Antibody, and drug conjugate and use thereof

Abstract

Provided are an antibody and an antibody-drug conjugate. More specifically, provided are an antibody and an antibody-drug conjugate (ADC) targeting TROP2 and / or NECTIN4, and a composition containing the antibody or the ADC and the therapeutic use thereof.

Description

Antibodies and drug conjugates thereof and their uses 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, as well as compositions containing the antibodies or ADCs and therapeutic applications thereof. Background Art

[0002] Trop2 (trophoblast cell-surface antigen 2), also known as tumor-associated calcium signal transducer 2 (TACSTD2), epithelial glycoprotein 1 (EGP-1), gastrointestinal antigen 733-1 (GA733-1), and 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 its signaling pathways primarily promote tumor cell growth, proliferation, and metastasis by regulating calcium signaling, cell cycle protein expression, and reducing fibronectin adhesion. Therefore, Trop2 has become an attractive therapeutic target for cancer treatment. However, because Trop2 is expressed not only in various cancer tissues, such as ovarian, pancreatic, gastric, and breast cancers, but also in a wide range of normal human tissues, including the breast, cervix, prostate, skin, stomach, and thymus, drugs targeting Trop2 have the potential for on-target toxicity. For example, when the skin and oral mucosa are particularly affected by drugs, side effects such as stomatitis and rash are more likely to occur.

[0003] Currently, the only Trop2 ADC drug on the market is Gilead’s In the ASCENT study, sacituzumab govitecan (SG) caused a 45% incidence of Grade 3 or higher adverse reactions, with neutropenia and diarrhea receiving a black box warning from the FDA. Furthermore, 9% of patients experienced rash, and 5% experienced ocular toxicity. Datopotamab Deruxtecan (DS-1062, Dato-DXd) is a Dxd-targeted antibody-drug conjugate (ADC) jointly developed by Daiichi Sankyo and AstraZeneca. To mitigate toxicity, Dato-DXd utilizes a moderate-affinity monoclonal antibody targeting TG2 linked to a less toxic DNA topoisomerase I inhibitor (DXd), achieving a DAR of 4. However, due to Dxd-induced pulmonary toxicity in cynomolgus monkeys, the HNSTD (highest no-serious-toxicity dose) of Dato-DXd was only 10 mg / kg, lower than the 30 mg / kg HNSTD of DS-8201, which has a DAR of 8, in monkeys.

[0004] Nectin4 (Nectin cell adhesion molecule 4) is a type I transmembrane cell adhesion molecule belonging to the Nectin family. This protein was formerly known as a homolog of the poliovirus receptor (PVR / CD155), also known as 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 essential for achieving 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. It 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.

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

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

[0007] Antibody-based drugs have advanced cancer treatment, but the safety and efficacy of many remain suboptimal. A major challenge facing anticancer drugs remains drug selectivity: ensuring effective cancer targeting while minimizing collateral damage to normal cells. To overcome this limitation of single-targeted drugs, strategies have been proposed that combine multiple monoclonal antibodies targeting different targets. However, published studies on different mAb combination therapies have shown significant variability in efficacy, depending on factors such as the target combination. For example, simultaneous administration 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) for the treatment of advanced colorectal cancer has not resulted in favorable outcomes. 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 this field for new drug molecules with improved tumor specificity and selectivity to reduce on-target toxicity, increase the drug window and expand the therapeutic index.

[0009] SUMMARY OF THE INVENTION

[0010] By analyzing immunohistochemistry and bioinformatics data from 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 are co-expressed 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, while the co-expression ratio in normal tissues is low. Therefore, the present inventors proposed and designed antibody molecules that simultaneously target Trop2 and Nectin4 to reduce the on-target toxicity of the two targets and improve the therapeutic effect. On this basis, the inventors further proposed an innovative antibody model based on nanobodies, resulting in an antibody molecular weight of only about 60% of that of conventional four-chain antibodies, thereby achieving a higher proportion of antibody molecules enriched in tumor tissue, thereby further improving efficacy. Based on these designs and discoveries, the inventors established the multispecific antibodies and drug conjugates of the present invention and their uses, particularly in cancer treatment.

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

[0013] In a third aspect, the present invention provides an antibody-drug conjugate (ADC) comprising 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 invention have one or more of the following advantages:

[0015] (1) Bind to target cells expressing human Nectin4 and / or Trop2 with high affinity;

[0016] (2) synergistic binding to target cells expressing human Nectin4 and Trop2;

[0017] (3) coordinated endocytosis in target cells expressing human Nectin4 and Trop2;

[0018] (4) synergistic killing effect on target cells expressing human Nectin4 and Trop2;

[0019] (5) There is a significant bystander effect;

[0020] (6) It has high anti-tumor efficacy, has a stronger killing effect on tumor cells, and has a stronger inhibitory effect on tumor growth, especially tumors with high expression of Nectin4 and / or Trop2; it has 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) Compared with single-target drugs of Nectin4 and Trop2, the ability to resist the emergence of tumor resistance is enhanced;

[0023] (8) Resisting tumor drug resistance caused by Nectin4 or Trop2 antigen epitope mutations;

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

[0025] (10) Improved tumor targeting distribution compared to single-target drugs such as 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 conjugates of the present invention also have one or more of the following advantages:

[0028] (11) Better product uniformity.

[0029] (12) Low toxicity;

[0030] (13) Having good physical and chemical stability;

[0031] (14) It has good drugability.

[0032] The present invention is further illustrated in the following drawings and specific embodiments. However, these drawings and specific embodiments should not be considered to limit the scope of the present invention, and changes that are readily apparent to those skilled in the art will be included within the spirit of the present invention and the protection scope of the appended claims.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG1A shows endocytosis detection of an exemplary anti-Trop2 VHH-Fc on BT-474 cells.

[0035] FIG1B shows endocytosis detection of an exemplary anti-Trop2 VHH-Fc on NCI-N87 cells.

[0036] FIG2A shows endocytosis detection of an exemplary anti-Nectin4 VHH-Fc on BT474 cells.

[0037] FIG2B shows endocytosis detection of an exemplary anti-Nectin4 VHH-Fc on NCI-N87 cells.

[0038] FIG3 shows the binding of an exemplary anti-Trop2 VHH-Fc to NCI-N87 cells.

[0039] FIG4A shows the binding of exemplary anti-Nectin4 VHH-Fc antibodies to NCI-N87 cells.

[0040] FIG4B shows the binding of exemplary anti-Nectin4 VHH-Fc antibodies to BT474 cells.

[0041] FIG5A shows the binding of an exemplary anti-Nectin4 VHH-His antibody to CHO-K1-Human Nectin4 cells.

[0042] FIG5B shows the binding of exemplary anti-Nectin4 VHH-His antibodies to CHO-K1-Cyno Nectin4 cells.

[0043] FIG5C shows the non-specific binding of an exemplary anti-Nectin4 VHH-His antibody to CHO-K1 cells.

[0044] FIG6 shows the expression of Trop2 antigen and Nectin4 antigen on various target cells.

[0045] FIG7A shows that the exemplary antibody V-hu21-Fc has no specific binding to the paralogous Nectin 1, 2, 3 and Trop 1 antigens.

[0046] FIG7B shows that the exemplary antibody V-hu23-Fc has no specific binding to the paralogous Nectin 1, 2, and 3 antigens but specifically binds to the target antigen Nectin 4.

[0047] FIG7C shows that the exemplary antibody V-hu23-Fc has no specific binding to the paralog Trop1 antigen but has specific binding to the target antigen Trop2.

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

[0049] Figure 8B shows binding of an exemplary two-chain form of a bispecific antibody to NCI-N87 cells.

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

[0051] Figure 9B shows binding of an exemplary two-chain form of a bispecific antibody to BT-474 cells.

[0052] Figure 10A shows binding of an exemplary two-chain form of a bispecific antibody to MDA-MB-468 cells.

[0053] Figure 10B shows binding of an exemplary two-chain form of a bispecific antibody to NCI-N87 cells.

[0054] Figure 10C shows binding of an exemplary two-chain form of a bispecific antibody to BT-474 cells.

[0055] FIG11 shows the binding of the exemplary antibody V-hu21-Fc to different tumor cells.

[0056] FIG12 shows the binding of the exemplary antibody V-hu23-Fc to different tumor cells.

[0057] FIG13A shows the results of BT-474 cells internalizing an exemplary bispecific anti-Trop2 / Nectin4 antibody molecule.

[0058] FIG13B shows the results of BT-474 cells internalizing an exemplary bispecific anti-Trop2 / Nectin4 antibody molecule.

[0059] FIG13C shows the results of internalization of an exemplary bispecific anti-Trop2 / Nectin4 antibody molecule by NCI-N87 cells.

[0060] FIG13D shows the results of internalization of an exemplary bispecific anti-Trop2 / Nectin4 antibody molecule by NCI-N87 cells.

[0061] FIG13E shows the results of endocytosis of exemplary bispecific antibodies V-hu21-Fc and V-hu23-Fc molecules by different target cells.

[0062] Figure 14A-E shows the comparison of cell binding ability of ADC and corresponding bispecific antibodies.

[0063] FIG15A shows that V-hu21-VA-Exd kills target cells MDA-MB-468.

[0064] FIG15B shows the killing effect of V-hu21-VA-Exd on non-target MKN45 cells.

[0065] FIG15C 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] FIG15D shows the killing effect of V-hu23-Glu-Exd and V-hu24-Glu-Exd on target MDA-MB-468 cells.

[0067] FIG15E 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] FIG16A shows the bystander effect of V-hu21-VA-Exd.

[0069] FIG16B shows the bystander effect of V-hu23-Glu-Exd.

[0070] FIG17 shows the efficacy of multiple administrations of ADCs in the mouse MDA-MB-468 subcutaneous transplantation model.

[0071] FIG18 shows the efficacy of a single dose of ADCs in a mouse MDA-MB-468 subcutaneous transplantation model.

[0072] FIG19 shows the efficacy of multiple administrations of ADCs in the mouse HT1376 subcutaneous transplantation model.

[0073] FIG20 shows the efficacy of a single dose of ADCs in the mouse HT1197 subcutaneous transplantation model.

[0074] FIG21 shows the efficacy of a single dose of ADCs in the MDA-MB-453 subcutaneous transplant model.

[0075] FIG22 shows the efficacy of multiple doses of ADC in a human head and neck cancer xenograft model.

[0076] FIG23 shows the efficacy of a single dose of ADC in an in vivo human endometrial cancer xenograft model.

[0077] FIG24 shows the efficacy of a single dose of ADC in an in vivo human cervical cancer xenograft model.

[0078] FIG25 shows the efficacy of ADC administered twice in an in vivo mouse colon cancer xenograft model.

[0079] Figure 26 shows the co-expression of Trop2 and Nectin4 in various tumors, as revealed by bioinformatics analysis. The figure uses TPM (Transcripts Per Million) to quantify gene expression levels, showing the expression abundance of Trop2 and Nectin4 in different tumor tissue types. Figure 26A shows exemplary tumor types that are dually positive for Trop2 and Nectin4 according to the median TPM of the patient population. Figure 26B shows the distribution of Trop2 and Nectin4 expression observed in patient populations of specific tumor types.

[0080] Figure 27 shows the biodistribution of the bispecific antibody in a mouse humanized HT1376 xenograft tumor model. Figure 27A: Tumor accumulation of the antibody over time was quantified by the tumor-to-background fluorescence ratio. Figure 27B: Organ tissue distribution of the antibody was assessed by the tumor-to-muscle fluorescence ratio 72 hours after injection of the fluorescently labeled antibody protein.

[0081] Detailed Description of the Invention

[0082] Unless otherwise limited, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present 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 merely illustrative and are not intended to be restrictive. Other features, objects and advantages of the present invention will become apparent from this specification and the accompanying drawings and from the appended claims.

[0083] definition

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

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

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

[0087] In this article, the term "antigen binding molecule" refers to a protein or polypeptide comprising an antigen binding domain or antigen binding site that can bind to a target antigen. When the target antigen is TROP2 and / or NECTIN4, the antigen binding molecules that bind to TROP2 and / or NECTIN4 are also referred to as TROP2 binding molecules, NECTIN4 binding molecules or TROP2 / NECTIN4 binding molecules. Antigen binding molecules include, for example, antibodies and antigen binding fragments thereof, and 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 a "complementary determining region" or "CDR".

[0088] As used herein, the term "antibody" refers to a polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen. The term encompasses various antibody structures, including but not limited to monoclonal antibodies, single-chain antibodies or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), single-domain antibodies, heavy-chain antibodies, chimeric or humanized antibodies, complete antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0089] As used herein, "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] Herein, the terms "antibody fragment" and "antigen-binding fragment" are used interchangeably and refer to molecules different from intact antibodies, which contain a portion of an intact antibody and are capable of binding to the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; 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 by antibody fragments. Unless otherwise specified herein or clearly contradicted by the context, the term "antibody" mentioned herein is equivalent to "antibody or antibody fragment thereof." In some embodiments according to the present invention, the antibody fragment comprises a cysteine ​​residue portion for forming an interchain disulfide bond between heavy chains, for example, a cysteine ​​residue in the hinge region of an antibody, to provide an amino acid residue site that can be used for sulfhydryl coupling 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 that can be used for sulfhydryl coupling chemistry.

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

[0092] As used herein, the term "multispecific" refers to an antigen-binding molecule (e.g., an antibody) that has at least two antigen-binding sites that each bind to a different antigenic epitope, e.g., 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 to two different epitopes.

[0093] As used herein, the term "valency" or "valency" with respect to antibodies refers to the total number of antigen-binding sites in an antibody molecule, or the number of antigen-binding sites with the same antigen-binding specificity. For example, a tetravalent antibody means that the antibody molecule contains a total of four antigen-binding sites; the antibody molecule can also be a "2+2 valency" bispecific antibody, i.e., the antibody has two different antigen-binding specificities, wherein two identical antigen-binding sites are present for each antigen-binding specificity.

[0094] The term "on-target / off tumor toxicity" refers to the fact that in addition to tumor cells, normal tissue cells also express tumor-associated antigens targeted by antibodies, thereby binding to the antibodies and causing damage.

[0095] In this article, the terms "TROP2" and "Trop2" are used interchangeably to refer to trophoblast cell surface glycoprotein antigen 2. Unless otherwise indicated, the term includes any variant of human TROP2, including sequence variants, especially naturally occurring variants, allelic variants, and post-translational modification variants and conformational variants, and covers its species homologs. In addition, it should be understood that the term not only covers TROP2 naturally or recombinantly expressed by cells or TROP2 expressed on natural or recombinant cells, but also covers recombinantly expressed fusion proteins comprising the extracellular domain of TROP2. An example of TROP2 is a human TROP2 protein comprising the amino acid sequence under UniProtKB-P09758, or a recombinant protein comprising the extracellular domain of the protein (especially, the amino acid sequence of amino acids 31-274). Another example of TROP2 is a monkey TROP2 protein comprising the amino acid sequence under NCBI-XP_005543292.2, or a recombinant protein comprising the extracellular domain of the protein. Herein, unless otherwise specified, the term "TROP2" or "Trop2" refers to human-derived Trop2. 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 a VHH domain.

