single-chain bispecific antibody to NCI-N87 cells.

[0049] Figure 8 Panel B shows the binding of an exemplary double-chain bispecific antibody to NCI-N87 cells.

[0050] Figure 9 Panel A shows the binding of an exemplary single-chain bispecific antibody to BT-474 cells.

[0051] Figure 9Panel B shows the binding of an exemplary dual-chain format bispecific antibody to BT-474 cells.

[0052] Figure 10 Panel A shows the binding of an exemplary dual-chain format bispecific antibody to MDA-MB-468 cells.

[0053] Figure 10 Panel B shows the binding of an exemplary dual-chain format bispecific antibody to NCI-N87 cells.

[0054] Figure 10 Panel C shows the binding of an exemplary dual-chain format bispecific antibody to BT-474 cells.

[0055] Figure 11 Shows the binding of the exemplary antibody V-hu21-Fc to different tumor cells.

[0056] Figure 12 Shows the binding of the exemplary antibody V-hu23-Fc to different tumor cells.

[0057] Figure 13A Shows the endocytosis results of BT-474 cells for the exemplary bispecific anti-Trop2 / Nectin4 antibody molecule.

[0058] Figure 13B Shows the endocytosis results of BT-474 cells for the exemplary bispecific anti-Trop2 / Nectin4 antibody molecule.

[0059] Figure 13C Shows the endocytosis results of NCI-N87 cells for the exemplary bispecific anti-Trop2 / Nectin4 antibody molecule.

[0060] Figure 13D Shows the endocytosis results of NCI-N87 cells for the exemplary bispecific anti-Trop2 / Nectin4 antibody molecule.

[0061] Figure 13E Shows the endocytosis results of different target cells for the exemplary bispecific antibody V-hu21-Fc and V-hu23-Fc molecules.

[0062] Figure 14 Panels A-E show the comparison of the cell binding ability of the ADC with the corresponding bispecific antibody.

[0063] Figure 15A Shows the targeted killing of the target cell MDA-MB-468 by V-hu21-VA-Exd.

[0064] Figure 15BShows the killing effect of V-hu21-VA-Exd on non-target cells MKN45 cells.

[0065] Figure 15C Shows the killing effect of V-hu21-VA-Exd, V-hu23-VA-Exd and V-hu24-VA-Exd on target cells MDA-MB-468.

[0066] Figure 15D Shows the killing effect of V-hu23-Glu-Exd and V-hu24-Glu-Exd on target cells MDA-MB-468 cells.

[0067] Figure 15E Shows the synergistic killing effect of the anti-Trop2 binding site and the anti-Nectin4 binding site of the antibody on double-positive NCI-H292 tumor cells.

[0068] Figure 16A Shows the bystander effect of V-hu21-VA-Exd.

[0069] Figure 16B Shows the bystander effect of V-hu23-Glu-Exd.

[0070] Figure 17 Shows the pharmacodynamic effect of multiple administrations of ADCs in a mouse MDA-MB-468 subcutaneous xenograft model.

[0071] Figure 18 Shows the pharmacodynamic effect of single administration of ADCs in a mouse MDA-MB-468 subcutaneous xenograft model.

[0072] Figure 19 Shows the pharmacodynamic effect of multiple administrations of ADCs in a mouse HT1376 subcutaneous xenograft model.

[0073] Figure 20 Shows the pharmacodynamic effect of single administration of ADCs in a mouse HT1197 subcutaneous xenograft model.

[0074] Figure 21 Shows the pharmacodynamic effect of single administration of ADCs in a MDA-MB-453 subcutaneous xenograft model.

[0075] Figure 22 Shows the pharmacodynamic effect of multiple administrations of ADCs in a human-derived head and neck cancer xenograft model in vivo.

[0076] Figure 23 Shows the pharmacodynamic effect of single administration of ADCs in a human-derived endometrial cancer xenograft model in vivo.

[0077] Figure 24Show the efficacy of a single administration of the ADC in a human-derived cervical cancer xenograft model in vivo.

[0078] Figure 25 Show the efficacy of two administrations of the ADC in a murine-derived colon cancer xenograft model in vivo.

[0079] Figure 26 Show the co-expression of Trop2 and Nectin4 in multiple tumors revealed by bioinformatics analysis. In the figure, TPM (Transcripts Per Million) is used to quantify the gene expression level, showing the expression abundance of Trop2 and Nectin4 in different types of tumor tissues. Figure 26 A shows exemplary tumor types with dual positive expression of Trop2 and Nectin4 according to the median TPM of the patient population. Figure 26 B shows the expression distribution of Trop2 and Nectin4 observed in the patient population of a specific tumor type.

[0080] Figure 27 Show the biodistribution of a bispecific antibody in a murine human-derived HT1376 xenograft model. Figure 27 A: Quantify the change in tumor accumulation of the antibody over time by the fluorescence ratio of tumor to background. Figure 27 B: Evaluate the distribution of the antibody in organ tissues by the fluorescence ratio of tumor to muscle 72 hours after injection of the fluorescently labeled antibody protein. Detailed description of the invention

[0082] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the present invention will be apparent from this specification, the drawings, and the appended claims.

[0083] Definitions

[0084] The term "about", when used in conjunction with a numerical value, means a numerical value that encompasses a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0085] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0086] In this text, when the terms "comprising" or "including" are used, unless otherwise specified, the situation consisting of the recited elements, integers or steps is also covered. For example, when referring to an antibody variable region "comprising" a specific sequence, an antibody variable region consisting of that specific sequence is also intended to be covered.

[0087] In this text, the term "antigen-binding molecule" refers to a protein or polypeptide comprising an antigen-binding domain or antigen-binding site capable of binding to a target antigen. When the target antigen is TROP2 and / or NECTIN4, the antigen-binding molecule that binds to TROP2 and / or NECTIN4 is also referred to as a TROP2-binding molecule, a NECTIN4-binding molecule or a TROP2 / NECTIN4-binding molecule. 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, chimeric antigen receptors (CARs). As will be appreciated by those skilled in the art, the antigen-binding site of an antibody typically comprises amino acid residues from "complementary determining regions" or "CDRs".

[0088] In this text, the term "antibody" refers to a polypeptide comprising at least a light chain or a heavy chain immunoglobulin variable region that specifically recognizes and binds an antigen. The term covers various antibody structures, including, but not limited to, monoclonal antibodies, single-chain antibodies or multi-chain antibodies, mono-specific or multi-specific antibodies (e.g., bispecific antibodies), single-domain antibodies, heavy-chain antibodies, chimeric antibodies or humanized antibodies, intact antibodies and antibody fragments, provided that they exhibit the desired antigen-binding activity.

[0089] In this text, an "intact antibody" refers to an immunoglobulin molecule comprising at least two heavy chains (H) and two light chains (L). Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.

[0090] As used herein, the terms "antibody fragment" and "antigen-binding fragment" are used interchangeably and refer to a molecule distinct from a full antibody that comprises a portion of a full antibody and is capable of binding an antigen to which the full antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; 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 clearly contradicted by context, the term "antibody" as used herein is equivalent to "antibody or its antibody fragment". In some embodiments according to the present invention, the antibody fragment comprises a cysteine residue moiety for forming an interchain disulfide bond between the heavy chains, e.g., a cysteine residue in the antibody hinge region, to provide an amino acid residue site available for thiol conjugation chemistry. In other embodiments according to the present invention, the antibody fragment comprises a cysteine residue introduced into the Fc region to provide an amino acid residue site available for thiol conjugation chemistry.

[0091] As used herein, the terms "antigen-binding site" and "antigen-binding domain" are used interchangeably and denote the region in an antibody molecule that actually binds to an antigen. The antigen-binding site for the antibodies of the present invention is preferably provided by the variable domain from a heavy-chain antibody (i.e., "VHH").

[0092] As used herein, the term "multispecific" refers to an antigen-binding molecule (e.g., an antibody) having at least two antigen-binding sites that bind to different antigen epitopes, e.g., binding to different epitopes on different antigens and / or different epitopes on the same antigen. Accordingly, "monospecific" refers to the ability to bind only one epitope. "Bispecific" refers to the ability to bind two different epitopes.

[0093] As used herein, the expression "valence" or "valency" in relation to an antibody refers to the total number of antigen-binding sites in the antibody molecule, or the number of antigen-binding sites having the same antigen-binding specificity. For example, a tetravalent antibody means that the antibody molecule comprises a total of 4 antigen-binding sites; the antibody molecule can be a "2+2 valence" type of bispecific antibody, i.e., the antibody has two different antigen-binding specificities, and for each antigen-binding specificity, there are 2 identical antigen-binding sites.

[0094] The term "on-target / off tumor toxicity" refers to the situation where, in addition to tumor cells, normal tissue cells also express tumor-associated antigens targeted by the antibody, resulting in damage due to binding to the antibody.

[0095] In this text, the terms "TROP2" and "Trop2" are used interchangeably and refer to trophoblast cell surface glycoprotein antigen 2. Unless otherwise specified, the term includes any variant of human Trop2, including sequence variants, especially naturally occurring variants, allelic variants, as well as post-translational modification variants and conformational variants, and encompasses its species homologs. In addition, it should be understood that the term not only covers Trop2 expressed natively or recombinantly by cells or expressed on native or recombinant cells, but also covers recombinantly expressed fusion proteins containing the extracellular domain of Trop2. An example of Trop2 is the human Trop2 protein containing the amino acid sequence under UniProtKB - P09758, or a recombinant protein containing the extracellular domain of said protein (especially, the amino acid sequence of amino acids 31 - 274). Another example of Trop2 is the monkey Trop2 protein containing the amino acid sequence under NCBI - XP_005543292.2, or a recombinant protein containing the extracellular domain of said protein. In this text, unless otherwise specified, the term "TROP2" or "Trop2" refers to Trop2 derived from humans. In the embodiments of the present disclosure, "antigen - binding specificity for Trop2", that is, "antigen - binding domain that specifically binds to TROP2", is preferably provided by the VHH domain.

[0096] In this text, the term "TROP2 - positive" cells refers to cells that are positive for Trop2 cell - surface expression, such as cancer cells, engineered cancer cells, or engineered non - tumor cells. The Trop2 expression level on the cell surface can be determined by any conventional method known in the art for determining the expression level of cell - surface antigens, such as, for example, FACS detection method or immunofluorescence staining method. Trop2 has a significantly higher expression level on a variety of tumor cells than on normal tissues / cells, such as, for example, HT1376 (human bladder cancer cells) and MDA - MB - 468 (human breast cancer cells). Preferably, in this text, TROP2 - positive cells are TROP2 - positive tumor cells.

[0097] In this text, the term "NECTIN4" is used interchangeably with "Nectin4" and "Nectin4", and refers to Nectin cell adhesion molecule 4. Unless otherwise specified, the term includes any variant of human NECTIN4, including sequence variants, especially naturally occurring variants, allelic variants, as well as post-translational modification variants and conformational variants, and encompasses its species homologs. In addition, it should be understood that the term not only covers NECTIN4 that is naturally or recombinantly expressed by cells or expressed on natural or recombinant cells, but also covers recombinantly expressed fusion proteins containing the extracellular domain of NECTIN4. An example of NECTIN4 is the human NECTIN4 protein containing the amino acid sequence under UniProtKB-Q96NY8, or a recombinant protein containing the extracellular domain of said protein (especially, the amino acid sequence of amino acids 32 - 349). Another example of NECTIN4 is the monkey NECTIN4 protein containing the amino acid sequence under NCBI-_XP_005541277.1, or a recombinant protein containing the extracellular domain of said protein. In this text, unless otherwise specified, the term "NECTIN4" refers to NECTIN4 derived from humans. In the exemplary embodiments of the present disclosure, "antigen-binding specificity for NECTIN4", that is, "antigen-binding domain that specifically binds to NECTIN4", is preferably provided by a pair of VHH domains.

[0098] In this text, the term "NECTIN4-positive" cells refers to cells that are positive for NECTIN4 cell surface expression, such as cancer cells, engineered cancer cells, or engineered non-tumor cells. The NECTIN4 expression level on the cell surface can be determined by any conventional method known in the art for determining the expression level of cell surface antigens, such as FACS detection method or immunofluorescence staining method. NECTIN4 has a significantly higher expression level on a variety of tumor cells than on normal tissues / cells, such as HT1376 (human bladder cancer cells) and BT474 (human breast ductal cancer cells). Preferably, NECTIN4-positive cells are NECTIN4-positive tumor cells.

[0099] In this text, 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 (such as an antibody) and its binding partner (such as an epitope). "Binding affinity" reflects the intrinsic binding affinity of the 1:1 interaction between the members of the binding pair. Binding affinity is usually expressed by the binding dissociation equilibrium constant (K D ) and can be measured by common methods known in the art, such as surface plasmon resonance (SPR) technology.

[0100] As used herein, the term "avidity" or "binding avidity" refers to the combined strength of the interaction of multiple binding sites of a molecule (antibody) with the same target. Thus, a necessary condition for avidity is the multivalency of the molecule (such as an antibody) for a target.

[0101] As used herein, the term "immunoglobulin" refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of approximately 150,000 daltons composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also referred to as the heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also referred to as the light chain variable domain, followed by a light chain constant domain (CL). The heavy chains of immunoglobulins can be assigned to one of five classes, designated α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), based on the type of their constant regions, and some of these classes can be further divided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can also be divided into one of two types, designated κ and λ, based on the amino acid sequence of their constant domains.

[0102] As used herein, the term "isotype" refers to the type of antibody determined by the constant region of the antibody heavy chain. For example, antibodies according to the invention can be IgA (such as IgA1 or IgA2), IgG1, IgG2 (such as IgG2a or IgG2b), IgG3, IgG4, IgE, IgM, and IgD antibodies and have a heavy chain constant region of the immunoglobulin type. In addition, the invention contemplates not only antibodies with naturally occurring sequence constant regions but also antibodies comprising variant sequence constant regions.

[0103] As used herein, the term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. In the case of heavy chain antibodies, such as those from camelid heavy chain antibodies, a single VH domain (also referred to herein as a VHH domain) can be sufficient to confer antigen-binding specificity. The VHH domain, like the variable regions of the heavy and light chains of a conventional IgG antibody, contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs), and is arranged in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In some aspects according to the present invention, one or more residues in the variable region of an antibody can be modified, e.g., residue modification of one or more CDR regions and / or of one or more framework regions, particularly conservative residue substitutions, to obtain an antibody variant that still substantially retains at least one biological property (e.g., antigen-binding ability) of the parental antibody. In still further aspects, the antibody variable region can be modified by CDR grafting. Since the CDR sequences are responsible for most antibody-antigen interactions, recombinant antibody variants can be constructed that mimic the properties of a known antibody. In such antibody variants, the CDR sequences from a known antibody are grafted onto the framework regions of a different antibody with different properties, and can be mutated at 1 to several residues as needed, e.g., back mutations to refine the desired properties of the antibody. The properties of the mutated and / or modified antibody or an ADC conjugate containing it, such as target antigen-binding properties or other desired functional properties, e.g., endocytic activity, pharmacokinetics, and in vivo tumor killing activity, can be evaluated in in vitro or in vivo assay experiments. Accordingly, the present invention also contemplates variants of any variable region (e.g., VHH) given herein.

[0104] As used herein, the terms "complementary determining region" or "CDR region" or "CDR" or "hypervariable region" refer to regions in the variable domain of an antibody that are highly variable in sequence and form structurally defined loops ("hypervariable loops") and / or contain antigen - contacting residues ("antigen - contact points"). CDRs are mainly responsible for binding to epitopes. In the VHH domain of the antibodies of the present invention, the CDRs are sequentially numbered starting from the N - terminus and are commonly referred to as CDR1, CDR2, and CDR3. One can use methods well - known in the art to determine the CDR sequences in a given VHH domain, for example, by the Kabat, AbM, Chothia, Contact, and IMGT methods to define the region and combinatorial ranges of the CDRs. Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the aforementioned ways and their combinations. Further, as is known in the art, although CDRs are different between antibodies, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. By using at least two of the Kabat, Chothia, AbM, and Contact methods, one can determine the minimal overlapping region, thus providing a "minimal binding unit" for antigen binding. Such a minimal binding unit can be a sub - part of a CDR. The residues of the remaining part of the CDR sequence, as will be apparent to those skilled in the art, can be determined by the structure and protein folding of the antibody. Accordingly, the present invention also contemplates variants of any CDR given herein. For example, in a variant of a CDR, the amino acid residues of the minimal binding unit can remain unchanged while the remaining CDR residues can be substituted.

[0105] Unless otherwise specified, in the present invention, when referring to residue positions in the antibody variable region and CDRs (including heavy - chain variable region residues), it refers to the numbered positions according to the Kabat numbering system.

[0106] As used herein, the terms "VHH" and "VHH domain" are used interchangeably and refer to the heavy - chain variable domain derived from a heavy - chain antibody lacking a light chain, sometimes also referred to as a single - variable - domain fragment (sVD). Thus, VHH is different from the conventional VH of a four - chain immunoglobulin in that it does not need to pair with a light - chain variable domain to form an antigen - binding site. Such VHH molecules can be derived from antibodies produced in camelid species (such as camels, alpacas, dromedaries, llamas, and guanacos). Other species besides camelids can also produce heavy - chain antibodies that are naturally lacking in light chains, and such VHHs are also within the scope of the present invention. In some cases, for the therapeutic application of an antibody or its derivative molecules, it is desirable to reduce its immunogenicity. Thus, preferably, in one embodiment, the antibodies of the present invention comprise humanized variable regions (such as VHH domains).

[0107] As used herein, the term "half-life extending domain" refers to a chemical structure that can confer an increased circulatory half-life to a molecule (such as an antibody) to which it is conjugated, upon administration to an animal. Such chemical structures include, for example, flexible hydrophilic molecules (such as carbohydrates or PEG (polyethylene glycol)), immunoglobulin Fc regions, serum albumin, serum albumin binding domains (such as small organic molecules, fatty acids, peptides, and proteins capable of binding to serum albumin), or serum albumin binding peptides. The half-life extending domain can be linked to the antibody of the invention by chemical conjugation or fusion, depending on its specific nature. Preferably, the half-life extending domain used in the present invention is a peptide or polypeptide structure.

[0108] As used herein, a "serum albumin binding peptide" refers to a peptide or polypeptide that can bind to serum albumin and thus act as a half-life extending domain. Examples of such peptides or polypeptides include, but are not limited to, fragments derived from anti-serum albumin antibodies, such as Fab, single-chain Fab, scFv, single variable domain fragments (sVD), VHH, as well as bacterial albumin binding domains and peptides, such as the albumin binding domain from Streptococcal Protein G (SPG). Such peptides or polypeptides can be linked to the antibody molecule of the invention by genetic fusion, thereby avoiding chemical conjugation. See, for example, Expert Opin Biol Ther. 2016 Jul;16(7):903-15. doi:10.1517 / 14712598.2016.1165661.

[0109] As used herein, the terms "immunoglobulin Fc region", "Fc region", and "Fc domain" are used interchangeably herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Fc regions that can be used in the antibodies of the invention include, but are not limited to, Fc regions of IgG1, IgG2, IgG3, or IgG4 having native or variant sequences. Unless otherwise specified herein, the amino acid residue numbering in the Fc region or heavy chain constant region is numbered according to the EU numbering system (also referred to as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, the terms "Fc region" or "Fc domain" do not include the heavy chain variable region VH and the light chain variable region VL of an immunoglobulin, and the heavy chain constant region CH1 and the light chain constant region CL; but may include the CH2 domain and the CH3 domain, and may or may not include the immunoglobulin hinge region. For example, in some instances, the Fc region may comprise or consist of the CH2 domain and the CH3 domain from the N-terminus to the C-terminus. In other instances, the Fc region may comprise or consist of the immunoglobulin hinge region or a portion of the immunoglobulin hinge region, the CH2 domain, and the CH3 domain from the N-terminus to the C-terminus.

[0110] As used herein, the term "native sequence Fc region" encompasses naturally occurring Fc region sequences of various immunoglobulins, such as Fc region sequences of various Ig subclasses and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520.). In some embodiments, the human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may be present or absent. In some embodiments, the human IgG heavy chain Fc region bears at the N-terminus the hinge sequence or a portion of the hinge sequence of a native immunoglobulin, such as the sequence from E216 to T225 or the sequence from D221 to T225 according to EU numbering.

[0111] As used herein, the term "variant sequence Fc region" refers to an Fc region polypeptide that contains modifications relative to the native sequence Fc region polypeptide. Such modifications can be additions, deletions, or substitutions of amino acid residues. Substitutions can include both naturally occurring and non-naturally occurring amino acids. The purpose of the modifications can be to alter the binding of the Fc region to its receptor and the effector functions thereby elicited, or the purpose of the modifications can be to prevent unwanted heavy chain mispairing, or to site-specifically introduce amino acid modifications that can be used for conjugating other active molecules.

[0112] As used herein, the term "effector function" refers to those biological activities attributed to the Fc region of an immunoglobulin 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 have altered effector functions relative to an antibody molecule having a wild-type Fc region, such as reduced or eliminated Fcγ receptor binding, etc.

[0113] As used herein, the terms "flexible linker peptide" or "peptide linker" or "linker peptide" are used interchangeably and refer to a short amino acid sequence composed 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.

[0114] As used herein, "percent identity (%)" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a specific amino acid sequence shown in this specification after aligning the candidate sequence with the specific amino acid sequence shown in this specification and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention that have a substantial degree of identity relative to the antibody molecules and their sequences specifically disclosed herein, such as an identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or higher. Such variants can contain conservative modifications.

[0115] For a polypeptide sequence, "conservative modification" includes substitution, deletion, or addition to the polypeptide sequence, which results in the replacement of an amino acid with a chemically similar amino acid. Tables of conservative substitutions providing functionally similar amino acids are well known in the art. The following 8 groups contain amino acids that are conservative substitutions 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, e.g., Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modification" is particularly used 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 an antibody containing the amino acid sequence.

[0116] As used herein, the term "binding" or "specifically binding" means that the binding interaction is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen-binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art. For example, the binding ability of an antibody to an antigen can be detected by the ELISA assay described in the examples, or the binding ability of an antibody to a cell expressing an antigen on the surface can be detected by the FACS assay described in the examples, or the affinity constant K can be detected by the SPR technique described in the examples. D .

[0117] As used herein, the term "epitope" refers to the portion of an antigen to which an antibody specifically binds. An epitope can consist of contiguous and / or non-contiguous amino acids that form a conformational space unit. Epitope grouping of different antibodies that bind to the same antigen can be performed by competitive binding assays. When the test antibody blocks the binding of a reference antibody to an antigen (e.g., TROP2 or NECTIN4) 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., TROP2 or NECTIN4) by 50% or more in a competitive binding assay, the test antibody and the reference antibody can be considered "competitive binding antibodies". Competitive binding antibodies can bind to the same epitope region as the reference antibody, such as the same epitope, adjacent epitopes, or overlapping epitopes. Competitive binding assays 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 competition assay, or by the methods described in the examples herein.

[0118] As used herein, a "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs (e.g., CDRs) in the humanized antibody correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody may optionally comprise at least a portion of the antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized. As used herein, in some embodiments, the humanized antibodies of the present invention have framework region sequences "derived from" a particular human germline sequence. Herein, "derived from" means that the amino acid sequence of the antibody framework region has at least 85%, 90% identity with the corresponding framework region amino acid sequence encoded by the human germline immunoglobulin gene, and the antibody retains antigen-binding activity.

[0119] As used herein, if an amino acid sequence (e.g., VHH) is specific for two different antigens or epitopes (e.g., TROP2 or NECTIIN4 from different mammalian species, e.g., human TROP2 or human NECTIIN4, cynomolgus monkey TROP2 or cynomolgus monkey NECTIIN4), it is said to be "cross-reactive" with these two different antigens or epitopes. It would be advantageous for an antibody to have human-monkey species cross-reactivity, especially with similar human-monkey antigen-binding affinities, which can contribute to the preclinical drug development of the antibody, such as the toxicology detection of ADC molecules composed of the antibody. In some embodiments, the antibodies of the present invention preferably have human-monkey species cross-reactivity.

[0120] As used herein, the terms "endocytosis" and "internalization" are used interchangeably and refer to the process by which a ligand / receptor complex is internalized and delivered into the cytosol or transferred to a suitable intracellular compartment triggered by the binding of a ligand to the corresponding receptor on the cell surface. In some embodiments, the antibodies of the present invention trigger endocytosis mediated by the TROP2 and / or NECTIIN4 receptor after binding to TROP2 and / or NECTIIN4 expressed on the cell surface. As used herein, endocytosis and the endocytosis rate can be measured by, for example, 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 to deliver anti-tumor drugs into cancer cells in the ADCs of the present invention.

[0121] As used herein, the term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", including primary transformed cells and progeny derived therefrom. A host cell is any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, such as mammalian cells, insect cells, yeast cells; and prokaryotic cells, such as Escherichia coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0122] As used herein, the term "expression vector" refers to a vector containing a recombinant polynucleotide that contains an expression control sequence operably linked to a 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 cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses) incorporated into the recombinant polynucleotide.

[0123] As used herein, when referring to "co-endocytosis", "co-binding" and "co-killing", the term "co-" means that the antibody of the present invention having dual-targeting binding sites for TROP2 and NECTIN4 or an ADC molecule based thereon has increased endocytosis, binding or killing effects when tested under the same conditions compared to a reference antibody having a corresponding single-targeting binding site for TROP2 or NECTIN4 (e.g., the parental antibody of TROP2 or NECTIN4) or an ADC molecule based thereon.

[0124] As used herein, the term "immunoconjugate" or "immunoconjugate" generally refers to a molecule formed by conjugating one or more immunoglobulin-related molecules or fragments thereof (e.g., an antibody or a fragment thereof) with one or more other molecules. Immunoconjugates typically contain at least one non-proteinaceous chemical moiety, such as a chemical linker used to effect the conjugation. In some cases, the other molecule may be a proteinaceous molecule, such as a peptide, polypeptide or protein. In some cases, the other molecule may also be a non-proteinaceous molecule, such as a chemical toxin. In some cases, the other molecule may be the same as the immunoglobulin-related molecule or fragment thereof. In some cases, the other molecule may be different from the immunoglobulin-related molecule or fragment thereof. The one or more other molecules may be the same or different from each other. For example, the other molecule may be a target-binding element and / or an effector element, such as a chemotherapeutic agent, toxin, drug (such as an immunotherapeutic agent), radioactive element, probe or signal molecule, etc.

[0125] As used herein, the terms "individual" or "subject" are used interchangeably and refer to a mammal. 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, the individual is a human.

[0126] As used herein, the term "treatment" refers to a clinical intervention that is intended to alter the natural course of a disease in an individual being treated. Desired therapeutic effects include, but are not limited to, preventing the appearance or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and relieving or improving the prognosis. In the context of treating a tumor or cancer, "treatment" encompasses anti-tumor biological effects that can be brought about by artificial intervention means (e.g., by the administration of a drug), including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0127] As used herein, the terms "cancer" and "tumor" are used interchangeably and refer to or describe a physiological disorder in a mammal that is typically characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinomas, solid tumors, and liquid tumors. In certain embodiments, cancers suitable for treatment by the antibodies or immunoconjugates or immunofusions of the present invention include TROP2-positive and / or NECTIN4-positive tumors / cancers, including also their metastatic forms.

[0128] As used herein, an "antibody-drug conjugate (ADC)" refers to a compound / molecule obtained by linking an antibody to a (small molecule) drug through a linker.

[0129] The term "linker" refers to a structural moiety that links a drug (e.g., a small molecule drug) to an antibody moiety. It should be understood that the linker has a functional group that can form a bond with a functional group of an antibody or its antigen-binding fragment before being linked to the antibody or its antigen-binding fragment.

[0130] The term "linker-payload" refers to a compound formed by linking a payload, such as a drug (e.g., a small molecule drug), to a linker.

[0131] As used herein, the term "alkyl" refers to a fully saturated, branched or unbranched hydrocarbon group. The alkyl group preferably contains 1 to 16 carbon atoms, such as 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms or 1 to 4 carbon atoms. 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.

[0132] The term "alkenyl" refers to a straight-chain or branched hydrocarbon group containing 2 to 16 carbon atoms and containing at least one double bond and no triple bonds. The alkenyl group preferably contains 2 to 12 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 2 to 6 carbon atoms or 2 to 4 carbon atoms. Representative examples of alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, hexenyl, etc.

[0133] The term "alkynyl" refers to a straight-chain or branched hydrocarbon group containing 2 to 16 carbon atoms and containing at least one triple bond. The alkynyl group preferably contains 2 to 12 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 2 to 6 carbon atoms or 2 to 4 carbon atoms. Representative examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc.

[0134] The term "halogen" or "halo" refers to fluorine (-F), chlorine (-Cl), bromine (-Br) or iodine (-I).

[0135] The term "haloalkyl" refers to an alkyl group as defined herein that is substituted with one or more halogen groups as defined herein. The haloalkyl group can preferably be a monohaloalkyl group, a dihaloalkyl group or a polyhaloalkyl group (including a perhaloalkyl group). The monohaloalkyl group can contain one iodine, bromine, chlorine or fluorine in the alkyl group. The dihaloalkyl and polyhaloalkyl groups can contain two or more of the same halogen atoms or a combination of different halo groups in the alkyl group. Preferably, the polyhaloalkyl group contains at most 12, 10, 8, 6, 4, 3 or 2 halogen groups. Non-limiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl. The perhaloalkyl group refers to an alkyl group in which all hydrogen atoms are replaced by halogen atoms.

[0136] The term "haloalkenyl" refers to an alkenyl group as defined herein that is substituted with one or more halogen groups as defined herein. The term "haloalkynyl" refers to an alkynyl group as defined herein that is substituted with one or more halogen groups as defined herein. The meaning of "halo" as defined for "haloalkyl" is applicable to "haloalkenyl" and "haloalkynyl".

[0137] The term "amino acid" refers to both naturally occurring and synthetic amino acids. Amino acids can be either L- or D-isomers. The conventional writing of amino acids involved herein follows the conventional usage. See, for example, Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. And in the present disclosure, amino acids are generally represented by the single-letter and three-letter abbreviations well-known in the art. For example, the amino acids can be selected from phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), citrulline (Cit), serine (Ser; S), glutamic acid (Glu; E), aspartic acid (Asp; D), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), and glutamine (Gln).

[0138] The term "penturonic acid" refers to a compound formed by oxidizing the primary hydroxyl group of a pentose to a carboxyl group. Examples of penturonic acids include, but are not limited to, xyluronic acid and arabinuronic acid.

[0139] The term "hexuronic acid" refers to a compound formed by oxidizing the primary hydroxyl group of a hexose to a carboxyl group. Examples of hexuronic acids include, but are not limited to, glucuronic acid, galacturonic acid, and mannuronic acid.

[0140] The term "pentose", also known as a five-carbon sugar, refers to a monosaccharide containing five carbon atoms. Pentoses include D-type and / or L-type pentoses. Examples of pentoses include, but are not limited to, xylose, arabinose, ribose, and deoxyribose.

[0141] The term "hexose", also known as a six-carbon sugar, refers to a monosaccharide containing six carbon atoms. Hexoses include D-type and / or L-type pentoses. Examples of hexoses include, but are not limited to, glucose, galactose, mannose, and fructose.

[0142] The term "optional" or "optionally": means that the subsequent described event or situation may or may not occur, and the description includes instances where the event or situation occurs and instances where the event or situation does not occur. For example, when a group or structure is "optionally substituted", the group or structure may be substituted or may not be substituted.

[0143] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the ADC conjugate of the present invention and is not biologically or otherwise undesirable. The ADC conjugates of the present invention may exist in their pharmaceutically acceptable salt forms, including acid addition salts and base addition salts. In the present invention, the pharmaceutically acceptable non-toxic acid addition salts refer to the salts formed by the ADC conjugates in the present invention with organic or inorganic acids, 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. The pharmaceutically acceptable non-toxic base addition salts refer to the salts formed by the ADC conjugates in 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 organic bases containing N groups.

[0144] The term "solvate" refers to an association formed by one or more solvent molecules with the ADC antibody-drug conjugate in the present invention. The solvents that form solvates include but are not limited to water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, etc.

[0145] Where there is no contradiction in the context, the terms "pharmaceutically acceptable" and "medicinal" are used interchangeably herein.