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

[0097] In this article, the term "NECTIN4" is used interchangeably with "Nectin4" and "Nectin4" to refer to Nectin cell adhesion molecule 4. Unless otherwise indicated, 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 covers species homologs thereof. In addition, it should be understood that the term covers not only NECTIN4 naturally or recombinantly expressed by cells or NECTIN4 expressed on natural or recombinant cells, but also recombinantly expressed fusion proteins comprising the extracellular domain of NECTIN4. An example of NECTIN4 is a human NECTIN4 protein comprising the amino acid sequence under UniProtKB-Q96NY8, or a recombinant protein comprising the extracellular domain of the protein (especially, the amino acid sequence of amino acids 32-349). Another example of NECTIN4 is a monkey NECTIN4 protein comprising the amino acid sequence under NCBI-_XP_005541277.1, or a recombinant protein comprising the extracellular domain of the protein. Herein, unless otherwise specified, the term "NECTIN4" refers to human-derived NECTIN4. In the 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 VHH domain pair.

[0098] As used herein, the term "NECTIN4-positive" cells refers to cells that express NECTIN4 on their cell surface, such as cancer cells, modified cancer cells, or modified non-tumor cells. The level of NECTIN4 expression on the cell surface can be determined by any conventional method known in the art for determining the level of cell surface antigen expression, such as FACS detection or immunofluorescence staining. NECTIN4 is expressed at a significantly higher level on a variety of tumor cells than on normal tissues / cells, such as HT1376 (human bladder cancer cells) and BT474 (human breast ductal carcinoma cells). Preferably, NECTIN4-positive cells are NECTIN4-positive tumor cells.

[0099] As used herein, the term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen epitope). "Binding affinity" reflects the intrinsic binding affinity of a 1:1 interaction between members of a binding pair. Binding affinity can often be expressed in terms of 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 affinity" refers to the combined strength of the interactions of multiple binding sites of a molecule (antibody) with the same target. Therefore, a necessary condition for avidity is the multivalency of a molecule (e.g., 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, the IgG class immunoglobulin is a heterotetrameric glycoprotein of approximately 150,000 daltons consisting of two light chains and two heavy chains bonded by disulfide bonds. From N-terminus to C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also known as a heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3). Similarly, from N-terminus to C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also known as a 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, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), based on the type of their constant region, some of which 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 assigned to one of two types, called κ and λ, based on the amino acid sequence of their constant domains.

[0102] As used herein, the term "isotype" refers to the antibody type determined by the constant region of the heavy chain of the antibody. For example, antibodies according to the present invention can be IgA (e.g., IgA1 or IgA2), IgG1, IgG2 (e.g., IgG2a or IgG2b), IgG3, IgG4, IgE, IgM, and IgD antibodies, and have a heavy chain constant region of said immunoglobulin type. In addition, the present invention contemplates not only antibodies employing native 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 the heavy or light chain of an antibody that participates in binding of an antibody to an antigen. In the case of a heavy chain antibody, for example, a heavy chain antibody from Camelidae, a single VH domain (also referred to herein as a VHH domain) may be sufficient to confer antigen binding specificity. The VHH domain, like the heavy and light chain variable regions of conventional IgG antibodies, comprises four conserved framework regions (FRs) and three complementary determining regions (CDRs), and is arranged in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In some aspects of the present invention, one or more residues in the variable region of an antibody may be modified, for example, by modifying one or more CDR regions and / or one or more framework regions, particularly by replacing conserved residues, to obtain an antibody variant that substantially retains at least one biological property (for example, antigen binding ability) of the parent antibody. In further aspects, the antibody variable region may be modified by CDR transplantation. Since CDR sequences are responsible for most antibody-antigen interactions, recombinant antibody variants that mimic the properties of known antibodies may be constructed. In such antibody variants, the CDR sequences from known antibodies are transplanted onto the framework regions of different antibodies with different properties, and one to several residue mutations, such as back mutations, can be performed as needed to refine the desired properties of the antibody. The properties of the mutated and / or modified antibody or ADC conjugate comprising it can be evaluated in in vitro or in vivo assays, such as target antigen binding properties or other desired functional properties, such as endocytosis activity, pharmacokinetics, and in vivo tumor killing activity. Therefore, the present invention also contemplates variants of any variable region (e.g., VHH) given herein.

[0104] As used herein, "complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" refers to a region in an antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. In the VHH domains of the antibodies of the present invention, CDRs are numbered sequentially starting from the N-terminus and are commonly referred to as CDR1, CDR2, and CDR3. The CDR sequences in a defined VHH domain can be determined using schemes well known in the art, such as the Kabat, AbM, Chothia, Contact, and IMGT schemes to define the regional extent of CDRs and their combined extents. Unless otherwise indicated, in the present invention, the terms "CDR" or "CDR sequence" encompass CDR sequences determined in any of the aforementioned ways and combinations thereof. Furthermore, it is known in the art that, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, and Contact methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. Such a minimum binding unit can be a subsection of a CDR. The residues of the remaining CDR sequence, as will be appreciated by those skilled in the art, can be determined by the structure and protein folding of the antibody. Therefore, the present invention also contemplates variants of any CDR described herein. For example, in a CDR variant, the amino acid residues of the minimum binding unit can remain unchanged, while the remaining CDR residues can be replaced.

[0105] Unless otherwise indicated, throughout the present invention, references to residue positions in antibody variable regions and CDRs (including heavy chain variable region residues) refer to positions numbered according to the Kabat numbering system.

[0106] Herein, the terms "VHH" and "VHH domain" are used interchangeably to refer to heavy chain variable domains derived from heavy chain antibodies lacking light chains, sometimes also referred to as single variable domain fragments (sVD). Therefore, VHH is different from the conventional VH of four-chain immunoglobulins in that it does not need to be paired with a light chain variable domain to form an antigen binding site. Such VHH molecules can be derived from antibodies produced in Camelidae species (e.g., camels, alpacas, dromedaries, llamas, and guanacos). Other species besides Camelidae can also produce heavy chain antibodies that naturally lack light chains, and such VHH are also within the scope of the present invention. In some cases, for therapeutic applications of antibodies or their derivative molecules, it is desirable to reduce their immunogenicity. Therefore, preferably, in one embodiment, the antibodies of the present invention comprise humanized variable regions (e.g., VHH domains).

[0107] As used herein, the term "half-life extension domain" refers to a chemical structure that can confer an increased circulating half-life to a molecule (e.g., an antibody) bound thereto after administration to an animal. Such chemical structures include, for example, flexible hydrophilic molecules (e.g., carbohydrates or PEG (polyethylene glycol)), immunoglobulin Fc regions, serum albumin, serum albumin binding domains (e.g., small organic molecules, fatty acids, peptides, and proteins capable of binding to serum albumin), or serum albumin binding peptides. The half-life extension domain can be connected to the antibody of the present invention by chemical conjugation or fusion, depending on its specific properties. Preferably, the half-life extension 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 thereby function as a half-life extension 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, and 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 molecules of the present invention by genetic fusion, thereby avoiding chemical coupling. See, for example, Expert Opin Biol Ther. 2016 Jul; 16 (7): 903-15. doi: 10.1517 / 14712598.2016.1165661.

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

[0110] In this article, the term "native sequence Fc region" encompasses naturally occurring various immunoglobulin Fc region sequences, such as various Ig subtypes and the Fc region sequences of 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, human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or from Pro230 to the heavy chain carboxyl terminus. However, the C-terminal terminal lysine (Lys447) in the Fc region may be present or absent. In some embodiments, human IgG heavy chain Fc region carries a hinge sequence or a partial hinge sequence of a natural immunoglobulin at the N-terminus, such as, according to EU numbering, a sequence from E216 to T225 or a sequence from D221 to T225.

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

[0112] As used herein, the term "effector function" refers to those biological activities attributable to the Fc region of an immunoglobulin that vary with the immunoglobulin isotype. Examples of immunoglobulin effector functions include Fc receptor binding, C1q binding, 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, such as reduced or abolished Fcγ receptor binding, relative to an antibody molecule having a wild-type Fc region.

[0113] As used herein, the terms "flexible linking peptide" or "peptide linker" or "connecting peptide" are used interchangeably to refer to a short amino acid sequence consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T), used alone or in combination, or from the hinge region of an immunoglobulin.

[0114] As used herein, the "percentage (%) identity" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific amino acid sequence set forth in this specification, after aligning the candidate sequence with the specific amino acid sequence set forth in this specification and, if necessary, introducing gaps to achieve the maximum percentage identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the invention that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein. The variants may comprise conservative modifications.

[0115] With respect to polypeptide sequences, "conservative modifications" include substitutions, deletions, or additions to a polypeptide sequence that result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following eight 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 the antibody containing the amino acid sequence.

[0116] As used herein, the term "binding" or "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, for example, by detecting the binding ability of an antibody to an antigen by the ELISA assay described in the Examples, or by detecting the binding ability of an antibody to cells expressing an antigen on their surface by the FACS assay described in the Examples, or by detecting the affinity constant K 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 be composed of continuous and / or discontinuous amino acids that form a conformational spatial unit. Different antibodies that bind to the same antigen can be grouped into epitopes by competitive binding assays. When the test antibody blocks the binding of the reference antibody to the 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 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 "competing antibodies for binding." Competing antibodies can bind to the same epitope region as the reference antibody, such as the same epitope, an adjacent epitope, or an overlapping epitope. 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] In this article, "humanized" antibody refers to a chimeric antibody comprising 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 non-human antibodies, and all or substantially all of the FRs correspond to those of human antibodies. Humanized antibodies optionally can comprise at least a portion of an 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. In this article, in some embodiments, the humanized antibody of the present invention has a framework region sequence "derived from" a specific human germline sequence. Here, "derived from" refers to a chimeric antibody that has at least 85%, 90% identity with the corresponding framework region amino acid sequence encoded by the human germline immunoglobulin gene, and the antibody maintains antigen binding activity.

[0119] Herein, if an amino acid sequence (e.g., VHH) is specific for two different antigens or antigenic determinants (e.g., TROP2 or NECTIN4 from different mammalian species, such as human TROP2 or human NECTIN4, cynomolgus monkey TROP2 or cynomolgus monkey NECTIN4), it is said to be "cross-reactive" to these two different antigens or antigenic determinants. It is advantageous for the antibody to have human-monkey species cross-reactivity, especially to have similar human-monkey antigen binding affinity, which can be helpful for preclinical drug development of antibodies, such as toxicological testing of ADC molecules composed of antibodies. In some embodiments, the antibodies of the present invention preferably have human-monkey species cross-reactivity.

[0120] In this article, the terms "endocytosis" and "internalization" are used interchangeably and refer to the process in which the ligand / receptor complex is internalized and delivered to the cytosol or transferred to the appropriate intracellular compartment, triggered by the binding of the ligand to the corresponding receptor on the cell surface. In some embodiments, the antibodies of the present invention trigger endocytosis mediated by TROP2 and / or NECTIN4 receptors after binding to TROP2 and / or NECTIN4 expressed on the cell surface. In this article, endocytosis and endocytosis rate can be measured by the methods described in the examples to characterize the endocytic activity of the antibody. In some embodiments, the antibodies of the present invention having endocytic activity can be used as a tool for carrying anti-tumor drugs into cancer cells in the ADC 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 the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0122] As used herein, the term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector comprises 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) into which the recombinant polynucleotide is incorporated.

[0123] Herein, when referring to "synergistic endocytosis", "synergistic binding" and "synergistic killing", the expression "synergistic" means that the antibodies of the present invention having dual targeting binding sites for TROP2 and NECTIN4 or ADC molecules based thereon have increased endocytosis, binding or killing effects when tested under the same conditions compared to reference antibodies having corresponding single targeting binding sites for TROP2 or NECTIN4 (e.g., the parent antibody of TROP2 or NECTIN4) or ADC molecules 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., antibodies or fragments thereof) to one or more other molecules. An immunoconjugate typically comprises at least one non-proteinaceous chemical moiety, for example, a chemical linker for achieving the conjugation. In some cases, the other molecules may be proteinaceous molecules, such as peptides, polypeptides, or proteins. In some cases, the other molecules may also be non-proteinaceous molecules, for example, chemical toxins. In some cases, the other molecules may be immunoglobulin-related molecules or fragments thereof. In some cases, the other molecules may be different from immunoglobulin-related molecules or fragments thereof. The one or more other molecules may be the same or different from each other. For example, the other molecules may be target binding elements and / or effector elements, such as chemotherapeutic agents, toxins, drugs (e.g., immunotherapeutic agents), radioactive elements, probes, or signaling molecules.

[0125] As used herein, the terms "individual" or "subject" are used interchangeably to refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual is a human.

[0126] As used herein, the term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual being treated. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of progression of the disease, improving or alleviating the disease state, and alleviating or improving prognosis. In the context of tumor or cancer treatment, "treatment" encompasses anti-tumor biological effects that can be induced by human intervention (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 to refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas, solid tumors, and liquid tumors. In certain embodiments, cancers suitable for treatment by the antibodies, immunoconjugates, or immunofusions of the present invention include TROP2-positive and / or NECTIN4-positive tumors / cancers, including metastatic forms thereof.

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

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

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

[0131] The term "alkyl" as used herein 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 or branched chain hydrocarbon group containing 2 to 16 carbon atoms and including at least one double bond and no triple bonds. Alkenyl groups preferably contain 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, ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like.

[0133] The term "alkynyl" refers to a straight or branched chain hydrocarbon group containing 2 to 16 carbon atoms and at least one triple bond. Alkynyl groups preferably contain 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, and the like.

[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. Halogenated alkyl groups may preferably be monohalogenated alkyl, dihalogenated alkyl, or polyhalogenated alkyl (including perhalogenated alkyl). Monohalogenated alkyl groups may contain one iodine, bromine, chlorine, or fluorine in the alkyl group. Dihalogenated alkyl and polyhalogenated alkyl groups may contain two or more identical halogen atoms or a combination of different halo groups in the alkyl group. Preferably, polyhalogenated alkyl groups contain up to 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. Perhalogenated alkyl groups refer 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, substituted with one or more halo groups, as defined herein. The term "haloalkynyl" refers to an alkynyl group, as defined herein, substituted with one or more halo groups, as defined herein. The meaning of "halo" as defined for "haloalkyl" applies to both "haloalkenyl" and "haloalkynyl."

[0137] The term "amino acid" refers to naturally occurring and synthetic amino acids. Amino acids can be L or D isomers. Conventional amino acids are referred to herein in accordance with 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. Also, in this disclosure, amino acids are generally referred to by single-letter and three-letter abbreviations known in the art. For example, the amino acid 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 oxidation of the primary hydroxyl group of a hexose to a carboxyl group. Examples of hexuronic acid include, but are not limited to, glucuronic acid, galacturonic acid, and mannuronic acid.

[0140] The term "pentose," also known as five-carbon sugar, refers to a monosaccharide containing five carbon atoms. Pentose includes D- and / or L-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- and / or L-pentoses. Examples of hexoses include, but are not limited to, glucose, galactose, mannose, and fructose.

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

[0143] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the ADC conjugates of the present invention and is not biologically or otherwise undesirable. The ADC conjugates of the present invention may exist as pharmaceutically acceptable salts thereof, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable, non-toxic acid addition salt refers to a salt formed between the ADC conjugates of the present invention and an organic or inorganic acid, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, and the like. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the ADC conjugates of the present invention with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed with organic bases containing an N group.

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

[0145] As used herein, "pharmaceutically acceptable" and "pharmaceutically acceptable" are used interchangeably unless there is any contradiction in the context.

[0146] The term "drug:antibody ratio" or "DAR" refers to the ratio of the drug moiety (D) coupled to the Ab moiety described herein to the Ab moiety in an antibody drug conjugate molecule. In some embodiments described herein, the DAR can be determined by p in Formula I, for example, the DAR can be 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 a population of molecules in a product, i.e., the overall ratio of the drug moiety (D) coupled to the Ab moiety described herein to the Ab moiety in a product as measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis, and / or HPLC), which DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the conjugate of the invention is 1 to 16, e.g., 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, e.g., 1.0-8.0, 2.0-6.0, e.g., 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, 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, and ranges having two of these values ​​as endpoints. It should be understood that when referring to an average DAR value, the ADC of the present invention refers to a population of ADC molecules or a mixture of ADC molecules comprising ADC molecules with the same and / or different DARs.

[0147] The term "therapeutic agent" as used 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 herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0149] "Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.

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

[0151] The term "small molecule drug" refers to low molecular weight organic compounds that can modulate biological processes, particularly alter or prevent pathological processes. A "small molecule" is defined as a molecule with a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 1 kD, more preferably less than 500 kD. Small molecule drugs include, but are not limited to, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than larger molecules.

[0152] The term "immunomodulator" as used herein refers to a natural or synthetic agent or drug that inhibits or regulates (e.g., activates) an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants or immune agonists. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0153] “Anti-tumor compounds” are pharmaceutically active compounds that have an effect on tumors, including but not limited to cytotoxic agents or chemotherapeutic agents, such as the cytotoxic agents disclosed in WO2021 / 173773 and US5658920, such as camptothecin compounds Exitecan (topoisomerase I inhibitor Exatecan), Dxd (a new topoisomerase I inhibitor Exatecan derivative), and auristatin compounds such as monomethyl auristatin E (MMAE), the structure of which is shown below:

[0154] The term "effective amount" refers to an amount or dosage of an antibody or ADC molecule or composition or combination of the present invention that, after single or multiple doses, produces the desired effect in a patient in need of treatment or prevention. Depending on the desired effect, both "therapeutically effective amount" and "prophylactically effective amount" may be included.

[0155] A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the required dosage and for the required period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or ADC molecule or composition or combination are outweighed by the therapeutically beneficial effects. 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% relative to an untreated subject.

[0156] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result, at the required dosage and for the required period of time. Typically, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0157] The term "anti-tumor effect" refers to a biological effect that can be demonstrated by various means, including but not limited to, for example, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0158] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0159] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0160] The terms "drug combination", "combination product", "drug association" or "combination product" refer to non-fixed combination products or fixed combination products, including but not limited to kits and pharmaceutical compositions. The term "non-fixed combination" means that the active ingredients (e.g., (i) the antibody molecule or ADC molecule of the present invention, and (ii) the other therapeutic agent) are administered to a patient as separate entities simultaneously, without specific time restrictions, or sequentially at the same or different time intervals, wherein such administration provides a prophylactically or therapeutically effective level of the two or more active agents in the patient. In some embodiments, the antibody molecule or ADC molecule of the present invention and the other therapeutic agent used in the drug combination are administered at levels no greater than when they are used alone. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosages and / or time intervals of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0161] The term "combination therapy" or "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. Such administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in 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 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.

[0162] As used herein, "prevention" includes the inhibition of the development or progression of a disease or condition, or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0163] I. Multispecific Antibodies of the Present Invention

[0164] Through in-depth research, the inventors discovered 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 to Trop2 and Nectin4, wherein the antibody comprises at least one antigen-binding domain that specifically binds to Trop2 and at least one antigen-binding domain that specifically binds to Nectin4. In some aspects, to increase the half-life of the antibodies of the present invention in animal circulation, the antibodies of the present invention further comprise a half-life-extending domain, such as a serum albumin-binding peptide or an immunoglobulin Fc region. The antibodies of the present invention can take 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.

[0165] The following describes in detail the components of the multispecific antibodies of the present invention. Those skilled in the art will appreciate that, unless the context clearly indicates otherwise, any combination of any technical features of these components is contemplated by the present invention. Furthermore, those skilled in the art will appreciate that, unless the context clearly indicates otherwise, the antibodies of the present invention (including any form of antibody) may include any such combination of features.

[0166] Antigen binding domain

[0167] Although high-affinity antibodies are favored in early antibody screening, more and more 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 quickly eliminated from the circulation. In addition, high affinity may also cause the antibody to be tightly bound or rapidly internalized by the peripheral cells of the tumor tissue initially encountered, thereby limiting the penetration of the antibody into the tumor. On the contrary, if the antibody affinity is too low, although it may increase the tumor penetration efficiency of the antibody, such antibodies will also have a low tumor retention rate because the antibody cannot effectively bind to the receptor. Therefore, depending on the specific application of the antibody or antibody-based molecule, it is more advantageous in some cases to screen and adopt antibodies with medium affinity that can allow the antibody to be effectively delivered throughout the tumor and allow the antibody to accumulate in the tumor. In this article, "medium affinity" refers to the binding affinity K of the antigen binding domain for its target epitope, for example, as measured by surface plasmon resonance technology (SPR). D The values ​​were equal to or higher than 1 nM, but less than 500 nM.

[0168] With respect to the antibodies targeting Trop2 and Nectin4 of the present invention, the inventors have discovered in their research that, in some cases (e.g., when using antibodies as ADC delivery vehicles), by selecting Trop2 and Nectin4 antigen-binding domains with intermediate binding affinity, and by adjusting the valency of the Trop2 and Nectin4 antigen-binding domains in the antibody, the multispecific antibodies of the present invention can advantageously be endowed with additional advantages in terms of efficacy and safety. 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 high valency (e.g., bivalent) of the intermediate affinity Nectin4 binding domain, the avidity is increased, which can balance the efficacy and toxicity of the antibody. In addition, by selecting the antigen-binding domain in the antibody as a VHH domain based on a nanobody, the enrichment rate and amount of the antibody in tumor tissue can be further improved.

[0169] 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 a moderate binding affinity for Trop2 (preferably human Trop2), for example, a binding affinity of K D The value is 50x10 -8 M to 0.5x10 -8 or 50x10 -8 M to 1x10 -8 M, optionally 15x10 -8 M to 1x10 -8In some embodiments, the Nectin4 antigen binding domain of the antibody according to the present invention has a medium binding affinity for Nectin4 (preferably human Nectin4), for example, a binding affinity K D The value is 50x10 -8 M to 0.5x10 -8 or 50x10 -8 M to 1x10 -8 M, optionally 30x10 - 8 M to 1x10 -8 In some embodiments, the antigen binding affinity of the Trop2 and Nectin4 antigen binding domains is K D The values ​​are 50x10 -8 M to 0.5x10 -8 In some embodiments, the binding affinity of the Trop2 binding domain to Trop2 is K D The value is 10x10 -8 M to 1x10 -8 M, and wherein the binding affinity of the Nectin4 binding domain to Nectin4 is 30x10 -8 M to 1x10 -8 M, optionally 20x10 -8 M to 1x10 -8 M.

[0170] 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 of the multispecific antibody according to the present invention (that is, the total number of antigen binding domains) is 2-5 valences, preferably not more than 4 valences. In some embodiments, the multispecific antibody according to the present invention is a trivalent antibody, which comprises one of the medium-affinity Trop2 binding domains and two of the medium-affinity Nectin4 binding domains. In other embodiments, the multispecific antibody according to the present invention is a tetravalent antibody, which comprises two of the medium-affinity Trop2 binding domains and two of the medium-affinity Nectin4 binding domains.

[0171] In some embodiments, the antibodies of the present invention are "isolated" antibodies. As used herein, "isolated" antibodies refer to artificial antibodies, recombinantly produced antibodies, and antibodies that have been at least partially separated from components of the natural environment in which they were produced. In some embodiments, the isolated antibodies are purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0172] Trop2 antigen-binding domain

[0173] In the multispecific antibodies according to the present invention, in some embodiments, preferably, the antigen binding site that specifically binds to TROP2 is composed of a VHH domain (also abbreviated herein as VHH Trop2 )supply.

[0174] In some embodiments, the VHH according to the invention Trop2 The domain comprises a CDR1, CDR2, and CDR3 sequence in a variable region having an amino acid sequence selected from the group consisting of 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 more preferably, the CDRs are defined according to AbM. However, it should be understood that the CDRs may also be defined using any other method known in the art.

[0175] In some embodiments, the VHH according to the invention TROP2 The domain comprises CDR1, CDR2 and CDR3 sequences, wherein the CDR1, CDR2 and CDR3 sequences:

[0176] (i) comprising or consisting of the amino acid sequences of SEQ ID Nos: 6, 7 and 8, respectively;

[0177] (ii) comprising or consisting of the amino acid sequences of SEQ ID Nos: 10, 11 and 12, respectively;

[0178] (iii) comprising or consisting of the amino acid sequences of SEQ ID Nos: 14, 15 and 16, respectively; or

[0179] (iv) comprising or consisting of the amino acid sequences of SEQ ID Nos: 31, 32 and 33, respectively.

[0180] In some embodiments, the VHH according to the invention TROP2The domain comprises: a VHH variable region sequence of any of the exemplary antibodies of the present invention or a variant thereof, for example, an antibody or fragment thereof having the same CDR sequence as one of the exemplary antibodies and having the same or different framework region sequence, such as a humanized antibody. In some cases, it is preferred to mutate one to several residues of the framework sequence, for example, to remove post-translational modifications (PTMs) and / or deimmunization. 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.

[0181] In some embodiments, the VHH according to the 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 that is at least 80%, 85%, 90%, 95% or 99% identical to an amino acid sequence selected from SEQ ID Nos: 5, 9, 13, 29, 30, 98 and 99 and that retains 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 that has 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 retains the ability to specifically bind to TROP2. Preferably, the amino acid additions, deletions, and / or substitutions do not occur in the CDR region.

[0182] In some preferred embodiments, the VHH according to the present invention TROP2 The VHH domain comprises the CDR1-3 sequences of SEQ ID Nos: 10-12. 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. TROP2 The domain comprises the amino acid sequence of SEQ ID NO: 9, or consists of the amino acid sequence shown in SEQ ID NO: 9.

[0183] In some preferred embodiments, the VHH according to the present invention TROP2 The VHH domain comprises the CDR1-3 sequences of SEQ ID Nos: 31-33. 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 thereto. TROP2 The domain comprises the amino acid sequence of SEQ ID NO: 29 or 30, or consists of the amino acid sequence shown in SEQ ID NO: 29 or 30.

[0184] Nectin4 antigen-binding domain

[0185] In the multispecific antibodies according to the present invention, in some embodiments, preferably, the antigen binding site that specifically binds to NECTIN4 consists of a VHH domain (also abbreviated herein as VHH Nectin4 )supply.

[0186] In some embodiments, the VHH according to the invention Nectin4 The domain comprises a variable region comprising a CDR1, CDR2, and CDR3 sequence selected from the group consisting of 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 more preferably, according to AbM. However, it should be understood that the CDRs may also be defined using any other method known in the art.

[0187] In some embodiments, the VHH according to the invention NECTIN4 The domain comprises CDR1, CDR2 and CDR3 sequences, wherein the CDR1, CDR2 and CDR3 sequences:

[0188] (i) comprising or consisting of the amino acid sequences of SEQ ID Nos: 18, 19 and 20, respectively;

[0189] (ii) comprising or consisting of the amino acid sequences of SEQ ID Nos: 22, 23 and 24, respectively;

[0190] (iii) comprising or consisting of the amino acid sequences of SEQ ID Nos: 26, 27, and 28, respectively;

[0191] (iv) comprising or consisting of the amino acid sequences of SEQ ID Nos: 18, 36 and 20, respectively; or

[0192] (v) comprising or consisting of the amino acid sequences of SEQ ID Nos: 18, 37 and 20, respectively.

[0193] In some embodiments, the VHH according to the invention NECTIN4 The domain comprises: a VHH variable region sequence of any of the exemplary antibodies of the present invention or a variant thereof, for example, an antibody or fragment thereof having the same CDR sequence as one of the exemplary antibodies and having the same or different framework region sequence, such as a humanized antibody. In some cases, it is preferred to mutate one to several residues of the framework sequence, for example, to remove post-translational modifications (PTMs) and / or deimmunization. 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.

[0194] In some embodiments, the VHH according to the invention NECTIN4 The 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 other embodiments, the VHH according to the present invention NECTIN4 The domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to an amino acid sequence selected from SEQ ID Nos: 17, 21, 25, 34, 35, 38 and 100-106 and that retains the ability to specifically bind to NECTIN4. In still other embodiments, the VHH according to the present invention NECTIN4 The domain comprises an amino acid sequence that has 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 retains the ability to specifically bind to NECTIN 4. Preferably, the amino acid additions, deletions, and / or substitutions do not occur in the CDR regions.

[0195] In some preferred embodiments, the VHH according to the present invention NECTIN4 The VHH domain comprises the CDR1-3 sequences of SEQ ID Nos: 22-24. NECTIN4 The 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. NECTIN4 The domain comprises the amino acid sequence of SEQ ID NO: 21, or consists of the amino acid sequence shown in SEQ ID NO: 21.

[0196] In some preferred embodiments, the VHH according to the present invention NECTIN4The VHH domain comprises a CDR1 sequence of SEQ ID NO: 18, a CDR2 sequence of SEQ ID NO: 36, and a CDR3 sequence of SEQ ID NO: 20. In some embodiments, the VHH NECTIN4 The domain comprises 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 thereto. NECTIN4 The domain comprises the amino acid sequence of SEQ ID NO: 34 or 38, or consists of the amino acid sequence shown in SEQ ID NO: 34 or 38.