[0146] The term "drug:antibody ratio" or "DAR" refers to the ratio of the drug moiety (D) conjugated to the Ab moiety in an antibody-drug conjugate molecule to the Ab moiety. In some embodiments described herein, the DAR can be determined by p in Formula I. For example, the DAR can be from 1 to 16, such as 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The DAR can also be calculated as the average DAR of the molecular population in the product, i.e., the overall ratio of the drug moiety (D) conjugated to the Ab moiety in the product measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and / or HPLC), and this DAR is referred to as the average DAR in the text. In some embodiments, the average DAR value of the conjugate of the present invention is from 1 to 16, such as 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 1.0-8.0, 2.0-6.0, such as 0.5, 0.6, 0.7, 0.8, 0.9, 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, 7.8.0, 7.9, 8, 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. It should be understood that when referring to the average DAR value, the ADC of the present invention refers to an ADC molecular population or an ADC molecular mixture, which contains ADC molecules having the same and / or different DARs.

[0147] The term "therapeutic agent" as described herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs or immunomodulators (e.g., immunosuppressants).

[0148] The term "cytotoxic agent" as used in the present invention refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction.

[0149] "Chemotherapeutic agent" includes chemical compounds useful in the treatment of cancer or immune system diseases.

[0150] The term "drug" refers to an organic compound capable of regulating biological processes, particularly of altering or preventing pathological processes.

[0151] The term "small molecule drug" refers to an organic compound of low molecular weight capable of regulating biological processes, particularly of altering or preventing pathological processes. "Small molecule" is defined as a molecule having a molecular weight less than 10 kD, typically less than 2 kD and preferably less than 1 kD, more preferably less than 500 D. Small molecule drugs include, but are not limited to, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptidomimetics, and antibody mimetics. As therapeutic agents, small molecules can be more cell permeable, less susceptible to degradation, and less likely to elicit an immune response than macromolecules.

[0152] 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. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressive agents or immunostimulatory agents. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0153] "Antitumor compound" is a pharmaceutically active compound that acts on tumors, which includes, but is not limited to, cytotoxic agents or chemotherapeutic agents, such as the cytotoxic agents disclosed in WO2021 / 173773, US5658920, such as the camptothecin compound exatecan (topoisomerase I inhibitor Exatecan), Dxd (a novel topoisomerase I inhibitor Exatecan derivative), auristatin compounds such as monomethyl auristatin E (MMAE), the structure of which is shown below:

[0154]

[0155]

[0156] The term "effective amount" refers to such an amount or dose of the antibody or ADC molecule or composition or combination of the present invention that, when administered to a patient in a single or multiple doses, produces the desired effect in a patient in need of treatment or prevention. Depending on the desired effect, it can include "therapeutically effective amount" and "preventively effective amount".

[0157] "Therapeutically effective amount" means an amount that, when administered in the required dose and for the required period of time, effectively achieves the desired therapeutic result. A therapeutically effective amount is also an amount in which any toxic or harmful effects of the antibody or ADC molecule or composition or combination are outweighed by the therapeutic beneficial effects. Relative to an untreated subject, a "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 30%, even more preferably by at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or even 100%.

[0158] "Prophylactically effective amount" means an amount that, when administered in the required dose and for the required period of time, effectively achieves the desired prophylactic result. Generally, since prophylactic doses are administered in a subject before or at an earlier stage of a disease, a prophylactically effective amount will be less than a therapeutically effective amount.

[0159] The term "antitumor effect" refers to a biological effect that can be demonstrated by a variety of means, including but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0160] The term "pharmaceutical excipient" refers to diluents, adjuvants (e.g., Freund's adjuvant (complete and incomplete)), excipients, carriers, stabilizers, etc. that are administered together with the active substance.

[0161] The term "pharmaceutical composition" refers to a composition that is in a form that permits the biological activity of the active ingredient(s) contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered.

[0162] The terms "pharmaceutical combination", "combination product", "drug combination" or "combination product" refer to non-fixed combination products or fixed combination products, including but not limited to kits, pharmaceutical compositions. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody molecule or ADC molecule of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or at the same or different time intervals, sequentially, in separate entities, wherein such administration provides a prophylactic or therapeutically effective level of two or more active agents in the patient. In some embodiments, the antibody molecule or ADC molecule of the present invention and other therapeutic agents used in the pharmaceutical combination are administered at levels not exceeding those at which they are used alone. The term "fixed combination" means that two or more active agents are administered to a patient in the form of a single entity. Preferably, the doses and / or time intervals of the two or more active agents are selected such that the combined use of the components results in an effect greater than that achieved by the use of any one of the components alone in the treatment of a disease or disorder. Each component may be in the form of a separate formulation, and the formulation forms may be the same or different.

[0163] The term "combination therapy" or "combination therapy" refers to the administration of two or more therapeutic agents or treatment methods (e.g., radiotherapy or surgery) to treat diseases described herein. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, such as a single capsule with a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of each active ingredient in a plurality of or in separate containers (e.g., tablets, capsules, powders, and liquids). The powder and / or liquid can be reconstituted or diluted to the desired dose before administration. In addition, such administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the treatment regimen will provide a beneficial effect of the drug combination in treating the disorders or conditions described herein.

[0164] As used herein, "prevention" includes inhibition of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the signs or symptoms of cancer occur, particularly in a subject at risk for cancer.

[0165] I. Multispecific Antibodies of the Present Invention

[0166] Through in-depth research, the inventors found that by designing antibody molecules that simultaneously target Trop2 and Nectin4, the on-target toxicity of the two targets can be reduced and the anti-tumor efficacy can be improved. Therefore, in a first aspect, the present invention provides a multispecific antibody that binds Trop2 and Nectin4, wherein the antibody comprises at least one antigen binding domain that specifically binds Trop2 and at least one antigen binding domain that specifically binds Nectin4. In some aspects, in order to increase the half-life of the antibody of the present invention in animal circulation, the antibody of the present invention also comprises a half-life extension domain, for example, a serum albumin binding peptide or an immunoglobulin Fc region. The antibody of the present invention can be in any suitable form, such as a single-chain or double-chain form, wherein the domains located on the same chain can be connected by a peptide linker or directly connected as needed.

[0167] The components of the multispecific antibodies of the present invention are described in detail below. A person 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 the present invention. Furthermore, a person skilled in the art will understand that, unless the context clearly indicates otherwise, the antibodies of the present invention (including antibodies of any form) may include any such combination of features.

[0168] Antigen-binding domain

[0169] Although high-affinity antibodies have been favored in early antibody screening, an increasing number of studies have shown that high-affinity antibodies are not equivalent to high efficacy. For example, high-affinity antibodies that bind to rapidly internalized targets may be rapidly eliminated from the circulation. In addition, high affinity may cause the antibody to be tightly bound or rapidly internalized by the peripheral cells of the tumor tissue encountered initially, thus limiting the penetration of the antibody into the tumor. Conversely, if the antibody affinity is too low, although it may improve the tumor penetration efficiency of the antibody, such an antibody will also have a low tumor retention rate due to its inability to effectively bind to the receptor. Therefore, depending on the specific application impact of the antibody or antibody-based molecule, screening and adopting medium-affinity antibodies that can allow the antibody to be effectively delivered throughout the tumor while allowing the antibody to accumulate in the tumor is more advantageous in some cases. In this article, "medium affinity" means that, for example, as determined by surface plasmon resonance technology (SPR), the binding affinity K D value is equal to or higher than 1 nM but less than 500 nM.

[0170] Regarding the antibodies targeting Trop2 and Nectin4 of the present invention, in the study, the inventors found that in some cases (such as using the antibody as an ADC carrier), by selecting Trop2 and Nectin4 antigen-binding domains with medium binding affinity, and by adjusting the valency of the Trop2 and Nectin4 antigen-binding domains in the antibody, additional advantages in efficacy and safety can be advantageously imparted to the multispecific antibodies of the present invention. For example, by reducing the valency and affinity of the Trop2-binding domain, it can help reduce the systemic toxicity of the antibody when administered to an individual; while by including a relatively higher valency (such as divalent) medium-affinity Nectin4-binding domain to increase avidity, it can play a role in balancing the efficacy and toxicity of the antibody. In addition, by selecting the antigen-binding domain in the antibody as a nanobody-based VHH domain, the enrichment rate and enrichment amount of the antibody in tumor tissue can be further improved.

[0171] Therefore, in some embodiments, the present invention provides a multispecific antibody comprising a Trop2-binding domain and a Nectin4-binding domain. In some embodiments, the Trop2 antigen-binding domain of the antibody according to the present invention has medium binding affinity for Trop2 (preferably human Trop2). For example, the binding affinity K D value is 50x10 -8 M to 0.5x10 -8 or 50x10 -8 M to 1x10 -8 M, optionally 15x10 -8 M to 1x10 -8M. In some embodiments, the Nectin4 antigen-binding domain of the antibody according to the present invention has a moderate binding affinity for Nectin4 (preferably human Nectin4). For example, the binding affinity K D value is 50x10 -8 M to 0.5x10 -8 or 50x10 -8 M to 1x10 -8 M, optionally 30x10 -8 M to 1x10 -8 M. In some embodiments, the antigen-binding affinities K D values of the Trop2 and Nectin4 antigen-binding domains are 50x10 -8 M to 0.5x10 -8 M respectively. In some embodiments, the binding affinity K D value of the Trop2-binding domain for Trop2 is 10x10 -8 M to 1x10 -8 M, and wherein the binding affinity of the Nectin4-binding domain for Nectin4 is 30x10 -8 M to 1x10 -8 M, optionally 20x10 -8 M to 1x10 -8 M.

[0172] In some embodiments, the present invention provides a multispecific antibody comprising a Trop2 antigen-binding domain and a Nectin4 antigen-binding domain, wherein the number ratio or valence ratio of the Trop2 antigen-binding domain to the Nectin4 antigen-binding domain is 1:1 or 1:2. In some embodiments, the valence (i.e., the total number of antigen-binding domains) of the multispecific antibody according to the present invention is 2-5 valent, preferably not exceeding 4 valent. In some embodiments, the multispecific antibody according to the present invention is a trivalent antibody, which comprises 1 medium-affinity Trop2-binding domain and 2 medium-affinity Nectin4-binding domains. In other embodiments, the multispecific antibody according to the present invention is a tetravalent antibody, which comprises 2 medium-affinity Trop2-binding domains and 2 medium-affinity Nectin4-binding domains.

[0173] In some embodiments, the antibodies of the invention are "isolated" antibodies. As used herein, an "isolated" antibody refers to an antibody that is artificial, recombinantly produced, and has been at least partially separated from the components in its natural environment in which it was produced. In some embodiments, the isolated antibody is purified to greater than 95% or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

[0174] Trop2 antigen-binding domain

[0175] In some embodiments of the multispecific antibodies according to the invention, preferably, the antigen-binding site that specifically binds to TROP2 is provided by a VHH domain (also abbreviated herein as VHH Trop2 ).

[0176] In some embodiments, the VHH Trop2 domain according to the invention comprises CDR1, CDR2, and CDR3 sequences in the variable region having an amino acid sequence selected from SEQ ID Nos: 5, 9, 13, 29, 30, 98, and 99. Preferably, the CDRs are defined according to AbM, Chothia, Kabat, IMGT, or any combination thereof. More preferably, the CDRs are defined according to Kabat or AbM or a combination thereof, and even more preferably, the CDRs are defined according to AbM. It should be understood, however, that the CDRs can also be defined in any other manner known in the art.

[0177] In some embodiments, the VHH TROP2 domain according to the invention comprises CDR1, CDR2, and CDR3 sequences, wherein the CDR1, CDR2, and CDR3 sequences:

[0178] (i) respectively comprise or consist of the amino acid sequences of SEQ ID Nos: 6, 7, and 8;

[0179] (ii) respectively comprise or consist of the amino acid sequences of SEQ ID Nos: 10, 11, and 12;

[0180] (iii) respectively comprise or consist of the amino acid sequences of SEQ ID Nos: 14, 15, and 16; or

[0181] (iv) respectively comprise or consist of the amino acid sequences of SEQ ID Nos: 31, 32, and 33.

[0182] In some embodiments, the VHH TROP2The domain comprises: the VHH variable region sequence of any of the exemplary antibodies of the present invention or a variant thereof, e.g., an antibody or a fragment thereof having the same CDR sequence as one of the exemplary antibodies and having the same or different framework region sequences, e.g., a humanized antibody. In some cases, preferably, one to several residue mutations are made to the framework sequence, e.g., to remove post-translational modifications (PTMs) and / or deimmunize. The maintenance or improvement of the antigen-binding properties or other functional properties of the mutated antibody can be evaluated in in vitro or in vivo assays.

[0183] In some embodiments, the VHH according to the present invention TROP2 The domain comprises a variable region sequence having an amino acid sequence selected from SEQ ID Nos: 5, 9, 13, 29, 30, 98, and 99. In still other embodiments, the VHH according to the present invention TROP2 The domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity to an amino acid sequence selected from SEQ ID Nos: 5, 9, 13, 29, 30, 98, and 99 and retaining the ability to specifically bind to TROP2. In still other embodiments, the VHH according to the present invention TROP2 The domain comprises an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions (e.g., conservative substitutions) compared to an amino acid sequence selected from SEQ ID Nos: 5, 9, 13, 29, 30, 98, and 99 and retaining the ability to specifically bind to TROP2. Preferably, the amino acid additions, deletions, and / or substitutions do not occur in the CDR regions.

[0184] In some preferred embodiments, the VHH according to the present invention TROP2 The domain comprises the CDR1-3 sequences of SEQ ID Nos: 10-12. In some embodiments, the VHH TROP2 The domain comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto. Preferably, the VHH TROP2 The domain comprises the amino acid sequence of SEQ ID NO: 9, or consists of the amino acid sequence shown by SEQ ID NO: 9.

[0185] In some preferred embodiments, the VHH according to the present invention TROP2 The domain comprises the CDR1-3 sequences of SEQ ID Nos: 31-33. In some embodiments, the VHH TROP2The domain comprises the amino acid sequence of SEQ ID NO:29, 30, 98 or 99, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity therewith. Preferably, the VHH TROP2 domain comprises the amino acid sequence of SEQ ID NO:29 or 30, or consists of the amino acid sequence shown by SEQ ID NO:29 or 30.

[0186] Nectin4 antigen-binding domain

[0187] In some embodiments of the multispecific antibody according to the present invention, preferably, the antigen-binding site that specifically binds NECTIN4 is provided by a VHH domain (also abbreviated as VHH in this article Nectin4 ).

[0188] In some embodiments, the VHH Nectin4 domain according to the present invention comprises CDR1, CDR2 and CDR3 sequences in the variable region having an amino acid sequence selected from SEQ ID Nos: 17, 21, 25, 34, 35, 38 and 100-106. Preferably, the CDRs are defined according to AbM, Chothia, Kabat, IMGT or any combination thereof. More preferably, the CDRs are defined according to Kabat or AbM or a combination thereof, and even more preferably, the CDRs are defined according to AbM. It should be understood, however, that the CDRs can also be defined in any other manner known in the art.

[0189] In some embodiments, the VHH NECTIN4 domain according to the present invention comprises CDR1, CDR2 and CDR3 sequences, wherein the CDR1, CDR2 and CDR3 sequences:

[0190] (i) respectively comprise or consist of the amino acid sequences of SEQ ID Nos: 18, 19 and 20;

[0191] (ii) respectively comprise or consist of the amino acid sequences of SEQ ID Nos: 22, 23 and 24;

[0192] (iii) respectively comprise or consist of the amino acid sequences of SEQ ID Nos: 26, 27 and 28;

[0193] (iv) respectively comprise or consist of the amino acid sequences of SEQ ID Nos: 18, 36 and 20; or

[0194] (v) respectively comprise or consist of the amino acid sequences of SEQ ID Nos: 18, 37 and 20.

[0195] In some embodiments, the VHH according to the present invention NECTIN4 domain comprises: the VHH variable region sequence of any of the exemplary antibodies of the present invention or a variant thereof, for example, an antibody or a fragment thereof, such as a humanized antibody, having the same CDR sequence as one of the exemplary antibodies and having the same or different framework region sequences. In some cases, preferably, 1 to several residue mutations are made to the framework sequence, for example, to remove post-translational modifications (PTMs) and / or deimmunize. The retention or improvement of the antigen-binding properties or other functional properties of the mutated antibody can be evaluated in in vitro or in vivo assay tests.

[0196] In some embodiments, the VHH according to the present invention NECTIN4 domain comprises a variable region sequence having an amino acid sequence selected from SEQ ID Nos: 17, 21, 25, 34, 35, 38 and 100 - 106. In still further embodiments, the VHH according to the present invention NECTIN4 domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% identity to an amino acid sequence selected from SEQ ID Nos: 17, 21, 25, 34, 35, 38 and 100 - 106 and retaining the ability to specifically bind NECTIN4. In still further embodiments, the VHH according to the present invention NECTIN4 domain comprises an amino acid sequence having one or more (preferably 1 - 10, more preferably 1 - 5) amino acid additions, deletions and / or substitutions (e.g., conservative substitutions) compared to an amino acid sequence selected from SEQ ID Nos: 17, 21, 25, 34, 35, 38 and 100 - 106 and retaining the ability to specifically bind NECTIN4. Preferably, the amino acid additions, deletions and / or substitutions do not occur in the CDR regions.

[0197] In some preferred embodiments, the VHH according to the present invention NECTIN4 domain comprises the CDR1 - 3 sequences of SEQ ID Nos: 22 - 24. In some embodiments, the VHH NECTIN4 domain comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto. Preferably, the VHH NECTIN4 domain comprises the amino acid sequence of SEQ ID NO: 21, or consists of the amino acid sequence shown by SEQ ID NO: 21.

[0198] In some preferred embodiments, the VHH according to the present invention NECTIN4The domain contains the CDR1 sequence of SEQ ID NO:18, the CDR2 sequence of SEQ ID NO:36, and the CDR3 sequence of SEQ ID NO:20. In some embodiments, the VHH NECTIN4 domain contains the amino acid sequence of SEQ ID NO:34, 38, 102, 104 or 106, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity therewith. Preferably, the VHH NECTIN4 domain contains the amino acid sequence of SEQ ID NO:34 or 38, or consists of the amino acid sequence shown by SEQ ID NO:34 or 38.

[0199] In some preferred embodiments, the VHH according to the present invention NECTIN4 domain contains the CDR1 sequence of SEQ ID NO:18, the CDR2 sequence of SEQ ID NO:37, and the CDR3 sequence of SEQ ID NO:20. In some embodiments, the VHH NECTIN4 domain contains the amino acid sequence of SEQ ID NO:35, 103 or 105, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity therewith. Preferably, the VHH NECTIN4 domain contains the amino acid sequence of SEQ ID NO:35, or consists of the amino acid sequence shown by SEQ ID NO:35.

[0200] Half-life extension domain

[0201] The multispecific antibody according to the present invention, in addition to the aforementioned antigen-binding domains, in some embodiments, may further comprise a half-life extension domain to adjust the circulating half-life time of the antibody after administration to an animal. Preferably, the half-life extension domain for the antibody of the present invention is a peptide or polypeptide structure, which can be conjugated to the antibody of the present invention by genetic fusion. Available peptide or polypeptide half-life extension domains include, but are not limited to, immunoglobulin Fc region, serum albumin or serum albumin-binding peptide. In some embodiments, preferably, the antibody of the present invention comprises an immunoglobulin Fc region, especially when the antibody adopts a double-chain configuration. In some embodiments, preferably, the antibody of the present invention comprises a serum albumin-binding peptide, especially when the antibody adopts a single-chain configuration. The half-life extension domain can be linked to the C- or N-terminus of the TROP2-binding domain or to the C- or N-terminus of NECTIN4.

[0202] Immunoglobulin Fc region

[0203] The half-life extension domain can comprise an immunoglobulin Fc region. 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. The immunoglobulin Fc region typically comprises two or three heavy chain constant domains (termed CH2, CH3, and CH4) and a hinge region, and is typically present in a dimeric form. The two chains in the dimeric Fc region can be linked by disulfide bonds within the hinge region. Fc regions from immunoglobulin isotypes IgG1, IgG2, and IgG4 are capable of binding to the FcRn receptor and undergoing FcRn-mediated recycling to provide a long circulatory half-life. The interaction site of IgG with FcRn has been determined to be in the Fc region covering portions of the CH2 and CH3 domains.

[0204] The immunoglobulin Fc region for use in the multispecific antibodies of the present invention can be from any immunoglobulin Fc region. In some embodiments, the immunoglobulin Fc region comprises at least the immunoglobulin CH2 domain and CH3 domain. In some embodiments, the immunoglobulin Fc region further comprises a hinge region or a partial hinge region. In some embodiments, the immunoglobulin Fc region comprises, from the N-terminus to the C-terminus, an immunoglobulin hinge region or a partial hinge region, the CH2 domain, and the CH3 domain, or consists of the foregoing. In some embodiments, the immunoglobulin Fc region comprises, from the N-terminus to the C-terminus, the CH2 domain and the CH3 domain, or consists of the foregoing. In some embodiments, the immunoglobulin Fc region is preferably from IgG1, IgG2, or IgG4, or a subtype thereof. Preferably, the immunoglobulin Fc region comprises a human Fc region sequence.

[0205] The immunoglobulin Fc region can be fused to the C- or N-terminus of other domains (i.e., the TROP2 or NECTIN4 binding domains, such as VHH TROP2 or VHH NECTIN4 ). The immunoglobulin Fc can be fused to other domains via a peptide linker or directly fused to other domains. In embodiments where the immunoglobulin Fc region is fused to other domains at its N-terminus, preferably, the fusion is carried out via an immunoglobulin hinge region sequence.

[0206] In some cases, an immunoglobulin Fc region comprising a hinge region sequence is preferred, which can, for example, promote dimerization of the antibody polypeptide chains and / or provide cysteine residues for coupling other active molecules. Such hinge sequences can substantially or partially correspond to the hinge regions of IgG1, IgG2, IgG3 or IgG4. For example, the hinge region sequence described can include all or part of the core hinge region and all or part of the 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 the two Fc chains. In some embodiments, the hinge region sequence comprises the hinge region sequence from E216 to T225 or from D221 to T225 of IgG1 (according to EU numbering), or the corresponding hinge region sequence from other immunoglobulin isotypes. In some embodiments, the hinge region sequence comprises the amino acid sequence of SEQ ID NO:96 or 97.

[0207] The immunoglobulin Fc region for the multispecific antibodies of the present invention can be a native Fc region sequence. Alternatively, the Fc region can contain mutations relative to the native Fc sequence. Mutations include substitutions, insertions and / or deletions. Such mutations can be made for the purpose of introducing desired therapeutic properties. For example, to promote heterodimerization, Knob-into-Hole (KiH) mutations can be introduced into the CH3 domain. In this case, one Fc chain is designed to contain a large protruding residue (i.e., Knob), while the other Fc chain is designed to contain a complementary pocket (i.e., Hole). Suitable positions for KiH mutations are known in the art. Exemplary KiH mutations include, but are not limited to, the Knob mutation T366W and the Hole mutations T366S, L368A, Y407V combination; and the Knob mutation T366Y and the Hole mutation Y407T combination. When the multispecific antibody of the present invention comprises an asymmetric double-stranded structure, preferably the Fc region contains KiH mutations that promote correct heterodimerization of the antibody polypeptide chains. Additionally or alternatively, cysteine mutations can be introduced into the Fc region to increase disulfide bond linkages in the dimerized Fc region. For example, the mutation S354C can be introduced into one Fc chain and the mutation Y349C can be introduced into the other Fc chain.

[0208] In addition, 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, in cases where effector function is not required, the Fc region may contain mutations that reduce or eliminate effector function. In some cases (such as when the antibody of the present invention is used as an ADC carrier), preferably, the Fc region contains mutations that reduce or eliminate the binding of the Fc region to Fcγ receptors, such as the LALA mutation in which lysine (L) at positions 234 and 235 of the Fc region is changed to alanine (A), to reduce Fcγ receptor-mediated off-target cytotoxicity. Additionally or alternatively, mutations can 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 conjugating active molecules. Additionally or alternatively, the Fc region may be mutated for antibody production reasons, such as removing or replacing amino acids that may undergo post-translational modifications (such as glycosylation), to provide improved drugability and developability of the therapeutic antibody.

[0209] In some embodiments, the multispecific antibody according to the present invention comprises an Fc region from IgG, such as the Fc region of IgG1, IgG2, or IgG4, preferably the Fc region from human IgG1 or human IgG4. Preferably, the Fc region comprises an amino acid sequence selected from SEQ ID NOs: 83-87 and 111-117 or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto.

[0210] Serum albumin-binding peptide

[0211] The half-life extension domain may comprise a serum albumin-binding peptide. As is known in the art, serum albumin has a long circulating half-life. Attachment to albumin is a well-known strategy for improving the in vivo circulating half-life of therapeutic molecules. Albumin can be non-covalently linked by using a specific albumin-binding domain, or covalently linked by conjugation or direct gene fusion.

[0212] The albumin-binding domain for the multispecific antibody of the present invention is preferably a peptide or polypeptide that binds serum albumin. Such a peptide or polypeptide is also referred to herein as a "serum albumin-binding peptide". Examples of serum albumin-binding peptides that can be used in the present invention include, for example, antibodies and antibody fragments that specifically bind serum albumin, such as single-domain antibody-based (O’Connor-Semmes et al., Clin Pharmacol Ther. December 2014; 96(6):704-12); and artificial proteins based on natural proteins that bind to serum albumin (such as streptococcal protein G), such as Other examples of serum albumin-binding peptides useful in the present invention include, for example, the many ISVDs that bind to human serum albumin described in WO 04 / 041865, WO 06 / 122787, WO 2012 / 175400, said ISVDs being selected from the serum albumin-binding portions of Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10 and Alb-23.

[0213] The serum albumin-binding peptide can be fused to other domains (i.e., TROP2 or NECTIN4-binding domains, such as VHH TROP2 or VHH NECTIN4 ) at the C- or N-terminus. The serum albumin-binding peptide can be fused to other domains via a peptide linker or directly fused to other domains. Preferably, the serum albumin-binding peptide is fused to other domains via a peptide linker. The sequence or optimal length of the available peptide linkers can be readily determined based on the site of attachment. In some embodiments, preferably, the peptide linker is 5-15 amino acids in length, more preferably the peptide linker comprises the amino acid sequence G4S or (G4S)2.

[0214] In some embodiments, the serum albumin-binding peptide for the multispecific antibodies of the present invention is an antibody or antibody fragment that specifically binds serum albumin, especially a single-chain antibody or a single-domain antibody. In some embodiments, the serum albumin-binding peptide is a VHH domain that specifically binds serum albumin (also abbreviated herein as VHH SA ). Preferably, the serum albumin is human serum albumin (HSA). Preferably, the serum albumin-binding peptide has a picomolar to micromolar binding affinity for human serum albumin.

[0215] In some embodiments, the multispecific antibody according to the present invention comprises a VHH domain that specifically binds serum albumin. In some embodiments, the VHH SA comprises CDR1, CDR2, and CDR3 in the variable region sequence having an amino acid sequence selected from SEQ ID NO: 39 or 40. The CDRs can be defined according to AbM, Chothia, Kabat, IMGT, or any combination thereof. In some embodiments, the VHH SA comprises the amino acid sequence of SEQ ID NO: 39 or 40 or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto. In some embodiments, the VHH SA comprises or consists of the amino acid sequence of SEQ ID NO: 40.

[0216] Peptide linker

[0217] In the multispecific antibodies according to the invention, the antibody components (i.e., antigen-binding domains and optionally half-life-binding domains) can be linked using a peptide linker.

[0218] There is no particular limitation on the peptide linker that can be used in the antibodies of the invention. The peptide linker sequence is generally flexible. It can mainly consist of amino acids such as glycine, alanine, and serine that do not have large side chains that may limit flexibility. Alternatively, it can consist of a sequence from an immunoglobulin hinge region. Depending on the connection position and the components to be connected, those skilled in the art can easily determine the sequence or optimal length of the available peptide linker.

[0219] Suitable peptide 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 in length, or longer. In some cases, the length of the peptide linker sequence can be shorter, for example less than about 20 or 15 amino acids in length, such as 2-15 amino acids in length or 5-10 amino acids in length.

[0220] Suitable peptide linker sequences include, but are not limited to, G4S (SEQ ID NO:88); (G4S)2 (SEQ ID NO:89); (G4S)3 (SEQ ID NO:90); GGGSG (SEQ ID NO:118); GGSGG (SEQ ID NO:119); GSGGG (SEQ ID NO:120); GSGGGP (SEQ ID NO:121); GGEPS (SEQ ID NO:122); GGEGGGP (SEQ ID NO:123); and GGEGGGSEGGGS (SEQ ID NO:124); and (G4S)n (SEQ ID NO:91), where n is an integer equal to or greater than 1; TS(G4S)n (SEQ ID NO:92), where n is an integer equal to or greater than 1; G(G4S)n (SEQ ID NO:93), where n is an integer equal to or greater than 1; (G4)n (SEQ ID NO:94), where n is an integer equal to or greater than 1; (GRPGS)n (SEQ ID NO:95), where n is an integer equal to or greater than 1. Peptide linkers that can be used in the antibody molecules of the present invention can also be, for example but not limited to, the following amino acid sequences: (G3S)2 (SEQ ID NO:125), (G4S)2 (SEQ ID NO:89), (G3S)3 (SEQ ID NO:126), (G4S)3 (SEQ ID NO:90), (G3S)4 (SEQ ID NO:127), (G4S)4 (SEQ ID NO:128), (G3S)5 (SEQ ID NO:129), (G4S)5 (SEQ ID NO:130), (G3S)6 (SEQ ID NO:131), (G4S) (SEQ ID NO:88), GGG (SEQ ID NO:132), DGGGS (SEQ ID NO:133), TGEKP (SEQ ID NO:134), GGRR (SEQ ID NO:135), EGKSSGSGSESKVD (SEQ ID NO:136), KESGSVSSEQLAQFRSLD (SEQ ID NO:137), GGRRGGGS (SEQ ID NO:138), LRQRDGERP (SEQ ID NO:139), LRQKDGGGSERP (SEQ ID NO:140), and GSTSGSGKPGSGEGSTKG (SEQ ID NO:141). Alternatively, computer programs can be used to simulate the three-dimensional structures of proteins and peptides, or phage display methods can be used to rationally design suitable flexible linker peptides.

[0221] In some embodiments, the peptide linker used in the antibodies of the present invention is a flexible linking peptide of 5 - 50 amino acids, preferably a linking peptide comprising glycine (G) and / or serine (S) and / or threonine residues (T). In one embodiment, the peptide linker has a length of 5 - 50 amino acids, for example, a length of 5, 10, 15, 20, 25, or 30 amino acids, or has an amino acid length falling between any two integers. In some embodiments, the peptide linker comprises the amino acid sequence (G4S) n (SEQ ID NO:91), 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. In still other embodiments, the peptide linker is from the hinge region of an immunoglobulin.

[0222] In some preferred embodiments, in the multispecific antibodies according to the present invention, the peptide linker for linking the antigen - binding domain and the half - life extension domain (if present) comprises the amino acid sequence G4S (SEQ ID NO:88) or (G4S)2 (SEQ ID NO:89).

[0223] Form of multispecific antibody

[0224] The multispecific antibodies according to the present invention can adopt any suitable form, such as single - chain or double - chain form.

[0225] In some embodiments, the present invention provides multispecific antibodies in single - chain form. In some embodiments, the multispecific antibodies according to the present invention comprise a single polypeptide chain, wherein the polypeptide chain comprises at least one (preferably 1 or 2) Nectin4 - binding domain and at least one (preferably 1 or 2) Trop2 - binding domain and optionally at least one half - life extension domain, wherein the half - life extension domain is a serum albumin - binding peptide, and preferably the domains are linked by a peptide linker.

[0226] In some embodiments, the present invention provides multispecific antibodies in double - chain form. In some embodiments, the multispecific antibodies according to the present invention comprise a first and a second polypeptide chain, wherein the first and second polypeptide chains respectively comprise at least one (preferably 1 or 2) Nectin4 - binding domain and / or at least one (preferably 1 or 2) Trop2 - binding domain and at least one (preferably 1) half - life extension domain, wherein the half - life extension domain is an immunoglobulin Fc region, and preferably the domains on the same chain are linked by a peptide linker or directly linked.

[0227] Single-chain form

[0228] In some embodiments, the present invention provides a multispecific antibody comprising a single polypeptide chain. In some embodiments, the polypeptide chain comprises, from the N-terminus to the C-terminus:

[0229] (VHH A ) n1 -(VHH B ) n2 -(HLE) n3 -(VHH A ) n4 -(VHH B ) n5 -(HLE) n6， (I)

[0230] wherein n1, n2, n3, n4, n5, and n6 are each independently selected from the integers 0, 1, or 2; wherein VHH A and VHH B represent VHH domains that bind to antigens A and B, respectively, wherein A and B are different from each other and are independently selected from Trop2 and Nectin4; wherein HLE represents a serum albumin-binding peptide as a half-life extension domain; and wherein the symbol "-" represents connection by a peptide linker or direct connection, preferably representing a peptide linker of 5-15 amino acids in length.

[0231] The multispecific antibody can have any suitable valence. In some cases, preferably the multispecific antibody has a valence of 2 to 6 (i.e., n1 + n2 + n4 + n5 = 2 to 6), more preferably not exceeding 4 (i.e., n1 + n2 + n4 + n5 = 2 to 4).

[0232] The number of VHH Nectin4 domains and VHH Trop2 domains in the multispecific antibody can be equal or unequal. In some embodiments, preferably, the number of VHH Trop2 domains does not exceed 4, preferably does not exceed 3, for example 1-2. In some embodiments, preferably the number of VHH Nectin4 domains does not exceed 4, preferably does not exceed 3, for example 1-2. In some embodiments, preferably, the ratio of the number of VHH Trop2 domains to the number of VHH Nectin4 domains is preferably 1:1 or 1:2. In one embodiment, the number of VHH Trop2 domains is 1 or 2, and the number of VHH Nectin4 domains is 1 or 2.

[0233] The multispecific antibody can optionally contain or not contain an HLE domain, for example the antibody can contain 0 or 1 HLE.

[0234] In some preferred embodiments, the present invention provides a multispecific antibody of formula I above, wherein n1, n2, n3, n4, n5 and n6 are each independently selected from the integers 0, 1 or 2; and wherein n1 + n4 = 1 or 2, n2 + n5 = 1 or 2, and n3 + n6 = 0 or 1.

[0235] In some more preferred embodiments, the present invention provides a multispecific antibody comprising a single polypeptide chain, wherein the polypeptide chain comprises, from the N-terminus to the C-terminus:

[0236] (i) VHH A -VHH B ,

[0237] (ii) VHH A -VHH B -HLE,

[0238] (iii) VHH B -VHH A -VHH B ,

[0239] (iv) VHH A -VHH B -VHH B ,

[0240] (v) VHH A -HLE-VHH B -VHH B , or

[0241] (vi) VHH A -VHH A -HLE-VHH B -VHH B , wherein preferably A represents Trop2 and B represents Nectin4.

[0242] In the above-described single-chain antibody embodiments, preferably, the HLE represents an anti-serum albumin VHH domain (VHH SA ). The VHH SA domain can be any VHH SA domain described herein or known in the art. For example, by way of example, the VHH SAThe domain may comprise an amino acid sequence selected from SEQ ID NOs: 39 and 40, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity therewith, or having additions, deletions and / or substitutions of one or more (preferably 1-10, more preferably 1-5) amino acids. In some embodiments, preferably, the HLE comprises or consists of the amino acid sequence of SEQ ID NO: 40.

[0243] In the above embodiments of the single-chain form of the antibody, preferably, the domains are linked by a peptide linker. Preferably, the peptide linker has a length of 5-15 amino acids and more preferably comprises the G4S amino acid sequence or the (G4S)2 amino acid sequence.

[0244] As understood by those skilled in the art, for the convenience of subsequent purification or ligation with other active molecules, the single-chain multispecific antibody of the present invention may optionally be added with a purification tag (such as His tag) or other auxiliary peptide sequences at the N-terminus or C-terminus of the polypeptide chain. The addition can be achieved through a peptide linker (such as AAA) or by direct ligation.

[0245] The multispecific antibody having the polypeptide chain of formula (I) is preferably a trivalent bispecific antibody or a tetravalent bispecific antibody.

[0246] Symmetric double-chain form

[0247] In some embodiments, the present invention provides a multispecific antibody comprising two identical polypeptide chains. In some embodiments, the present invention provides a multispecific antibody comprising the following polypeptide chain, wherein the polypeptide chain comprises, from the N-terminus to the C-terminus:

[0248] (VHH A ) n1 -(VHH B ) n2 -HLE-(VHH A ) n3 -(VHH B ) n4， (II)

[0249] wherein n1, n2, n3 and n4 are each independently an integer selected from 0, 1 or 2;

[0250] wherein VHH A and VHH BRespectively represent VHH domains that bind to antigens A and B, where A and B are different from each other and independently selected from Trop2 and Nectin4; where HLE represents the immunoglobulin Fc region as a half-life extension domain, especially the human IgG1 or human IgG4 Fc region; where the symbol "-" represents connection by a peptide linker or direct connection, preferably a peptide linker with a length of 5-15 amino acids.

[0251] The multispecific antibody can have any suitable valence. In some cases, preferably, the multispecific antibody has a valence of 2 to 6 (i.e., 2x(n1 + n2 + n3 + n4) = 2 to 6), and more preferably, no more than 4 valence.

[0252] In the multispecific antibody, the VHH Nectin4 domain and the VHH Trop2 domain can be equal or unequal in number. In some embodiments, preferably, the number of VHH Trop2 domains does not exceed 4, preferably does not exceed 3, for example, it is 1-2. In some embodiments, preferably, the number of VHH Nectin4 domains does not exceed 4, preferably does not exceed 3, for example, it is 1-2. In some embodiments, preferably, the number of VHH Trop2 domains and the number of VHH Nectin4 domains preferably have a ratio of 1:1 or 1:2. In one embodiment, the number of VHH Trop2 domains is 1 or 2, and the number of VHH Nectin4 domains is 1 or 2.

[0253] In some preferred embodiments, n3 and n4 are 0, and the polypeptide chain of formula (II) from the N-terminus to the C-terminus includes: (VHH A ) n1 -(VHH B ) n2 -HLE, where n1 and n2 are independently selected from integers of 1 or 2.

[0254] In some more preferred embodiments, n3 and n4 are 0, and n1 and n2 are 1. The polypeptide chain of formula (II) from the N-terminus to the C-terminus includes: VHH A -VHH B -HLE, for example, VHH Nectin4 -VHH Trop2 -Fc or VHH Trop2 -VHH Nectin4 -Fc.

[0255] In some further preferred embodiments, the polypeptide chain of formula (II) from the N-terminus to the C-terminus includes VHH Nectin4 -VHHTrop2 -Fc.

[0256] In the above-mentioned multispecific antibody, preferably, each antigen-binding domain on the polypeptide chain (i.e., the VHH Trop2 domain and the VHH Nectin4 domain) is connected by a peptide linker. Preferably, the peptide linker has a length of 5-15 amino acids, and more preferably contains the G4S amino acid sequence or the (G4S)2 amino acid sequence. In the above-mentioned multispecific antibody, preferably, the Fc region contains a hinge region or a partial hinge region and is directly connected to the domain located at its N-terminus.

[0257] The multispecific antibody having the polypeptide chain of formula (II) can associate to form a homodimer due to the dimerization of the immunoglobulin Fc region, thereby generating a double-stranded form of the multispecific binding molecule. In some embodiments, the Fc region contains the amino acid sequence 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.

[0258] The multispecific antibody having the polypeptide chain of formula (II) is preferably a tetravalent bispecific antibody.

[0259] Asymmetric double-chain form

[0260] In some embodiments, the present invention provides a multispecific antibody comprising two different polypeptide chains. In some embodiments, the present invention provides a multispecific antibody comprising a first polypeptide chain and a second polypeptide chain, wherein

[0261] The first polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE-(VHH A ) n3 -(VHH B ) n4， (III)

[0262] The second polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH B ) m1 -(VHH A ) m2 -HLE-(VHH B ) m3 -(VHH A ) m4， (IV)

[0263] wherein n1, n2, n3 and n4, and m1, m2, m3 and m4 are independently selected from the integers 0, 1 or 2;

[0264] wherein VHH A and VHH B respectively represent VHH domains that bind to antigens A and B, where A and B are different from each other and independently selected from Trop2 and Nectin4; wherein HLE represents an immunoglobulin Fc region as a half-life extension domain, particularly a human IgG1 or IgG4 Fc region; wherein the symbol "-" represents connection by a peptide linker or direct connection, preferably a peptide linker having a length of 5 to 15 amino acids.

[0265] The multispecific antibody can have any suitable valence. In some cases, preferably the multispecific antibody has a valence of 2 to 6 (i.e., n1 + n2 + n3 + n4 + m1 + m2 + m3 + m4 = 2 to 6), more preferably not exceeding 4 valences.

[0266] In the multispecific antibody, the number of VHH Nectin4 domains and VHH Trop2 domains can be equal or unequal. In some embodiments, preferably, the number of VHH Trop2 domains does not exceed 4, preferably does not exceed 3, for example 1 to 2. In some embodiments, preferably the number of VHH Nectin4 domains does not exceed 4, preferably does not exceed 3, for example 1 to 2. In some embodiments, preferably, the number of VHH Trop2 domains to the number of VHH Nectin4 domains is preferably 1:1 or 1:2. In one embodiment, the number of VHH Trop2 domains is 1 or 2, and the number of VHH Nectin4 domains is 1 or 2.

[0267] In some preferred embodiments, n3, n4, m3, and m4 are 0, and the multispecific antibody comprises a first and a second polypeptide chain, wherein

[0268] The first polypeptide chain from the N-terminus to the C-terminus comprises: (VHH A ) n1 -(VHH B ) n2 -HLE ，

[0269] The second polypeptide chain from the N-terminus to the C-terminus comprises: (VHH B ) m1 -(VHH A ) m2 -HLE ，

[0270] wherein n1, n2, and m1, m2 are independently selected from the integers 0, 1, or 2, provided that n1 and n2 are not both 0, m1 and m2 are not both 0, and the sum of n1 and m2 and the sum of n2 and m1 are greater than or equal to 1.

[0271] In some more preferred embodiments, n3, n4, m3, and m4 are 0, and m1 = 0 and m2 = 1 or m2 = 0 and m1 = 1, and the multispecific antibody comprises a first and a second polypeptide chain, wherein

[0272] The first polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE ，

[0273] The second polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH B ) m1 -HLE ； Or (VHH A ) m2 -HLE ，

[0274] wherein n1, m1, and m2 are independently selected from the integers 1 or 2, and n2 is an integer of 0 or 1.

[0275] In some further preferred embodiments, n3, n4, m3, and m4 are 0, n1 + m2 = 1 or 2, n2 + m1 = 1 or 2, and m1 = 0 and m2 = 1 or m2 = 0 and m1 = 1, and the multispecific antibody comprises a first and a second polypeptide chain, wherein

[0276] The first polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH A )-HLE ，

[0277] The second polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH B )-HLE ；

[0278] Or,

[0279] The first polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH A )-(VHH B )-HLE ，

[0280] The second polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH A )-HLE ；

[0281] Or,

[0282] The first polypeptide chain contains, from the N-terminus to the C-terminus: (VHH A )-(VHH A )-HLE ，

[0283] The second polypeptide chain contains, from the N-terminus to the C-terminus: (VHH B )-HLE ；

[0284] Alternatively,

[0285] The first polypeptide chain contains, from the N-terminus to the C-terminus: (VHH A )-(VHH B )-HLE ，

[0286] The second polypeptide chain contains, from the N-terminus to the C-terminus: (VHH B )-HLE.

[0287] In some further preferred embodiments, the multispecific antibody comprises a first and a second polypeptide chain, wherein:

[0288] The first polypeptide chain contains, from the N-terminus to the C-terminus: (VHH A )-HLE ，

[0289] The second polypeptide chain contains, from the N-terminus to the C-terminus: (VHH B )-HLE ；

[0290] Alternatively,

[0291] The first polypeptide chain contains, from the N-terminus to the C-terminus: (VHH A )-(VHH B )-HLE ，

[0292] The second polypeptide chain contains, from the N-terminus to the C-terminus: (VHH A )-HLE ；

[0293] Alternatively,

[0294] The first polypeptide chain contains, from the N-terminus to the C-terminus: (VHH A )-(VHH A )-HLE ，

[0295] The second polypeptide chain contains, from the N-terminus to the C-terminus: (VHH B )-HLE ；

[0296] wherein A represents NECTIN4, and B represents TROP2.

[0297] In some of the most preferred embodiments, the multispecific antibody comprises first and second polypeptide chains, wherein:

[0298] The first polypeptide chain comprises, from the N-terminus to the C-terminus: VHH Nectin4 -VHH Trop2 -Fc; and

[0299] The second polypeptide chain comprises, from the N-terminus to the C-terminus: VHH Nectin4 -Fc.

[0300] Due to the dimerization of the immunoglobulin Fc region, the first and second polypeptide chains of the above multispecific antibody can associate to form a heterodimer, thereby generating a multispecific binding molecule in a double-chain form. Preferably, in order to promote the heterodimerization of the first and second polypeptide chains, Knob-into-hole mutations can be introduced into the Fc regions of the first and second polypeptide chains, such as (T366W / T366S, L368A, Y407V) mutations or (T366Y / Y407T) mutations. Preferably, the Fc region comprises an amino acid sequence from human IgG1 or IgG4, and preferably, the Fc region further comprises mutations that reduce or eliminate Fcγ receptor binding, such as LALA mutations.

[0301] In some embodiments, the multispecific antibody according to the present invention comprises a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region comprise amino acid mutations that promote the formation of the Fc dimer. In some embodiments, the first Fc region comprises T336W and S354C, and the second Fc region comprises T366S, L368A, Y407V and Y349C, or vice versa; or, the first Fc region comprises T336W, and the second Fc region comprises T366S, L368A and Y407V, or vice versa; or, the first Fc region comprises T366Y, and the second Fc region comprises Y407T, or vice versa. In some further embodiments, the first Fc region and the second Fc region further respectively comprise mutations that reduce or eliminate the binding of the Fc region to FcγR, such as, L234AL235A mutations. In some embodiments, preferably, the first and second Fc regions comprise amino acid sequences selected from SEQ ID NOs: 111-116 or amino acid sequences having at least 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto.

[0302] In the above multispecific antibody, preferably, each antigen-binding domain on the polypeptide chain (i.e., the VHH Trop2 domain and the VHH Nectin4The domains are connected by a peptide linker. Preferably, the peptide linker has a length of 5-15 amino acids, and more preferably, it contains the G4S amino acid sequence or the (G4S)2 amino acid sequence. In the above-mentioned multispecific antibody, preferably, the Fc region contains a hinge region or a partial hinge region and is directly connected to the domain located at its N-terminus.

[0303] The multispecific antibody having the first and second polypeptide chains of formula (III) and formula (IV) is preferably a trivalent bispecific antibody or a tetravalent bispecific antibody.

[0304] Example antigen domain combinations

[0305] In some embodiments of the single-chain and double-chain form multispecific antibodies according to the present invention described above, preferably, the multispecific antibody according to the present invention comprises the following combinations of TROP2 and NECTIN4 antigen-binding domains:

[0306] (a) The antigen-binding domain that specifically binds Nectin4 contains 3 CDRs in the amino acid sequence of SEQ ID NO:25, and the antigen-binding domain that specifically binds Trop2 contains 3 CDRs in the amino acid sequence of SEQ ID NO:13;

[0307] (b) The antigen-binding domain that specifically binds Nectin4 contains 3 CDRs in the amino acid sequence of SEQ ID NO:21, and the antigen-binding domain that specifically binds Trop2 contains 3 CDRs in the amino acid sequence of SEQ ID NO:9;

[0308] (c) The antigen-binding domain that specifically binds Nectin4 contains 3 CDRs in the amino acid sequence of SEQ ID NO:25, and the antigen-binding domain that specifically binds Trop2 contains 3 CDRs in the amino acid sequence of SEQ ID NO:9;

[0309] (d) The antigen-binding domain that specifically binds Nectin4 contains 3 CDRs in the amino acid sequence of SEQ ID NO:35, and the antigen-binding domain that specifically binds Trop2 contains 3 CDRs selected from the amino acid sequences of SEQ ID NO:29 and 30; or

[0310] (e) The antigen-binding domain that specifically binds Nectin4 contains 3 CDRs selected from the amino acid sequences of SEQ ID NO:34 and 38, and the antigen-binding domain that specifically binds Trop2 contains 3 CDRs selected from the amino acid sequences of SEQ ID NO:29 and 30.

[0311] In some embodiments of the single-chain and double-chain forms of the multispecific antibodies according to the present invention, more preferably, the multispecific antibodies according to the present invention comprise the following combinations of TROP2 and NECTIN4 antigen-binding domains:

[0312] (a) The antigen-binding domain that specifically binds Nectin4 comprises the amino acid sequence of SEQ ID NO:21, and the antigen-binding domain that specifically binds Trop2 comprises the amino acid sequence of SEQ ID NO:9;

[0313] (b) The antigen-binding domain that specifically binds Nectin4 comprises the amino acid sequence of SEQ ID NO:35 and the antigen-binding domain that specifically binds Trop2 comprises an amino acid sequence selected from SEQ ID NO:29 and 30; or

[0314] (c) The antigen-binding domain that specifically binds Nectin4 comprises an amino acid sequence selected from SEQ ID NO:34 and 38 and the antigen-binding domain that specifically binds Trop2 comprises an amino acid sequence selected from SEQ ID NO:29 and 30.

[0315] In some embodiments of the single-chain and double-chain forms of the multispecific antibodies according to the present invention, even more preferably, the multispecific antibodies according to the present invention comprise the following combinations of TROP2 and NECTIN4 antigen-binding domains:

[0316] (a) The antigen-binding domain that specifically binds Nectin4 comprises an amino acid sequence selected from SEQ ID NO:34 and the antigen-binding domain that specifically binds Trop2 comprises an amino acid sequence selected from SEQ ID NO:29; or

[0317] (b) The antigen-binding domain that specifically binds Nectin4 comprises an amino acid sequence selected from SEQ ID NO:38 and the antigen-binding domain that specifically binds Trop2 comprises an amino acid sequence selected from SEQ ID NO:30.

[0318] Exemplary multispecific antibody

[0319] In some embodiments, the present invention provides a multispecific antibody, wherein the antibody is in a symmetric double-chain form, and wherein the polypeptide chain of the antibody comprises the amino acid sequence of SEQ ID NO:53 or 54 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, preferably comprising the amino acid sequence of SEQ ID NO:53.

[0320] In some embodiments, the present invention provides multispecific antibodies, wherein the antibodies are in an asymmetric double-chain form, and wherein the antibodies comprise first and second polypeptide chains selected from the group consisting of:

[0321] (i) a first and a second polypeptide chain comprising SEQ ID NOs: 55 and 56, respectively, or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity thereto,

[0322] (ii) a first and a second polypeptide chain comprising SEQ ID NOs: 57 and 58, respectively, or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity thereto,

[0323] (iii) a first and a second polypeptide chain comprising SEQ ID NOs: 59 and 60, respectively, or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity thereto,

[0324] (iv) a first and a second polypeptide chain comprising SEQ ID NOs: 61 and 62, respectively, or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity thereto,

[0325] (v) a first and a second polypeptide chain comprising SEQ ID NOs: 63 and 64, respectively, or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity thereto,

[0326] (vi) a first and a second polypeptide chain comprising SEQ ID NOs: 65 and 66, respectively, or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity thereto,

[0327] (vii) a first and a second polypeptide chain comprising SEQ ID NOs: 67 and 68, respectively, or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity thereto,

[0328] (viii) a first and a second polypeptide chain comprising SEQ ID NOs: 69 and 70, respectively, or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity thereto,

[0329] (ix) a first and a second polypeptide chain comprising SEQ ID NOs: 71 and 72, respectively, or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity thereto,

[0330] (x) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 73 and 74 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, and

[0331] (xi) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 75 and 76 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto,

[0332] Preferably, the first and second polypeptide chains each comprise the amino acid sequences of SEQ ID NOs: 61 / 62, 69 / 70 or 71 / 72.

[0333] In some embodiments, the invention provides a multispecific antibody, wherein the antibody is in single-chain form and the polypeptide chain of the antibody comprises an amino acid sequence selected from SEQ ID NOs: 41 - 52 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.

[0334] Properties of the multispecific antibody of the present invention

[0335] The multispecific antibodies of the invention may have one or more of the following properties:

[0336] (i) Bind to tumor cells co-expressing TROP2 and NECTIN4 with high avidity;

[0337] (ii) Have species cross-reactivity with cynomolgus monkey TROP2 and NECTIN4;

[0338] (iii) Have TROP2 and / or NECTIN4 receptor-mediated endocytic activity;

[0339] (iv) Exhibit cooperative binding on tumor cells co-expressing TROP2 and NECTIN4 antigens;

[0340] (v) Exhibit cooperative endocytosis on tumor cells co-expressing TROP2 and NECTIN4 antigens.

[0341] In some aspects, the multispecific antibodies of the present invention have high binding affinity for tumor cells co-expressing TROP2 and NECTIN4. The binding EC50 value and / or the maximum binding amount of the antibodies of the present invention to TROP2 and / or NECTIN4 positive tumor cells can be determined by FACS or ELISA assays (such as the assays described in the examples), and optionally compared with a reference antibody to reflect the cellular binding affinity of the antibody. In some embodiments, compared with TROP2 and NECTIN4 monospecific binding molecules, the multispecific antibodies of the present invention exhibit a synergistic binding effect on tumor cells co-expressing TROP2 and NECTIN4.

[0342] In some embodiments, the multispecific antibodies of the present invention exhibit cross-reactivity to human and monkey TROP2 and NECTIN4. In some embodiments, the KD value of the antibody of the present invention binding to human TROP2 is approximately equivalent to the KD value of the antibody binding to monkey TROP2. For example, the KD ratio between the two is between 1 and 10, such as between 1 and 5, and more preferably between approximately 1 and 3. In still some embodiments, the KD value of the antibody binding to human TROP2 is approximately equivalent to the KD value of the antibody binding to monkey TROP2. For example, the KD ratio (human / monkey KD ratio or monkey / human KD ratio) between the two is between 1 and 10, such as between 1 and 5, and more preferably between approximately 1 and 3. In some embodiments, the KD value of the antibody of the present invention binding to human NECTIN4 is approximately equivalent to the KD value of the antibody binding to monkey NECTIN4. For example, the KD ratio between the two is between 1 and 5, and more preferably between approximately 1 and 3. In still some embodiments, the KD value of the antibody binding to human NECTIN4 is approximately equivalent to the KD value of the antibody binding to monkey NECTIN4. For example, the KD ratio (human / monkey KD ratio or monkey / human KD ratio) between the two is between 1 and 10, such as between 1 and 5, and more preferably between approximately 1 and 3.

[0343] In some embodiments, the multispecific antibodies of the present invention have endocytic activity mediated by the TROP2 and / or NECTIN4 receptors. The endocytic activity of the antibodies can be evaluated in cell-based assays, such as those described in the Examples. In some embodiments, in an assay based on TROP2 and / or NECTIN4 positive cells, after incubating the antibody to be tested with TROP2 and / or NECTIN4 positive cells (especially TROP2 and / or NECTIN4 positive tumor cells) at 37°C for a period of time (e.g., 2 hours or 4 hours), compared to a negative control maintained at 4°C for the same time, the change in the amount of antibody bound to the cell surface is detected by fluorescence using flow cytometry to determine the endocytosis and / or endocytic rate of the antibody. In some embodiments, when compared with a TROP2 monospecific binding molecule or a NECTIN4 monospecific binding molecule, the internalization mediated by the multispecific antibodies of the present invention shows greater selectivity for tumor cells co-expressing both targets, whereby the adverse effects of the antibody on normal tissues that do not show a significant level of co-expression of TROP2 and NECTIN4 or only high expression of TROP2 or only high expression of NECTIN4 can be minimized. In some embodiments, compared with TROP2 and NECTIN4 monospecific binding molecules, the multispecific antibodies of the present invention exhibit synergistic endocytosis for tumor cells co-expressing TROP2 and NECTIN4.

[0344] In still further embodiments, the multispecific antibodies of the present invention further have one or more of the following characteristics: (iv) good developability; (v) good stability; and (vi) favorable pharmacokinetic properties.

[0345] In some embodiments, after one-step purification from an antibody product produced by recombinant mammalian cells using protein A affinity chromatography, the multispecific antibodies of the present invention can achieve a purity of 90% or more than 95% as determined by SEC-HPLC.

[0346] In still further embodiments, the multispecific antibodies of the present invention have good stability. In some embodiments, as determined by the assay in the Examples, after being placed at room temperature for 14 days, the antibodies of the present invention show no change in antigen-binding activity compared to before storage.

[0347] In still further embodiments, the multispecific antibodies of the present invention have serum stability. The serum stability of the antibodies can be determined by measuring the circulating half-life of the antibodies in animals according to the animal experiments in the Examples.

[0348] II. The anti-Trop2 VHH domain and anti-Trop2 antibody of the present invention

[0349] In a second aspect, the present invention provides an anti-Trop2 VHH domain that binds to Trop2 and an anti-Trop2 antibody comprising said VHH domain. In some embodiments, the anti-Trop2 VHH domain according to the present invention comprises CDR1, CDR2, and CDR3 sequences selected from the amino acid sequences of SEQ ID NOs: 5, 9, 13, 29, 30, 98, and 99;

[0350] More preferably, the CDR1, CDR2, and CDR3 sequences:

[0351] (i) comprise or consist of the amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively;

[0352] (ii) comprise or consist of the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively;

[0353] (iii) comprise or consist of the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively; or

[0354] (iv) comprise or consist of the amino acid sequences of SEQ ID NOs: 31, 32, and 33, respectively;

[0355] More preferably, the VHH domain comprises an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5, 9, 13, 29, 30, 98, and 99, or an amino acid sequence having at least 85%, 90%, 95%, or 99% identity to said amino acid sequence, or having an addition, deletion, and / or substitution of one or more (preferably 1-10, more preferably 1-5) amino acids,

[0356] Most preferably, the VHH domain comprises an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5, 9, 13, 29, 30, 98, and 99, or consists of an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5, 9, 13, 29, 30, 98, and 99.

[0357] The anti-Trop2 antibody according to the present invention may comprise or consist of an anti-Trop2 VHH domain according to the present invention and may have any suitable antibody structure, including, but not limited to, single-chain antibodies or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), linear antibodies, single-domain antibodies, heavy-chain antibodies, chimeric antibodies or humanized antibodies. In some embodiments, the anti-Trop2 antibody according to the present invention comprises an immunoglobulin Fc region linked to the VHH domain (i.e., has the form of VHH-Fc). In some embodiments, the anti-Trop2 antibody according to the present invention is a heavy-chain antibody. As used herein, the term "heavy-chain antibody (hcAb)" refers to an antibody that does not have a light chain and may comprise VHH-CH2-CH3 or VHH-CH1-CH2-CH3 from the N-terminus to the C-terminus; it may form a homodimer, such as a heavy-chain dimer antibody without a light chain. In some embodiments, the anti-Trop2 antibody according to the present invention further comprises an anti-Nectin4 binding domain and is preferably the anti-Trop2 and NECTIN4 multispecific antibody according to the present invention described in Section I above.

[0358] The anti-Trop2 VHH domain according to the present invention has good tumor targeting, tumor tissue penetration ability and target cell endocytosis ability, and has human-monkey species immune cross-reactivity. Therefore, the anti-Trop2 VHH domain according to the present invention or the anti-Trop2 antibody according to the present invention can be used as a targeting module for conjugates or conjugates and conjugated or coupled with, such as chemotherapeutic agents, toxins, drugs (such as immunotherapeutic agents), radioactive elements, probes or signal molecules, etc., to provide better tumor killing, immune regulation or disease detection and other applications.

[0359] III. Anti-NETIN4 VHH Domain and Anti-NETIN4 Antibody of the Present Invention

[0360] In a third aspect, the present invention provides an anti-Nectin4 VHH domain that binds Nectin4 and an anti-Nectin4 antibody comprising the VHH domain. In some embodiments, the anti-Nectin4 VHH domain according to the present invention comprises CDR1, CDR2 and CDR3 sequences selected from the amino acid sequences of SEQ ID NOs: 17, 21, 25, 34, 35, 38 and 100-106;

[0361] Preferably, the CDR1, CDR2 and CDR3 sequences:

[0362] (i) comprise or consist of the amino acid sequences of SEQ ID NOs: 18, 19 and 20, respectively;

[0363] (ii) Comprising or consisting of the amino acid sequences set forth in SEQ ID NOs: 22, 23 and 24, respectively;

[0364] (iii) Comprising or consisting of the amino acid sequences set forth in SEQ ID NOs: 26, 27 and 28, respectively;

[0365] (iv) Comprising or consisting of the amino acid sequences set forth in SEQ ID NOs: 18, 36 and 20, respectively; or

[0366] (v) Comprising or consisting of the amino acid sequences set forth in SEQ ID NOs: 18, 37 and 20, respectively;

[0367] More preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35, 38 and 100 - 106, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity to the amino acid sequence, or having an addition, deletion and / or substitution of one or more (preferably 1 - 10, more preferably 1 - 5) amino acids.

[0368] Most preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35, 38 and 100 - 106, or consists of an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35, 38 and 100 - 106.

[0369] The anti - NECTIN4 antibody according to the present invention may comprise or consist of an anti - Nectin4 VHH domain according to the present invention and may have any suitable antibody structure, including, but not limited to, single - chain antibodies or multi - chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), linear antibodies, single - domain antibodies, heavy - chain antibodies, chimeric antibodies or humanized antibodies. In some embodiments, the anti - NECTIN4 antibody according to the present invention comprises an immunoglobulin Fc region linked to the VHH domain (i.e., having the VHH - Fc form). In some embodiments, the anti - NECTIN4 antibody according to the present invention is a heavy - chain antibody. In some embodiments, the anti - NECTIN4 antibody according to the present invention further comprises an anti - TROP2 binding domain and is preferably the anti - Trop2 and NECTIN4 multispecific antibody according to the present invention described in Section I above.

[0370] The anti-NECTIN4 VHH domain according to the present invention has good tumor targeting, tumor tissue penetration ability, and target cell endocytosis ability, and has human-monkey species immune cross-reactivity. Therefore, the anti-NECTIN4 VHH domain according to the present invention or the anti-NECTIN4 antibody according to the present invention can be used as a targeting module of a conjugate or conjugate, and conjugated or coupled with, for example, chemotherapeutic agents, toxins, drugs (such as immunotherapeutic agents), radioactive elements, probes or signal molecules, etc., to provide better applications such as tumor killing, immune regulation or disease detection.

[0371] IV. Production and purification of the antibody of the present invention

[0372] In a fourth aspect, the present invention provides a method for producing the antibody of the present invention. To produce the antibody of the present invention, the polypeptide chain of the antibody of the present invention can be obtained, for example, by solid-phase peptide synthesis (such as Merrifield solid-phase synthesis) or recombinant production, and assembled under suitable conditions.

[0373] For recombinant production, the polynucleotide encoding any one polypeptide chain and / or multiple polypeptide chains of the antibody can be isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Using conventional methods, the polynucleotide can be easily isolated and sequenced. In one embodiment, a polynucleotide encoding one or more polypeptide chains of the antibody of the present invention is provided. In yet another embodiment, the present invention provides a vector, preferably an expression vector, comprising one or more polynucleotides of the present invention. Thus, in one embodiment, the present invention provides a method for producing the antibody of the present invention, the method comprising: culturing a host cell comprising a polynucleotide encoding the polypeptide chain under conditions suitable for expressing the polypeptide chain of the antibody; and assembling the polypeptide chain under conditions suitable for the polypeptide chain to assemble into the antibody to produce the antibody.

[0374] Methods well known to those skilled in the art can be used to construct the expression vector. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, λ phages, or yeast artificial chromosomes (YACs).