[0197] In some preferred embodiments, the VHH according to the present invention NECTIN4 The VHH domain comprises a CDR1 sequence of SEQ ID NO: 18, a CDR2 sequence of SEQ ID NO: 37, and a CDR3 sequence of SEQ ID NO: 20. In some embodiments, the VHH NECTIN4 The domain comprises 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 thereto. NECTIN4 The domain comprises the amino acid sequence of SEQ ID NO: 35, or consists of the amino acid sequence shown in SEQ ID NO: 35.

[0198] Half-life extension domain

[0199] According to the multispecific antibodies of the present invention, in addition to the aforementioned antigen-binding domain, in some embodiments, a half-life extension domain may also be included to adjust the circulating half-life of the antibody after administration to an animal. Preferably, the half-life extension domain used in the antibody of the present invention is a peptide or polypeptide structure, which can be coupled 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 regions, serum albumin or serum albumin binding peptides. In some embodiments, preferably, the antibody of the present invention comprises an immunoglobulin Fc region, especially when the antibody adopts a two-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 connected to the C or N terminus of the TROP2 binding domain or to the C or N terminus of NECTIN4.

[0200] Immunoglobulin Fc region

[0201] 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, interacting with some proteins of the cell surface Fc receptors and the complement system. The immunoglobulin Fc region generally comprises two or three heavy chain constant domains (referred to as CH2, CH3 and CH4) and a hinge region, and generally exists in a dimerized form. The two chains in the dimerized Fc region can be connected by the disulfide bond in the hinge region. The Fc region from immunoglobulin isotypes IgG1, IgG2 and IgG4 can bind to FcRn receptors and undergo FcRn-mediated recycling to provide a long circulation half-life. The interaction position of IgG and FcRn has been determined in the Fc region of the CH2 and CH3 domains of the covering portion.

[0202] The immunoglobulin Fc region used 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 an immunoglobulin CH2 domain and a CH3 domain. In some embodiments, the immunoglobulin Fc region further comprises a hinge region or a portion of a hinge region. In some embodiments, the immunoglobulin Fc region comprises, or consists of, an immunoglobulin hinge region or a portion of a hinge region, a CH2 domain, and a CH3 domain from the N-terminus to the C-terminus. In some embodiments, the immunoglobulin Fc region comprises, or consists of, a CH2 domain and a CH3 domain from the N-terminus to the C-terminus. In some embodiments, the immunoglobulin Fc region is preferably derived from IgG1, IgG2, or IgG4, or a subtype thereof. Preferably, the immunoglobulin Fc region comprises an Fc region sequence from a human.

[0203] The immunoglobulin Fc region 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 immunoglobulin Fc region 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, the fusion is preferably performed via the immunoglobulin hinge region sequence.

[0204] In some cases, an immunoglobulin Fc region comprising a hinge region sequence is preferred, which can, for example, promote dimerization of the antibody polypeptide chain and / or provide cysteine ​​residues for coupling to other active molecules. Such a hinge region may correspond substantially or partially to the hinge region of IgG1, IgG2, IgG3 or IgG4. For example, the hinge region sequence may 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 two Fc chains. In some embodiments, the hinge region sequence comprises the hinge region sequence from E216 to T225 of IgG1 or the hinge region sequence from D221 to T225 (according to EU numbering), or corresponding hinge region sequences from other immunoglobulin isotypes. In some embodiments, the hinge region sequence comprises the amino acid sequence of SEQ ID NO: 96 or 97.

[0205] The immunoglobulin Fc region used in 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, a Knob-into-Hole (KiH) mutation 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, a combination of Knob mutation T366W and Hole mutations T366S, L368A, and Y407V; and a combination of Knob mutation T366Y and Hole mutation Y407T. When the multispecific antibodies of the present invention comprise an asymmetric double-chain structure, it is preferred that the Fc region contain a KiH mutation that promotes proper heterodimerization of the antibody polypeptide chains. Additionally or alternatively, cysteine ​​mutations can be introduced in the Fc region to increase disulfide linkages in the dimerized Fc region, for example, introducing the mutation S354C in one Fc chain and the mutation Y349C in the other Fc chain.

[0206] In addition, according to the specific application of the antibody or antibody-based molecule, the Fc region may also include mutations that change the effector function. For example, in the case where the effector function is not required, the Fc region may include mutations that reduce or eliminate the effector function. In some cases (for example, in the case of using the antibody of the present invention as an ADC carrier), preferably, the Fc region includes mutations that reduce or eliminate the Fc region and Fcγ receptors, such as LALA mutations 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 coupling 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 modification (such as glycosylation) to provide improved drugability and developability of therapeutic antibodies.

[0207] In some embodiments, the multispecific antibody according to the present invention comprises an Fc region from an IgG, such as an IgG1, IgG2, or IgG4 Fc region, preferably an 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.

[0208] serum albumin binding peptide

[0209] 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 attached non-covalently through the use of specific albumin binding domains, or covalently by conjugation or direct genetic fusion.

[0210] The albumin binding domain used in the multispecific antibodies of the present invention is preferably a peptide or polypeptide that binds to serum albumin. Such peptides or polypeptides are also referred to herein as "serum albumin binding peptides." Examples of serum albumin binding peptides that can be used in the present invention include, for example, antibodies and antibody fragments that specifically bind to serum albumin, such as single domain antibody-based (O'Connor-Semmes et al. Clin Pharmacol Ther. 2014 Dec;96(6):704-12); and artificial proteins based on natural proteins that bind to serum albumin (e.g., Streptococcus G protein), e.g. Other examples of serum albumin binding peptides that can be used in the present invention include, for example, the many ISVDs that bind to human serum albumin described in WO 04 / 041865, WO 06 / 122787, and WO 2012 / 175400, wherein the ISVD is selected from the serum albumin binding portion of Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10, and Alb-23.

[0211] 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 of the peptide. 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 linker can be easily determined based on the connection position. In some embodiments, the peptide linker is preferably 5-15 amino acids in length, and more preferably, the peptide linker comprises the amino acid sequence G4S or (G4S)2.

[0212] In some embodiments, the serum albumin binding peptide used in the multispecific antibody of the present invention is an antibody or antibody fragment that specifically binds to serum albumin, in particular a single-chain antibody or a single-domain antibody. In some embodiments, the serum albumin binding peptide is a VHH domain (also abbreviated herein as VHH) that specifically binds to serum albumin. 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.

[0213] In some embodiments, a multispecific antibody according to the present invention comprises a VHH domain that specifically binds to serum albumin. SA comprising CDR1, CDR2, and CDR3 in a variable region sequence having an amino acid sequence selected from SEQ ID NO: 39 or 40. The CDRs may be defined according to AbM, Chothia, Kabat, IMGT, or any combination thereof. In some embodiments, the VHH SA comprising 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 Comprising or consisting of the amino acid sequence of SEQ ID NO: 40.

[0214] Peptide linker

[0215] In the multispecific antibodies according to the present invention, the antibody components (ie, the antigen binding domain and optionally the half-life binding domain) may be connected using a peptide linker.

[0216] There are no specific restrictions on the peptide linkers that can be used in the antibodies of the present invention. The peptide linker sequence is generally flexible. It can be mainly composed of amino acids that do not have large side chains that may limit flexibility, such as glycine, alanine and serine. Alternatively, it can be composed of sequences from the hinge region of an immunoglobulin. Depending on the connection position and the assembly to be connected, those skilled in the art can easily determine the sequence or optimal length of an available peptide linker.

[0217] Suitable peptide linker length 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 acid lengths, or longer.In some cases, the length of peptide linker sequence can be shorter, for example, less than about 20 or 15 amino acid lengths, for example 2-15 amino acid lengths or 5-10 amino acid lengths.

[0218] 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), wherein n is an integer equal to or greater than 1; TS(G4S)n (SEQ ID NO: 92), wherein n is an integer equal to or greater than 1; G(G4S)n (SEQ ID NO: 93), wherein n is an integer equal to or greater than 1; (G4)n (SEQ ID NO: 94); NO:94), wherein n is an integer equal to or greater than 1; (GRPGS)n(SEQ ID NO:95), wherein 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). NO: 137), GGRRGGGS (SEQ ID NO: 138), LRQRDGERP (SEQ ID NO: 139), LRQKDGGGSERP (SEQ ID NO: 140), and GSTSGSGKPGSGEGSTKG (SEQ ID NO: 141). Alternatively, suitable flexible linker peptides can be rationally designed by using computer programs to simulate the three-dimensional structure of proteins and peptides, or by phage display methods.

[0219] In some embodiments, the peptide linker used in the antibody of the present invention is a flexible connecting peptide of 5-50 amino acids, preferably a connecting 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, 5, 10, 15, 20, 25 or 30 amino acids, or 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), wherein 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 a hinge region from an immunoglobulin.

[0220] In some preferred embodiments, in the multispecific antibodies according to the present invention, the peptide linker used to connect 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).

[0221] Multispecific antibody formats

[0222] The multispecific antibodies according to the present invention may be in any suitable form, such as single-chain or two-chain form.

[0223] In some embodiments, the present invention provides a multispecific antibody in single-chain form. In some embodiments, the multispecific antibody according to the present invention comprises a single polypeptide chain, wherein the polypeptide chain comprises at least one (preferably one or two) Nectin4 binding domains and at least one (preferably one or two) Trop2 binding domains 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 connected by a peptide linker.

[0224] In some embodiments, the present invention provides a two-chain multispecific antibody. In some embodiments, the multispecific antibody according to the present invention comprises a first and a second polypeptide chain, wherein the first and second polypeptide chains respectively comprise at least one (preferably one or two) Nectin4 binding domains and / or at least one (preferably one or two) Trop2 binding domains and at least one (preferably one) half-life extension domain, wherein the half-life extension domain is an immunoglobulin Fc region, and preferably the domains on the same chain are connected by a peptide linker or directly connected.

[0225] Single-chain form

[0226] In some embodiments, the present invention provides a multispecific antibody comprising a single polypeptide chain. In some embodiments, the polypeptide chain comprises, from N-terminus to C-terminus:

[0227] (VHH A ) n1 -(VHH B ) n2 -(HLE) n3 -(VHH A ) n4 -(VHH B ) n5 -(HLE) n6， (I)

[0228] wherein n1, n2, n3, n4, n5 and n6 are independently selected from integers of 0, 1 or 2; wherein VHH A and VHH B wherein the VHH domains respectively represent the 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 a serum albumin binding peptide as a half-life extension domain; wherein the symbol "-" represents connection via a peptide linker or direct connection, preferably represents a peptide linker of 5-15 amino acids in length.

[0229] The multispecific antibody may have any suitable valency. In some cases, it is preferred that the multispecific antibody has 2 to 6 valencies (i.e., n1+n2+n4+n5=2 to 6), more preferably no more than 4 valencies (i.e., n1+n2+n4+n5=2 to 4).

[0230] VHH in the multispecific antibody Nectin4 Domains and VHH Trop2 The number of domains can be equal or unequal. In some embodiments, preferably, VHH Trop2 The number of domains is no more than 4, preferably no more than 3, for example 1-2. In some embodiments, preferably VHH Nectin4 The number of domains is no more than 4, preferably no more than 3, for example 1-2. In some embodiments, preferably, VHH Trop2 Domain structure and VHH Nectin4 The ratio of the number of domains is preferably 1:1 or 1:2. In one embodiment, VHH Trop2 The number of domains is 1 or 2, and VHH Nectin4 The number of domains is 1 or 2.

[0231] The multispecific antibody may or may not contain an HLE domain as desired, for example, the antibody may contain 0 or 1 HLE.

[0232] In some preferred embodiments, the present invention provides a multispecific antibody of the above-mentioned formula I, wherein n1, n2, n3, n4, n5 and n6 are independently selected from integers of 0, 1 or 2; and wherein n1+n4=1 or 2, n2+n5=1 or 2, and n3+n6=0 or 1.

[0233] In some more preferred embodiments, the present invention provides a multispecific antibody comprising a single polypeptide chain, wherein the polypeptide chain comprises, from N-terminus to C-terminus:

[0234] (i) VHH A -VHH B ,

[0235] (ii) VHH A -VHH B -HLE,

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

[0237] (iv) VHH A -VHH B -VHH B ,

[0238] (v)VHH A -HLE-VHH B -VHH B ,or

[0239] (vi)VHH A -VHH A -HLE-VHH B -VHH B , wherein preferably A represents Trop2 and B represents Nectin4.

[0240] In the above single-chain antibody embodiment, preferably, the HLE represents an anti-serum albumin VHH domain (VHH SA ). The VHH SA The domain may be any VHH described herein or known in the art SA For 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 thereto, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions. In some embodiments, preferably, the HLE comprises or consists of the amino acid sequence of SEQ ID NO: 40.

[0241] In the above single-chain antibody embodiment, preferably, each domain is connected by a peptide linker. Preferably, the peptide linker has a length of 5-15 amino acids, and more preferably comprises a G4S amino acid sequence or a (G4S)2 amino acid sequence.

[0242] As those skilled in the art will appreciate, to facilitate subsequent purification or linkage to other active molecules, the single-chain multispecific antibodies of the present invention may optionally have a purification tag (e.g., a His tag) or other auxiliary peptide sequence added to the N-terminus or C-terminus of the polypeptide chain. Such addition can be achieved via a peptide linker (e.g., AAA) or by direct linkage.

[0243] The multispecific antibody having a polypeptide chain of formula (I) is preferably a trivalent bispecific antibody or a tetravalent bispecific antibody.

[0244] Symmetrical double-stranded form

[0245] In some embodiments, the present invention provides multispecific antibodies comprising two identical polypeptide chains. In some embodiments, the present invention provides multispecific antibodies comprising the following polypeptide chains, wherein the polypeptide chains comprise, from N-terminus to C-terminus:

[0246] (VHH A ) n1 -(VHH B ) n2 -HLE-(VHH A ) n3 -(VHH B ) n4， (II)

[0247] wherein n1, n2, n3 and n4 are independently selected from an integer of 0, 1 or 2;

[0248] Among them VHH A and VHH Bwherein the VHH domains respectively represent the 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 extending domain, in particular a human IgG1 or human IgG4 Fc region; wherein the symbol "-" represents connection via a peptide linker or direct connection, preferably a peptide linker of 5-15 amino acids in length.

[0249] The multispecific antibody may have any suitable valency. In some cases, it is preferred that the multispecific antibody has 2 to 6 valencies (ie, 2x(n1+n2+n3+n4)=2 to 6), more preferably no more than 4 valencies.