[0375] In one embodiment, the present invention also provides a host cell comprising one or more polynucleotides of the present invention. In some embodiments, a host cell comprising an 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), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), dog kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), CHO cells, NSO cells, myeloma cell lines such as YO, NS0, P3X63, and Sp2 / 0, etc. In a preferred embodiment, the host cell is a CHO or HEK293 cell.

[0376] The antibodies prepared by the methods described herein can be purified by known prior art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. After purification, the purity of the antibodies of the present invention can be determined by any one of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc. The physical / chemical properties and / or biological activities of the antibodies provided herein can be identified, screened, or characterized by a variety of assays known in the art.

[0377] In a preferred embodiment, the antibodies of the present invention exhibit good production properties, especially good expression yields and good by-product profiles, when recombinantly produced in mammalian host cells such as CHO cells.

[0378] V. Antigen-binding molecules, immunofusions, immunoconjugates, and antibody-drug conjugates (ADCs)

[0379] In a fifth aspect, the present invention provides antigen-binding molecules, immunofusions, or immunoconjugates produced by fusing or conjugating the antibodies of the present invention to heterologous molecules.

[0380] In one embodiment, the antigen-binding molecule according to the present invention comprises at least one (e.g., 1, 2, or 3) of the present invention's VHHs that specifically bind Trop2 described herein Trop2 domains. In one embodiment, the antigen-binding molecule according to the present invention comprises at least one (e.g., 1, 2, or 3) of the present invention's VHHs that specifically bind Nectin4 described herein NECTIN4Domain. The antigen-binding molecule according to the present invention can be, including but not limited to, a monospecific or multispecific protein comprising the VHH domain. As an example, the antigen-binding molecule is a chimeric antigen receptor.

[0381] In one embodiment, the present invention provides an immunoconjugate comprising the antibody of the present invention. In one embodiment of the immunoconjugate according to the present invention, the antibody (or its antigen-binding fragment) of the present invention is directly or indirectly linked to a heterologous peptide or polypeptide molecule through an amino acid peptide linker. Heterologous peptides or polypeptides that can be mentioned include but are not limited to proteins or polypeptides that confer another functional activity to the fusion, or tag peptides that facilitate the purification or detection of the immunoconjugate.

[0382] In one embodiment, the present invention provides an immunoconjugate comprising the antibody or antigen-binding molecule of the present invention. In one embodiment of the immunoconjugate according to the present invention, the antibody (or its antigen-binding fragment) or the antigen-binding molecule of the present invention is conjugated to a therapeutic agent, a diagnostic agent, or a detectable agent. In the conjugate, a linker can be used to covalently link the 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 after delivery to the target site. For example, acid-labile linkers, peptidase-sensitive linkers, photo-labile linkers, dimethyl linkers, or disulfide-containing linkers can be used.

[0383] In the embodiment conjugated to a therapeutic agent, the therapeutic agents applicable to the conjugate include but are not limited to cytotoxins (such as cell growth inhibitors or cell killers), drugs, or radioisotopes.

[0384] In the embodiment conjugated to a diagnostic agent or a detectable agent, such conjugates can be used as part of a clinical test method (such as determining the efficacy of a specific therapy) for monitoring or predicting the onset, formation, progression, and / or severity of a disease or disorder. Such diagnosis and detection can be achieved by conjugating the antibody to a detectable agent, which includes but is not limited to various enzymes, such as horseradish peroxidase; cofactors, such as streptavidin / biotin and avidin / biotin; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metals and non-radioactive paramagnetic metal ions used in various positron emission imaging techniques.

[0385] In some embodiments, the therapeutic agents applicable to the conjugate include but are not limited to drugs (such as anti-tumor drugs); in other embodiments, the diagnostic agents applicable to the conjugate include but are not limited to radio-diagnostic agents, fluorescent substances, or luminescent substances.

[0386] Antibody-drug conjugate (ADC)

[0387] In some preferred embodiments, the immunoconjugates according to the invention are antibody-drug conjugates (ADCs).

[0388] In some embodiments, the invention provides an antibody-drug conjugate having formula (I):

[0389] Ab-(L-D) p (I)

[0390] or a pharmaceutically acceptable salt or solvate thereof,

[0391] wherein:

[0392] Ab is an antibody or antigen-binding molecule of the invention, such as an antibody or fragment thereof (such as an antigen-binding fragment) that specifically binds to Trop2 and / or Nectin4 (such as human Trop2 and / or human Nectin4);

[0393] L is a linker;

[0394] D is a drug, such as an anti-tumor compound; and

[0395] 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. In some embodiments, Ab is a multispecific antibody that binds Trop2 and Nectin4 according to the present disclosure. In some particularly preferred embodiments, the Ab is a double-stranded multispecific antibody, wherein:

[0396] (a) the antibody is in a symmetric double-stranded form, and wherein the polypeptide chain of the antibody comprises the amino acid sequence of SEQ ID NO:53 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98% or 99% identity thereto; or

[0397] (b) the antibody is in an asymmetric double-stranded form, and wherein the antibody comprises a first and a second polypeptide chain, the first and second polypeptide chains containing the amino acid sequences of SEQ ID NOs:61 / 62, 69 / 70 or 71 / 72, respectively, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0398] In some embodiments, D in formula (I) of the present invention can be any anti-tumor compound, as long as it is a compound with anti-tumor effect and having a substituent or partial structure capable of connecting to the linker structure, without particular limitation. The anti-tumor compound can be a pharmaceutically active compound having an effect on tumors. For the anti-tumor compound, preferably part or all of the linker can be cleaved within tumor cells to release the anti-tumor compound moiety, thereby showing anti-tumor effect. When cleaving the linker at the connection part with the drug, the anti-tumor compound is released in an unmodified structure, and its original anti-tumor effect can be exerted.

[0399] In some embodiments, the anti-tumor compound can be, for example, a cytotoxic agent, such as camptothecin compounds such as Exatecan, Dxd, auristatin compounds such as monomethyl auristatin E (MMAE).

[0400] In some embodiments, D has the structure shown in formula (D-1a) or formula (D-1b):

[0401]

[0402] wherein R 1a is selected from H and C1-C6 alkyl;

[0403] R 2a is selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and -SR 5a ;

[0404] R 3a is selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ; and

[0405] R 4a and R 5a are independently selected from H and C1-C4 alkyl;

[0406]

[0407] wherein R 1b 、R 2b 、R 3b 、R 4b 、R 5b and R 8b are each independently selected from C 1-8 alkyl; preferably C 1-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl;

[0408] R 6b and R 7bEach independently selected from C 1-8 alkoxy groups, such as methoxy, ethoxy or propoxy;

[0409] R 9b is selected from C 1-8 alkyl and COOH; preferably C 1-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl; and

[0410] R 10b is selected from OH and H.

[0411] In some embodiments, R 1a is H; R 2a is C1-C6 alkyl; R 3a is halogen, preferably -F; R 4a is C1-C4 alkyl, preferably ethyl.

[0412] In some embodiments, R 1b , R 4b and R 8b each independently selected from C 1-2 alkyl; preferably methyl;

[0413] R 2b , R 3b and R 5b each independently selected from C 3-4 alkyl;

[0414] R 6b and R 7b each independently selected from C 1-2 alkoxy; and

[0415] R 9b is selected from C 1-4 alkyl and R 10b is OH; or R 9b is COOH and R 10b is H.

[0416] In some embodiments, D has the structure shown in formula (D-2a) or formula (D-2b):

[0417]

[0418] wherein R 1a , R 2a , R 3a and R 4a are as defined above; or

[0419]

[0420] wherein R 1b , R2b , R 3b , R 4b , R 5b , R 6b , R 7b , R 8b , R 9b and R 10b as defined above.

[0421] In some embodiments, D has the structure shown in formula (D-3a) or (D-3b):

[0422]

[0423] In some embodiments, D has the structure shown in formula (D-4a) or (D-4b):

[0424]

[0425] In some embodiments, -L- has the following structure: -Z-L1-L2-L3-

[0426] where

[0427] Z is selected from where m is independently an integer selected from 1 - 10, such as 1, 2, 3, 4, 5, 6, 7, or 8;

[0428] L1 is selected from absent, where n1 and m1 are each independently an integer selected from 0 - 20, such as an integer selected from 0 - 12, such as 1, 2, 3, 4, 5, 6, 7, or 8;

[0429] L2 is an amino acid residue or a peptide residue composed of 2 - 8 amino acids; and

[0430]

[0431] in X is selected from -NH-, -O-, and -S-; R 1c are each independently selected from C 1-8 alkyl, C 1-8 haloalkyl-, C 1-8 alkoxy, halogen, nitro, and cyano; Su are each independently selected from pentose, penturonic acid, hexose, and hexuronic acid; n2 is 0, 1, 2, 3, or 4; n5 is 0, 1, 2, or 3; n3 and n4 are independently 1, 2, 3, 4, 5, or 6;

[0432] It should be understood that in the above -Z-L1-L2-L3-, Z is connected to the S on Ab, and L3 is connected to D.

[0433] In some embodiments,

[0434] Z is selected from wherein m is an integer selected from 1 - 10, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0435] L1 is selected from absent, wherein n1 is independently an integer selected from 0 - 12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0436] L2 is a peptide residue composed of 2 - 8 amino acids; and

[0437] L3 is selected from: wherein R 1c is selected from: H and C1 - C6 alkyl; n2 is 1, 2, 3 or 4; and n3 and n4 are independently 1, 2, 3, 4, 5 or 6.

[0438] In some embodiments, Z is selected from wherein m is 1, 2, 3, 4, 5, 6, 7 or 8.

[0439] In some embodiments, Z is selected from wherein m is 1, 2, 3, 4, 5, 6, 7 or 8.

[0440] In some embodiments, Z is selected from

[0441] In some embodiments, Z is selected from

[0442] In some embodiments, L1 is selected from absent, wherein n1 is independently an integer selected from 0 - 12, such as 1, 2, 3, 4, 5, 6, 7 or 8.

[0443] In some embodiments, L1 is absent or

[0444] In some embodiments, wherein L2 is an amino acid residue or a peptide residue composed of 2, 3, 4, 5, 6, 7 or 8 amino acids.

[0445] In some embodiments, L2 is a peptide residue composed of 2, 3, 4, 5, 6 or 7 amino acids.

[0446] In some embodiments, L2 is an amino acid residue or a peptide residue composed of 2, 3 or 4 amino acids.

[0447] There are no particular restrictions on the amino acids constituting L2. For example, they can be L- or D-amino acids, preferably L-amino acids. In addition to α-amino acids, they can also be amino acids with structures such as β-alanine, ε-aminohexanoic acid, γ-aminobutyric acid, etc. Furthermore, they can also be non-natural amino acids such as N-methylated amino acids, etc.

[0448] In some embodiments, the amino acid residues or the amino acids constituting the peptide residues are each independently selected from glycine (Gly), valine (Val), alanine (Ala), lysine (Lys), citrulline (Cit), glutamine (Gln), glutamate (Glu), phenylalanine (Phe), leucine (Leu), tyrosine (Tyr), serine (Ser), aspartic acid (Asp), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), methionine (Met), tryptophan (Trp), cysteine (Cys), histidine (His), and threonine (Thr), wherein the amino acid residues or amino acids are optionally substituted with one or more C 1-6 alkyl groups.

[0449] In some embodiments, the amino acid residues or the amino acids constituting the peptide residues are each independently selected from glycine (Gly), valine (Val), alanine (Ala), lysine (Lys), citrulline (Cit), glutamine (Gln), glutamate (Glu), phenylalanine (Phe), aspartic acid (Asp), asparagine (Asn), arginine (Arg), and threonine (Thr).

[0450] In some embodiments, the amino acid residues or the amino acids constituting the peptide residues are each independently selected from glycine (Gly), valine (Val), alanine (Ala), phenylalanine (Phe), glutamate (Glu), and citrulline (Cit).

[0451] In some embodiments, L2 is selected from -Ala-, -Val-, -Gly-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Val-Cit-, and -Glu-Val-Cit-.

[0452] In some embodiments, L2 is selected from -Gly-, -Val-Ala-, and -Gly-Gly-Phe-Gly-.

[0453] In some embodiments, L2 is selected from -Val-Ala-, -Gly-Gly-Phe-Gly-, -Val-Cit-, and -Glu-Val-Cit-.

[0454] It should be understood that L2 is linked to L1 or Z through the amino group of the left - hand amino acid and to L3 through the carbonyl group of the right - hand amino acid, which is consistent with the following explanation.

[0455] In some embodiments, L3 is selected from:

[0456]

[0457] wherein R 1c are each independently selected from C 1-8 alkyl, C 1-8 haloalkyl -, C 1-8 alkoxy, halogen, nitro and cyano; Su are each independently selected from 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.

[0458] In some embodiments, L3 is selected from:

[0459]

[0460] wherein each variable is as defined herein.

[0461] In some embodiments, L3 is selected from:

[0462]

[0463] wherein each variable is as defined herein.

[0464] In some embodiments, L3 is selected from: wherein each variable is as defined herein.

[0465] In some embodiments, Su is selected from xylose, arabinose, xyluronic acid, arabinuronic acid, glucose, galactose, mannose, glucuronic acid, galacturonic acid and mannuronic acid.

[0466] In some embodiments, Su is

[0467] In some embodiments, Su are each independently:

[0468]

[0469] In some embodiments, Su are each independently

[0470] In some embodiments, Su are each independently

[0471] In some embodiments, L3 is selected from: wherein R 1c is selected from: H and C1-C6 alkyl; n2 is 1, 2, 3 or 4; n3 and n4 are independently 1, 2, 3, 4, 5 or 6.

[0472] In some embodiments, L3 is selected from:

[0473] In some embodiments, L3 is selected from:

[0474] In some embodiments, L3 is selected from:

[0475] It should be understood that L3 is connected to L2 through the amino group on the left side and to D through the carbonyl group on the right side, which is consistent with the following explanation.

[0476] In some embodiments, Z is selected from where m is 1, 2, 3, 4, 5, 6, 7 or 8;

[0477] L1 is selected from absent, where n1 is independently an integer selected from 0-12; and

[0478] L3 is selected from: wherein R 1c is selected from: H and C1-C6 alkyl; n2 is 1, 2, 3 or 4; n3 and n4 are independently 1, 2, 3, 4, 5 or 6.

[0479] In some embodiments, -Z-L1-L2-L3- is selected from the following structures

[0480]

[0481]

[0482] It should be understood that without special instructions and without contradiction according to the context, for the ADC of the present invention, the bond on the left side of the divalent group shown herein is connected to Ab or the group near the Ab end, and the bond on the right side of the divalent group is connected to D or the group near the D end. For example, when L2 is wherein the amino group on the left side is connected to L1 and the carbonyl group on the right side is connected to L3;

[0483] In some embodiments, the antibody-drug conjugate has an average DAR of 2-10, 6-10, 4-8, 7-9 or 2-4.

[0484] In some embodiments, formula (I) is as shown in formula (I’),

[0485] Ab-(S-L-D)p (I’)

[0486] wherein Ab, L, D, and p are as defined above for formula (I);

[0487] It should be understood that S in formula (I') is sulfur from the antibody Ab.

[0488] In some embodiments, the antibody-drug conjugate is selected from

[0489]

[0490]

[0491] wherein Ab is an antibody of the present invention, preferably V-15, V-hu-21, V-hu-23, or V-hu-24; p is as defined above, and preferably, the antibody-drug conjugate has an average DAR of, for example, 2-10, 6-10, 4-8, 7-9, or 2-4.

[0492] It should be understood that the S atom connected to Ab in the above ADC is from the antibody Ab. Ab opens the disulfide bond under the action of a reducing agent such as TCEP to generate a mercapto group -SH, which then connects to the terminal functional group of the linker, such as a maleimide moiety.

[0493] VI. Pharmaceutical Compositions, Drug Combinations, and Kits

[0494] In one aspect, the present invention provides a composition, for example, a pharmaceutical composition, which comprises an antibody described herein formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic agents, absorption delaying agents, etc. that are physiologically compatible. 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 antibody of the present invention or the immunoconjugate or immunofusion of the present invention is the sole active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may comprise an antibody described herein or the immunoconjugate or immunofusion of the present invention and more than one therapeutic agent.

[0495] In another aspect, the present invention also provides a drug combination comprising an antibody described herein or the immunoconjugate or immunofusion of the present invention and more than one therapeutic agent.

[0496] The therapeutic agents suitable for the pharmaceutical compositions and drug combinations of the present invention can be therapeutic agents selected from any one 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; (iv) drugs that have an inhibitory effect on tumors.

[0497] The pharmaceutical composition of the present invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of the antibody described in the present invention. A "therapeutically effective amount" refers to the amount that, at the required dosage and for the required period of time, effectively achieves the desired therapeutic outcome. The therapeutically effective amount may vary according to various factors such as the disease state, the age, sex, and weight of the individual, etc. The therapeutically effective amount is an amount in which any toxic or harmful effects are less than the therapeutic beneficial effects. Relative to untreated subjects, the "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80%. The ability of the antibody of the present invention to inhibit a measurable parameter (e.g., tumor volume) can be evaluated in an animal model system predictive of efficacy in human tumors. A "prophylactically effective amount" refers to the amount that, at the required dosage and for the required period of time, effectively achieves the desired prophylactic outcome. Generally, since the prophylactic dose is used in a subject before or at an earlier stage of the disease, the prophylactically effective amount is less than the therapeutically effective amount.

[0498] Kits containing the antibodies described herein are also within the scope of the present invention. The kit may comprise one or more other elements, such as, for example, instructions for use; other reagents, such as markers or reagents for conjugation; a pharmaceutically acceptable carrier; and devices or other materials for administration to a subject.

[0499] VII. Uses and Methods

[0500] Based on the good targeting of the antibody of the present invention or the immunoconjugate or fusion of the present invention to tumor cells expressing TROP2 and / or NECTIN4 and the above other excellent properties, the present invention also provides the application and method of the antibody of the present invention or the immunoconjugate or fusion of the present invention in the treatment and prevention of TROP2- and / or NECTIN4-related diseases.

[0501] TROP2 and NECTIN4 are overexpressed on the cell surface of cancer tissues from multiple sources, and thus are suitable targets for developing cancer immunotherapy. In one aspect, therefore, the present invention provides the use of the immunoconjugate or conjugate of the present invention, or a pharmaceutically acceptable salt or solvate thereof, and the antibody of the present invention or an antigen-binding fragment thereof for preventing and / or treating TROP2- and / or NECTIN4-positive tumors (such as cancers) in a subject. In such use, the immunoconjugate or conjugate of the present invention, or a pharmaceutically acceptable salt or solvate thereof, or the antibody or antigen-binding fragment of the present invention can be administered to the subject as the sole active agent, or can be administered to the subject in combination with other therapies or therapeutic agents. The other therapies and therapeutic agents include, for example, drugs that target antigens on the surface of tumor cells and eliminate tumors by binding to and / or blocking these molecules; drugs that activate the immune system of the subject and prompt it to spontaneously eliminate tumors.

[0502] In yet another aspect, the present invention also provides a method for preventing or treating TROP2- and / or NECTIN4-positive tumors (such as cancers) in a subject, comprising administering to a subject in need thereof the immunoconjugate or conjugate of the present invention, or a pharmaceutically acceptable salt or solvate thereof, or administering to a subject in need thereof the antibody of the present invention or an antigen-binding fragment thereof.

[0503] The TROP2- and / or NECTIN4-positive tumors suitable for the methods and uses of the present invention can be selected from various solid tumors.

[0504] Using TCGA tumor transcriptome data, an analysis of expression level correlation (Pearson correlation analysis) performed on clinical pathological samples from human tumor patients has revealed the expression patterns of TROP2 and NECTIN4 proteins in various human tumor tissues. Tumor types showing a dual positive expression trend of TROP2 and NECTIN4 include, but are not limited to, bladder cancer (e.g., BLCA (Bladder Urothelial Carcinoma)); cervical cancer (e.g., CESC (Cervical squamous cell carcinoma and adenocarcinoma)); head and neck cancer (e.g., HNSC (Head and neck squamous cell carcinoma)); lung cancer (e.g., LUSC (Lung Squamous Cell Carcinoma) and LUAD (Lung Adenocarcinoma)); breast cancer (e.g., BRCA (Breast Invasive Carcinoma)); pancreatic cancer (e.g., PAAD (Pancreatic adenocarcinoma)); esophageal cancer (e.g., ESCA (Esophageal carcinoma)); prostate cancer (e.g., PRAD (Prostate adenocarcinoma)); cholangiocarcinoma (e.g., CHOL (Cholangiocarcinoma)); endometrioid carcinoma (e.g., UCEC (Uterine Corpus Endometrial Carcinoma)); thyroid cancer (e.g., THCA (Thyroid carcinoma)); ovarian cancer (e.g., OV (Ovarian serous cystadenocarcinoma)); colorectal cancer (e.g., COAD (Colon adenocarcinoma) and READ (Rectum adenocarcinoma)); and gastric cancer (e.g., STAD (Stomach adenocarcinoma)). See Figure 26 These cancer types are all within the scope of consideration of the present disclosure.

[0505] In some embodiments, the tumors for use in the methods and applications of the present invention are selected from; urothelial carcinoma; bladder cancer (e.g., BLCA); cervical cancer (e.g., CESC); head and neck cancer (e.g., HNSC); lung cancer (e.g., LUSC and LUAD); breast cancer (e.g., BRCA); pancreatic cancer (e.g., PAAD); esophageal cancer (e.g., ESCA); prostate cancer (e.g., PRAD); cholangiocarcinoma (e.g., CHOL); endometrioid carcinoma (e.g., UCEC); thyroid cancer (e.g., THCA); ovarian cancer (e.g., OV); colorectal cancer (e.g., COAD and READ); and gastric cancer (e.g., STAD).

[0506] In some embodiments, the tumors for use in the methods and applications of the present invention are selected from; cervical cancer, endometrial cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma), breast cancer (e.g., triple negative breast cancer), gastric cancer, bladder cancer (e.g., muscle-invasive bladder cancer), thyroid cancer, ovarian cancer, lung cancer (e.g., non-small cell lung cancer), colorectal cancer, and urothelial cancer (e.g., metastatic urothelial cancer).

[0507] TROP2 and / or NECTIN4 positive tumors suitable for the methods and applications of the present invention can be carcinomas at an early, middle, or late stage or metastatic. In addition, TROP2 and / or NECTIN4 positive tumors suitable for the methods and applications of the present invention can be tumors that have undergone immune escape after prior treatment.

[0508] In some embodiments, the TROP2 and / or NECTIN4 positive tumors treated according to the methods of the present invention have at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100% TROP2 and / or NECTIN4 positive cells. The expression levels of TROP2 and / or NECTIN4 on tumor biopsies can be evaluated by immunohistochemistry. High percentages of TROP2 and / or NECTIN4 positive cells have been detected on biopsy specimens of various cancers, e.g., bladder cancer.

[0509] Preferably, in some embodiments, the methods according to the present invention are used to treat tumors having a high percentage of TROP2 and / or NECTIN4 positive cells, e.g., tumors having at least 25%, 50%, 75%, or 100% TROP2 and / or NECTIN4 positive cells. In some embodiments, the application of the methods of the present invention in said cancers induces tumor regression.

[0510] In some embodiments, the method according to the present invention can also be used to treat cancers with a percentage of TROP2 and / or NECTIN4 positive cells below 25% or 20%. In some embodiments, the use of the method of the present invention in said cancer results in tumor growth inhibition.

[0511] In any of the above embodiments of the methods of the invention, the administration of antibodies or binding fragments thereof according to the invention and immunoconjugates or couplings according to the invention or pharmaceutically acceptable salts or solvates thereof may include 1) therapeutic measures that cure, slow down, alleviate symptoms of a diagnosed pathological condition or disorder and / or stop the progression of the diagnosed pathological condition or disorder; or 2) preventive or prophylactic measures that prevent and / or slow the development of a pathological condition or disorder. Thus, in the methods of the invention, the subject may be an individual who has suffered from a disease, an individual who is susceptible to a disease, or an individual who wants to prevent a disease. The individual will benefit from the therapeutic or prophylactic measures and show a reduction or improvement in the occurrence, recurrence or development of a disease, disorder, condition, and / or symptom compared to an individual who has not received the treatment. In some embodiments, the invention relates to the treatment of a disease or disorder; in other embodiments, the invention relates to the prevention of a disease or disorder.

[0512] The antibodies or binding fragments thereof according to the present invention and the immunoconjugates or pharmaceutically acceptable salts or solvates thereof according to the present invention, and other therapeutic agents optionally used in combination therewith, can be administered by any suitable method, including parenteral administration, intratumoral administration and intranasal administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration and pulse infusion.

[0513] For the prevention or treatment of disease, the appropriate dosage of the antibodies or binding fragments thereof according to the invention and the immunoconjugates or couplings according to the invention or pharmaceutically acceptable salts or solvates thereof, when used alone or in combination with one or more other therapeutic agents, will depend on the type of disease being 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 treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician.

[0514] In some embodiments, the present invention also provides the use of the immunoconjugate or conjugate of the present invention or its pharmaceutically acceptable salt or solvate and the antibody or antibody fragment of the present invention as a medicament or for the preparation of a medicament. In some embodiments, the medicament is a medicament for the aforementioned treatment and prevention methods.

[0515] VIII. Preparation of ADC molecules of the present invention

[0516] Another aspect of the present invention provides a method for preparing an ADC using the antibody of the present invention. The "ADC" in the present invention is defined as an antibody conjugated to a biologically and / or pharmaceutically active substance (D) via a linker (L). The method includes conjugating the antibody (Ab) of the present invention to one or more active substances D via one or more linkers (L) defined in the present invention. Preferably, the linker-active substance is site-specifically conjugated to the antibody.

[0517] In some embodiments, the method includes preparing an Ab for the ADC, which includes culturing a host cell containing a nucleic acid encoding the Ab (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector containing the nucleic acid under conditions suitable for the expression of the Ab or its chain, as provided above, and optionally recovering the Ab from the host cell (or host cell culture medium).

[0518] In some embodiments, the method includes the following steps:

[0519] (a) Adding the antibody Ab to a buffer solution, adding a reducing agent, and then incubating;

[0520] (b) Adding a linker-payload to the reaction solution in step (a) for conjugation to obtain a crude product; and

[0521] (c) Optionally purifying the crude product to obtain the antibody-drug conjugate of the present invention;

[0522] wherein Ab is as defined above.

[0523] It should be understood that the reaction of the linker-payload with Ab provides the -L-D moiety in the compound of formula I, and when -L-D is clearly defined, the structure of the linker-payload can be determined according to the prior art.

[0524] In some embodiments, the buffer solution in step a) is a PBS buffer, preferably having a pH of 5.0 - 9.0, such as 6.0 - 8.0.

[0525] In some embodiments, the reducing agent in step a) is TCEP.

[0526] In some embodiments, the linker-payload has the following structure: Z'-L1-L2-L3-D, where L1, L2, L3, D are as defined above, and Z' is m is as defined above.

[0527] In some embodiments, for the method of synthesizing an ADC in which Z is it further includes an additional hydrolysis step for ring-opening of the maleimide.

[0528] In some embodiments, the steps are carried out under the specific reaction conditions disclosed in the examples.

[0529] It should be noted that embodiments obtained by floating the range or specific values of the specific reaction conditions disclosed in the examples by 100%, 80%, 60%, 40%, 20% or 10% are also contemplated in the present invention.

[0530] In one aspect, the present invention also provides the following embodiments:

[0531] 1. A multispecific antibody that binds Trop2 and Nectin4, wherein the antibody comprises at least one antigen-binding domain that specifically binds Trop2 and at least one antigen-binding domain that specifically binds Nectin4.

[0532] 2. The multispecific antibody of embodiment 1, wherein the antibody has one or more of the following characteristics:

[0533] (a) The Trop2 and Nectin4 antigen-binding domains are respectively VHH domains;

[0534] (b) The antigen-binding affinities K D values of the Trop2 and Nectin4 antigen-binding domains are respectively 50x10 -8 M to 0.5x10 -8 M;

[0535] (c) The valence ratio of the Trop2 antigen-binding domain to the Nectin4 antigen-binding domain in the antibody is 1:1 or 1:2; and

[0536] (d) The antibody is a trivalent or tetravalent bispecific antibody; and

[0537] Preferably, the antibody also has one or more of the following characteristics: exhibits (e) synergistic binding, (f) synergistic endocytosis, and (g) improved tumor cell targeting distribution on target tumor cells expressing human Nectin4 and Trop2, as compared to Nectin4 and Trop2 single-target drugs.

[0538] 3. The multispecific antibody according to any one of embodiments 1-2, wherein: the antigen-binding domain that specifically binds Trop2 is a VHH domain, and

[0539] wherein, the VHH domain comprises the CDR1, CDR2, and CDR3 sequences selected from the amino acid sequences of SEQ ID NOs: 5, 9, 13, 29, 30, 98, and 99;

[0540] More preferably, the CDR1, CDR2 and CDR3 sequences:

[0541] (i) respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 6, 7 and 8;

[0542] (ii) respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 10, 11 and 12;

[0543] (iii) respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 14, 15 and 16; or

[0544] (iv) respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 31, 32 and 33;

[0545] More preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 5, 9, 13, 29, 30, 98 and 99, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity to the amino acid sequence, or having an addition, deletion and / or substitution of one or more (preferably 1-10, more preferably 1-5) amino acids,

[0546] Most preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 5, 9, 13, 29, 30, 98 and 99, or consists of an amino acid sequence selected from SEQ ID NOs: 5, 9, 13, 29, 30, 98 and 99.

[0547] 4. The multispecific antibody according to any one of embodiments 1-3, wherein: the antigen-binding domain that specifically binds Nectin4 is a VHH domain, and

[0548] wherein the VHH domain comprises the CDR1, CDR2 and CDR3 sequences in an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35, 38 and 100-106;

[0549] More preferably, the CDR1, CDR2 and CDR3 sequences:

[0550] (i) respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 18, 19 and 20;

[0551] (ii) respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 22, 23 and 24;

[0552] (iii) respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 26, 27 and 28;

[0553] (iv) Comprising or consisting of the amino acid sequences of SEQ ID NOs: 18, 36 and 20, respectively; or

[0554] (v) Comprising or consisting of the amino acid sequences of SEQ ID NOs: 18, 37 and 20, respectively;

[0555] More preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35, 38 and 100 - 106, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity to the amino acid sequence, or having an addition, deletion and / or substitution of one or more (preferably 1 - 10, more preferably 1 - 5) amino acids,

[0556] Most preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35, 38 and 100 - 106, or consists of an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35, 38 and 100 - 106.

[0557] 5. The multispecific antibody according to any one of embodiments 1 - 4, wherein the antibody further comprises:

[0558] (a) A half - life extension domain, preferably, the half - life extension domain is selected from a serum albumin - binding peptide and an immunoglobulin Fc region; and / or

[0559] (b) A peptide linker, preferably, the peptide linker is 5 - 15 amino acids in length, more preferably, the peptide linker comprises the amino acid sequence (G4S) n , where n = 1, 2 or 3.

[0560] 6. The multispecific antibody according to any one of embodiments 1 - 5, wherein the antibody is a single - chain or double - chain antibody comprising at least one (preferably 1 or 2) Nectin4 - binding domain and at least one (preferably 1 or 2) Trop2 - binding domain.

[0561] 7. The multispecific antibody according to any one of embodiments 1 - 6, wherein the antibody comprises two identical polypeptide chains, and the polypeptide chain comprises, from the N - terminus to the C - terminus: (VHH A ) n1 -(VHH B ) n2 -HLE-(VHH A ) n3 -(VHH B ) n4，

[0562] wherein n1, n2, n3, and n4 are independently selected from the integers 0, 1, or 2, and preferably n3 and n4 are 0, and both n1 and n2 are 1;

[0563] wherein VHH A and VHH B respectively represent VHH domains that bind to antigen A and antigen B, where A and B are different from each other and are independently selected from Nectin4 and Trop2; wherein HLE represents the immunoglobulin Fc region as a half-life extension domain; wherein the symbol "-" represents connection by a peptide linker or direct connection.

[0564] 8. The multispecific antibody of embodiment 7, wherein the polypeptide chain comprises, from the N-terminus to the C-terminus: VHH A -VHH B -HLE, and wherein VHH A represents the Nectin4-binding domain and VHH B represents the Trop2-binding domain.

[0565] 9. The multispecific antibody of any one of embodiments 1-6, wherein the antibody comprises two different polypeptide chains, wherein:

[0566] The first polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE-(VHH A ) n3 -(VHH B ) n4，

[0567] The second polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH B ) m1 -(VHH A ) m2 -HLE-(VHH B ) m3 -(VHH A ) m4，

[0568] wherein n1, n2, n3, and n4 and m1, m2, m3, and m4 are independently selected from the integers 0, 1, or 2, and preferably wherein n3, n4, m3, and m4 are 0, and n1 + m2 = 1 or 2, n2 + m1 = 1 or 2, and more preferably m1 = 0 and m2 = 1, or m2 = 0 and m1 = 1;

[0569] wherein VHH A and VHH BSeparate VHH domains that bind antigen A and antigen B respectively, where A and B are different from each other and are independently selected from Nectin4 and Trop2; where HLE represents the immunoglobulin Fc region as a half-life extension domain, and preferably contains a knob-into-hole mutation; where the symbol "-" represents connection by a peptide linker or direct connection.

[0570] 10. The multispecific antibody of embodiment 9, wherein:

[0571] The first polypeptide chain contains, from the N-terminus to the C-terminus: VHH A -VHH B -HLE ，

[0572] The second polypeptide chain contains, from the N-terminus to the C-terminus: VHH A -HLE ；

[0573] And where VHH A represents the Nectin4 binding domain and VHH B represents the Trop2 binding domain.

[0574] 11. The multispecific antibody of any one of embodiments 7-10, wherein the immunoglobulin Fc region is the Fc region from IgG, for example, the Fc region from human IgG1 or IgG4, and preferably the Fc region contains mutations that reduce or eliminate the binding of the Fc region to FcγR.

[0575] 12. The multispecific antibody of any one of embodiments 1-6, wherein the antibody comprises a single polypeptide chain, and the polypeptide chain contains, from the N-terminus to the C-terminus: (VHH A ) n1 -(VHH B ) n2 -(HLE) n3 -(VHH A ) n4 -(VHH B ) n5 -(HLE) n6，

[0576] where n1, n2, n3, n4, n5 and n6 are independently selected integers from 0, 1 or 2, and preferably n1 + n4 = 1 or 2, n2 + n5 = 1 or 2, and n3 + n6 = 0 or 1;

[0577] where VHH A and VHH BSeparate VHH domains that bind antigen A and antigen B respectively, where A and B are different from each other and are independently selected from Nectin4 and Trop2; where HLE represents a serum albumin-binding peptide as a half-life extension domain; where the symbol "-" represents connection through a peptide linker or direct connection,

[0578] Preferably, the polypeptide chain comprises, from the N-terminus to the C-terminus:

[0579] (i) VHH A -VHH B ,

[0580] (ii) VHH A -VHH B -HLE,

[0581] (iii) VHH B -VHH A -VHH B ,

[0582] (iv) VHH A -VHH B -VHH B ,

[0583] (v) VHH A -HLE-VHH B -VHH B , or

[0584] (vi) VHH A -VHH A -HLE-VHH B -VHH B , where preferably A represents Trop2 and B represents Nectin4,

[0585] More preferably, the HLE represents an anti-serum albumin VHH domain, optionally selected from the amino acid sequences of SEQ ID NOs: 39 and 40, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity thereto, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions.

[0586] 13. A multispecific antibody according to any one of embodiments 1-12, wherein:

[0587] (a) The antigen-binding domain that specifically binds Nectin4 comprises the amino acid sequence of SEQ ID NO: 25 and the antigen-binding domain that specifically binds Trop2 comprises the amino acid sequence of SEQ ID NO: 13;

[0588] (b) The antigen - binding domain that specifically binds Nectin4 comprises the amino acid sequence of SEQ ID NO:21, and the antigen - binding domain that specifically binds Trop2 comprises the amino acid sequence of SEQ ID NO:9;

[0589] (c) The antigen - binding domain that specifically binds Nectin4 comprises the amino acid sequence of SEQ ID NO:25, and the antigen - binding domain that specifically binds Trop2 comprises the amino acid sequence of SEQ ID NO:9;

[0590] (d) The antigen - binding domain that specifically binds Nectin4 comprises the amino acid sequence of SEQ ID NO:35, and the antigen - binding domain that specifically binds Trop2 comprises an amino acid sequence selected from SEQ ID NOs:29 and 30; or

[0591] (e) The antigen - binding domain that specifically binds Nectin4 comprises an amino acid sequence selected from SEQ ID NOs:34 and 38, and the antigen - binding domain that specifically binds Trop2 comprises an amino acid sequence selected from SEQ ID NOs:29 and 30.

[0592] 14. The multispecific antibody of embodiments 1 - 6, wherein the antibody is in a double - chain form, wherein:

[0593] (a) The antibody is in a symmetric double - chain form, and the polypeptide chain of the antibody comprises the amino acid sequence of SEQ ID NO:53 or 54 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity therewith, preferably comprises the amino acid sequence of SEQ ID NO:53; or

[0594] (b) The antibody is in an asymmetric double - chain form, and the antibody comprises a first and a second polypeptide chain selected from the group consisting of:

[0595] (i) A first and a second polypeptide chain respectively comprising SEQ ID NOs:55 and 56 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity therewith,

[0596] (ii) A first and a second polypeptide chain respectively comprising SEQ ID NOs:57 and 58 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity therewith,

[0597] (iii) A first and a second polypeptide chain respectively comprising SEQ ID NOs:59 and 60 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity therewith,

[0598] (iv) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 61 and 62 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto,

[0599] (v) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 63 and 64 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto,

[0600] (vi) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 65 and 66 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto,

[0601] (vii) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 67 and 68 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto,

[0602] (viii) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 69 and 70 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto,

[0603] (ix) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 71 and 72 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto,

[0604] (x) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 73 and 74 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, and

[0605] (xi) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 75 and 76 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto,

[0606] Preferably, the first and second polypeptide chains each comprise the amino acid sequences of SEQ ID NOs: 61 / 62, 69 / 70 or 71 / 72.

[0607] 15. The multispecific antibody of embodiments 1 - 6, wherein the antibody is in single-chain form, and wherein the polypeptide chain of the antibody comprises an amino acid sequence selected from SEQ ID NOs: 41 - 52 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.

[0608] 16. A VHH domain that specifically binds to Trop2, and

[0609] wherein the VHH domain comprises CDR1, CDR2 and CDR3 sequences selected from the amino acid sequences of SEQ ID NOs: 5, 9, 13, 29, 30, 98 and 99;

[0610] More preferably, the CDR1, CDR2 and CDR3 sequences:

[0611] (i) respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 6, 7 and 8;

[0612] (ii) respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 10, 11 and 12;

[0613] (iii) respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 14, 15 and 16; or

[0614] (iv) respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 31, 32 and 33;

[0615] More preferably, the VHH domain comprises an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5, 9, 13, 29, 30, 98 and 99, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity to the amino acid sequence, or having an addition, deletion and / or substitution of one or more (preferably 1-10, more preferably 1-5) amino acids,

[0616] Most preferably, the VHH domain comprises an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5, 9, 13, 29, 30, 98 and 99, or consists of an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5, 9, 13, 29, 30, 98 and 99.

[0617] 17. A VHH domain that specifically binds to Nectin4, and

[0618] wherein the VHH domain comprises CDR1, CDR2 and CDR3 sequences selected from the amino acid sequences of SEQ ID NOs: 17, 21, 25, 34, 35, 38 and 100-106;

[0619] Preferably, the CDR1, CDR2 and CDR3 sequences:

[0620] (i) respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 18, 19 and 20;

[0621] (ii) Comprising or consisting of the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively;

[0622] (iii) Comprising or consisting of the amino acid sequences set forth in SEQ ID NOs: 26, 27, and 28, respectively;

[0623] (iv) Comprising or consisting of the amino acid sequences set forth in SEQ ID NOs: 18, 36, and 20, respectively; or

[0624] (v) Comprising or consisting of the amino acid sequences set forth in SEQ ID NOs: 18, 37, and 20, respectively;

[0625] More preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35, 38, and 100 - 106, or an amino acid sequence having at least 85%, 90%, 95%, or 99% identity to the amino acid sequence, or having one or more (preferably 1 - 10, more preferably 1 - 5) amino acid additions, deletions, and / or substitutions;

[0626] Most preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35, 38, and 100 - 106, or consists of an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35, 38, and 100 - 106.

[0627] 18. An antibody comprising the VHH domain that specifically binds Trop2 of embodiment 16 and / or the VHH domain that specifically binds Nectin4 of embodiment 17.

[0628] 19. An antigen - binding molecule comprising the antibody of embodiment 18.

[0629] 20. A polynucleotide encoding the antibody of any one of embodiments 1 - 15, the VHH domain of embodiment 16 or 17, the antibody of embodiment 18, or the antigen - binding molecule of embodiment 19.

[0630] 21. A vector, preferably an expression vector, comprising the polynucleotide of embodiment 20.

[0631] 22. A host cell comprising the polynucleotide of embodiment 20 or the vector of embodiment 21, for example, the host cell is a mammalian cell.

[0632] 23. A method for producing the antibody of any one of embodiments 1 - 15 or 18, the method comprising:

[0633] Culturing a host cell comprising a polypeptide chain encoding the antibody under conditions suitable for expressing the polypeptide chain; and assembling the polypeptide chain under conditions suitable for the polypeptide chain to assemble into the antibody to produce the antibody.

[0634] 24. An immunoconjugate or immunofusion comprising the antibody according to any one of embodiments 1-15 or 18.

[0635] 25. An antibody-drug conjugate of formula (I):

[0636] Ab-(L-D) p (I)

[0637] or a pharmaceutically acceptable salt or solvate thereof,

[0638] wherein:

[0639] Ab is the antibody according to any one of embodiments 1-15 or 18 or the antigen-binding molecule of embodiment 19;

[0640] L is a linker;

[0641] D is a drug, preferably an anti-tumor compound; and

[0642] p is an integer selected from 1 to 16, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0643] 26. The antibody-drug conjugate according to embodiment 25 or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-tumor compound is a cytotoxic agent, such as a camptothecin compound, an auristatin compound, such as irinotecan, Dxd or MMAE.

[0644] 27. The antibody-drug conjugate according to embodiment 25 or a pharmaceutically acceptable salt or solvate thereof, wherein D has the structure shown in formula (D-1a) or formula (D-1b):

[0645]

[0646] wherein R 1a is selected from H and C1-C6 alkyl;

[0647] R 2a is selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and -SR 5a ;

[0648] R 3a is selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ; and

[0649] R 4a and R 5a are each independently selected from H and C1-C4 alkyl;

[0650] or

[0651]

[0652] wherein R 1b 、R 2b 、R 3b 、R 4b 、R 5b and R 8b are each independently selected from C 1-8 alkyl; such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl;

[0653] R 6b and R 7b are each independently selected from C 1-8 alkoxy, such as methoxy, ethoxy or propoxy;

[0654] R 9b is selected from C 1-8 alkyl and COOH; such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl; and

[0655] R 10b is selected from OH and H.

[0656] 28. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to embodiment 27, wherein

[0657] R 1a is H; R 2a is C1-C6 alkyl; R 3a is halogen, preferably -F; R 4a is C1-C4 alkyl, preferably ethyl;

[0658] R 1b 、R 4b and R 8b are each independently selected from C 1-2 alkyl; preferably methyl;

[0659] R 2b 、R 3b and R 5b are each independently selected from C 3-4 alkyl;

[0660] R 6b and R 7b are each independently selected from C 1-2 alkoxy; and

[0661] R 9b is selected from C 1-4 alkyl and R 10b is OH; or R 9b is COOH and R 10b is H.

[0662] 29. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to embodiment 27, wherein D has the structure shown in formula (D-2a) or formula (D-2b):

[0663]

[0664] wherein R 1a , R 2a , R 3a and R 4a are as defined in embodiment 27 or 28;

[0665]

[0666] wherein R 1b , R 2b , R 3b , R 4b , R 5b , R 6b , R 7b , R 8b , R 9b and R 10b are as defined in embodiment 27 or 28.

[0667] 30. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to embodiment 27, wherein D has the structure shown in formula (D-3a) or (D-3b):

[0668]

[0669] 31. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to embodiment 27, wherein D has the structure shown in formula (D-4a) or (D-4b):

[0670]

[0671] 32. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of embodiments 25-31, wherein -L- has the following structure -Z-L1-L2-L3-

[0672] wherein

[0673] Z is selected from wherein m is independently an integer selected from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0674] L1 is selected from absent, wherein n1 and m1 are each independently an integer selected from 0 to 20, such as an integer selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0675] L2 is an amino acid residue or a peptide residue composed of 2 to 8 amino acids; and

[0676] L3 is selected from: wherein X is selected from -NH-, -O- and -S-; R 1c are each independently selected from C 1-8 alkyl, C 1-8 haloalkyl-, C 1-8 alkoxy, halogen, nitro and cyano; Su are each independently selected from pentose, penturonic acid, hexose and hexuronic acid; n2 is 0, 1, 2, 3 or 4; n5 is 0, 1, 2 or 3; n3 and n4 are independently 1, 2, 3, 4, 5 or 6;.

[0677] 33. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to embodiment 32,

[0678] wherein Z is selected from wherein m is 1, 2, 3, 4, 5, 6, 7 or 8;

[0679] Preferably, Z is selected from

[0680] 34. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of embodiments 32-33,

[0681] wherein L1 is selected from absent, wherein n1 is independently an integer selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0682] Preferably, L1 is absent or

[0683] 35. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of embodiments 32-34,

[0684] wherein L2 is an amino acid residue or a peptide residue composed of 2, 3, 4, 5, 6 or 7 amino acids; preferably, each of the amino acid residues or the amino acids constituting the peptide residue is independently selected from glycine (Gly), valine (Val), alanine (Ala), lysine (Lys), citrulline (Cit), glutamine (Gln), glutamate (Glu), phenylalanine (Phe), leucine (Leu), tyrosine (Tyr), serine (Ser), aspartic acid (Asp), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), methionine (Met), tryptophan (Trp), cysteine (Cys), histidine (His) and threonine (Thr), wherein the amino acid residue or amino acid is optionally substituted by one or more C 1-6 alkyl groups;

[0685] More preferably, each of the amino acid residues or the amino acids constituting the peptide residue is independently selected from glycine (Gly), valine (Val), alanine (Ala), phenylalanine (Phe), glutamate (Glu) and citrulline (Cit);

[0686] Even more preferably, L2 is selected from -Ala-, -Val-, -Gly-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Val-Cit- and -Glu-Val-Cit-.

[0687] 36. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of embodiments 32-35,

[0688] wherein L3 is selected from:

[0689]

[0690] wherein R 1c are each independently selected from C 1-8 alkyl, C 1-8 haloalkyl-, C 1-8 alkoxy, halogen, nitro and cyano; Su are each independently selected from 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;

[0691] Preferably, L3 is selected from:

[0692] as defined for each variable above.

[0693] 37. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of embodiments 32-36, wherein each Su is independently:

[0694]

[0695] More preferably, each Su is independently Even more preferably, each Su is independently

[0696] 38. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of embodiments 32-36, wherein L3 is selected from:

[0697] Preferably, L3 is selected from:

[0698] 39. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to embodiment 35,

[0699] Z is selected from L1 is selected from absent and L2 is selected from -Gly-, -Val-Ala-, and -Gly-Gly-Phe-Gly; and

[0700] L3 is selected from:

[0701] 40. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to embodiment 35,

[0702] wherein -Z-L1-L2-L3- is selected from the following structures

[0703]

[0704] 41. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of embodiments 25-40, which has an average DAR value of 2-10, such as 4-8.

[0705] 42. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to embodiment 25, wherein the antibody-drug conjugate is selected from

[0706]

[0707] where Ab and p are as defined in embodiment 25,

[0708] Preferably, Ab is a diabody, wherein:

[0709] (a) The antibody is in a symmetric double-stranded form, and the polypeptide chain of the antibody comprises the amino acid sequence of SEQ ID NO:53; or

[0710] (b) The antibody is in an asymmetric double-stranded form, and the antibody comprises first and second polypeptide chains containing the amino acid sequences of SEQ ID NOs: 61 / 62, 69 / 70 or 71 / 72, respectively:

[0711] Preferably, the antibody-drug conjugate has an average DAR of 2-10 or 4-8.

[0712] 43. A pharmaceutical composition comprising the antibody according to any one of embodiments 1-15 or 18, the antigen-binding molecule of embodiment 19 or the immunoconjugate or immunofusion of embodiment 24, or the antibody-drug conjugate according to any one of embodiments 25-42 or a pharmaceutically acceptable salt or solvate thereof and a pharmaceutically acceptable carrier.

[0713] 44. Use of the antibody according to any one of embodiments 1-15 or 18, the antigen-binding molecule of embodiment 19 or the immunoconjugate or immunofusion of embodiment 24, or the antibody-drug conjugate according to any one of embodiments 25-42 or a pharmaceutically acceptable salt or solvate thereof as a medicament or for the preparation of a medicament.

[0714] 45. The use of embodiment 44, wherein the medicament is used for treating and / or preventing cancer in an individual, wherein the cancer is a Trop2-positive, Nectin4-positive, or Trop2- and Nectin4-double-positive solid tumor, selected from, for example, urothelial carcinoma; bladder cancer (e.g., BLCA); cervical cancer (e.g., CESC); head and neck cancer (e.g., HNSC); lung cancer (e.g., LUSC and LUAD); breast cancer (e.g., BRCA); pancreatic cancer (e.g., PAAD); esophageal cancer (e.g., ESCA); prostate cancer (e.g., PRAD); cholangiocarcinoma (e.g., CHOL); endometrioid carcinoma (e.g., UCEC); thyroid cancer (e.g., THCA); ovarian cancer (e.g., OV); colorectal cancer (e.g., COAD and READ); and gastric cancer (e.g., STAD), including its primary, recurrent, and refractory cancers.

[0715] Any and all of the features described above and throughout this application can be combined in various embodiments of the present invention. The following examples further illustrate the present invention. However, it should be understood that the examples are for illustrative purposes only and should not be construed as constituting any limitation. Examples

[0716] Materials and Methods

[0717] Reference antibody and its preparation

[0718] In this example, reference antibody Datopotamab (also known as V-BMK1) and reference antibody Enfortumab (also known as V-BMK2) are used.

[0719] "Reference antibody Datopotamab" is an anti-Trop2 antibody constructed based on the amino acid sequences of the heavy and light chain variable regions of the monospecific bivalent antibody Datopotamab (FDA UNII: BA3HQU7PO9) published by NCATS Inxight Drugs. In the context of reference antibody Datopotamab, unless otherwise clearly stated, the reference antibody will have a Fab antigen-binding domain, as well as a human IgG1 heavy chain constant region and a kappa light chain.

[0720] "Reference antibody Enfortumab" is an anti-Nectin4 antibody constructed based on the amino acid sequences of the heavy and light chain variable regions of the monospecific bivalent antibody Enfortumab (FDA UNII: U1HUE4W970) published by NCATS Inxight Drugs. In the context of reference antibody Enfortumab, unless otherwise clearly stated, the reference antibody will have a Fab antigen-binding domain, as well as a human IgG1 heavy chain constant region and a kappa light chain.

[0721] The heavy and light chain variable region sequences of the two reference antibodies are shown in SEQ ID NOs: 1-4, respectively.

[0722] The reference antibodies are prepared as follows. The coding sequences of the reference antibodies are gene synthesized by GenScript (Shanghai, China), cloned into the pcDNA 3.4 expression vector, and transfected into Expi293F cells. The culture supernatant of the transfected cells is collected, and the antibody is separated and purified by a protein A column. The concentration of the purified antibody is measured using Nano Drop; and the protein purity is determined by SDS-PAGE and analytical HPLC-SEC, and then it is stored at -80 °C for later use.

[0723] Antigen and its preparation

[0724] Retrieve the extracellular domain sequence information (Gln31 - Tyr274) of human Trop2 (UniProt_P09758 - 1) and cynomolgus monkey Trop2 (XP_005543292.2), and the extracellular domain sequence information (Gly32 - Ser349) of human Nectin4 (UniProt_Q96NY8 - 1) and cynomolgus monkey Nectin4 (XP_005541277.1) from the database. Add His tags to the C - terminus respectively, optimize according to human codon preference, then perform gene synthesis and sub - clone them into the pcDNA3.4 vector. After verification by Sanger sequencing, extract the plasmids for standby. Transiently transfect Expi293F cells with these constructed eukaryotic expression vectors of proteins. Collect the protein expression supernatant from the transfected cell culture, purify the target protein using nickel column, and perform SDS - PAGE experiment to detect the protein purity, with the purity > 95%. Use the above - mentioned reference anti - Trop2 antibody (Datopotamab) and reference anti - Nectin 4 antibody (Enfortumab), and confirm the activity of the prepared recombinant antigen proteins of human Trop2 - His and human Nectin4 - His by ELISA method.

[0725] Engineered cell line for recombinant expression of antigen and its preparation

[0726] Prepare CHO - K1 engineering cell lines expressing human Trop2 and CHO - K1 engineering cell lines expressing human Nectin4 according to the following steps.

[0727] Insert the full - length genes of human Trop2 and human Nectin4 into the lentiviral expression vector pLVX - puro respectively. Package lentivirus using 293T cells and infect CHO - K1 cells. The cells are cultured in a medium containing 8 μg / ml puromycin screening pressure to obtain polyclonal cell strains. Through limited dilution, select stable transfection CHO - K1 - human Trop2 cell lines and CHO - K1 - human Nectin4 cell lines with high expression of Trop2 and Nectin4. Perform FACS detection using reference antibodies Datopotamab and Enfortumab, and the positive rate of antigen expression in the stable transfection cell lines > 90%.

[0728] ELISA binding assay

[0729] Dilute the antigen to 0.5 - 1.0 μg / ml with PBS, coat the plate at 100 μl / well, and incubate overnight at 2 - 8°C or for 2 h at 37°C. Wash the plate 3 times with 0.05% PBST using a plate washer, add 250 μl / well of blocking solution (1% BSA or 8% skim milk powder in PBS), and let it stand at room temperature for more than 1 h for blocking. Dilute the sample to be tested to an appropriate concentration with the blocking solution. Wash the plate 3 times with 0.05% PBST using a plate washer, add 100 μl / well of the diluted sample, and let it stand at room temperature for about 1 h. Wash the plate 3 times with 0.05% PBST using a plate washer, add 100 μl / well of the secondary antibody - HRP diluted to the target dilution factor, and let it stand at room temperature for about 0.5 or 1 h. Wash the plate 3 times with 0.05% PBST using a plate washer, add 100 μl / well of TMB, observe the color change, and immediately add 100 μl / well of stop solution when the color is appropriate. Read OD450 - OD650.

[0730] FACS binding assay

[0731] Seed the target cells in a 96 - well plate at a density of 1 - 5×10 5 cells per well, and centrifuge at 300 g for 5 minutes at 4°C. Add the sample to be tested diluted to an appropriate concentration, and incubate for about 1 h at 2 - 8°C. Centrifuge at 4°C, remove the supernatant, wash 2 times with 200 μl / well of FACS buffer (1% BSA or 2% FBS in PBS), and centrifuge at 4°C. Add 100 μl / well of the flow - cytometry secondary antibody diluted to the target dilution factor, resuspend the cells, and incubate for about 0.5 h or 1 h at 2 - 8°C in the dark. After washing 2 times with 200 μl / well of FACS buffer, resuspend the cells with 100 μl / well of FACS buffer and analyze on the machine. Measure the MFI of the cells using a flow cytometer (Beckman Coulter).

[0732] Endocytosis assay

[0733] Seed the target cells in a 96 - well plate at a density of 1 - 5×10 5Plate cells, add the test sample diluted to an appropriate concentration, incubate at 2 - 8°C for about 0.5 h to allow the test sample to bind to the cells. Centrifuge at 4°C, 800 x g for 3 min, discard the supernatant, and wash the cells 2 - 3 times with pre - cooled FACS buffer at 200 μl / well to remove excess unbound test sample. Resuspend the cells with pre - cooled FACS buffer at 100 μl / well. Divide the cells into two groups equally and incubate at 4°C and 37°C for 4 h respectively. Immediately after incubation, add ice - cold FACS buffer to terminate the endocytosis experiment. Centrifuge at 4°C, 800 x g for 3 min, and wash the cells 2 - 3 times with pre - cooled FACS buffer at 200 μl / well. Immediately add 100 μl / well of the secondary antibody diluted to the target dilution factor, resuspend the cells, and incubate in the dark at 2 - 8°C for about 30 minutes to 1 h. After washing the cells 2 - 3 times with 200 μL / well of FACS buffer, add 100 μl / well of FACS buffer to resuspend the cells and perform machine detection. Use a flow cytometer (Beckman Coulter) to measure the MFI of the cells. Calculate the internalization level of the antibody bound to the cell surface using the following formula:

[0734] Endocytosis = MFI of the sample incubated at 4°C - MFI of the sample incubated at 37°C.

[0735] Endocytosis rate % = 100% - (MFI of the sample incubated at 37°C / MFI of the sample incubated at 4°C) × 100%.

[0736] SPR assay

[0737] Detect the binding affinity between the antibody and the antigen by Surface Plasmon Resonance (SPR) technology.

[0738] Fix the antibody to detect the antigen: Fix the antibody at 10 μg / mL on the Protein A chip. Inject the diluted antigen at a flow rate of 30 μL / min, with a binding time of 120 seconds, and then a dissociation time of 200 seconds. After each dissociation stage, 10 mM glycine (pH 2.0) is used for chip regeneration. The experimental data is analyzed using a 1:1 binding model.

[0739] Fix the antigen to detect the antibody: The antigen concentration is 10 μg / mL and is fixed on the CM5 chip. The diluted antibody is injected at a flow rate of 30 uL / min, with a binding time of 120 seconds, and then a dissociation time of 200 seconds. After each dissociation, 10 mM glycine (pH 2.0) is used for regeneration. The experimental data is analyzed using a 1:1 binding model.

[0740] SEC-HPLC

[0741] At ambient column temperature, an appropriate amount of protein sample was loaded onto a TSK-gel G3000SWxL column (Tosoh Corporation) or a Zenix-CSEC-300 column (Sepax Technologies). Using an Agilent 1260 HPLC system, the sample was isocratically eluted for 20 minutes at a flow rate of 0.8 mL / min using a mobile phase consisting of 0.05 M sodium phosphate, 0.3 M sodium chloride, pH 6.8 ± 0.1. The eluted protein was detected using UV absorbance at 280 nm.

[0742] CEX-HPLC

[0743] At ambient column temperature, an appropriate amount of protein sample was loaded onto a ProPac TM WCX-10 BioLC column (Thermo SCIENTIFIC). Using an Agilent 1260 HPLC system, mobile phase A consisting of 2-methylpiperazine, imidazole, Tris, pH 5.0 ± 0.1 and mobile phase B consisting of 100 mM sodium chloride and 2-methylpiperazine, imidazole and Tris, pH 10.8 ± 0.1 were used to gradient elute the sample for 70 minutes at a flow rate of 0.9 mL / min. The eluted protein was detected using UV absorbance at 280 nm.

[0744] HIC-HPLC

[0745] At ambient column temperature, an appropriate amount of protein sample was loaded onto a MAbPac TM HIC-Butyl column (Thermo SCIENTIFIC). Using an Agilent 1260 HPLC system, mobile phase A and mobile phase B were used to gradient elute the sample for 35 minutes at a flow rate of 0.8 mL / min, where mobile phase A consisted of 1.5 M ammonium sulfate, 0.05 M sodium phosphate and 5% isopropanol, pH 6.0 ± 0.1, and mobile phase B consisted of 0.05 M sodium phosphate and 5% isopropanol, pH 6.0 ± 0.1. The eluted protein was detected using UV absorbance at 280 nm.

[0746] Determination of thermal stability by DSF

[0747] Based on the micro differential scanning fluorimetry (nanoDSF) technique, with a temperature range of 25 - 95 °C and a heating rate of 1 °C / min, the denaturation temperature (Tm and Tonset) of the antibody protein and the onset temperature of protein aggregation (Tagg) were accurately determined by changes in the fluorescence spectrum from 280 - 450 nm and the intensity of laser scattered light at 266 nm or 473 nm to evaluate the thermal stability of the antibody protein.

[0748] Example 1.1 VHH Screening

[0749] Using alpaca immunization and magnetic sorting techniques, candidate VHH sequences that bind to Trop2 and Nectin4 were screened through preliminary property characterization. Briefly, using the recombinant antigen proteins human Trop2-His and human Nectin4-His prepared above, alpacas were immunized separately, with an immunization interval of 14 days. Starting from the second immunization, peripheral blood was collected seven days after each immunization, and the titer of immune serum was monitored through ELISA binding experiments. After the serum immune titer reached the standard for blood collection and library construction, peripheral blood of immunized alpacas was collected, and peripheral blood mononuclear cells (PBMCs) were isolated. Total RNA was extracted from PBMCs, and using RNA as a template, reverse transcription was performed using the PrimeScript TM II 1st Strand cDNA Synthesis Kit (Takara) to prepare cDNA. Using cDNA as a template, the first-round PCR amplification generated nucleic acid fragments of conventional IgG (containing VH) and pure heavy-chain IgG lacking the CH1 domain (containing VHH). These two types of nucleic acids were separated on an agarose gel, and the coding nucleic acid containing VHH was extracted, purified, and then subjected to the second-round PCR amplification. The VHH fragment was separated by gel, purified, and recovered. The recovered VHH gene fragment was mixed with the linearized yeast display vector pDisplay and co-transformed into yeast competent cells by electroporation to generate a yeast display library that displays VHH antibodies on the surface of yeast cells. Yeast cells that bind to the target antigen were enriched from the constructed library through magnetic sorting using streptavidin magnetic beads that had been pre-incubated with the target antigen and thus bound the antigen.

[0750] The yeast cell solution obtained after magnetic bead sorting was spread on SDCAA plates, and monoclonal cells were picked for culture. After 48 hours of induced expression, the monoclonal cell culture was incubated successively with Biotin-antigen and PE-Streptavidin. After the incubation was completed, flow cytometry (FACS) was performed to determine the positive monoclonal yeast cells that bound to the target antigen. Genomic DNA was extracted from the cultures of the obtained positive yeast cell clones for PCR amplification of the antibody sequence and sequencing.

[0751] Based on the sequencing results, candidate VHH sequences with large sequence differences were selected and ligated into the expression vector pcDNA3.4 in the form of C-terminal fusion with the human IgG1-Fc sequence. After the vector was verified by sequencing, it was transiently transfected into HEK-293F cells (hereinafter also abbreviated as "293F cells"). The supernatant of the culture was taken for characterization of the binding properties and endocytosis properties of the expressed antibody, and finally the anti-Trop2 VHH antibody and anti-Nectin4 VHH antibody in Table 1 below were screened and obtained:

[0752] Table 1. VHH antibodies and their variable region sequences

[0753] Anti-Trop2 antibody VHH sequence Anti-Nectin4 antibody VHH sequence A04 SEQ ID NO:5 F09 (or F9) SEQ ID NO:17 G08 SEQ ID NO:9 F12 SEQ ID NO:21 H03 SEQ ID NO:13 D9 SEQ ID NO:25

[0754] Example 1.2. In vitro characterization of candidate VHHs

[0755] Expression and purification of candidate VHH-Fc antibodies

[0756] The coding gene of the above VHH antibody sequence was synthesized and inserted into the expression vector pcDNA3.4 to fuse the hIgG1 Fc sequence (SEQ ID NO:85) at the C-terminus. The constructed expression vector was transiently transfected into 293F cells. After culturing the transfected cells continuously for 7 days, the culture supernatant was collected and filtered through a 0.45 μm filter membrane. The filtrate was transferred to a sterile centrifuge tube, and the antibody was purified using a Protein A column, and the purity of the antibody product was determined by SEC-HPLC method.