[0250] VHH in the multispecific antibody Nectin4 Domains and VHH Trop2 The number of domains can be equal or unequal. In some embodiments, preferably, VHH Trop2 The number of domains is no more than 4, preferably no more than 3, for example 1-2. In some embodiments, preferably VHH Nectin4 The number of domains is no more than 4, preferably no more than 3, for example 1-2. In some embodiments, preferably, VHH Trop2 Domain structure and VHH Nectin4 The ratio of the number of domains is preferably 1:1 or 1:2. In one embodiment, VHH Trop2 The number of domains is 1 or 2, and VHH Nectin4 The number of domains is 1 or 2.

[0251] In some preferred embodiments, n3 and n4 are 0, and the polypeptide chain of formula (II) comprises from N-terminus to C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE, wherein n1 and n2 are independently selected from an integer of 1 or 2.

[0252] In some more preferred embodiments, n3 and n4 are 0, and n1 and n2 are 1, and the polypeptide chain of formula (II) comprises, from N-terminus to C-terminus: VHH A -VHH B -HLE, such as VHH Nectin4 -VHH Trop2 -Fc or VHH Trop2 -VHH Nectin4 -Fc.

[0253] In some further preferred embodiments, the polypeptide chain of formula (II) comprises VHH from N-terminus to C-terminus Nectin4 -VHHTrop2 -Fc.

[0254] In the above multispecific antibodies, preferably, each antigen-binding domain on the polypeptide chain (ie, VHH Trop2 Domain structure and VHH Nectin4 The Fc region is connected to the N-terminal domain by a peptide linker. Preferably, the peptide linker has a length of 5-15 amino acids, and more preferably comprises a G4S amino acid sequence or a (G4S)2 amino acid sequence. In the above-mentioned multispecific antibody, preferably, the Fc region comprises a hinge region or a portion of the hinge region and is directly connected to the N-terminal domain.

[0255] Multispecific antibodies having polypeptide chains of formula (II) above can associate to form homodimers due to dimerization of the immunoglobulin Fc region, thereby generating a two-chain multispecific binding molecule. In some embodiments, the Fc region comprises an amino acid sequence from human IgG1 or IgG4. In some embodiments, the Fc region comprises a mutation that reduces or eliminates Fcγ receptor binding, such as a LALA mutation.

[0256] The multispecific antibody having a polypeptide chain of formula (II) is preferably a tetravalent bispecific antibody.

[0257] Asymmetric double-stranded form

[0258] 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

[0259] The first polypeptide chain comprises from N-terminus to C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE-(VHH A ) n3 -(VHH B ) n4， (III)

[0260] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH B ) m1 -(VHH A ) m2 -HLE-(VHH B ) m3 -(VHH A ) m4， (IV)

[0261] wherein n1, n2, n3 and n4 and m1, m2, m3 and m4 are independently selected from an integer of 0, 1 or 2;

[0262] Among them VHH A and VHH B wherein the VHH domains respectively represent the 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, in particular a human IgG1 or IgG4 Fc region; wherein the symbol "-" represents connection via a peptide linker or direct connection, preferably a peptide linker of 5-15 amino acids in length.

[0263] The multispecific antibody may have any suitable valency. In some cases, it is preferred that the multispecific antibody has 2 to 6 valencies (i.e., n1+n2+n3+n4+m1+m2+m3+m4=2 to 6), more preferably no more than 4 valencies.

[0264] VHH in the multispecific antibody Nectin4 Domains and VHH Trop2 The number of domains can be equal or unequal. In some embodiments, preferably, VHH Trop2 The number of domains is no more than 4, preferably no more than 3, for example 1-2. In some embodiments, preferably VHH Nectin4 The number of domains is no more than 4, preferably no more than 3, for example 1-2. In some embodiments, preferably, VHH Trop2 Domain structure and VHH Nectin4 The ratio of the number of domains is preferably 1:1 or 1:2. In one embodiment, VHH Trop2 The number of domains is 1 or 2, and VHH Nectin4 The number of domains is 1 or 2.

[0265] In some preferred embodiments, n3, n4, m3 and m4 are 0, and the multispecific antibody comprises a first and a second polypeptide chain, wherein

[0266] The first polypeptide chain comprises from N-terminus to C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE ，

[0267] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH B ) m1 -(VHH A ) m2 -HLE ，

[0268] wherein n1, n2 and m1, m2 are independently selected from integers of 0, 1 or 2, but n1 and n2 are not simultaneously 0, m1 and m2 are not simultaneously 0, and the sum of n1 and m2 and the sum of n2 and m1 are greater than or equal to 1.

[0269] In some more preferred embodiments, n3, n4, m3 and m4 are 0, and m1 = 0 and m2 = 1 or m2 = 0 and m1 = 1, the multispecific antibody comprises a first and a second polypeptide chain, wherein

[0270] The first polypeptide chain comprises from N-terminus to C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE ，

[0271] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH B ) m1 -HLE ； Or (VHH A ) m2 -HLE ，

[0272] wherein n1, m1 and m2 are independently selected from an integer of 1 or 2, and n2 is an integer of 0 or 1.

[0273] 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, the multispecific antibody comprises a first and a second polypeptide chain, wherein

[0274] The first polypeptide chain comprises from N-terminus to C-terminus: (VHH A )-HLE ，

[0275] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH B )-HLE ；

[0276] or,

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

[0278] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH A )-HLE ；

[0279] or,

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

[0281] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH B )-HLE ；

[0282] or,

[0283] The first polypeptide chain comprises from N-terminus to C-terminus: (VHH A )-(VHH B )-HLE ，

[0284] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH B )-HLE.

[0285] In some further preferred embodiments, the multispecific antibody comprises a first and a second polypeptide chain, wherein:

[0286] The first polypeptide chain comprises from N-terminus to C-terminus: (VHH A )-HLE ，

[0287] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH B )-HLE ；

[0288] or,

[0289] The first polypeptide chain comprises from N-terminus to C-terminus: (VHH A )-(VHH B )-HLE ，

[0290] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH A )-HLE ；

[0291] or,

[0292] The first polypeptide chain comprises from N-terminus to C-terminus: (VHH A )-(VHH A )-HLE ，

[0293] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH B )-HLE ；

[0294] Wherein A represents NECTIN4, and B represents TROP2.

[0295] In some most preferred embodiments, the multispecific antibody comprises a first and a second polypeptide chain, wherein:

[0296] The first polypeptide chain comprises from N-terminus to C-terminus: VHH Nectin4 -VHH Trop2 -Fc; and

[0297] The second polypeptide chain comprises from N-terminus to C-terminus: VHH Nectin4 -Fc.

[0298] Due to the dimerization of the immunoglobulin Fc region, the first and second polypeptide chains of the above-mentioned multispecific antibody can associate to form a heterodimer, thereby producing a two-chain multispecific binding molecule. Preferably, in order to promote the heterodimerization of the first and second polypeptide chains, a knob-into-hole mutation can be introduced into the Fc region of the first and second polypeptide chains, such as a (T366W / T366S, L368A, Y407V) mutation or a (T366Y / Y407T) mutation. Preferably, the Fc region comprises an amino acid sequence from human IgG1 or IgG4, and preferably, the Fc region further comprises a mutation that reduces or eliminates Fcγ receptor binding, such as a LALA mutation.

[0299] 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 comprise mutations that reduce or eliminate the binding of the Fc region to FcγR, for example, L234AL235A mutations. In some embodiments, preferably, the first and second Fc regions comprise an amino acid sequence selected from SEQ ID NOs: 111-116, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto.

[0300] In the above multispecific antibodies, preferably, each antigen-binding domain on the polypeptide chain (ie, VHH Trop2 Domain structure and VHH Nectin4The Fc region is connected to the N-terminal domain by a peptide linker. Preferably, the peptide linker has a length of 5-15 amino acids, and more preferably comprises a G4S amino acid sequence or a (G4S)2 amino acid sequence. In the above-mentioned multispecific antibody, preferably, the Fc region comprises a hinge region or a portion of the hinge region and is directly connected to the N-terminal domain.

[0301] 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.

[0302] Example antigenic domain combinations

[0303] In some embodiments of the above-mentioned single-chain and two-chain multispecific antibodies according to the present invention, preferably, the multispecific antibodies according to the present invention comprise the following combination of TROP2 and NECTIN4 antigen-binding domains:

[0304] (a) the antigen-binding domain that specifically binds to Nectin4 comprises the three CDRs in the amino acid sequence of SEQ ID NO: 25, and the antigen-binding domain that specifically binds to Trop2 comprises the three CDRs in the amino acid sequence of SEQ ID NO: 13;

[0305] (b) the antigen-binding domain that specifically binds to Nectin4 comprises the three CDRs in the amino acid sequence of SEQ ID NO: 21 and the antigen-binding domain that specifically binds to Trop2 comprises the three CDRs in the amino acid sequence of SEQ ID NO: 9;

[0306] (c) the antigen-binding domain that specifically binds to Nectin4 comprises the three CDRs in the amino acid sequence of SEQ ID NO: 25, and the antigen-binding domain that specifically binds to Trop2 comprises the three CDRs in the amino acid sequence of SEQ ID NO: 9;

[0307] (d) the antigen-binding domain that specifically binds to Nectin4 comprises three CDRs in the amino acid sequence of SEQ ID NO: 35 and the antigen-binding domain that specifically binds to Trop2 comprises three CDRs selected from the amino acid sequences of SEQ ID NOs: 29 and 30; or

[0308] (e) the antigen-binding domain that specifically binds to Nectin4 comprises three CDRs selected from the amino acid sequences of SEQ ID NOs: 34 and 38, and the antigen-binding domain that specifically binds to Trop2 comprises three CDRs selected from the amino acid sequences of SEQ ID NOs: 29 and 30.

[0309] In some embodiments of the single-chain and two-chain multispecific antibodies according to the present invention, more preferably, the multispecific antibodies according to the present invention comprise the following combination of TROP2 and NECTIN4 antigen binding domains:

[0310] (a) the antigen-binding domain that specifically binds to Nectin4 comprises the amino acid sequence of SEQ ID NO: 21, and the antigen-binding domain that specifically binds to Trop2 comprises the amino acid sequence of SEQ ID NO: 9;

[0311] (b) the antigen-binding domain that specifically binds to Nectin4 comprises the amino acid sequence of SEQ ID NO: 35 and the antigen-binding domain that specifically binds to Trop2 comprises an amino acid sequence selected from SEQ ID NOs: 29 and 30; or

[0312] (c) the antigen-binding domain that specifically binds to Nectin4 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 and 38, and the antigen-binding domain that specifically binds to Trop2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 and 30.

[0313] In some embodiments of the single-chain and two-chain multispecific antibodies according to the present invention, more preferably, the multispecific antibodies according to the present invention comprise the following combination of TROP2 and NECTIN4 antigen binding domains:

[0314] (a) the antigen-binding domain that specifically binds to Nectin4 comprises an amino acid sequence selected from SEQ ID NO: 34 and the antigen-binding domain that specifically binds to Trop2 comprises an amino acid sequence selected from SEQ ID NO: 29; or

[0315] (b) the antigen-binding domain that specifically binds to Nectin4 comprises an amino acid sequence selected from SEQ ID NO: 38, and the antigen-binding domain that specifically binds to Trop2 comprises an amino acid sequence selected from SEQ ID NO: 30.

[0316] Exemplary multispecific antibodies

[0317] In some embodiments, the present invention provides a multispecific antibody, wherein the antibody is in a symmetrical two-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 that is at least 95%, 96%, 97%, 98% or 99% identical thereto, preferably comprises the amino acid sequence of SEQ ID NO: 53.

[0318] In some embodiments, the present invention provides a multispecific antibody, wherein the antibody is in an asymmetric two-chain format, and wherein the antibody comprises a first and a second polypeptide chain selected from the group consisting of:

[0319] (i) a first and a second polypeptide chain comprising SEQ ID NOs: 55 and 56, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0320] (ii) a first and a second polypeptide chain comprising SEQ ID NOs: 57 and 58, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0321] (iii) a first and a second polypeptide chain comprising SEQ ID NOs: 59 and 60, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0322] (iv) a first and a second polypeptide chain comprising SEQ ID NOs: 61 and 62, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0323] (v) a first and a second polypeptide chain comprising SEQ ID NOs: 63 and 64, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0324] (vi) a first and a second polypeptide chain comprising SEQ ID NOs: 65 and 66, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0325] (vii) a first and a second polypeptide chain comprising SEQ ID NOs: 67 and 68, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0326] (viii) a first and a second polypeptide chain comprising SEQ ID NOs: 69 and 70, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0327] (ix) a first and a second polypeptide chain comprising SEQ ID NOs: 71 and 72, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0328] (x) a first and a second polypeptide chain comprising SEQ ID NOs: 73 and 74, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto, and

[0329] (xi) a first and a second polypeptide chain comprising SEQ ID NOs: 75 and 76, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0330] Preferably, the first and second polypeptide chains comprise the amino acid sequences of SEQ ID NOs: 61 / 62, 69 / 70 or 71 / 72, respectively.

[0331] In some embodiments, the present invention provides a multispecific antibody, wherein the antibody is in a single chain form, wherein the polypeptide chain of the antibody comprises an amino acid sequence selected from SEQ ID NOs:41-52 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical thereto.

[0332] Characteristics of the multispecific antibodies of the present invention

[0333] The multispecific antibodies of the present invention may have one or more of the following properties:

[0334] (i) Binds with high avidity to tumor cells co-expressing TROP2 and NECTIN4;

[0335] (ii) has species cross-reactivity with cynomolgus monkey TROP2 and NECTIN4;

[0336] (iii) having TROP2 and / or NECTIN4 receptor-mediated endocytosis activity;

[0337] (iv) exhibited synergistic binding on tumor cells co-expressing TROP2 and NECTIN4 antigens;

[0338] (v) Synergistic endocytosis was observed in tumor cells co-expressing TROP2 and NECTIN4 antigens.

[0339] In some aspects, the multispecific antibodies of the present invention have high binding affinity for tumor cells that co-express TROP2 and NECTIN4. The EC50 value and / or maximum binding amount of the antibodies of the present invention for binding to TROP2 and / or NECTIN4-positive tumor cells can be determined by FACS or ELSA assays (e.g., the assays described in the Examples), and optionally compared with a reference antibody to reflect the cell binding affinity of the antibodies. In some embodiments, the multispecific antibodies of the present invention exhibit synergistic binding to tumor cells that co-express TROP2 and NECTIN4 compared to the monospecific binding molecules for TROP2 and NECTIN4.