[0757] Detection of ELISA antigen-binding properties

[0758] The binding of candidate VHH-Fc antibodies to human and monkey antigens was detected by ELISA binding assay. In the Trop2 ELISA binding assay, the ELISA plate was coated with human Trop2-his antigen or cynomolgus monkey Trop2-his antigen, and the anti-hFc-HRP secondary antibody (Abcam, CAT#ab97225) was used for ELSIA determination. In the Nectin4 ELISA binding assay, the ELISA plate was coated with human Nectin4-his antigen or cynomolgus monkey Nectin4-his antigen, and the anti-hFc-HRP secondary antibody (Abcam, CAT#ab97225) was used for ELSIA determination. The ELISA detection results are shown in Table 2 below.

[0759] Table 2. Detection results of anti-Trop2 VHH-Fc binding ELISA

[0760]

[0761] As shown in the above table, in the Trop2 ELISA binding assay, all candidates showed good binding to human and monkey Trop2 antigens, and the EC50 ratio of the binding of all candidates to monkey and human Trop2 antigens was between 0.7 and 0.8, indicating that the affinity of the candidates to human and monkey Trop2 antigens was close.

[0762] Similarly, as shown in the above table, in the Nectin4 ELISA binding assay, all candidates showed good binding to human and monkey Nectin4 antigens, and the binding EC50 ratios of all candidates to monkey and human Nectin4 were 0.8 - 0.9, indicating that the affinity of the candidates to human and monkey Nectin4 antigens was close.

[0763] Detection of endocytosis properties

[0764] According to the preliminary FACS binding experiment, three concentrations of 100 nM, 10 nM, and 2 nM were selected as the concentrations for endocytosis detection. Endocytosis assays were performed on the candidate anti-Trop2 VHH-Fc antibody and anti-Nectin4 VHH-Fc antibody on tumor cells NCI-N87 and BT474. In this assay, 100 nM, 10 nM, or 2 nM of the VHH-Fc antibody to be tested was added to 2×10 5 / well target cells and incubated at 4 °C for 0.5 h; then the cells were evenly divided into two groups and incubated at 4 °C and 37 °C for 4 h respectively; after incubation, anti-hIgG-Fc-PE (diluted 1:500) was used to incubate in the dark at 4 °C for 30 minutes, and 1% paraformaldehyde (PFA) was used to fix the cells for FACS detection.

[0765] As Figure 1A and Figure 1B shown, for the candidate anti-Trop2 VHH-Fc antibodies tested, the trends of the endocytosis detection results were basically the same at the three measured concentrations. Compared with V-BMK1, candidates A04 and G08 had better binding to target cells and endocytosis not inferior to V-BMK1. At a concentration of 2 nM, the endocytosis rates in NCI-87 cells with high Trop2 expression levels (Trop2 hi ) and BT474 cells with low Trop2 expression levels (Trop2 low ) were both above 50%. The binding and endocytosis of H03 to target cells were slightly inferior to V-BMK1. At a concentration of 2 nM, the endocytosis rate in NCI-87 cells with high Trop2 expression levels (Trop2 hi ) was 54% and the endocytosis rate in BT474 cells with low Trop2 expression levels (Trop2 low ) was 38%.

[0766] As Figure 2A and 2B shown, for the candidate anti-Nectin4 VHH-Fc antibodies tested, the trends of the endocytosis detection results were basically the same at the three measured concentrations. All candidates F9-Fc, F12-Fc, and D9-Fc could bind to target cells, but there were significant differences in the target binding levels and endocytosis rates among the candidates.

[0767] Epitope binning

[0768] The epitope grouping of the candidate anti-VHH-Fc antibodies and related reference antibodies (Datopotamab or Enfortumab) was detected by ELISA binding assay.

[0769] In the ELISA binding assay, the antibody to be tested diluted to 1.0 μg / ml was used to coat the plate. The antigen (Trop2-his or Nectin4-his) was diluted to 300 ng / ml with the blocking solution, and the competitor antibody was diluted to 30 μg / ml with the blocking solution. Equal volumes of the antigen and the competitor were mixed and incubated at room temperature for 1 h. Additionally, a mixture of equal volumes of the antigen and the blocking solution was used as a control for the competition experiment to calculate the inhibition rate of the competitor against the antibody to be tested. The prepared mixture was added to the ELISA plate at 100 μl / well and incubated at room temperature for 1 h. Anti-His antibody-HRP (Sino Biological / 105327-MM02T-H) was used as the secondary antibody, and after incubation at room temperature for 1 h, it was detected on the machine.

[0770] As shown in the ELISA results in Table 3, the anti-Trop2 candidates A04-Fc, G08-Fc, and H0-Fc do not bind to the same epitope as Datopotamab.

[0771] As shown in the ELISA results in Table 4, for the binding of Enfortumab to the Nectin4 antigen, the anti-Nectin4 antibody F09-Fc has a weak signal inhibition, indicating that its binding epitope may be adjacent to Enfrotumab; while the anti-Nectin4 antibodies D9-Fc and F12-Fc are in different epitope groups from Enfortumab.

[0772] Table 3 Anti-Trop2 Epitope Competition

[0773]

[0774] Table 4 Anti-Nectin4 Epitope Competition

[0775]

[0776] Detection of FACS antigen-binding properties

[0777] The binding of the anti-Trop2 VHH-Fc candidate antibody molecules A04-Fc, G08-Fc, and H03-Fc to target cells was detected using FACS binding assay. The experiment was carried out under the following conditions: NCI-N87 target cells (2×10 5 / (well) + VHH-Fc or reference antibody (375 nM, 6× dilution, 4 °C for 1 h) + anti-hIgG Fc-PE secondary antibody (1:500, 4 °C for 0.5 h). The FACS binding results are as Figure 3 shown, and all three candidate antibodies exhibited good target cell binding properties.

[0778] Using the FACS binding assay, the binding of anti-Nectin4 VHH-Fc candidate antibody molecules D9-Fc, F09-Fc, and F12-Fc to target cells was detected. The assay was performed under the following conditions: NCI-N87 target cells (2×10 5 / (well) + D9-Fc or F12-Fc antibody or reference antibody (375 nM, 6× dilution, 4 °C for 1 h) + anti-hIgG Fc-PE secondary antibody (1:500, 4 °C for 0.5 h); or BT474 target cells (2×10 5 / (well) + F09-Fc or reference antibody (350 nM, 5× dilution, 4 °C for 1 h) + anti-hIgG Fc-PE secondary antibody (1:500, 4 °C for 1 h). The FACS binding results showed ( Figure 4 (A) and (B)), and all three candidate antibodies exhibited good target cell binding properties.

[0779] Example 1.3. Sequence optimization and characterization of VHH constructs

[0780] Sequence optimization of anti-Trop2 VHH construct

[0781] The anti-Trop2 VHH sequence A04 was selected and its sequence was optimized.

[0782] The original VHH sequence was humanized using the "best match method". The amino acid sequence of the VHH framework region was aligned and analyzed using the human germline V gene database to select the best germline sequence. The VHH CDR sequences were used to replace the best-matched human CDR sequences to generate humanized VHH sequences. Multiple residues in the framework region were subjected to back-mutation and post-translational modification (PTM) removal. The humanized sequences were reverse-translated and sent to GenScript (Shanghai, China) for gene synthesis. Then they were cloned into the pcDNA 3.4 expression vector and expressed as human IgG4 Half body by fusing the human IgG4 sequence (SEQ ID NO:87) at the C-terminus to obtain VHH antibody proteins.

[0783] The PTM removal and the VHH sequences of the humanized antibodies of A04 are shown in Table 5 below, where the CDR sequences defined according to the AbM protocol are underlined.

[0784] Table 5. Anti-Trop2 VHH sequences

[0785]

[0786] Table 6 below shows the expression and purification results of the A04 humanized antibody expressed in the form of VHH-hIgG4 half antibody.

[0787] Table 6. Expression and purification results of A04.m1 humanized antibody

[0788] Antibody Concentration (mg / ml) Purity (SEC-HPLC) % Yield (mg / L) A04.m1-hIgG4 1.19 96.5 119 A04m1.m10-hIgG4 1.04 96.2 104 A04m1.m11-hIgG4 1.87 95.7 187 A04m1.m12-hIgG4 1.44 95.3 144

[0789] The obtained antibodies A04.m1-hIgG4, A04m1.m10-hIgG4, A04m1.m11-hIgG4 and A04.m1.m12-hIgG4 were subjected to FACS detection. The binding of both antibodies to the target cell NCI-N87 was similar to that of the parental antibody (Table 7). The conditions for the FACS binding assay were as follows: target cells (1×10 5 / well) + sample (200 nM, 5X dilution, incubated at 4°C for 1 h) + anti-Fc-PE (1:500, incubated at 4°C for 0.5 h).

[0790] Table 7. Binding activity of A04 humanized antibody to NCI-N87

[0791]

[0792] SPR detection was performed to verify the human-monkey cross-reactivity of the selected A04 humanized antibodies (Table 8).

[0793] Table 8. Binding affinity kinetics of A04 humanized antibody to human and cynomolgus monkey Trop2 antigens

[0794]

[0795] The physicochemical properties of the VHH half antibody prepared from 293F host cells as described above were analyzed using HIC-HPLC and DSF assays. The results are shown in Table 9 below.

[0796] Table 9. Physicochemical property DA data of A04 humanized antibody

[0797]

[0798] Sequence optimization of anti-Nectin 4 VHH construct

[0799] The anti-Nectin4 VHH sequence F09 (hereinafter referred to as "F09") was selected and its sequence was optimized.

[0800] The original VHH sequences were humanized using the "best match method". The amino acid sequences of the VHH framework regions were aligned and analyzed using the human germline V gene database to select the best germline sequences. The VHH CDR sequences were used to replace the best-matched human CDR sequences to generate humanized VHH sequences. Multiple residues in the framework region were subjected to back-mutation and post-translational modification (PTM) removal. The humanized sequences were reverse-translated and sent to GenScript (Shanghai, China) for gene synthesis. Then they were constructed into the pcDNA 3.4 expression vector and expressed as humanized VHHs with a His tag fused to the C-terminus. The antibodies were purified using nickel columns, and the product purity was determined by SEC-HPLC to obtain the VHH-His antibody protein.

[0801] The humanized and optimized VHH sequences and their corresponding original VHH sequences are shown in Table 10 below, where the CDR sequences defined according to the AbM protocol are underlined.

[0802] Table 10 Humanized and optimized VHH sequences

[0803]

[0804]

[0805] The expression and purification results of the optimized F09 antibody expressed in the form of VHH-his are shown in Table 11 below.

[0806] Table 11. Expression and purification results of the optimized F09 antibody

[0807]

[0808] The obtained antibodies were subjected to FACS binding assays, and the resulting antibodies had similar antigen-binding affinities to the parental antibody F09. Through SPR assay experiments, the binding affinity kinetics of the obtained antibodies to human and cynomolgus monkey Nectin4 antigens were examined, and they exhibited binding affinities comparable to those of the parental and similar human-monkey cross-reactivity (Table 12).

[0809] Table 12. Binding affinity kinetics of the optimized F09 antibody to human and cynomolgus monkey Nectin4 antigens

[0810] Immobilized ligand Analyte ka (1 / Ms) kd (1 / s) KD (M) Human Nectin4-his F09.m1.his 3.44E+05 1.82E-02 5.29E-08 Human Nectin4-his F09.m2.his 2.88E+05 1.15E-02 3.99E-08 Human Nectin4-his F09.m5.his 2.91E+05 7.89E-03 2.71E-08 Human Nectin4-his F09.m6.his 5.36E+05 7.47E-03 1.39E-08 Human Nectin4-his F09.m1.m5.his 6.57E+04 1.20E-02 1.82E-07 Human Nectin4-his F09.m1.m6.his 7.54E+04 1.14E-02 1.51E-07 Human Nectin4-his F09.m2.m5.his 9.50E+04 7.54E-03 7.94E-08 Human Nectin4-his F09.m2.m6.his 1.43E+05 7.73E-03 5.40E-08 Human Nectin4-his F09.his 9.96E+04 1.14E-02 1.15E-07 Cyno Nectin4-His F09.m1.m5.his 8.19E+04 1.48E-02 1.80E-07 Cyno Nectin4-His F09.m1.m6.his 1.26E+05 1.34E-02 1.06E-07 Cyno Nectin4-His F09.m2.m5.his 6.95E+04 9.43E-03 1.36E-07 Cyno Nectin4-His F09.m2.m6.his 1.15E+05 7.97E-03 6.91E-08 Cyno Nectin4-His F09.his 9.11E+04 1.44E-02 1.58E-07

[0811] After converting F09.m2m5 into the Fc bivalent format, through FACS binding assays, when binding to the target cells BT474, F09.m2m5-Fc still exhibited binding similar to that of F09-Fc. The FACS assay was performed using the following conditions: BT474 target cells (2×10 5+(350 nM, 5x dilution, 4 °C for 1 h) + anti-hIgG Fc-PE secondary antibody (1:500, 4 °C for 1 h).

[0812] Based on the above data, F09.m2m5 was selected, and the immunogenicity of the humanized VHH sequence was analyzed by WeMol software. Mutation residues were introduced into F09.m2m5 to obtain the deimmunized sequences F09.m2m5-2 and F09.m2m5-4.

[0813] Table 13. Deimmunized VHH sequences of F09.m2m5

[0814]

[0815] For the deimmunized samples of F09.m2m5, cross-reactivity SPR and FACS assays were performed. The SPR assay results are shown in Table 14. The FACS assay results are as Figure 5 shown. This sample maintained cross-reactivity with human Nectin4 and cyno Nectin4 engineered cell lines and did not cross-react with CHO-K1 cells. Physicochemical property analysis of F09.m2m5.his, F09.m2m5-2.his, and F09.m2m5-4.his was performed by SEC-HPLC, CEX-HPLC, HIC-HPLC, and DLS assays. The results are shown in Table 15 below, and both samples showed physicochemical properties with good developability.

[0816] Table 14. Binding affinity kinetics of deimmunized samples of F09.m2m5 to human and cynomolgus monkey Nectin4 antigens

[0817] Immobilized ligand Analyte ka (1 / Ms) kd (1 / s) KD (M) Human Nectin4-his F09m2m5-2.his 8.75E+04 7.58E-03 8.66E-08 Human Nectin4-his F09m2m5-4.his 1.03E+05 7.91E-03 7.71E-08 Cyno Nectin4-His F09m2m5-2.his 6.48E+04 1.23E-02 1.90E-07 Cyno Nectin4-His F09m2m5-4.his 2.16E+05 1.05E-02 4.88E-08

[0818] Table 15. Physicochemical property data of F09.m2m5.his and its deimmunized antibodies

[0819]

[0820] Example 1.4 Generation of multispecific anti-Trop2 / Nectin4 antibody molecules

[0821] This example describes the design and construction of the structure and expression vector of an exemplary anti-Trop2 / Nectin4 bispecific antibody (BsAb).

[0822] Multi-specific antibody molecule design

[0823] The following bispecific antibody molecule constructs were designed:

[0824] A. Single-chain form, containing from N-terminus to C-terminus: (VHHA ) n1 -(VHH B ) n2 -(HLE) n3 -(VHH A ) n4 -(VHH B ) n5 -(HLE) n6，

[0825] wherein n1, n2, n3, n4, n5 and n6 are independently selected from the integers 0, 1 or 2; wherein VHH A and VHH B respectively represent VHH domains that bind to antigens A and B, wherein A and B are different from each other and independently selected from Trop2 and Nectin4; wherein HLE (Half-life extension) represents a serum albumin (SA) binding peptide as a half-life extension domain, in particular an anti-serum albumin VHH domain VHH SA ; wherein the symbol "-" represents connection by a peptide linker or direct connection, preferably a peptide linker having a length of 5-15 amino acids,

[0826] Preferably, the ratio of the VHH Trop2 domain to the VHH Nectin4 domain is 1:1 or 1:2,

[0827] Preferably, there are 1-2 VHH Trop2 domains and 1-2 VHH Nectin4 domains;

[0828] Optionally, there are 0 or 1 HLE domains;

[0829] For example, the single-chain form in Table 16 below:

[0830] Table 16. Single-chain antibody forms against Trop2 and Nectin4

[0831] Format Single polypeptide chain 1 <![CDATA[VHH Trop2 -VHH Nectin4 > 2 <![CDATA[VHH Trop2 -VHH Nectin4 -VHH SA > 3 <![CDATA[VHH Nectin4 -VHH Trop2 -VHH Nectin4 > 4 <![CDATA[VHH Trop2 -VHH Nectin4 -VHH Nectin4 > 5 <![CDATA[VHH Trop2 -VHH SA -VHH Nectin4 -VHH Nectin4 > 6 <![CDATA[VHH Trop2 -VHH Trop2 -VHH SA -VHH Nectin4 -VHH Nectin4 >

[0832] For single-chain form antibodies, in order to facilitate subsequent purification, a purification tag (such as a His tag) can optionally be added to the C-terminus of the antibody molecule, and the addition is optionally achieved through a small peptide linker (such as AAA) or by direct connection.

[0833] B. Symmetric double-chain form: Composed of two identical polypeptide chains, wherein: each chain from the N-terminus to the C-terminus contains: (VHH A ) n1 -(VHH B )n2 -HLE-(VHH A ) n3 -(VHH B ) n4， wherein n1, n2, n3 and n4 are independently selected from integers of 0, 1 or 2;

[0834] wherein VHH A and VHH B respectively represent VHH domains that bind to antigens A and B, wherein A and B are different from each other and are independently selected from Trop2 and Nectin4; wherein HLE represents an immunoglobulin Fc region as a half-life extension domain, particularly a human IgG1 or human IgG4 Fc region; wherein the symbol "-" represents connection by a peptide linker or direct connection, preferably a peptide linker with a length of 5-15 amino acids,

[0835] Preferably, the ratio of the VHH Trop2 domain to the VHH Nectin4 domain is 1:1 or 1:2,

[0836] Preferably, there are 1-2 VHH Trop2 domains, and 1-2 VHH Nectin4 domains;

[0837] More preferably, each chain contains, from the N-terminus to the C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE, wherein n1 and n2 are independently selected from integers of 1 or 2, and more preferably n1 and n2 are 1, such as the symmetric double-stranded form in Table 17 below:

[0838] Table 17. Symmetric double-stranded antibody forms against Trop2 and Nectin4

[0839] Format Antibody polypeptide chain 1 <![CDATA[(VHH Nectin4 -VHH Trop2 -Fc)x2]]> 2 <![CDATA[(VHH Trop2 -VHH Nectin4 -Fc)x2]]>

[0840] Due to the dimerization of the immunoglobulin Fc region, the first and second polypeptide chains can associate to form a homodimer, thereby generating a multi-specific binding molecule in a double-stranded form.

[0841] C. Asymmetric double-stranded form: composed of two different polypeptide chains, wherein

[0842] the first polypeptide chain contains, from the N-terminus to the C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE-(VHH A ) n3 -(VHHB ) n4，

[0843] The second polypeptide chain contains, from the N-terminus to the C-terminus: (VHH B ) m1 -(VHH A ) m2 -HLE-(VHH B ) m3 -(VHH A ) m4，

[0844] wherein n1, n2, n3 and n4, and m1, m2, m3 and m4 are independently selected from the integers 0, 1 or 2;

[0845] wherein VHH A and VHH B represent VHH domains that bind to antigens A and B, respectively, where A and B are different from each other and are independently selected from Trop2 and Nectin4; wherein HLE represents an immunoglobulin Fc region as a half-life extension domain, particularly a human IgG1 or IgG4 Fc region; wherein the symbol "-" represents connection by a peptide linker or direct connection, preferably a peptide linker having a length of 5-15 amino acids,

[0846] Preferably, the ratio of the VHH Trop2 domain to the VHH Nectin4 domain is 1:1 or 1:2,

[0847] Preferably, there are 1-2 VHH Trop2 domains, and 1-2 VHH Nectin4 domains;

[0848] More preferably, wherein

[0849] The first polypeptide chain contains, from the N-terminus to the C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE ，

[0850] The second polypeptide chain contains, from the N-terminus to the C-terminus: (VHH B ) m1 -(VHH A ) m2 -HLE ，

[0851] wherein n1, n2, m1, and m2 are independently selected from the integers 0, 1, or 2, provided that n1 and n2 cannot both be 0, m1 and m2 cannot both be 0, and the sum of n1 and m2 and the sum of n2 and m1 shall be greater than or equal to 1;

[0852] More preferably,

[0853] The first polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE ，

[0854] The second polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH B ) m1 -HLE ； Or (VHH A ) m2 -HLE ，

[0855] wherein n1, m1, and m2 are independently selected from the integers 1 or 2, and n2 is an integer of 0 or 1;

[0856] Even more preferably,

[0857] The first polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH A )-HLE ，

[0858] The second polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH B )-HLE ；

[0859] Or,

[0860] The first polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH A )-(VHH B )-HLE ，

[0861] The second polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH A )-HLE ；

[0862] Or,

[0863] The first polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH A )-(VHH A )-HLE ，

[0864] The second polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH B )-HLE；

[0865] Alternatively,

[0866] The first polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH A )-(VHH B )-HLE ，

[0867] The second polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH B )-HLE ；

[0868] For example, the double-stranded form in Table 18 below:

[0869] Table 18. Asymmetric double-stranded antibody forms against Trop2 and Nectin4

[0870]

[0871] Due to the dimerization of the immunoglobulin Fc region, the first and second polypeptide chains can associate to form a heterodimer, thereby generating a multispecific binding molecule in double-stranded form. Preferably, in order to promote the heterodimerization of the first and second polypeptide chains, Knob-into-hole mutations can be introduced into the Fc regions of the first and second polypeptide chains, such as the (T366W / T366S, L368A, Y407V) mutation or the (T366Y / Y407T) mutation.

[0872] Single-chain multi-specific antibody molecule construction

[0873] Exemplary single-chain forms of bispecific constructs as shown in Tables 19 and 20 below were constructed, and their corresponding amino acid sequences are provided in the sequence listing.

[0874] In Table 19, non-humanized VHH sequences were used as building blocks, where the VHH Trop2 amino acid sequences are from G08 (hereinafter referred to as T1) and H03 (hereinafter referred to as T2), and their amino acid sequences are shown in SEQ ID NO:9 and SEQ ID NO:13, respectively; the VHH Nectin4 amino acid sequences are from F12 (hereinafter referred to as N1) and D9 (hereinafter referred to as N2), and their amino acid sequences are shown in SEQ ID NO:21 and SEQ ID NO:25; the serum albumin-binding peptide as the half-life extension domain (HLE) is from the VHH domain VHH SA , more specifically from VHH SASequence MSA21 (hereinafter referred to as HLE1, SEQ ID NO: 39); the symbol "-" indicates that the two VHH domains are connected by a peptide linker.

[0875] Table 19. Single-chain multispecific antibodies

[0876]

[0877]

[0878] * Peptide linker = G4S

[0879] In Table 20, humanized VHH sequences were used as building blocks, where the VHH Trop2 amino acid sequence is from A04.m1 (hereinafter referred to as S1), and its amino acid sequence is shown in SEQ ID NO: 29; the VHH Nectin4 amino acid sequence is from F09.m2m5 (hereinafter referred to as F1), and its amino acid sequence is shown in SEQ ID NO: 34; the serum albumin (SA) binding peptide as the half-life extension domain (HLE) is from the VHH domain VHH SA and more specifically from the VHH sequence ALB8 (hereinafter referred to as HLE2, SEQ ID NO: 40); the symbol "-" indicates that the two VHH domains are connected by a peptide linker.

[0880] Table 20. Single-chain multispecific antibodies

[0881]

[0882] * Peptide linker = (G4S)2

[0883] The single-chain antibodies in Tables 19 and 20 were constructed into the pcDNA 3.4 expression vector in the form of adding a 6xHis tag at the C-terminus. Transfected into Expi293F cells, the cells were cultured for 3 days, and the culture supernatant of the transfected cells was collected. The expressed antibody was purified using a nickel column. The antibody concentration was measured using Nano Drop. The protein purity was determined by SDS-PAGE and analytical HPLC-SEC, and then it was stored at -80 °C.

[0884] Multi-chain multi-specific antibody molecule construction

[0885] Constructs were made of the exemplary multichain forms of bispecific antibodies as shown in Tables 21 and 22 below, and their corresponding amino acid sequences are provided in the sequence listing.

[0886] In Table 21, non-humanized VHH sequences were used as building blocks, where the VHH Trop2The amino acid sequences are from G08 (hereinafter referred to as T1) and H03 (hereinafter referred to as T2), and their amino acid sequences are shown in SEQ ID NO:9 and SEQ ID NO:13 respectively; VHH Nectin4 The amino acid sequences are from F12 (hereinafter referred to as N1) and D9 (hereinafter referred to as N2), and their amino acid sequences are shown in SEQ ID NO:21 and SEQ ID NO:25 respectively; the immunoglobulin Fc region as the half-life extension domain (HLE) is from human IgG, specifically the hIgG1 Fc sequence containing knob mutation and hole mutation ((T366Y / Y407T) or (T366W / T366S, L368A, Y407V)) respectively; the symbol "-" indicates that the two domains are connected by a peptide linker or directly connected.

[0887] Table 21. Fc dimer form multi-chain multispecific antibodies

[0888]

[0889]

[0890] Note: The two VHH domains are connected by the peptide linker G4S, and the VHH domain and the Fc domain are directly connected.

[0891] In Table 22, humanized VHH sequences are used as building blocks, where VHH Trop2 The amino acid sequences are from A04.m1 (hereinafter referred to as S1) or A04.m1m12 (hereinafter referred to as S2), and their amino acid sequences are shown in SEQ ID NO:29 or SEQ ID NO:30 respectively; VHH Nectin4 The amino acid sequences are from F09.m2m5 (hereinafter referred to as F1) or F09.m2m5-2 (hereinafter referred to as F2), and their amino acid sequences are shown in SEQ ID NO:34 and SEQ ID NO:38 respectively; the immunoglobulin Fc region as the half-life extension domain (HLE) is from human IgG, specifically the hIgG1 sequence containing or not containing knob mutation and hole mutation ((T366Y / Y407T) or (T366W / T366S, L368A, Y407V)); the symbol "-" indicates that the two domains are connected by a peptide linker or directly connected. Also shown in Table 22 are the Parental anti-Nectin4 antibody and Parental anti-Trop2 antibody as controls, where Parental anti-Nectin4 contains VHH Nectin4Domain (F2) with the amino acid sequence shown in SEQ ID NO: 109; Parental anti-Trop2 contains VHH trop2 Domain (S2) and VHH targeting rotavirus RV Domain (from DOI: 10.1053 / j.gastro.2013.06.053) with the amino acid sequences of the first and second chains shown in SEQ ID NO: 107 and 108, respectively.

[0892] Table 22. Fc dimer form multi-chain multispecific antibodies

[0893]

[0894]

[0895] * The two VHH domains are connected by a peptide linker (G4S)2, and the connection between the VHH domain and the Fc domain is either direct or through G4S

[0896] In the present invention, V-hu21-Fc, V-hu22-Fc, V-hu23-Fc, V-hu24-Fc, V-hu25-Fc, V-hu26-Fc, V-hu27-Fc, V-hu28-Fc, V-hu29-Fc are also respectively referred to as V-hu21, V-hu22, V-hu23, V-hu24, V-hu25, V-hu26, V-hu27, V-hu28, V-hu29.

[0897] The first / second polypeptide chains of the Fc dimer form of the bispecific antibody shown in Tables 21 and 22 are constructed into the pcDNA3.4 expression vector (if the antibody is a symmetric structure, one vector is generated; if the antibody is an asymmetric structure, two vectors respectively containing the coding genes of the first and second polypeptide chains are generated), and transfected into HEK293F cells. The cells are cultured for 3 days, and the culture supernatant of the transfected cells is collected and loaded onto a Protein A column (MabSelect PrismA, Cytiva) for purification. The antibody is eluted with an acetic acid-sodium acetate solution (pH 3.5) and then immediately neutralized with 2M Tris. The antibody concentration is measured using Nano Drop. The protein purity is determined by SDS-PAGE and analytical HPLC-SEC, and then it is stored at -80°C.

[0898] Control bispecific antibody construction

[0899] Using the variable region amino acid sequences from the reference antibodies Datopotamab and Enfortumab, control bispecific antibodies V-11 and V-12 were constructed. Antibody V-11 consists of the Fab portion of Datopotamab with an Fc region linked to the C-terminus and the scFv portion of Enfortumab with an Fc region linked to the C-terminus. The amino acid sequences of its two heavy chains and one light chain (SEQ ID NOs: 77 - 79) are provided in the sequence listing. Antibody V-12 consists of the scFv portion of Datopotamab with an Fc region linked to the C-terminus and the Fab portion of Enfortumab with an Fc region linked to the C-terminus. The amino acid sequences of its two heavy chains and one light chain (SEQ ID NOs: 80 - 82) are provided in the sequence listing. In substantially the same manner as described above, control antibodies V-11 and V-12 were produced and purified. The antibody concentration was measured using NanoDrop. Protein purity was determined by SDS-PAGE and analytical HPLC-SEC and then stored at -80°C.

[0900] Example 1.5 Characterization of Multispecific Anti-Trop2 / Nectin4 Antibody Molecules

[0901] Analysis of target antigen expression level on tumor cells

[0902] Using the reference antibodies Datopotamab and Enfortumab, the expression of Trop2 antigen and Nectin4 antigen on various target tumor cells was detected by FACS binding assay.

[0903] The detection results are as Figure 6 shown. On the tested tumor cell MKN45, both antigens showed extremely low expression levels and were antigen-negative cells; among the remaining tested tumors, except for BT474 cells with extremely low Trop2 expression, the remaining tumor cells showed high Trop2 expression levels, which were significantly higher than the expression level of Nectin4. Comparison of Nectin4 expression levels among different tumors showed that HT1197 and MDA-MB-231 had extremely low Nectin4 expression, NCI-N87, SK-BR-3, and BT474 had relatively moderate Nectin4 expression, while the remaining tumors had relatively high Nectin4 expression levels.

[0904] Non-specific binding analysis

[0905] The binding of the above-constructed and prepared multispecific antibody molecules to antigen homologs was detected by ELISA binding assay. The ELISA binding assay was performed under the following conditions: Antigen Trop1 / 2 or Nectin1 / 2 / 3 / 4 (1 μg / ml, overnight coating at 2-8 °C) + antibody sample to be tested (200 nM, 6-fold dilution, 1 h at RT) + Anti-Fc-HRP secondary antibody (1:5000, 1 h at RT).

[0906] The results showed that the multispecific antibody molecules had no specific binding to the homologs Trop1 and Nectin1 / 2 / 3. The results of the non-specific binding analysis for antibodies V-hu21-Fc and V-hu23-Fc are shown in Figure 7 .

[0907] Tumor cell binding activity

[0908] Based on the above analysis of antigen expression on tumor cells, cell lines NCI-N87 (Trop2 hi , Nectin4 med ), BT474 (Trop2 low , Nectin4 hi ), and MDA-MB-468 (Trop2 hi , Nectin4 hi ) with different target antigen expression levels were selected. The binding ability of the bispecific antibody (abbreviated as "V BsAb") of the present invention to different target cells was tested by FACS binding assay.

[0909] The single-chain antibody was tested under the following FACS binding assay conditions: Target cells (1-2×10 5 / well) + sample (200 nM, 3-fold dilution, 1 h at 4 °C) + anti-His-APC secondary antibody (Biolegend, 1:200, 1 h at 4 °C)

[0910] The double-chain antibody in the form of Fc dimer was tested under the following FACS binding assay conditions: Target cells (2×10 5 / well) + sample (200 nM, 3-fold dilution, 1 h at 4 °C) + anti-human IgG Fc-PE secondary antibody (Thermo, 1:500, 0.5 h at 4 °C).

[0911] On the NCI-N87 cell line with high Trop2 expression, the binding of the non-humanized V BsAb and the V BsAb containing Fc (V-10, V-13–V-18) of the present invention is shown in Figure 8As shown in A-8B. The binding of the non-humanized V BsAb and the Fc-containing V BsAb (V-10, V-13–V-18) of the present invention to the BT474 cell line with low Trop2 expression and relatively high Nectin4 expression is as Figure 9 shown in A-9B.

[0912] In the tumor cells MDA-MB-468 and NCI-87 with high Trop2 expression, the symmetric humanized V BsAb of the present invention (V-hu24-Fc and V-hu25-Fc) with 2 Trop2 binding sites and 2 Nectin4 binding sites has comparable high binding activity to the control antibody Datopotamab; the asymmetric humanized V BsAb of the present invention (V-hu21-Fc, hu23-Fc and hu28-Fc) with 1 Trop2 binding site and 2 Nectin4 binding sites shows comparable binding activity, both higher than the control Enfortumab antibody, and the difference from Enfortumab is more obvious in the target tumor cell NCI-87 with lower Nectin4 level; in addition, the humanized V BsAb (V-hu29-Fc) with only 1 Trop2 binding site and 1 Nectin4 binding site shows low binding activity similar to Enfortumab ( Figure 10 A and 10B). In the BT474 cells with low Trop2 expression and high Nectin4 expression, the humanized V BsAb of the present invention shows binding behavior similar to the control Enfortumab ( Figure 10 C).

[0913] The humanized V-hu21-Fc in the form of Fc dimer was selected, and the binding activity of the antibody to various different tumor cells was further detected by FACS binding assay. In the assay, the anti-Nectin4 monospecific bivalent antibody Parental anti-Nectin4 was used as a control. The conditions of the FACS binding assay were as follows: target cells (2×10 5 / well) + sample (200 nM, 3× dilution, 4°C for 1 h) + anti-human IgG Fc-PE secondary antibody (Thermo, 1:500, 4°C for 0.5 h). The results are shown in Figure 11 this figure.

[0914] The humanized V-hu23-Fc in the form of an Fc dimer was selected, and the binding activity of the antibody to HT1376 and NCI-H292 (human mucoepidermoid carcinoma of the lung, CBP60187, Nanjing Kebai Biotechnology Co., Ltd.) tumor cells was further detected by FACS binding assay. In the experiment, Parental anti-Trop2 and Parental anti-Nectin4 were used as controls. The FACS binding assay conditions were as follows: target cells (3×10 5 / well) + sample (200 nM, 3× dilution, 4°C for 1 h) + anti-human IgG Fc-PE secondary antibody (Thermo, 1:500, 4°C for 0.5 h). The results are shown in Figure 12 .

[0915] The results showed that V-21-Fc and V-23-Fc showed a synergistic binding effect on the cells that were positive for both targets compared with the parental control molecules.

[0916] Internalization assay

[0917] The endocytosis assay was used to detect the endocytosis ability of the bispecific antibody molecule of the present invention on different target cells NCI-N87, BT-474, HT1376, MDA-MB-468 and NCI-H292 cells.

[0918] The above cell lines with different target antigen expressions were selected to test the endocytosis ability of the V BsAb of the present invention. As shown in Figure 13A -E, all the tested V BsAbs showed good endocytosis activity, and V-21-Fc and V-hu23-Fc showed a synergistic endocytosis effect on the tumor cells that were positive for both targets (HT1376 and NCI-H292) compared with their parental control molecules Parental anti-Trop2 and Parental anti-Nectin4.

[0919] Binding kinetics SPR assay

[0920] The affinity of the bispecific antibody molecule V-hu21 for human and cynomolgus monkey Trop2 antigens and Nectin4 antigens (Sino Biological) was detected by SPR method. 10 μg / mL antibody was captured on a Protein A chip for 15 s, and then antigens diluted 2-fold in gradient were injected at a flow rate of 30 μL / min. The binding time was 120 seconds, and then the dissociation time was 200 seconds. After each dissociation stage, 10 mM glycine (pH 2.0) was used for chip regeneration. The experimental data was analyzed using a 1:1 binding model.

[0921] The affinity results of the candidate molecule V-hu21-Fc with human and cynomolgus macaque Trop2 are shown in Table 23. The results indicate that the candidate molecule V-hu21-Fc has a cross-reaction with cynomolgus macaque Trop2 and exhibits similar binding and dissociation rates with human Trop2 antigen and cynomolgus macaque Trop2 antigen.

[0922] Table 23. Affinity of V-hu21-Fc for human and cynomolgus macaque Trop2 antigens

[0923] Immobilized Analyte ka (1 / Ms) kd (1 / s) KD (M) V-hu21-Fc Human Trop2 3.45E+05 2.09E-02 6.06E-08 V-hu21-Fc Cyno Trop2 1.58E+05 9.81E-03 6.21E-08 V-BMK1 (Datopotamab) Human Trop2 1.32E+05 3.15E-03 2.38E-08 V-BMK1 (Datopotamab) Cyno Trop2 1.10E+05 3.57E-03 3.25E-08

[0924] The affinity results of the candidate molecule V-hu21-Fc with human and cynomolgus macaque Nectin4 are shown in Table 24. The results indicate that the candidate molecule V-hu21-Fc has a cross-reaction with cynomolgus macaque Nectin4 and exhibits similar binding and dissociation rates with human Nectin4 antigen and cynomolgus macaque Nectin4 antigen.

[0925] Table 24. Affinity of V-hu21-Fc for human and cynomolgus macaque Nectin4 antigens

[0926] Capture Analyte ka (1 / Ms) kd (1 / s) KD (M) V-hu21-Fc Human Nectin4 5.08E+04 9.86E-03 1.94E-07 V-hu21-Fc Cyno Nectin4 2.54E+05 1.50E-02 5.89E-08 V-BMK2 (Enfortumab) Human Nectin4 4.12E+05 4.72E-03 1.14E-08 V-BMK2 (Enfortumab) Cyno Nectin4 3.83E+05 8.31E-03 2.17E-08

[0927] In addition, the affinity of the bispecific antibody molecule V-hu23-Fc for human and cynomolgus macaque Trop2 antigens or human and cynomolgus macaque Nectin4 antigens was also detected by SPR method. The results are shown in Tables 25 and 26 below.

[0928] Table 25. Affinity of V-hu23-Fc for human and cynomolgus macaque Trop2 antigens

[0929] Immobilized Analyte ka (1 / Ms) kd (1 / s) KD (M) V-hu23-Fc Human Trop2-His 1.14E+05 9.14E-03 8.05E-08 V-hu23-Fc Cyno Trop2-His 9.95E+04 7.99E-03 8.03E-08 V-BMK1 (Datopotamab) Human Trop2-His 1.31E+05 3.59E-03 2.75E-08 V-BMK1 (Datopotamab) Cyno Trop2-His 1.10E+05 3.54E-03 3.23E-08

[0930] Table 26. Affinity of V-hu23-Fc for human and cynomolgus macaque Nectin4 antigens

[0931] Immobilized Analyte ka (1 / Ms) kd (1 / s) KD (M) V-hu23-Fc Human Nectin4-His 4.34E+04 5.94E-03 1.37E-07 V-hu23-Fc Cyno Nectin4-His 3.77E+04 7.18E-03 1.90E-07 V-BMK2-Enfortumab Human Nectin4-His 3.13E+05 3.93E-03 1.26E-08 V-BMK2-Enfortumab Cyno Nectin4-His 3.26E+05 7.38E-03 2.27E-08

[0932] Developability assessment

[0933] The candidate molecules V-hu21, hu23, hu24-Fc were analyzed for their physicochemical properties by SEC-HPLC, CEX-HPLC, HIC-HPLC and DLS measurements. The physicochemical properties showing developability are shown in Table 27.

[0934] Table 27. Physicochemical property DA data of V-hu21, hu23, hu24-Fc

[0935]

[0936] Example 2. Preparation and Characterization of Antibody-Drug Conjugates (ADCs)

[0937] The linker-payload used in the examples of the present invention is known in the prior art and / or commercially available. When the drawn structure is inconsistent with the actual situation, it should be allowed to modify the structure according to the actual situation.

[0938] Materials and Methods

[0939] Name Manufacturer Catalog Number Mal-Gly-EXD-D-glucuronic acid MedChemExpress HY-153179 Mal-PEG8-Val-Ala-PAB-Exatecan MedChemExpress HY-147271 Deruxtecan MedChemExpress HY-13631E Constant Temperature Oscillation Metal Bath Dlab HCM100-pro Centrifuge Thermo Fisher Scientific Cat#75002420 Amicon Utral-15 Millipore UFC903096 TECP aladdin T107252

[0940] General Synthesis Method A

[0941] Place 5.45 mg / ml antibody (e.g., the corresponding bispecific antibody prepared according to the above antibody preparation examples) in 20 mM His-hac, 150 mM NaCl, pH 5.5 buffer in an Eppendorf tube. Add 6 molar equivalents of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, 5 mM concentration) to the antibody buffer (TCEP:antibody = 6:1 molar ratio). Place the Eppendorf tube containing the reaction mixture on an oscillator (x500 rpm) and react at 37 °C for 2 hours. Add another 6 molar equivalents of TCEP (5 mM) to the mixture. Place the reaction mixture on an oscillator (500 rpm) and shake at 37 °C for another 2 hours. Then, ultrafiltration (MWCO 30 kd) is used to remove TECP and replenish to the original volume with 20 mM His-hac buffer. Add 6 molar equivalents of linker-payload (i.e., linker-payload) (5 mg / ml, in DMA) dropwise to the fully reduced antibody while keeping the DMA concentration below 20% (v / v). Keep the reaction on an oscillator at room temperature for 2 hours (linker-payload:antibody = 6:1). Detect the conversion rate by HIC-HLPC and purify when the conversion is complete. Transfer the reaction mixture to an ultrafiltration tube (MWCO 30 kd), add 10 mM His-hac (pH 6) to a total of 450 μl. Centrifuge the sample at 10,000 rpm for 5 minutes, leaving approximately 200 μl of solution, and replenish to 450 μl with His-hac. Discard the flow-through. Repeat the washing step 6 times. Transfer the remaining solution, add 200 μl of His-hac, rinse the filter membrane by pipetting up and down, and combine to obtain the ADC conjugate product.

[0942] Perform Pro A resin purification (40 g / L resin loading capacity). Wash the gravity column filled with resin with 10 CV of PBS, and then load the sample onto the column. First, wash with at least 10 CV of PBS buffer containing 10% DMSO, and then wash with at least 50 CV of PBS buffer to completely remove the free payload. Elute with acetic acid solution (concentration 50 mM) at pH 3 and immediately neutralize to pH 5.5 - pH 6.0 with 2 M tris buffer (pH 12.0). Repeat the elution step and measure the breakthrough concentration (Nanodrop, A280) until the conjugate is completely eluted. Combine the ADC solutions in Eppendorf tubes. If the final concentration is less than expected, concentrate to the desired concentration using an ultrafiltration membrane (MWCO 30 kd).

[0943] Determine the purity by SEC-HPLC method, the average DAR value by HIC-HPLC method, and the free linker-payload by RP-HPLC method.

[0944] General synthesis method B

[0945] Place the antibody in PBS at pH 7.4 (e.g., the corresponding bispecific antibody prepared according to the above antibody preparation examples) in an Eppendorf tube. Add 5.7 molar equivalents of TCEP (5 mM concentration) and 2.85 molar equivalents of ZnCl2 (5 mM concentration) to the antibody buffer (TCEP: antibody: ZnCl2 = 5.7:1:2.85). Place the Eppendorf tube containing the reaction mixture on an oscillator at 6°C (x500 rpm) for 16 hours. Add 12 molar equivalents of linker-payload (5 mg / ml, in DMA) dropwise to the fully reduced antibody while maintaining the DMA concentration at 10% (v / v). Keep the reaction on the oscillator at 6°C for 2 hours (linker-payload: antibody = 12:1). Detect the conversion rate by HIC-HLPC, and perform purification immediately when the conversion is complete. Transfer the reaction mixture to an ultrafiltration tube (MWCO 30 kd), and add PBS (pH 7.4) to 450 μl. Centrifuge the sample at 10,000 rpm for 5 minutes, leaving approximately 200 μl of solution, and supplement to the original volume with PBS. Discard the flow-through liquid. Repeat the washing step 10 times. Finally, add 200 μl of PBS, rinse the filter membrane by pipetting up and down, and combine the remaining solution to obtain the ADC product. If the final concentration is less than expected, concentrate to the desired concentration using an ultrafiltration membrane (MWCO 30 kd). Determine the ADC purity by SEC-HPLC method, the average DAR value by HIC-HPLC method, and detect the free linker-payload content by RP-HPLC method.

[0946] General synthesis method C

[0947] Place 5.45 mg / ml antibody (e.g., the corresponding bispecific antibody prepared according to the above antibody preparation examples) in 20 mM His-hac, 150 mM NaCl, pH 5.5 buffer in an Eppendorf tube. Add 6 molar equivalents of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, 5 mM concentration) to the antibody buffer (TCEP:antibody = 6:1). Place the Eppendorf tube containing the reaction mixture on an oscillator (x500 rpm) and react at 37 °C for 2 hours. Add another 6 molar equivalents of TCEP (5 mM) to the mixture. Place the reaction mixture on an oscillator (500 rpm) and shake at 37 °C for another 2 hours. Then, ultrafilter (MWCO 30 kd) to remove TECP and replenish to the original volume with 20 mM His-hac buffer. Add 8 molar equivalents of linker-payload (i.e., linker-payload) (5 mg / ml, in DMA) dropwise to the fully reduced antibody while keeping the DMA concentration below 20% (v / v). Keep the reaction on an oscillator at room temperature for 2 hours (linker-payload:antibody = 8:1). Detect the conversion rate by HIC-HLPC and purify when the conversion is complete. Transfer the reaction mixture to an ultrafiltration tube (MWCO 30 kd), add 10 mM His-hac (pH 6) containing 10% DMA to a total of 450 μl. Centrifuge the sample at 10,000 rpm for 5 minutes, leaving approximately 200 μl of solution, and replenish to 450 μl with His-hac. Discard the flow-through. Repeat the washing step 10 times. Then add 10 mM His-hac (pH 6) to a total of 450 μl, repeat the washing step 5 times, finally transfer the remaining solution, and add 200 μl of His-hac, and rinse the filter membrane by pipetting up and down. Combine the transferred solution and the filter membrane washings to obtain the ADC product.

[0948] Determine the ADC purity by SEC-HPLC method, the average DAR value by HIC-HPLC method, and detect the free linker-payload content by RP-HPLC method.

[0949] General methods and / or parameters for determining or detecting ADC

[0950] Size exclusion chromatography (SEC) method (for ADC purity detection).

[0951] SEC-HPLC method parameters

[0952]

[0953] Reverse-phase HPLC (RP HPLC) method (for free drug detection)

[0954] RP HPLC method parameters

[0955]

[0956] Perform elution according to the following table

[0957] Time (Min) Mobile Phase A (%) Mobile Phase B (%) 0.0 75.0 25.0 2.0 75.0 25.0 10.0 5.0 95.0 12.0 5.0 95.0 14.0 5.0 95.0 16.0 80.0 20.0 18.0 80.0 20.0

[0958] HIC-HPLC method (for free antibody and DAR distribution detection) HIC-HPLC conditions.

[0959]

[0960] Perform elution according to the following table

[0961] Time (Min) Mobile Phase A (%) Mobile Phase B (%) 0.0 100.0 0.0 7.0 100.0 0.0 23.0 0.0 100.0 26.0 0.0 100.0 26.5 100.0 0.0 35.0 100.0 0.0

[0962] Example 2.1 Preparation of V-hu21-VA-Exd

[0963]

[0964] Where Ab is the bispecific antibody V-hu21; p is mainly 4, and the obtained V-hu21-VA-Exd has an average DAR of 4.0 when measured.

[0965] According to the general synthesis method A, using the bispecific antibody V-hu21 prepared according to the method of the above example and the linker-payload Mal-PEG8-Val-Ala-PAB-Exatecan (CAS No.: 2679821-39-5; catalog number HY-147271), V-hu21-VA-Exd was prepared.

[0966] Example 2.2 Preparation of V-hu21-GGFG-Dxd

[0967]

[0968] Where Ab is the bispecific antibody V-hu21; p is mainly 4, and the obtained V-hu21-GGFG-Dxd has an average DAR of 4.0 when measured.

[0969] According to the general synthesis method A, using the bispecific antibody V-hu21 prepared according to the method of the above example and the linker-payload Deruxtecan (CAS No.: 1599440-13-7; MCE, HY-13631E), V-hu21-GGFG-Dxd was prepared. The product purity is 95%.

[0970] Preparation of Example 2.3V-15-GGFG-Dxd

[0971]

[0972] Where Ab is the bispecific antibody V-15; p is mainly 4, and the obtained V-15-GGFG-Dxd has an average DAR of 4.0 as measured.

[0973] According to the general synthesis method A, using the bispecific antibody V-15 prepared by the method of the above example and the linker-payload Deruxtecan (CAS No.: 1599440-13-7; MCE, HY-13631E), V-15-GGFG-Dxd was prepared. The product has an average DAR value of 4.0, a yield of 60%, and an SEC-HPLC purity of 95%.

[0974] Preparation of Example 2.4V-hu23-Glu-Exd

[0975]

[0976] Where Ab is the bispecific antibody V-hu23; p is mainly 4, and the obtained V-hu23-Glu-Exd has an average DAR of 4.0 as measured.

[0977] According to the general synthesis method C, using the bispecific antibody V-hu23 prepared by the method of the above example and the linker-payload Mal-Gly-EXD-D-glucuronic acid (CAS No.: 2763252-25-9; MCE, HY-153179), V-hu23-Glu-Exd was prepared. The product has an average DAR value of 4.0 and a yield of 86%.

[0978] Preparation of Example 2.5 Other ADCs

[0979] According to the method described in Example 2.1, but using different antibodies (such as V-15, V-hu23 or V-hu24) instead of V-hu21, V-15-VA-Exd, V-hu23-VA-Exd or V-hu24-VA-Exd were prepared respectively.

[0980] According to the method described in Example 2.4, but using a different antibody (V-hu24) instead of V-hu23, V-hu24-Glu-Exd was prepared.

[0981] Some relevant information of the ADC prepared in the embodiments of the present application and the control ADC (commercially available Dato-Dxd) is provided in the following table (Table 28). Among them, Dato-Dxd can also be prepared according to the aforementioned general synthesis method B.

[0982] Table 28 Basic Information of ADC

[0983]

[0984] In the present disclosure, when it comes to the linker-payload, Glu is also referred to as Gluc. For example, Glu-Exd is also referred to as Gluc-Exd.

[0985] Example 3. In Vitro Experiments of ADC

[0986] Example 3.1 In Vitro Binding Experiment of ADC

[0987] Method:

[0988] 1) The target cells NCI-N87 cells (TCHu130, Cell Bank of the Chinese Academy of Sciences), or MDA-MB-468 (TCHu136, Cell Bank of the Chinese Academy of Sciences) are plated at 1-3×10 5 cells per well, and the previously prepared antibody or ADC to be tested (100 nM, 3-fold dilution) is added and incubated at 2-8 °C for about 1 h

[0989] 2) Centrifuge at 4 °C to remove the supernatant, resuspend the cells with 200 μl / well of FACS Buffer (1% BSA in PBS), centrifuge at 4 °C, and resuspend the cells again with 200 μl / well of FACS Buffer, then centrifuge at 4 °C

[0990] 3) Resuspend the cells with 100 μl / well of the flow cytometry secondary antibody Anti-Hu IgG Fc-PE (ThermoFisher Scientific) diluted to the target dilution factor, and incubate at 2-8 °C in the dark for about 1 h

[0991] 4) After washing 2 times with 200 μl / well of FACS Buffer, resuspend the cells with 100 μl / well of FACS Buffer;

[0992] 5) Measure the MFI of the cells using a flow cytometer (Beckman Coulter).

[0993] The results are shown in Figure 14 A-E. Each ADC molecule showed a cell binding ability similar to its corresponding naked antibody.

[0994] Example 3.2 In Vitro Killing Experiment

[0995] Method 1

[0996] 1) Seed the target cells at 3000 - 6000 MDA - MB - 468 tumor cells (TCHu136, Cell Bank of the Chinese Academy of Sciences) or MKN45 tumor cells (CBP60488, Nanjing Kebai Biotechnology Co., Ltd.) per well, 100 μL per well, and incubate overnight in a 37°C, 5% CO₂ incubator; Dilute the test samples to an appropriate concentration (starting concentration 400 nM) with complete medium, add 100 μL per well to the seeded 96 - well culture plate, and incubate in a 37°C, 5% CO₂ incubator for 3 - 6 days. 2) Add 20 μL per well of CCK - 8 and incubate in a 37°C, 5% CO₂ incubator for 1 - 5 h. 3) Read the OD₄₅₀ value.

[0997] V - hu21 - VA - Exd and EV (i.e., Enfortumab Vedotin) on Nectin4 + Trop2 + MDA - MB - 468 cells have cytotoxic effects, but have no cytotoxicity on MNK45 cells (Nectin4 low Trop2 - ). The payloads Exatecan and MMAE can effectively kill these two cell lines. The results are shown in Figure 15A and B. V - hu21 - VA - Exd, V - hu23 - VA - Exd and V - hu24 - VA - Exd have cytotoxic effects on MDA - MB - 468 cells ( Figure 15C ). V - hu23 - Glu - Exd and V - hu24 - Glu - Exd have cytotoxic effects on MDA - MB - 468 cells ( Figure 15D ).

[0998] Method 2

[0999] Target cells NCI-H292 (human mucoepidermoid carcinoma lung, CBP60187, Nanjing Kebai Biotechnology Co., Ltd.) were plated at 5000 cells / well, 100 μL / well, and incubated overnight in a 37 °C, 5% CO2 incubator. rProtein G-MMAE (Levena; LEV-GME-100) was diluted to 200 nM with complete medium, and the target antibodies (including the V-hu23-Fc antibody, Parental anti-Nectin4 antibody, and Isotype antibody involved in Table 22) were diluted to 200 nM with complete medium. The target antibody and rProtein G-MMAE were mixed at a volume ratio of 1:1 (molar concentration ratio of 1:1) and incubated at room temperature for about 1 h. After the target antibody and rProtein G-MMAE mixture was diluted 1:2.5 with complete medium, it was serially diluted, and 100 μL / well was added to the pre-plated 96-well culture plate and incubated in a 37 °C, 5% CO2 incubator for 3 - 6 days; 20 μL / well of CCK-8 (Cell Counting Kit-8) was added and incubated in a 37 °C, 5% CO2 incubator for 1 - 5 h; the OD 450 was read.

[1000] From Figure 15E It can be seen that the killing ability of V-hu23-Protein G-MMAE on the Trop2 and Nectin4 double-positive tumor cell line NCI-H292 is superior to that of the anti-Nectin4 parental control molecule, demonstrating the synergistic killing effect of anti-Trop2 and anti-Nectin4 on double-positive tumor cells.

[1001] Example 3.3 Bystander effect

[1002] Experiment 1: ADC at a specified concentration was added to target antigen-positive (Ag+) cells (MDA-MB-468) or the culture medium and cultured for 4 days. 100 μL of the culture supernatant was transferred to target antigen-negative (Ag-) cells (MKN45) in 100 μL of culture medium. The Ag- cells were cultured for another 3 - 4 days in a 37 °C, 5% CO2 incubator. The viability of the Ag- cells was measured by adding CCK8.

[1003] For target antigen-negative tumor cells (MKN45), V-hu21-VA-Exd has a stronger bystander effect than EV (i.e., Enfortumab Vedotin). At 2.5 nM - 20 nM, the killing ratio is 60 - 80% VS <10%. The results are shown in Figure 16AIn the figure, "EV in medium" and "V-hu21-VA-Exd in medium" respectively represent the supernatants from the culture media supplemented with the corresponding ADC drugs; "EV cultured w / MDA-MB-468" and "V-hu21-VA-Exd cultured w / MDA-MB-468" respectively represent the supernatants from the culture media of MDA-MB-468 cells supplemented with the corresponding ADC drugs.

[1004] Experiment 2: Ag+ cells T47D (TCHu 87, Cell Bank of the Committee for Type Culture Collection, Chinese Academy of Sciences) and Ag- cells MKN45 were stained with fluorescent dyes CFSE (Invitrogen, 65-0850-84) and CellTrace Violet (Thremo, C34557 A) respectively. The positive cells T47D were plated at 75,000 cells / 250 μL / well, and the negative cells MKN45 were plated at 25,000 cells / 250 μL / well. The two types of cells were co-cultured and incubated overnight in a 37°C, 5% CO2 incubator. The test samples were diluted to 4 nM and 2 nM with complete medium and added to the pre-plated 24-well culture plates at 500 μL / well, and then incubated in a 37°C, 5% CO2 incubator for 3 days. After that, all the cells were collected into deep-well plates, counting beads (invitrogen, C36995) were added at 3 μL / well, and centrifuged at 400 g for 5 minutes. Then the cells were washed twice with DPBS. The collected cells were stained with Live / Dead dye (Thremo, L34976). After staining, the cells were washed twice with 2% FBS-DPBS buffer, and then resuspended. The cells were detected on a flow cytometer (Beckman, CytoFLEX), and the fluorescence signals in the V450 / B525 / R780 channels were recorded.

[1005] The results are as Figure 16B shown. The toxic small molecules released by enzymatic cleavage after the endocytosis of V-hu23-Glu-Exd by the positive cells T47D showed an obvious bystander killing effect. The bystander killing effect of V-hu23-Glu-Exd was significantly better than that of the control enfortumab vedotin (EV).

[1006] Example 4. In Vivo Effect Experiment of ADC

[1007] Unless otherwise specified and not conflicting with the context, the abbreviations used have the following meanings

[1008]

[1009]

[1010] Example 4.1 Antitumor Effect of ADCs in a Subcutaneous Xenograft Model of Human Breast Cancer MDA-MB-468 in Mice

[1011] This experiment was used to evaluate the pharmacodynamic effects of the test articles (including the ADCs of the present invention) in a subcutaneous xenograft model of human breast cancer MDA-MB-468 in NCG mice.

[1012] Experiment A

[1013] Formulation Information of Test Articles

[1014]

[1015] Model Establishment and Grouping

[1016] Model establishment: Female NCG mice (Balb / c nu / nu, from Jiangsu Jicui Biotechnology Co., Ltd.) at 7 - 8 weeks of age were used. After 1 week of adaptation, a cell suspension of MDA-MB-468 (TCHu136, from the Cell Bank of the Chinese Academy of Sciences) resuspended in PBS (1×10 7 cells / animal) was injected into the right scapular region. When the tumor volume grew to 100 - 200 cm 3 , the mice were randomly divided into 5 groups (n = 4) based on the average tumor volume. The day of grouping was defined as D0, and the test articles were administered intravenously on D1 once a week for a total of 3 times as expected.

[1017] Administration volume: Adjusted according to the body weight of the mice (mouse administration volume = 5 μL / g or 10 μL / g × mouse body weight (g))

[1018] Data collection: After the start of administration, the body weight of the mice was measured twice a week, the tumor volume was measured twice a week, and the animals were observed twice a day.

[1019] Endpoint of the experiment:

[1020] The endpoint was determined based on the tumor volume (1500 - 2000 cm 3 ) or the animal status. At the endpoint, all surviving animals were euthanized and the tumors were collected. The tumors were photographed, weighed, and then processed for further analysis.

[1021] Endpoint analysis

[1022] At the end of the experiment, the following indicators were analyzed:

[1023] Change in tumor volume (TGI TV )

[1024] Tumor weight (TGI TW )

[1025] Change in body weight

[1026] TGI TV Calculation formula:

[1027] TGI TV = {1 - [(V t - V0) / (C t - C0)]} × 100%

[1028] V t : The average tumor volume of the mice in the test article administration group on the t-th day;

[1029] V0: The average tumor volume of the mice in the test article administration group on the 0-th day;

[1030] C t : The average tumor volume of the mice in the vehicle group on the t-th day;

[1031] C0: The average tumor volume of the mice in the vehicle group on the 0-th day.

[1032] Statistical analysis

[1033] Analysis, processing, and reporting. Quantitative indicators are described as mean ± standard error (Mean ± SEM / SD). Quantitative indicators are analyzed using one-way ANOVA or two-way ANOVA, etc. For comparison between groups, a t-test is used, and p < 0.05 is considered statistically significant. Both statistical significance and biological significance are considered during the result analysis. The graphing software is GraphPad Prism 8.

[1034] The V-hu21-GGFG-Dxd 5mpk group showed efficacy comparable to that of the control drugs EV (EV- (Enfortumab vedotin)), SG (SG-

[1035] (Sacituzumab govitecan)) + EV combination administration and Dato-Dxd. The experimental results are shown in Figure 17 . It should be understood that unless otherwise specified, the control drugs EV and SG in other experiments of this disclosure are the same products as EV (EV-Padcev TM (enfortumab vedotin); manufacturer: Astellas / Seagen; batch number: 3PA0031) and SG (Trodelvy TM (Sacituzumab govitecan); manufacturer: Gilead; batch number: S22B002D) in this experiment.

[1036] Experiment B

[1037] Model construction and grouping

[1038] Model construction: Female NCG mice at 7 - 8 weeks of age (Balb / c nu / nu, from Jiangsu Jicui Biotechnology Co., Ltd.). After 1 week of adaptation, a suspension of MDA-MB-468 cells in PBS (1×10 7 cells / animal) was injected into the right scapular region. When the tumor volume grew to 100 - 200 cm 3 , the mice were randomly divided into 9 groups (n = 4) according to the average tumor volume. The day of grouping was defined as D0, and the test article was administered intravenously on D1, as a single dose. The dosing doses for each group are shown in Figure 18 .

[1039] Dosing volume: Adjusted according to the body weight of the mice (mouse dosing volume = 5 μL / g or 10 μL / g × mouse body weight (g))

[1040] Data collection: After starting the dosing, the body weight of the mice was measured 2 times a week, the tumor volume was measured 2 times a week, and the animals were observed 2 times a day.

[1041] Data processing was carried out with reference to Experiment A, and the experimental results are shown in Figure 18 .

[1042] At the same dose, compared with the control drug SG, V-hu21-VA-Exd, V-hu23-VA-Exd, and V-hu24-VA-Exd all showed better pharmacodynamic effects.

[1043] Example 4.2 Antitumor effect of V-ADCs in the HT1376 model

[1044] Model construction: Female NCG mice at 7 - 8 weeks of age (Balb / c nu / nu, from Jiangsu Jicui Biotechnology Co., Ltd.). After 1 week of adaptation, a suspension of HT-1376 (CBP60310, Nanjing Kebai Biopharmaceutical Technology Co., Ltd.) cells in PBS (5×10 6 cells / animal) was injected into the right scapular region. When the tumor volume grew to 100 - 200 mm 3 , the mice were randomly divided into 5 groups (n = 4) according to the average tumor volume. The day of grouping was defined as D0, and the test article was administered intravenously on D1, once a week for a total of 4 times. The dosing doses for each group are shown in Figure 19 .

[1045] Dosing volume: Adjusted according to the body weight of the mice (mouse dosing volume = 5 μL / g or 10 μL / g × mouse body weight (g))

[1046] Data collection: After starting the dosing, the body weight of the mice was measured 2 times a week, the tumor volume was measured 2 times a week, and the animals were observed 2 times a day.

[1047] Data processing was carried out with reference to Example 4.1, and the experimental results are shown inFigure 19 。

[1048] Both V-hu21-GGFG-Dxd and V-hu21-VA-Exd showed better effects than the control drugs EV (EV- (Enfortumab vedotin)), SG (SG- (Sacituzumab govitecan)) and the combined administration of EV.

[1049] Example 4.3 Antitumor effect of V-ADCs in the HT1197 model

[1050] Construction of the HT1197 model: Female BALB / c nu / nu mice at 7-8 weeks of age (from Shanghai Lingchang Biotechnology Co., Ltd.). After 3-7 days of adaptation, HT-1197 (CBP60673, Nanjing Kebai Biopharmaceutical Technology Co., Ltd.) PBS accompanied by a cell suspension resuspended in 30% Matrigel (1×10^7 cells / animal) was injected into the right scapular region. When the tumor volume grew to 100-200 mm^3, the mice were randomly divided into 6 groups (n = 4) according to the average tumor volume. The day of grouping was defined as D0, and the test article was administered intravenously on D0, with a total of 1 administration. The dosing doses for each group are as Figure 20 shown. For drug administration and data collection and processing, refer to Example 4.1. The experimental results showed that the cell line HT1197 used in this model expressed low levels of Nectin4 antigen. Therefore, this model also showed resistance to EV. The efficacy of V-hu23-Glu-Exd at 4 mpk was equivalent to that of Dato-Dxd at 8 mpk, and both were superior to EV at 3 mpk (MTD dose) and SG at 8 mpk.