[0340] 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 of the two is between 1-10, for example, between 1-5, more preferably between about 1-3. In further 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 of the two (human / monkey KD ratio or monkey / human KD ratio) is between 1-10, for example, between 1-5, more preferably between about 1-3. 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 of the two is between 1-5, more preferably between about 1-3. In still other 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 of the two (human / monkey KD ratio or monkey / human KD ratio) is between 1-10, such as between 1-5, more preferably between about 1-3.

[0341] In some embodiments, the multispecific antibodies of the present invention have endocytic activity mediated by TROP2 and / or NECTIN4 receptors. The endocytic activity of the antibody can be evaluated in a cell-based assay, such as the assay described in the Examples. In some embodiments, in an assay based on TROP2 and / or NECTIN4-positive cells, the antibody to be tested is incubated 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), and then the amount of antibody bound to the cell surface is detected by fluorescence by flow cytometry relative to a negative control maintained at 4°C for the same period of time to determine the endocytosis and / or endocytosis rate of the antibody. In some embodiments, when compared to a TROP2 monospecific binding molecule or a NECTIN4 monospecific binding molecule, internalization mediated by the multispecific antibodies of the present invention exhibits greater selectivity for tumor cells that co-express both targets, thereby minimizing adverse effects of the antibodies on normal tissues that do not exhibit significant levels of TROP2 and NECTIN4 co-expression, or that only express high levels of TROP2, or only express high levels of NECTIN4. In some embodiments, compared to a TROP2 and NECTIN4 monospecific binding molecule, the multispecific antibodies of the present invention exhibit synergistic endocytosis of tumor cells that co-express TROP2 and NECTIN4.

[0342] In still other embodiments, the multispecific antibodies of the invention further have one or more of the following properties: (iv) good developability; (v) good stability; and (vi) favorable pharmacokinetic properties.

[0343] In some embodiments, after one-step purification from the 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 greater than 95% as determined by SEC-HPLC.

[0344] In some further embodiments, the multispecific antibodies of the present invention have good stability. In some embodiments, as determined in the examples, after 14 days at room temperature, the antibodies of the present invention have no change in antigen binding activity compared to before storage.

[0345] In still other embodiments, the multispecific antibodies of the present invention are serum stable. 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 described in the Examples.

[0346] II. Anti-Trop2 VHH Domains and Anti-Trop2 Antibodies of the Invention

[0347] In a second aspect, the present invention provides an anti-Trop2 VHH domain that binds to Trop2 and an anti-Trop2 antibody comprising the VHH domain. In some embodiments, the anti-Trop2 VHH domain according to the present invention comprises a CDR1, CDR2, and CDR3 sequence selected from the amino acid sequences of SEQ ID NOs: 5, 9, 13, 29, 30, 98, and 99;

[0348] More preferably, the CDR1, CDR2 and CDR3 sequences are:

[0349] (i) comprising or consisting of the amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively;

[0350] (ii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively;

[0351] (iii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 14, 15 and 16, respectively; or

[0352] (iv) an amino acid sequence comprising or consisting of SEQ ID NOs: 31, 32, and 33, respectively;

[0353] Still 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 with said amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions,

[0354] Most preferably, the VHH domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 29, 30, 98 and 99, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 29, 30, 98 and 99.

[0355] 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, a single-chain antibody or a multi-chain antibody, a monospecific or multispecific antibody (e.g., a bispecific antibody), a linear antibody, a single-domain antibody, a heavy chain antibody, a chimeric antibody, or a humanized antibody. In some embodiments, the anti-Trop2 antibody according to the present invention comprises an immunoglobulin Fc region connected to the VHH domain (i.e., having a VHH-Fc format). 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 without a light chain, which may comprise VHH-CH2-CH3 from N-terminus to C-terminus, or may comprise VHH-CH1-CH2-CH3; it may constitute 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.

[0356] The anti-Trop2 VHH domains according to the present invention have excellent tumor targeting, tumor tissue penetration, and target cell endocytosis capabilities, and exhibit human and monkey immune cross-reactivity. Therefore, the anti-Trop2 VHH domains according to the present invention or the anti-Trop2 antibodies according to the present invention can be used as targeting modules in conjugates or coupled to agents such as chemotherapeutic agents, toxins, drugs (such as immunotherapeutic agents), radioactive elements, probes, or signaling molecules to provide applications such as improved tumor killing, immune regulation, or disease detection.

[0357] III. Anti-NETIN4 VHH Domains and Anti-NETIN4 Antibodies of the Invention

[0358] In a third aspect, the present invention provides an anti-NECTIN4 VHH domain that binds to NECTIN4 and an anti-NECTIN4 antibody comprising the VHH domain. In some embodiments, the anti-NECTIN4 VHH domain according to the present invention comprises a CDR1, CDR2, and CDR3 sequence selected from the amino acid sequences of SEQ ID NOs: 17, 21, 25, 34, 35, 38, and 100-106;

[0359] Preferably, the CDR1, CDR2 and CDR3 sequences are:

[0360] (i) comprising or consisting of the amino acid sequences of SEQ ID NOs: 18, 19 and 20, respectively;

[0361] (ii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 22, 23, and 24, respectively;

[0362] (iii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 26, 27, and 28, respectively;

[0363] (iv) an amino acid sequence comprising or consisting of SEQ ID NOs: 18, 36, and 20, respectively; or

[0364] (v) an amino acid sequence comprising or consisting of SEQ ID NOs: 18, 37, and 20, respectively;

[0365] Still 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 with said amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions,

[0366] Most preferably, the VHH domain comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 21, 25, 34, 35, 38 and 100-106.

[0367] 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, a single-chain antibody or a multi-chain antibody, a monospecific or multispecific antibody (e.g., a bispecific antibody), a linear antibody, a single-domain antibody, a heavy chain antibody, a chimeric antibody, or a humanized antibody. In some embodiments, the anti-NECTIN4 antibody according to the present invention comprises an immunoglobulin Fc region connected to the VHH domain (i.e., having a VHH-Fc format). 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 an anti-TROP2 and NECTIN4 multispecific antibody according to the present invention described in Section I above.

[0368] The anti-NECTIN4 VHH domains according to the present invention have excellent tumor targeting, tumor tissue penetration, and target cell endocytosis capabilities, and exhibit human and monkey immune cross-reactivity. Therefore, the anti-NECTIN4 VHH domains according to the present invention or the anti-NECTIN4 antibodies according to the present invention can be used as targeting modules in conjugates or coupled to agents such as chemotherapeutic agents, toxins, drugs (such as immunotherapeutic agents), radioactive elements, probes, or signaling molecules to provide applications such as improved tumor killing, immune regulation, or disease detection.

[0369] IV. Production and Purification of Antibodies of the Invention

[0370] In a fourth aspect, the present invention provides a method for producing an antibody of the present invention. To produce an antibody of the present invention, the polypeptide chains of the antibody of the present invention can be obtained, for example, by solid-state peptide synthesis (e.g., Merrifield solid-phase synthesis) or recombinant production and assembled under suitable conditions.

[0371] 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 a host cell. Using conventional methods, the polynucleotide can be easily isolated and sequenced. In one embodiment, a polynucleotide encoding one or more polypeptide chains of an antibody of the present invention is provided. In another embodiment, the present invention provides a vector comprising one or more polynucleotides of the present invention, preferably an expression vector. Therefore, in one embodiment, the present invention provides a method for producing an antibody of the present invention, the method comprising: culturing a host cell comprising encoding the polypeptide chain under conditions suitable for expressing the polypeptide chain of the antibody; and assembling the polypeptide chains to produce the antibody under conditions suitable for the assembly of the polypeptide chains into the antibody.

[0372] Expression vectors can be constructed using methods well known to those skilled in the art. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs).

[0373] 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 monkey kidney CV1 line (COS-7) transformed by SV40, 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), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), CHO cells, NSO cells, myeloma cell lines such as YO, NSO, P3X63 and Sp2 / 0, etc. In a preferred embodiment, the host cell is a CHO or HEK293 cell.

[0374] The antibodies prepared by the methods described herein can be purified by known prior art techniques 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 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.

[0375] In a preferred embodiment, the antibodies of the present invention exhibit good production properties when recombinantly produced in mammalian host cells, such as CHO cells, in particular, good expression yields and a good by-product profile.

[0376] V. Antigen Binding Molecules, Immunofusions, Immunoconjugates, and Antibody Drug Conjugates (ADCs)

[0377] In a fifth aspect, the invention provides antigen-binding molecules, immunofusions, or immunoconjugates produced by fusing or conjugating an antibody of the invention to a heterologous molecule.

[0378] In one embodiment, an antigen binding molecule according to the invention comprises at least one (eg, 1, 2, or 3) VHH of the invention as described herein that specifically binds to Trop2. Trop2 In one embodiment, the antigen binding molecule according to the present invention comprises at least one (eg, 1, 2, or 3) VHH of the present invention that specifically binds to Nectin 4 as described herein. NECTIN4The antigen binding molecule according to the present invention may be, but is not limited to, a monospecific or multispecific protein comprising the VHH domain. As an example, the antigen binding molecule is a chimeric antigen receptor.

[0379] In one embodiment, the present invention provides an immunofusion comprising an antibody of the present invention. In one embodiment of the immunofusion according to the present invention, the antibody of the present invention (or its antigen-binding fragment) is linked to a heterologous peptide or polypeptide molecule directly or via an amino acid peptide linker. Heterologous peptides or polypeptides that may be mentioned include, but are not limited to, proteins or polypeptides that confer another functional activity to the fusion, or tag peptides that facilitate purification or detection of the immunofusion.

[0380] In one embodiment, the invention provides immunoconjugates comprising antibodies of the present invention or antigen binding molecules of the present invention. In one embodiment of the immunoconjugates according to the present invention, antibodies of the present invention (or its antigen binding fragment) or antigen binding molecules of the present invention are conjugated to therapeutic agents or diagnostic agents or detectable agents. In conjugates, joints can be used to covalently link the different entities of the conjugate. Suitable joints include chemical joints or peptide joints. Advantageously, joints are "cleavable joints" that are beneficial to polypeptides released after being delivered to the target site. For example, acid labile joints, peptidase-sensitive joints, light labile joints, dimethyl joints or joints containing disulfide can be used.

[0381] In embodiments where conjugated to a therapeutic agent, therapeutic agents suitable for use in the conjugate include, but are not limited to, cytotoxins (eg, cytostatic or cell-killing agents), drugs, or radioisotopes.

[0382] In embodiments conjugated to diagnostic or detectable agents, such conjugates can be used as part of a clinical test method (e.g., to determine the efficacy of a particular therapy) to monitor or predict the onset, development, progression, and / or severity of a disease or condition. Such diagnosis and detection can be achieved by coupling the antibody to a detectable agent, including but not limited to a variety of enzymes, such as horseradish peroxidase; prosthetic groups, such as streptavidin / biotin and avidin / biotin; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metals and non-radioactive paramagnetic metal ions used in various positron emission tomography techniques.

[0383] In some embodiments, therapeutic agents suitable for use in the conjugates include, but are not limited to, drugs (e.g., anti-tumor drugs); in other embodiments, diagnostic agents suitable for use in the conjugates include, but are not limited to, radioactive diagnostic agents, fluorescent substances, or luminescent substances.

[0384] Antibody-drug conjugates (ADCs)

[0385] In some preferred embodiments, the immunoconjugate according to the present invention is an antibody drug conjugate (ADC).

[0386] In some embodiments, the present invention provides an antibody-drug conjugate having formula (I):

[0387] Ab-(LD) p (I)

[0388] or a pharmaceutically acceptable salt or solvate thereof,

[0389] in:

[0390] Ab is an antibody or antigen-binding molecule of the present invention, such as the above-mentioned 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);

[0391] L is a linker;

[0392] D is a drug, such as an anti-tumor compound; and

[0393] p is an integer selected from 1 to 16, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 12. In some embodiments, Ab is a multispecific antibody that binds to Trop2 and Nectin4 according to the present disclosure. In some particularly preferred embodiments, the Ab is a two-chain multispecific antibody, wherein:

[0394] (a) the antibody is in a symmetrical two-chain form, and wherein the polypeptide chain of the antibody comprises the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0395] (b) The antibody is in an asymmetric two-chain form, and wherein the antibody comprises a first and a second polypeptide chain, wherein the first and second polypeptide chains contain the amino acid sequence of SEQ ID NOs: 61 / 62, 69 / 70 or 71 / 72, respectively, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0396] In some embodiments, D in formula (I) of the present invention can be any anti-tumor compound, as long as it has an anti-tumor effect and has a substituent or partial structure that can be connected to the linker structure, without particular limitation. The anti-tumor compound can be a pharmaceutically active compound that has an effect on tumors. For anti-tumor compounds, part or all of the linker is preferably cleavable within tumor cells, freeing the anti-tumor compound portion, thereby exhibiting an anti-tumor effect. When the linker is cut from the connecting portion of the drug, the anti-tumor compound is released in an unmodified structure, and its original anti-tumor effect can be exerted.

[0397] In some embodiments, the anti-tumor compound can be, for example, a cytotoxic agent, such as a camptothecin compound, eg, Exatecan, Dxd, or an auristatin compound, eg, monomethyl auristatin E (MMAE).

[0398] In some embodiments, D has the structure shown in Formula (D-1a) or Formula (D-1b):

[0399] where R 1a Selected from H and C1-C6 alkyl;

[0400] R 2a Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and-SR 5a ;

[0401] R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ;and

[0402] R 4a and R 5a Independently selected from H and C1-C4 alkyl;

[0403] where R 1b 、R 2b 、R 3b 、R 4b 、R 5b and R 8b Each independently selected from C 1-8 Alkyl; preferably C 1-4 Alkyl, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl;

[0404] R 6b and R 7b Each independently selected from C 1-8 alkoxy, such as methoxy, ethoxy or propoxy;

[0405] R 9b 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

[0406] R 10b Selected from OH and H.

[0407] In some embodiments, R 1a H; R 2a is a C1-C6 alkyl group; R 3a is halogen, preferably -F; R 4a It is a C1-C4 alkyl group, preferably an ethyl group.

[0408] In some embodiments, R 1b 、R 4b and R 8b Each independently selected from C 1-2 Alkyl; preferably methyl;

[0409] R 2b 、R 3b and R 5b Each independently selected from C 3-4 alkyl;

[0410] R 6b and R 7b Each independently selected from C 1-2 alkoxy; and

[0411] R 9b Selected from C 1-4 Alkyl and R 10b OH; or R 9b is COOH and R 10b For H.

[0412] In some embodiments, D has the structure shown in Formula (D-2a) or Formula (D-2b):

[0413] where R 1a 、R 2a 、R 3a and R 4a as defined above; or

[0414] where R 1b 、R 2b 、R 3b 、R 4b 、R 5b 、R 6b 、R 7b 、R 8b、R 9b and R 10b As defined above.

[0415] In some embodiments, D has the structure shown in formula (D-3a) or (D-3b):

[0416] In some embodiments, D has the structure shown in formula (D-4a) or (D-4b):

[0417] In some embodiments, -L- has the structure: -Z-L1-L2-L3-

[0418] in

[0419] Z is selected from wherein m is independently an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7 or 8;

[0420] L1 is selected from the group consisting of: wherein n1 and m1 are each independently an integer selected from 0-20, for example an integer selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0421] L2 is an amino acid residue or a peptide residue consisting of 2-8 amino acids; and

[0422] L3 is selected from: wherein X is selected from -NH-, -O- and -S-; R 1c Each independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl-, C 1-8 Alkoxy, halogen, nitro and cyano; Su is each independently selected from pentose, pentose uronic acid, hexose and hexose uronic 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;

[0423] It should be understood that in the above -Z-L1-L2-L3-, Z is connected to S on Ab, and L3 is connected to D.

[0424] In some embodiments,

[0425] Z is selected from wherein m is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7 or 8;

[0426] L1 is selected from the group consisting of: wherein n1 is independently an integer selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0427] L2 is a peptide residue consisting of 2-8 amino acids; and

[0428] L3 is selected from: where 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.

[0429] In some embodiments, Z is selected from wherein m is 1, 2, 3, 4, 5, 6, 7 or 8.

[0430] In some embodiments, Z is selected from wherein m is 1, 2, 3, 4, 5, 6, 7 or 8.

[0431] In some embodiments, Z is selected from

[0432] In some embodiments, Z is selected from

[0433] 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.

[0434] In some embodiments, L1 is absent or

[0435] In some embodiments, wherein L2 is an amino acid residue or a peptide residue consisting of 2, 3, 4, 5, 6, 7 or 8 amino acids.

[0436] In some embodiments, L2 is a peptide residue consisting of 2, 3, 4, 5, 6, or 7 amino acids.

[0437] In some embodiments, L2 is an amino acid residue or a peptide residue consisting of 2, 3, or 4 amino acids.

[0438] The amino acids constituting L2 are not particularly limited and may be, for example, L- or D-amino acids, preferably L-amino acids. Furthermore, in addition to α-amino acids, amino acids having structures such as β-alanine, ε-aminocaproic acid, and γ-aminobutyric acid may also be used. Furthermore, non-natural amino acids such as N-methylated amino acids may also be used.

[0439] 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), 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 residues or amino acids are optionally replaced by one or more C 1-6 Alkyl substitution.

[0440] In some embodiments, 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), aspartic acid (Asp), asparagine (Asn), arginine (Arg) and threonine (Thr).

[0441] In some embodiments, 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).

[0442] In some embodiments, L2 is selected from -Ala-, -Val-, -Gly-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Val-Cit-, and -Glu-Val-Cit-.

[0443] In some embodiments, L2 is selected from -Gly-, -Val-Ala-, and -Gly-Gly-Phe-Gly-.

[0444] In some embodiments, L2 is selected from -Val-Ala-, -Gly-Gly-Phe-Gly-, -Val-Cit-, and -Glu-Val-Cit-.

[0445] It should be understood that L2 is linked to L1 or Z via the amino group of the left amino acid and to L3 via the carbonyl group of the right amino acid, which is consistent with the following explanation.

[0446] In some embodiments, L3 is selected from:

[0447] where R1c 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.

[0448] In some embodiments, L3 is selected from:

[0449] wherein the variables are as defined herein.

[0450] In some embodiments, L3 is selected from:

[0451] wherein the variables are as defined herein.

[0452] In some embodiments, L3 is selected from: wherein the variables are as defined herein.

[0453] In some embodiments, Su is selected from the group consisting of xylose, arabinose, xyluronic acid, arabinuronic acid, glucose, galactose, mannose, glucuronic acid, galacturonic acid, and mannuronic acid.

[0454] In some embodiments, Su is

[0455] In some embodiments, Su are each independently:

[0456] In some embodiments, Su are each independently

[0457] In some embodiments, Su are each independently

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

[0459] In some embodiments, L3 is selected from:

[0460] In some embodiments, L3 is selected from:

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

[0462] It should be understood that L3 is connected to L2 via the amino group on the left and to D via the carbonyl group on the right, which is consistent with the following explanation.

[0463] In some embodiments, Z is selected from Wherein m is 1, 2, 3, 4, 5, 6, 7 or 8;

[0464] L1 is selected from the group consisting of: wherein n1 is independently an integer selected from 0-12; and

[0465] L3 is selected from: where 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.

[0466] In some embodiments, -Z-L1-L2-L3- is selected from the following structures

[0467] It should be understood that, unless otherwise specified and not contradictory 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 a group near the end of Ab, and the bond on the right side of the divalent group is connected to D or a group near the end of D. For example, when L2 is When , the amino group on the left is connected to L1, and the carbonyl group on the right is connected to L3;

[0468] In some embodiments, the antibody-drug conjugate has an average DAR of 2-10, 6-10, 4-8, 7-9, or 2-4.

[0469] In some embodiments, Formula (I) is as shown in Formula (I'),

[0470] Ab-(SLD)p(I')

[0471] wherein Ab, L, D and p are as defined above for formula (I);

[0472] It will be understood that S in formula (I') is the sulfur from the antibody Ab.

[0473] In some embodiments, the antibody-drug conjugate is selected from

[0474] wherein Ab is an antibody of the invention, preferably V-15, V-hu-21, V-hu-23 or V-hu-24; p is as defined above, preferably, the antibody-drug conjugate has an average DAR of, for example, 2-10, 6-10, 4-8, 7-9 or 2-4.

[0475] It should be understood that the S atom connected to Ab in the above ADC comes from the antibody Ab. Ab opens the disulfide bond under the action of a reducing agent such as TCEP to generate a sulfhydryl -SH, which is then connected to the terminal functional group of the linker, such as the maleimide portion.

[0476] VI. Pharmaceutical Compositions, Drug Combinations, and Kits

[0477] In one aspect, the present invention provides compositions, e.g., pharmaceutical compositions, comprising an antibody as described herein formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic agents, and absorption delaying agents 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, an antibody of the present invention or an immunoconjugate of the present invention or an immunofusion is the sole active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may comprise an antibody as described herein or an immunoconjugate of the present invention or an immunofusion with one or more therapeutic agents.

[0478] In another aspect, the present invention also provides a pharmaceutical combination comprising an antibody as described herein or an immunoconjugate or immunofusion of the invention and one or more therapeutic agents.

[0479] The therapeutic agents suitable for use in the pharmaceutical compositions and drug combinations of the present invention can be selected from any of the following categories (i)-(iv): (i) drugs that enhance antigen presentation (e.g., tumor antigen presentation); (ii) drugs that enhance effector cell responses (e.g., B cell and / or T cell activation and / or mobilization); (iii) drugs that reduce immunosuppression; (iv) drugs that have tumor inhibitory effects.

[0480] The pharmaceutical compositions of the present invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of the antibodies of the present invention. A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. The therapeutically effective amount may vary depending on a variety of factors, such as the disease state, age, sex, and weight of the individual. A therapeutically effective amount is an amount in which any toxic or deleterious effects are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80% relative to an untreated subject. The ability of the antibodies of the present invention to inhibit a measurable parameter (e.g., tumor volume) can be evaluated in an animal model system that is predictive of efficacy in human tumors. A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic outcome at the desired dosage and for the desired period of time. Typically, because prophylactic doses are used in subjects before or at an earlier stage of the disease, the prophylactically effective amount is less than the therapeutically effective amount.

[0481] Kits comprising the antibodies described herein are also within the scope of the present invention. The kits may include one or more other elements, including, for example, instructions for use; other reagents, such as labels or reagents for coupling; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

[0482] VII. Uses and Methods

[0483] Based on the good targeting properties of the antibodies or immunoconjugates or fusions of the present invention to tumor cells expressing TROP2 and / or NECTIN4 and the other excellent properties mentioned above, the present invention also provides the antibodies or immunoconjugates or fusions of the present invention and their uses and methods in the treatment and prevention of TROP2 and / or NECTIN4-related diseases.

[0484] TROP2 and NECTIN4 are overexpressed on the surface of cancer tissue cells from various sources and are therefore suitable targets for the development of cancer immunotherapy. In one aspect, therefore, the present invention provides the use of the immunoconjugates or conjugates of the present invention or their pharmaceutically acceptable salts or solvates and the antibodies or antigen-binding fragments thereof of the present invention for preventing and / or treating TROP2 and / or NECTIN4 positive tumors (e.g., cancer) in a subject. In the application, the immunoconjugates or conjugates of the present invention or their pharmaceutically acceptable salts or solvates, or the antibodies or antigen-binding fragments 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 subject's immune system and promote its spontaneous elimination of tumors.

[0485] In another aspect, the present invention also provides a method for preventing or treating TROP2 and / or NECTIN4-positive tumors (e.g., cancer) in a subject, comprising administering to a subject in need thereof an immunoconjugate or coupling of the present invention or a pharmaceutically acceptable salt or solvate thereof, or administering to a subject in need thereof an antibody or antigen-binding fragment thereof of the present invention.

[0486] TROP2 and / or NECTIN4 positive tumors suitable for the methods and uses of the present invention can be selected from various solid tumors.

[0487] Using TCGA tumor transcriptome data, expression level correlation analysis (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 that show a trend of double positive expression of TROP2 and NECTIN4 include, but are not limited to, bladder cancer (e.g., BLCA (Bladder Urothelial Carcinoma, bladder urothelial carcinoma)); cervical cancer (e.g., CESC (Cervical squamous cell carcinoma, 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, 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); bile duct cancer (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 contemplated by the present disclosure.

[0488] In some embodiments, the tumor used in the methods and uses of the present invention is selected from the group consisting of: 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); bile duct cancer (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).

[0489] In some embodiments, the tumor used in the methods and uses of the present invention is selected from the group consisting of: 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 carcinoma).

[0490] TROP2 and / or NECTIN4 positive tumors suitable for the methods and uses of the present invention can be early, mid or late stage cancers or metastatic cancers. In addition, TROP2 and / or NECTIN4 positive tumors suitable for the methods and uses of the present invention can be tumors that have previously received treatment and have escaped immune responses.

[0491] In some embodiments, a TROP2 and / or NECTIN4 positive tumor treated according to the methods of the present invention has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% TROP2 and / or NECTIN4 positive cells. TROP2 and / or NECTIN4 expression levels can be assessed on tumor biopsies by immunohistochemistry. High percentages of TROP2 and / or NECTIN4 positive cells have been detected on biopsies of various cancers, for example, bladder cancer.

[0492] 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, for example, tumors having at least 25%, 50%, 75%, or 100% TROP2 and / or NECTIN4-positive cells. In some embodiments, the use of the methods of the present invention for such cancers induces tumor regression.

[0493] In some embodiments, the methods according to the present invention can also be used to treat cancers in which the percentage of TROP2 and / or NECTIN4 positive cells is less than 25% or 20%. In some embodiments, the use of the methods of the present invention in such cancers results in tumor growth inhibition.

[0494] In any of the above embodiments of the methods of the present invention, the administration of an antibody or binding fragment thereof according to the present invention and an immunoconjugate or conjugate according to the present invention, or a pharmaceutically acceptable salt or solvate thereof, may comprise 1) a therapeutic measure that cures, slows down, alleviates the symptoms of, and / or stops the progression of, a diagnosed pathological condition or disorder; or 2) a preventative or prophylactic measure that prevents and / or slows the development of a pathological condition or disorder. Thus, in the methods of the present invention, the subject may be an individual already suffering from a disease, an individual susceptible to a disease, or an individual for whom a disease is to be prevented. The individual will benefit from the therapeutic or prophylactic measure and exhibit a reduction or improvement in the occurrence, recurrence, or development of the disease, disorder, condition, and / or symptom compared to an individual who has not received the treatment. In some embodiments, the present invention relates to the treatment of a disease or disorder; in other embodiments, the present invention relates to the prevention of a disease or disorder.

[0495] 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 infusion includes 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.

[0496] 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 conjugates 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 being administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician.

[0497] In some embodiments, the present invention also provides the use of the immunoconjugates or conjugates of the present invention or their pharmaceutically acceptable salts or solvates and the antibodies or antibody fragments of the present invention as a medicament or for the preparation of a medicament. In some embodiments, the medicament is a medicament for use in the aforementioned treatment and prevention methods.

[0498] VIII. Preparation of ADC molecules of the present invention

[0499] Another aspect of the present invention provides a method for preparing an ADC using the antibody of the present invention. "ADC" in the present invention is defined as an antibody coupled to a biologically and / or pharmaceutically active active substance (D) via a linker (L). The method comprises coupling an antibody of the present invention (Ab) to one or more active substances D via one or more linkers (L) as defined herein. Preferably, the linker-active substance is site-specifically coupled to the antibody.

[0500] In some embodiments, the method includes preparing an Ab for ADC, comprising culturing a host cell comprising a nucleic acid encoding the Ab (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector comprising the nucleic acid, as provided above, under conditions suitable for expression of the Ab or its chains, and optionally recovering the Ab from the host cell (or host cell culture medium).

[0501] In some embodiments, the method comprises the steps of:

[0502] (a) adding the antibody Ab to a buffer solution, adding a reducing agent, and then incubating;

[0503] (b) adding a linker-payload to the reaction solution in step (a) for coupling to obtain a crude product; and

[0504] (c) optionally purifying the crude product to obtain the antibody drug conjugate of the present invention;

[0505] wherein Ab is as defined above.

[0506] It should be understood that the linker-payload reacts with Ab to provide the -LD portion in the compound of Formula I. In the case where -LD is clearly defined, the structure of the linker-payload can be determined based on the prior art.

[0507] 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.

[0508] In some embodiments, the reducing agent of step a) is TCEP.

[0509] In some embodiments, the linker-payload has the following structure: Z'-L1-L2-L3-D, wherein L1, L2, L3, and D are as defined above, and Z' is m is as defined above.

[0510] In some embodiments, for the synthesis of wherein Z is The method for producing an ADC further comprises an additional hydrolysis step to open the maleimide ring.

[0511] In some embodiments, the steps are performed under the specific reaction conditions disclosed in the Examples.

[0512] It should be noted that embodiments in which the ranges or specific values ​​of the specific reaction conditions disclosed in the examples are varied by 100%, 80%, 60%, 40%, 20% or 10% are also contemplated by the present invention.

[0513] In one aspect, the present invention also provides the following Group A embodiments:

[0514] A1. A multispecific antibody that binds to Trop2 and Nectin-4, wherein the antibody comprises at least one antigen-binding domain that specifically binds to Trop2 and at least one antigen-binding domain that specifically binds to Nectin-4.