[1051] Example 4.4 Antitumor effect of V-ADCs in the MDA-MB-453 model

[1052] Construction of the MDA-MB-453 model: Female NSG female mice at 7-8 weeks of age (from Shanghai Model Organisms Center, Inc.). After 3-7 days of adaptation, MDA-MB-453 (CBP60386, Nanjing Kebai Biopharmaceutical Technology Co., Ltd.) PBS accompanied by a cell suspension resuspended in 30% Matrigel (1×10^7 cells / animal) was injected into the right scapular region. When the tumor volume grew to 110-210 mm^3, the mice were randomly divided into 8 groups (n = 4) according to the average tumor volume. The day of grouping was defined as D0, and the test article was administered intravenously on D0, with a total of 1 administration. The dosing doses for each group are shown in Figure 21。Data collection and processing refer to Example 4.1. The experimental results show that at a dose of 4 mpk, the pharmacodynamic effects of V-hu23-VA-Exd, V-hu23-Glu-Exd, and V-hu24-Glu-Exd are all superior to those of EV at 3 mpk (MTD dose), SG at 8 mpk, and EV+SG (1.25+8 mpk, clinical use dose).

[1053] Example 4.5 Antitumor effect of V-ADCs in the LD1-0023-200615 model of human head and neck cancer xenografts in vivo

[1054] Model construction: Female NU / NU mice, 7-8 weeks old (Beijing Vital River Laboratory Animal Technology Co., Ltd.). After 1 week of adaptation, the established human tumor tissue (Shanghai Lide Biotechnology Co., Ltd.) was inoculated subcutaneously into immunodeficient mice and continuously observed until tumors formed and the tumor volume reached approximately 500-800 mm 3 At this time, the tumor tissue was dissected and evenly cut into tissue pieces approximately 3 mm×3 mm×3 mm. Then, the tissue pieces were inoculated subcutaneously into mice, and the growth of the tumors was observed. When the average tumor volume reached approximately 100-200 mm 3 (average value 120±30 mm 3 ), the tumor-bearing mice were randomly divided into 2 groups (n = 5). The day of grouping was defined as D0, and the test article was administered intravenously on D0, QWx3. The dosing doses for each group are shown in Figure 22 .

[1055] Data processing was carried out with reference to Example 4.1. The experimental results are shown in Figure 22 . At a dose of 8 mg / kg, on the 28th day after dosing, the pharmacodynamic effect of V-hu23-VA-Exd could reach TGI 94.56%, and the tumor suppression effect could be maintained for at least 49 days.

[1056] Example 4.6 Antitumor effect of V-ADCs in the LD1-0014-200628 model of human endometrial cancer xenografts in vivo

[1057] Model construction: Female NU / NU mice, 7-8 weeks old (Beijing Vital River Laboratory Animal Technology Co., Ltd.). After 1 week of adaptation, the established human tumor tissue (Shanghai Lide Biotechnology Co., Ltd.) was inoculated subcutaneously into immunodeficient mice and continuously observed until tumors formed and the tumor volume reached approximately 500-800 mm 3 At this time, the tumor tissue was dissected and evenly cut into tissue pieces approximately 3 mm×3 mm×3 mm. Then, the tissue pieces were inoculated subcutaneously into mice, and the growth of the tumors was observed. When the average tumor volume reached approximately 100-200 mm 3 (average value 120±30 mm 3) The tumor-bearing mice were randomly divided into 2 groups (n = 5). The day of grouping was defined as D0. The test article was administered intravenously on D0 as a single dose. The dosing doses for each group were as Figure 23 .

[1058] Data processing was performed with reference to Example 4.1. The experimental results are shown in Figure 23 . At a dose of 8 mg / kg, on the 28th day after dosing, the efficacy of V-hu23-VA-Exd could reach TGI 130.47%.

[1059] Example 4.7 Anti-tumor effect of V-ADCs in the human-derived cervical cancer xenograft LD1-0010-200615 model

[1060] Model construction: Female NU / NU mice at 7-8 weeks of age (Beijing Vital River Laboratory Animal Technology Co., Ltd.). After 1 week of adaptation, the established human tumor tissue (Shanghai Leady Biotechnology Co., Ltd.) was inoculated subcutaneously into immunodeficient mice. Continuous observation was carried out until tumor formation and the tumor volume reached approximately 500-800 mm 3 . Then, the tumor tissue was dissected and evenly cut into tissue pieces of approximately 3 mm × 3 mm × 3 mm. After that, the tumor tissue pieces were inoculated subcutaneously into mice, and the growth of the tumor was observed. When the average tumor volume reached approximately 100-200 mm 3 (average value 120 ± 30 mm 3 ), the tumor-bearing mice were randomly divided into 2 groups (n = 5). The day of grouping was defined as D0. The test article was administered intravenously on D0 as a single dose. The dosing doses for each group were as Figure 24 .

[1061] Data processing was performed with reference to Example 4.1. The experimental results are shown in Figure 24 . At a dose of 8 mg / kg, on the 28th day after dosing, the efficacy of V-hu23-Glu-Exd could reach TGI 94.37%.

[1062] Example 4.8 Anti-tumor effect of V-ADCs in the murine MC38-hNectin4 model

[1063] Model construction: Female BALB / c nude mice at 7-8 weeks of age (Beijing Vital River Laboratory Animal Technology Co., Ltd.). After 1 week of adaptation, MC38-hNectin4 (MC38 cells stably expressing human Nectin4 recombinantly, CBPG0035, Nanjing Kebai Biopharmaceutical Technology Co., Ltd.) PBS resuspended cell suspension (1×10 6 cells / animal) was injected into the right scapular region. When the tumor volume grew to 100-250 mm 3 , grouping was performed according to the average tumor volume.

[1064] The formulation information of the test article is as follows:

[1065] Drug Preparation Method Dosing Concentration (mg / mL) Vehicle 10 mM glutamate, 8% (w / v) sucrose, pH 5.0 -- V-hu23-Glu-Exd Dilute to the target concentration with the vehicle 0.1, 0.3 and 0.8 EV Dilute to the target concentration with 10 mM His buffer (pH 6.2) 0.3 Isotype-control ADC Dilute to the target concentration with the vehicle 0.3 V-hu23-Fc Dilute to the target concentration with the vehicle 0.8 Exatecan 10% DMSO / 5% Tween80 / 85% dd water 0.03

[1066] Administration volume: Adjusted according to the body weight of the mice (administration volume for mice = 5 or 10 μL / g × body weight of mice (g)). Data collection: After starting the administration, weigh the mice 2 - 3 times a week, measure the tumor volume 2 - 3 times a week, and observe the animals 2 times a day.

[1067] The tumor-bearing mice were randomly divided into 8 groups (n = 5). Define the day of grouping as D0. Administer the test article intravenously on D0 and D4, for a total of 2 administrations. The administration doses for each group are shown in Figure 25 .

[1068] Refer to Example 4.1 for data processing. The experimental results are shown in Figure 25 . After administration, all animals in the dose groups tolerated well; MC38-hNectin4 cells express high levels of the multidrug resistance protein MDR1 and are resistant to EV. Therefore, EV did not show obvious anti-tumor effects. At the same time, neither isotype control ADC, the small molecule exatecan (product number HY-13631, MedChemExpress LLC. Equivalent amount of exatecan contained in V-hu23-Glu-Exd 8mpk) nor the naked anti-V-hu23-Fc showed obvious anti-tumor effects. However, all high, medium, and low dose groups of the ADC V-hu23-Glu-Exd of the present invention had significant tumor inhibitory effects, and showed a dose correlation. Under the conditions of this experiment, V-hu23-Glu-Exd was significantly more potent than EV.

[1069] Example 5 Conduct in vivo imaging to study the biodistribution of the fluorescently labeled bispecific antibody

[1070] Use the IVIS Lumina Series III system to conduct fluorescence in vivo imaging experiments to evaluate the differences in biodistribution and tumor targeting ability between the bispecific antibody of the present invention and conventional monoclonal antibodies. These experiments were carried out in Balb / c nu / nu nude mice carrying a subcutaneously implanted human urothelial tumor cell line HT1376 on one side. During the experiment, female mice (25 - 32 g) were 6 to 8 weeks old, and the tumor volume was about 500 mm 3 or so.

[1071] Preparation of test article: The buffer of the test drug was replaced with PBS at pH = 8.5 using a centrifugal filter (Millipore). Then, the labeling reaction was carried out by immediately mixing Sulfo-Cy7 NHS Ester (Duofluor, 10 mg / mL) with the antibodies (V-15 and Enfortumab) at a molar ratio of 6:1 (dye / protein), and reacting in the dark at 20 - 25 °C for 5 hours. After labeling, the conjugate was purified using a centrifugal filter (3K) to remove unbound free dye. After the conjugation reaction, the dye / protein molar ratios of V-15-Cy7 and Enfortumab-Cy7 were 1.2 and 2.4, respectively. The in vitro binding affinity was tested by FACS, and it was evaluated that the binding did not affect the protein properties after the drug was incubated with the cells for 1 hour. Then, 1 nmol of the dye-labeled protein was injected into tumor-bearing mice at a concentration based on the dye concentration. At time points of 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 24 hours, 48 hours, and 72 hours, in vivo imaging was performed using an IVIS Lumina III system. The data was analyzed using in vivo imaging software (version: 4.7.4).

[1072] The results are as Figure 27 shown in A. As time extended, the distribution of V-15-Cy7 in the tumor tissues of mice accumulated over time and was more than that of Enfortumab-Cy7 in the tumor tissues of mice. Figure 27 B shows that 72 hours after injecting the fluorescent dye-labeled protein into tumor-bearing mice, the drug was distributed into multiple tissues with different tissue penetration abilities. The tumor-to-muscle tissue accumulation ratios of V-15-Cy7 and Enfortumab-Cy7 at 72 hours were 233 and 162, respectively, indicating that V-15-Cy7 had better tumor accumulation. However, in other normal tissues (except kidney tissue), the accumulation of V-15-Cy7 was slightly lower than that of Enfortumab-Cy7, meaning that the molecule of the present invention had better tumor targeting.

[1073] Overview of the Sequence Listing

[1074]

[1075]

[1076]

[1077]

[1078]

[1079]

[1080]

[1081]

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[1085]

Claims

1. A multispecific antibody that binds Trop2 and Nectin4, wherein the antibody comprises at least one antigen-binding domain that specifically binds Trop2 and at least one antigen-binding domain that specifically binds Nectin4.

2. The multispecific antibody of claim 1, wherein the antibody has one or more of the following characteristics: (a) The Trop2 and Nectin4 antigen-binding domains are VHH domains, respectively; (b) The antigen-binding affinities K D values of the Trop2 and Nectin4 antigen-binding domains are respectively 50x10 -8 M to 0.5x10 -8 M; (c) The valence ratio of the Trop2 antigen-binding domain to the Nectin4 antigen-binding domain in the antibody is 1:1 or 1:2; and (d) The antibody is a trivalent or tetravalent bispecific antibody; and Preferably, the antibody further has one or more of the following characteristics: (e) synergistic binding; (f) synergistic endocytosis; and (g) improved tumor cell targeting distribution on target tumor cells expressing human Nectin4 and Trop2, compared to Nectin4 and Trop2 single-target drugs.

3. The multispecific antibody according to any one of claims 1-2, wherein: The antigen-binding domain that specifically binds Trop2 is a VHH domain, and wherein the VHH domain comprises the CDR1, CDR2, and CDR3 sequences selected from the amino acid sequences of SEQ ID NOs: 5, 9, 13, 29, 30, 98, and 99; More preferably, the CDR1, CDR2, and CDR3 sequences: (i) respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 6, 7, and 8; (ii) respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 10, 11, and 12; (iii) respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 14, 15, and 16; or (iv) respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 31, 32, and 33; More preferably, the VHH domain comprises the amino acid sequences selected from SEQ ID NOs: 5, 9, 13, 29, 30, 98, and 99, or an amino acid sequence having at least 85%, 90%, 95%, or 99% identity to the amino acid sequence, or having an addition, deletion, and / or substitution of one or more (preferably 1-10, more preferably 1-5) amino acids, Most preferably, the VHH domain comprises the amino acid sequences selected from SEQ ID NOs: 5, 9, 13, 29, 30, 98, and 99, or consists of the amino acid sequences selected from SEQ ID NOs: 5, 9, 13, 29, 30, 98, and 99.

4. The multispecific antibody according to any one of claims 1-3, wherein: The antigen-binding domain that specifically binds Nectin4 is a VHH domain, and wherein the VHH domain comprises the CDR1, CDR2, and CDR3 sequences selected from the amino acid sequences of SEQ ID NOs: 17, 21, 25, 34, 35, 38, and 100-106; More preferably, the CDR1, CDR2, and CDR3 sequences: (i) Comprising or consisting of the amino acid sequences of SEQ ID NOs: 18, 19, and 20, respectively; (ii) Comprising or consisting of the amino acid sequences of SEQ ID NOs: 22, 23, and 24, respectively; (iii) Comprising or consisting of the amino acid sequences of SEQ ID NOs: 26, 27, and 28, respectively; (iv) Comprising or consisting of the amino acid sequences of SEQ ID NOs: 18, 36, and 20, respectively; or (v) Comprising or consisting of the amino acid sequences of SEQ ID NOs: 18, 37, and 20, respectively; More preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35, 38, and 100 - 106, or an amino acid sequence having at least 85%, 90%, 95%, or 99% identity to the amino acid sequence, or having an addition, deletion, and / or substitution of one or more (preferably 1 - 10, more preferably 1 - 5) amino acids; Most preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35, 38, and 100 - 106, or consists of an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35, 38, and 100 - 106.

5. The multispecific antibody of any one of claims 1 - 4, wherein the antibody further comprises: (a) A half - life extending domain, preferably, the half - life extending domain is selected from serum albumin - binding peptides and immunoglobulin Fc regions; and / or (b) Peptide linker, preferably, the peptide linker has a length of 5-15 amino acids, more preferably, the peptide linker comprises the amino acid sequence (G4S) n , where n = 1, 2 or 3.

6. The multispecific antibody of any one of claims 1 - 5, wherein the antibody is a single - chain or double - chain antibody comprising at least one (preferably 1 or 2) Nectin4 - binding domain and at least one (preferably 1 or 2) Trop2 - binding domain.

7. A multispecific antibody according to any one of claims 1-6, wherein the antibody comprises two identical polypeptide chains, and wherein the polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE-(VHH A ) n3 -(VHH B ) n4， Wherein n1, n2, n3, and n4 are independently selected from the integers 0, 1, or 2, and preferably n3 and n4 are 0, and both n1 and n2 are 1; Wherein VHH A and VHH B respectively represent the VHH domains that bind to antigen A and antigen B, where A and B are different from each other and are independently selected from Nectin4 and Trop2; wherein HLE represents the immunoglobulin Fc region as a half-life extension domain; and the symbol "-" represents connection by a peptide linker or direct connection.

8. The multispecific antibody of claim 7, wherein the polypeptide chain comprises, from the N-terminus to the C-terminus: VHH A -VHH B -HLE, and wherein VHH A represents the Nectin4 binding domain and VHH B represents the Trop2 binding domain.

9. The multispecific antibody of any one of claims 1 - 6, wherein the antibody comprises two different polypeptide chains, wherein: The first polypeptide chain contains, from the N-terminus to the C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE-(VHH A ) n3 -(VHH B ) n4， The second polypeptide chain contains, from the N-terminus to the C-terminus: (VHH B ) m1 -(VHH A ) m2 -HLE-(VHH B ) m3 -(VHH A ) m4， Wherein n1, n2, n3, n4, m1, m2, m3, and m4 are independently selected from the integers 0, 1, or 2, and preferably wherein n3, n4, m3, and m4 are 0, and n1 + m2 = 1 or 2, n2 + m1 = 1 or 2, more preferably m1 = 0 and m2 = 1, or m2 = 0 and m1 = 1; Wherein VHH A and VHH B respectively represent VHH domains that bind to antigen A and antigen B, where A and B are different from each other and are independently selected from Nectin4 and Trop2; wherein HLE represents an immunoglobulin Fc region as a half-life extension domain, and preferably contains a knob-into-hole mutation; wherein the symbol "-" represents connection by a peptide linker or direct connection.

10. The multispecific antibody of claim 9, wherein: The first polypeptide chain contains from the N-terminus to the C-terminus: VHH A - VHH B - HLE ， The second polypeptide chain contains from the N-terminus to the C-terminus: VHH A -HLE ； and wherein the VHH A represents a Nectin4 binding domain and the VHH B represents a Trop2 binding domain.

11. The multispecific antibody of any one of claims 7 - 10, wherein the immunoglobulin Fc region is an Fc region from IgG, for example, an Fc region from human IgG1 or IgG4, preferably the Fc region comprises a mutation that reduces or eliminates the binding of the Fc region to FcγR.

12. A multispecific antibody according to any one of claims 1-6, wherein said antibody comprises a single polypeptide chain, and wherein said polypeptide chain comprises, from the N-terminus to the C-terminus: (VHH A ) n1 -(VHH B ) n2 -(HLE) n3 -(VHH A ) n4 -(VHH B ) n5 -(HLE) n6， wherein n1, n2, n3, n4, n5 and n6 are each independently selected from the integers 0, 1 or 2, and preferably n1 + n4 = 1 or 2, n2 + n5 = 1 or 2, and n3 + n6 = 0 or 1; Wherein VHH A and VHH B respectively represent VHH domains that bind to antigen A and antigen B, where A and B are different from each other and are independently selected from Nectin4 and Trop2; wherein HLE represents a serum albumin-binding peptide as a half-life extension domain; wherein the symbol "-" represents connection by a peptide linker or direct connection, Preferably, wherein said polypeptide chain comprises, from the N-terminus to the C-terminus: (i)VHH A -VHH B , (ii)VHH A -VHH B -HLE, (iii)VHH B -VHH A -VHH B , (iv)VHH A -VHH B -VHH B , (v)VHH A -HLE-VHH B -VHH B ,or (vi)VHH A -VHH A -HLE-VHH B -VHH B , wherein preferably A represents Trop2 and B represents Nectin4, More preferably, said HLE represents an anti-serum albumin VHH domain, optionally selected from the amino acid sequences of SEQ ID NOs: 39 and 40, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity thereto, or having an addition, deletion and / or substitution of one or more (preferably 1-10, more preferably 1-5) amino acids.

13. The multispecific antibody according to any one of claims 1-12, wherein: (a) the antigen-binding domain specifically binding Nectin4 comprises the amino acid sequence of SEQ ID NO: 25 and the antigen-binding domain specifically binding Trop2 comprises the amino acid sequence of SEQ ID NO: 13; (b) the antigen-binding domain specifically binding Nectin4 comprises the amino acid sequence of SEQ ID NO: 21 and the antigen-binding domain specifically binding Trop2 comprises the amino acid sequence of SEQ ID NO: 9; (c) the antigen-binding domain specifically binding Nectin4 comprises the amino acid sequence of SEQ ID NO: 25 and the antigen-binding domain specifically binding Trop2 comprises the amino acid sequence of SEQ ID NO: 9; (d) the antigen-binding domain specifically binding Nectin4 comprises the amino acid sequence of SEQ ID NO: 35 and the antigen-binding domain specifically binding Trop2 comprises an amino acid sequence selected from SEQ ID NOs: 29 and 30; or (e) the antigen-binding domain specifically binding Nectin4 comprises an amino acid sequence selected from SEQ ID NOs: 34 and 38 and the antigen-binding domain specifically binding Trop2 comprises an amino acid sequence selected from SEQ ID NOs: 29 and 30.

14. The multispecific antibody according to claims 1-6, wherein the antibody is in a double-chain form, wherein: (a) the antibody is in a symmetric double-chain form, and wherein the polypeptide chain of the antibody comprises the amino acid sequence of SEQ ID NO: 53 or 54 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, preferably comprises the amino acid sequence of SEQ ID NO: 53; or (b) the antibody is in an asymmetric double-chain form, and wherein the antibody comprises a first and a second polypeptide chain selected from the group consisting of: (i) a first and a second polypeptide chain respectively comprising the amino acid sequences of SEQ ID NOs: 55 and 56 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, (ii) a first and a second polypeptide chain respectively comprising the amino acid sequences of SEQ ID NOs: 57 and 58 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, (iii) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 59 and 60 or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity thereto, (iv) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 61 and 62 or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity thereto, (v) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 63 and 64 or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity thereto, (vi) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 65 and 66 or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity thereto, (vii) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 67 and 68 or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity thereto, (viii) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 69 and 70 or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity thereto, (ix) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 71 and 72 or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity thereto, (x) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 73 and 74 or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity thereto, and (xi) a first and a second polypeptide chain each comprising the amino acid sequences of SEQ ID NOs: 75 and 76 or amino acid sequences having at least 95%, 96%, 97%, 98% or 99% identity thereto, Preferably, the first and second polypeptide chains each comprise the amino acid sequences of SEQ ID NOs: 61 / 62, 69 / 70 or 71 / 72.

15. The multispecific antibody of claims 1-6, wherein the antibody is in single-chain form, and wherein the polypeptide chain of the antibody comprises an amino acid sequence selected from SEQ ID NOs: 41-52 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.

16. A VHH domain that specifically binds to Trop2, and wherein the VHH domain comprises CDR1, CDR2 and CDR3 sequences selected from the amino acid sequences of SEQ ID NOs: 5, 9, 13, 29, 30, 98 and 99; More preferably, the CDR1, CDR2 and CDR3 sequences: (i) each comprise the amino acid sequences of SEQ ID NOs: 6, 7 and 8 or consist thereof; (ii) each comprise the amino acid sequences of SEQ ID NOs: 10, 11 and 12 or consist thereof; (iii) Comprising or consisting of the amino acid sequences of SEQ ID NOs: 14, 15 and 16, respectively; or (iv) Comprising or consisting of the amino acid sequences of SEQ ID NOs: 31, 32 and 33, respectively; More preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 5, 9, 13, 29, 30, 98 and 99, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity to the amino acid sequence, or having one or more (preferably 1 - 10, more preferably 1 - 5) amino acid additions, deletions and / or substitutions. Most preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 5, 9, 13, 29, 30, 98 and 99, or consists of an amino acid sequence selected from SEQ ID NOs: 5, 9, 13, 29, 30, 98 and 99.

17. A VHH domain that specifically binds Nectin4, and wherein the VHH domain comprises the CDR1, CDR2 and CDR3 sequences of an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35, 38 and 100 - 106; Preferably, the CDR1, CDR2 and CDR3 sequences: (i) Comprising or consisting of the amino acid sequences of SEQ ID NOs: 18, 19 and 20, respectively; (ii) Comprising or consisting of the amino acid sequences of SEQ ID NOs: 22, 23 and 24, respectively; (iii) Comprising or consisting of the amino acid sequences of SEQ ID NOs: 26, 27 and 28, respectively; (iv) Comprising or consisting of the amino acid sequences of SEQ ID NOs: 18, 36 and 20, respectively; or (v) Comprising or consisting of the amino acid sequences of SEQ ID NOs: 18, 37 and 20, respectively; More preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35, 38 and 100 - 106, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity to the amino acid sequence, or having one or more (preferably 1 - 10, more preferably 1 - 5) amino acid additions, deletions and / or substitutions. Most preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35, 38 and 100 - 106, or consists of an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35, 38 and 100 - 106.

18. An antibody comprising the VHH domain that specifically binds Trop2 as claimed in claim 16 and / or the VHH domain that specifically binds Nectin4 as claimed in claim 17.

19. An antigen - binding molecule comprising the antibody of claim 18.

20. A polynucleotide encoding the antibody according to any one of claims 1-15, the VHH domain of claim 16 or 17, the antibody of claim 18, or the antigen-binding molecule of claim 19.

21. A vector, preferably an expression vector, comprising the polynucleotide of claim 20.

22. A host cell comprising the polynucleotide of claim 20 or the vector of claim 21, for example, the host cell is a mammalian cell.

23. A method for producing the antibody according to any one of claims 1-15 or 18, the method comprising: culturing a host cell comprising a polynucleotide encoding the polypeptide chain of the antibody under conditions suitable for expressing the polypeptide chain of the antibody; and assembling the polypeptide chain to produce the antibody under conditions suitable for the assembly of the polypeptide chain into the antibody.

24. An immunoconjugate or immunofusion comprising the antibody according to any one of claims 1-15 or 18.

25. An antibody-drug conjugate of formula (I): Ab-(L-D) p (I) or a pharmaceutically acceptable salt or solvate thereof, wherein: Ab is the antibody according to any one of claims 1-15 or 18 or the antigen-binding molecule of claim 19; L is a linker; D is a drug, preferably an anti-tumor compound; and p is an integer selected from 1 to 16, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

26. The antibody-drug conjugate according to claim 25 or a pharmaceutically acceptable salt or solvate thereof, wherein the anti-tumor compound is a cytotoxic agent, such as a camptothecin compound, an auristatin compound, such as irinotecan, Dxd, or MMAE.

27. The antibody-drug conjugate according to claim 25 or a pharmaceutically acceptable salt or solvate thereof, wherein D has a structure represented by formula (D-1a) or formula (D-1b): wherein R 1a is selected from H and C1-C6 alkyl; 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 are independently selected from H and C1-C4 alkyl; or formula (D-1b) wherein R 1b , R 2b , R 3b , R 4b , R 5b and R 8b are each independently selected from C 1-8 alkyl; such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl; R 6b and R 7b each independently selected from C 1-8 alkoxy groups such as methoxy, ethoxy or propoxy; R 9b Selected from C 1-8 alkyl and COOH; such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl; and R 10b Selected from OH and H.

28. The antibody-drug conjugate according to claim 27 or a pharmaceutically acceptable salt or solvate thereof, wherein R 1a is H; R 2a is a C1-C6 alkyl group; R 3a is a halogen, preferably -F; R 4a is a C1-C4 alkyl group, preferably ethyl; R 1b 、 R 4b and R 8b each independently selected from C 1-2 alkyl; preferably methyl; R 2b , R 3b and R 5b Each independently selected from C 3-4 alkyl; R 6b and R 7b each independently selected from C 1-2 alkoxy; and R 9b selected from C 1-4 alkyl and R 10b is OH; or R 9b is COOH and R 10b is H.

29. The antibody-drug conjugate according to claim 27 or a pharmaceutically acceptable salt or solvate thereof, wherein D has a structure represented by formula (D-2a) or formula (D-2b): wherein R 1a 、R 2a 、R 3a and R 4a are as defined in claim 27 or 28; wherein R 1b , R 2b , R 3b , R 4b , R 5b , R 6b , R 7b , R 8b , R 9b and R 10b are as defined in claim 27 or 28.

30. The antibody-drug conjugate according to claim 27 or a pharmaceutically acceptable salt or solvate thereof, wherein D has a structure represented by formula (D-3a) or (D-3b):

31. The antibody-drug conjugate according to claim 27 or a pharmaceutically acceptable salt or solvate thereof, wherein D has a structure represented by formula (D-4a) or (D-4b):

32. The antibody-drug conjugate according to any one of claims 25-31 or a pharmaceutically acceptable salt or solvate thereof, wherein -L- has the following structure -Z-L1-L2-L3- wherein Z is selected from wherein m is independently an integer selected from 1 to 10, for example 1, 2, 3, 4, 5, 6, 7 or 8; L1 is selected from non-existence, wherein n1 and m1 are each independently an integer selected from 0 to 20, such as an integer selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7 or 8; L2 is an amino acid residue or a peptide residue composed of 2-8 amino acids; and L3 is selected from: wherein X is selected from -NH-, -O- and -S-; R 1c are each independently selected from C 1-8 alkyl, C 1-8 haloalkyl-, C 1-8 alkoxy, halogen, nitro and cyano; Su are each independently selected from pentose, penturonic acid, hexose and hexuronic acid; n2 is 0, 1, 2, 3 or 4; n5 is 0, 1, 2 or 3; n3 and n4 are independently 1, 2, 3, 4, 5 or 6;.

33. The antibody-drug conjugate according to claim 32 or a pharmaceutically acceptable salt or solvate thereof, Among them, Z is selected from where m is 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, Z is selected from 34. The antibody-drug conjugate according to any one of claims 32-33 or a pharmaceutically acceptable salt or solvate thereof, Among them, L1 is selected from non-existent, wherein n1 is independently an integer selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, L1 is absent or 35. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 32-34, wherein L2 is an amino acid residue or a peptide residue composed of 2, 3, 4, 5, 6 or 7 amino acids; preferably, the amino acid residue or the amino acids constituting the peptide residue are each independently selected from glycine (Gly), valine (Val), alanine (Ala), lysine (Lys), citrulline (Cit), glutamine (Gln), glutamic acid (Glu), phenylalanine (Phe), leucine (Leu), tyrosine (Tyr), serine (Ser), aspartic acid (Asp), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), methionine (Met), tryptophan (Trp), cysteine (Cys), histidine (His) and threonine (Thr), wherein the amino acid residue or amino acids are optionally substituted by one or more C 1-6 alkyl groups; More preferably, the amino acid residue or the amino acids constituting the peptide residue are each independently selected from glycine (Gly), valine (Val), alanine (Ala), phenylalanine (Phe), glutamic acid (Glu), and citrulline (Cit); Even more preferably, L2 is selected from -Ala-, -Val-, -Gly-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Val-Cit-, and -Glu-Val-Cit-.

36. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 32-35, Among them, L3 is selected from: wherein R 1c is independently selected from C 1-8 alkyl, C 1-8 haloalkyl-, C 1-8 alkoxy, halogen, nitro and cyano; Su is independently selected from 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; Preferably, L3 is selected from: as defined for each variable.

37. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 32-36, Among them, Su are each independently: More preferably, each Su is independently Even more preferably, each Su is independently 38. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 32-36, Among them, L3 is selected from: Preferably, L3 is selected from:

39. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 35, Z is selected from L1 is selected from non-existence and L2 is selected from -Gly-, -Val-Ala-, and -Gly-Gly-Phe-Gly; and L3 is selected from:

40. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 35, wherein -Z-L1-L2-L3- is selected from the following structures 41. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 25-40, which has an average DAR value of 2-10, for example 4-8.

42. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 25, wherein the antibody-drug conjugate is selected from where Ab and p are as defined in claim 25, Preferably, Ab is a bispecific antibody, wherein: (a) the antibody is in a symmetric bispecific form, and wherein the polypeptide chain of the antibody comprises the amino acid sequence of SEQ ID NO:53; or (b) the antibody is in an asymmetric bispecific form, and wherein the antibody comprises a first and a second polypeptide chain containing the amino acid sequences of SEQ ID NOs:61 / 62, 69 / 70, or 71 / 72, respectively: Preferably, the antibody-drug conjugate has an average DAR of 2-10 or 4-8.

43. A pharmaceutical composition comprising the antibody according to any one of claims 1-15 or 18, the antigen-binding molecule according to claim 19, or the immunoconjugate or immunofusion according to claim 24, or the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 25-42 and a pharmaceutically acceptable carrier. Use of an antibody according to any one of claims 1-15 or 18, an antigen-binding molecule according to claim 19, an immunoconjugate or immunofusion according to claim 24, or an antibody-drug conjugate according to any one of claims 25-42, or a pharmaceutically acceptable salt or solvate thereof, as a medicament or for the preparation of a medicament. Use according to claim 44, wherein the medicament is for treating and / or preventing cancer in an individual, wherein the cancer is a Trop2-positive, Nectin4-positive, or Trop2- and Nectin4-double-positive solid tumor, selected from, for example, urothelial carcinoma; bladder cancer (e.g., BLCA); cervical cancer (e.g., CESC); head and neck cancer (e.g., HNSC); Lung cancer (e.g., LUSC and LUAD); breast cancer (e.g., BRCA); pancreatic cancer (e.g., PAAD); esophageal cancer (e.g., ESCA); Prostate cancer (e.g., PRAD); cholangiocarcinoma (e.g., CHOL); endometrioid carcinoma (e.g., UCEC); thyroid cancer (e.g., THCA); ovarian cancer (e.g., OV); colorectal cancer (e.g., COAD and READ); and gastric cancer (e.g., STAD), including primary, recurrent, and refractory cancers thereof.

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