[0515] A2. The multispecific antibody of embodiment A1, wherein the antibody has one or more characteristics selected from the group consisting of:

[0516] (a) the Trop2 and Nectin-4 antigen binding domains are respectively VHH domains;

[0517] (b) Antigen binding affinity K of the Trop2 and Nectin-4 antigen binding domains D The values ​​are 50x10 -8 M to 0.5x10 -8 M;

[0518] (c) the valency ratio of the Trop2 antigen-binding domain to the Nectin-4 antigen-binding domain in the antibody is 1:1 or 1:2; and

[0519] (d) The antibody is a trivalent or tetravalent bispecific antibody.

[0520] A3. The multispecific antibody of embodiments A1-A2, wherein: the antigen binding domain that specifically binds to Trop2 is a VHH domain, and

[0521] wherein the VHH domain comprises a CDR1, CDR2, and CDR3 sequence selected from the amino acid sequences of SEQ ID NOs: 5, 9, 13, 29, and 30;

[0522] More preferably, the CDR1, CDR2 and CDR3 sequences are:

[0523] (i) comprising or consisting of the amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively;

[0524] (ii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively;

[0525] (iii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 14, 15 and 16, respectively; or

[0526] (iv) an amino acid sequence comprising or consisting of SEQ ID NOs: 31, 32, and 33, respectively;

[0527] Still preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 5, 9, 13, 29 and 30, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity with said amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions,

[0528] Most preferably, the VHH domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 29 and 30, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 29 and 30.

[0529] A4. The multispecific antibody of embodiments A1-A3, wherein: the antigen binding domain that specifically binds to nectin-4 is a VHH domain, and

[0530] wherein the VHH domain comprises a CDR1, CDR2, and CDR3 sequence selected from the amino acid sequences of SEQ ID NOs: 17, 21, 25, 34, 35, and 38;

[0531] More preferably, the CDR1, CDR2 and CDR3 sequences are:

[0532] (i) comprising or consisting of the amino acid sequences of SEQ ID NOs: 18, 19 and 20, respectively;

[0533] (ii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 22, 23, and 24, respectively;

[0534] (iii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 26, 27, and 28, respectively;

[0535] (iv) an amino acid sequence comprising or consisting of SEQ ID NOs: 18, 36, and 20, respectively; or

[0536] (v) an amino acid sequence comprising or consisting of SEQ ID NOs: 18, 37, and 20, respectively;

[0537] Still preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35 and 38, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity with said amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions,

[0538] Most preferably, the VHH domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 21, 25, 34, 35 and 38, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 21, 25, 34, 35 and 38.

[0539] A5. The multispecific antibody of embodiments A1-A4, wherein the antibody further comprises:

[0540] (a) a half-life prolonging domain, preferably, the half-life prolonging domain is selected from a serum albumin binding peptide and an immunoglobulin Fc region; and / or

[0541] (b) a linker, preferably, the linker is 5-15 amino acids in length, more preferably, the linker comprises the amino acid sequence (G4S) n , where n = 1, 2 or 3.

[0542] A6. The multispecific antibody of embodiments A1-A5, 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.

[0543] A7. The multispecific antibody of embodiments A1-A6, wherein the antibody comprises two identical polypeptide chains, wherein the polypeptide chains comprise from N-terminus to C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE-(VHH A ) n3 -(VHH B ) n4，

[0544] wherein n1, n2, n3 and n4 are independently selected from integers of 0, 1 or 2, and preferably n3 and n4 are 0, and n1 and n2 are both 1;

[0545] Among them VHH A and VHH Bwherein the VHH domains binding to antigen A and antigen B, respectively, wherein 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; wherein the symbol "-" indicates connection via a linker or direct connection.

[0546] A8. The multispecific antibody of embodiment A7, wherein the polypeptide chain comprises from N-terminus to C-terminus: VHH A -VHH B -HLE, and VHH A Nectin4 binding domain and VHH B Represents the Trop2 binding domain.

[0547] A9. The multispecific antibody of embodiments A1-A6, wherein the antibody comprises two different polypeptide chains, wherein:

[0548] The first polypeptide chain comprises from N-terminus to C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE-(VHH A ) n3 -(VHH B ) n4，

[0549] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH B ) m1 -(VHH A ) m2 -HLE-(VHH B ) m3 -(VHH A ) m4，

[0550] wherein n1, n2, n3 and n4 and m1, m2, m3 and m4 are independently selected from an integer of 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, further preferably m1=0 and m2=1 or m2=0 and m1=1;

[0551] Among them VHH A and VHH B wherein the VHH domains respectively bind to antigen A and antigen B, wherein 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 comprises a knob-into-hole mutation; wherein the symbol "-" indicates connection via a linker or direct connection.

[0552] A10. The multispecific antibody of embodiment A9, wherein:

[0553] The first polypeptide chain comprises from N-terminus to C-terminus: VHH A -VHH B -HLE ，

[0554] The second polypeptide chain comprises from N-terminus to C-terminus: VHH A -HLE ；

[0555] And among them VHH A Nectin4 binding domain and VHH B Represents the Trop2 binding domain.

[0556] A11. The multispecific antibody of embodiments A7-A11, wherein the immunoglobulin Fc region is an Fc region from IgG, for example, an Fc region from human IgG1 or IgG4, and preferably the Fc region comprises a mutation that reduces or eliminates the binding of the Fc region to FcγR.

[0557] A12. The multispecific antibody of embodiments A1-A6, wherein the antibody comprises a single polypeptide chain, wherein the polypeptide chain comprises from N-terminus to C-terminus: (VHH A ) n1 -(VHH B ) n2 -(HLE) n3 -(VHH A ) n4 -(VHH B ) n5 -(HLE) n6，

[0558] wherein n1, n2, n3, n4, n5 and n6 are independently selected from integers of 0, 1 or 2, and preferably n1+n4=1 or 2, n2+n5=1 or 2, and n3+n6=0 or 1;

[0559] Among them VHH A and VHH B wherein the VHH domains bind to antigen A and antigen B, respectively, wherein 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 via a linker or direct connection,

[0560] Preferably, the polypeptide chain comprises from N-terminus to C-terminus:

[0561] (i) VHH A-VHH B ,

[0562] (ii) VHH A -VHH B -HLE,

[0563] (iii) VHH B -VHH A -VHH B ,

[0564] (iv) VHH A -VHH B -VHH B ,

[0565] (v)VHH A -HLE-VHH B -VHH B ,or

[0566] (vi)VHH A -VHH A -HLE-VHH B -VHH B , wherein preferably A represents Trop2 and B represents Nectin4,

[0567] Still 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.

[0568] A13. The multispecific antibody of embodiments A1-A12, wherein:

[0569] (a) the antigen-binding domain that specifically binds to Nectin4 comprises the amino acid sequence of SEQ ID NO: 25 and the antigen-binding domain that specifically binds to Trop2 comprises the amino acid sequence of SEQ ID NO: 13;

[0570] (b) the antigen-binding domain that specifically binds to Nectin4 comprises the amino acid sequence of SEQ ID NO: 21 and the antigen-binding domain that specifically binds to Trop2 comprises the amino acid sequence of SEQ ID NO: 9;

[0571] (c) the antigen-binding domain that specifically binds to Nectin4 comprises the amino acid sequence of SEQ ID NO: 25 and the antigen-binding domain that specifically binds to Trop2 comprises the amino acid sequence of SEQ ID NO: 9;

[0572] (d) the antigen-binding domain that specifically binds to Nectin4 comprises the amino acid sequence of SEQ ID NO: 35 and the antigen-binding domain that specifically binds to Trop2 comprises an amino acid sequence selected from SEQ ID NOs: 29 and 30; or

[0573] (e) the antigen-binding domain that specifically binds to Nectin4 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 and 38, and the antigen-binding domain that specifically binds to Trop2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 and 30.

[0574] A14. The multispecific antibody of embodiments A1-A6, wherein the antibody is in a two-chain form, wherein:

[0575] (a) the antibody is in a symmetrical two-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 that is at least 95%, 96%, 97%, 98% or 99% identical thereto, preferably comprises the amino acid sequence of SEQ ID NO: 53; or

[0576] (b) the antibody is in an asymmetric two-chain form, and wherein the antibody comprises a first and a second polypeptide chain selected from the group consisting of:

[0577] (i) a first and a second polypeptide chain comprising SEQ ID NOs: 55 and 56, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0578] (ii) a first and a second polypeptide chain comprising SEQ ID NOs: 57 and 58, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0579] (iii) a first and a second polypeptide chain comprising SEQ ID NOs: 59 and 60, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0580] (iv) a first and a second polypeptide chain comprising SEQ ID NOs: 61 and 62, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0581] (v) a first and a second polypeptide chain comprising SEQ ID NOs: 63 and 64, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0582] (vi) a first and a second polypeptide chain comprising SEQ ID NOs: 65 and 66, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0583] (vii) a first and a second polypeptide chain comprising SEQ ID NOs: 67 and 68, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0584] (viii) a first and a second polypeptide chain comprising SEQ ID NOs: 69 and 70, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0585] (ix) a first and a second polypeptide chain comprising SEQ ID NOs: 71 and 72, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0586] (x) a first and a second polypeptide chain comprising SEQ ID NOs: 73 and 74, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto, and

[0587] (xi) a first and a second polypeptide chain comprising SEQ ID NOs: 75 and 76, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0588] Preferably, the first and second polypeptide chains comprise the amino acid sequences of SEQ ID NOs: 61 / 62, 69 / 70 or 71 / 72, respectively.

[0589] A15. The multispecific antibody of embodiments A1-A6, wherein the antibody is in single-chain form, wherein the polypeptide chain of the antibody comprises an amino acid sequence selected from SEQ ID NOs: 41-52 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical thereto.

[0590] A16. An anti-Trop2 antibody comprising a VHH domain that specifically binds to Trop2, and

[0591] wherein the VHH domain comprises a CDR1, CDR2, and CDR3 sequence selected from the amino acid sequences of SEQ ID NOs: 5, 9, 13, 29, and 30;

[0592] More preferably, the CDR1, CDR2 and CDR3 sequences are:

[0593] (i) comprising or consisting of the amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively;

[0594] (ii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively;

[0595] (iii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 14, 15 and 16, respectively; or

[0596] (iv) an amino acid sequence comprising or consisting of SEQ ID NOs: 31, 32, and 33, respectively;

[0597] Still preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 5, 9, 13, 29 and 30, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity with said amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions,

[0598] Most preferably, the VHH domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 29 and 30, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 29 and 30.

[0599] A17. An anti-Necti-4 antibody comprising a VHH domain that specifically binds to Nectin-4, and

[0600] wherein the VHH domain comprises a CDR1, CDR2, and CDR3 sequence selected from the amino acid sequences of SEQ ID NOs: 17, 21, 25, 34, 35, and 38;

[0601] Preferably, the CDR1, CDR2 and CDR3 sequences are:

[0602] (i) comprising or consisting of the amino acid sequences of SEQ ID NOs: 18, 19 and 20, respectively;

[0603] (ii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 22, 23, and 24, respectively;

[0604] (iii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 26, 27, and 28, respectively;

[0605] (iv) an amino acid sequence comprising or consisting of SEQ ID NOs: 18, 36, and 20, respectively; or

[0606] (v) an amino acid sequence comprising or consisting of SEQ ID NOs: 18, 37, and 20, respectively;

[0607] Still preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35 and 38, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity with said amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions,

[0608] Most preferably, the VHH domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 21, 25, 34, 35 and 38, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 21, 25, 34, 35 and 38.

[0609] A18. A polynucleotide encoding the antibody of any one of embodiments A1-A17.

[0610] A19. A vector, preferably an expression vector, comprising the polynucleotide of embodiment A18.

[0611] A20. A host cell comprising the polynucleotide of embodiment A18 or the vector of embodiment A19, for example, wherein the host cell is a mammalian cell.

[0612] A21. A method for producing the antibody of any one of embodiments A1-A17, comprising:

[0613] The antibody is produced by culturing host cells containing proteins encoding the polypeptide chains under conditions suitable for expression of the polypeptide chains; and assembling the polypeptide chains under conditions suitable for assembly of the polypeptide chains into the antibody.

[0614] A22. An immunoconjugate or immunofusion comprising the antibody of any one of embodiments A1-A17.

[0615] A23. The immunoconjugate according to embodiment A22, which is an antibody-drug conjugate represented by formula (I):

[0616] Ab-(LD) p (I)

[0617] or a pharmaceutically acceptable salt or solvate thereof,

[0618] in:

[0619] Ab is the antibody of any one of embodiments A1-A17;

[0620] L is the connector;

[0621] D is a drug, preferably an anti-tumor compound; and

[0622] p is an integer selected from 1 to 16, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0623] A24. The antibody-drug conjugate according to embodiment A23, 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 exitecan, Dxd or MMAE.

[0624] A25. The antibody-drug conjugate according to embodiment A23, or a pharmaceutically acceptable salt or solvate thereof, wherein D has the structure shown in Formula (D-1a) or Formula (D-1b):

[0625] where R 1a Selected from H and C1-C6 alkyl;

[0626] R 2a Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and-SR 5a ;

[0627] R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ;and

[0628] R 4a and R 5a Independently selected from H and C1-C4 alkyl;

[0629] or

[0630] where R 1b 、R 2b 、R 3b 、R 4b 、R 5b and R 8b Each independently selected from C 1-8 Alkyl; for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl;

[0631] R 6b and R 7b Each independently selected from C 1-8 alkoxy, such as methoxy, ethoxy or propoxy;

[0632] R 9b Selected from C 1-8alkyl and COOH; for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl; and

[0633] R 10b Selected from OH and H.

[0634] A26. The antibody-drug conjugate according to embodiment A25, or a pharmaceutically acceptable salt or solvate thereof, wherein

[0635] R 1a H; R 2a is a C1-C6 alkyl group; R 3a is halogen, preferably -F; R 4a is a C1-C4 alkyl group, preferably an ethyl group;

[0636] R 1b 、R 4b and R 8b Each independently selected from C 1-2 Alkyl; preferably methyl;

[0637] R 2b 、R 3b and R 5b Each independently selected from C 3-4 alkyl;

[0638] R 6b and R 7b Each independently selected from C 1-2 alkoxy; and

[0639] R 9b Selected from C 1-4 Alkyl and R 10b OH; or R 9b is COOH and R 10b For H.

[0640] A27. The antibody-drug conjugate according to embodiment A25, or a pharmaceutically acceptable salt or solvate thereof, wherein D has a structure represented by formula (D-2a) or formula (D-2b):

[0641] where R 1a 、R 2a 、R 3a and R 4a As defined in embodiment A25 or A26;

[0642] where R 1b 、R 2b 、R 3b 、R 4b 、R 5b 、R 6b、R 7b 、R 8b 、R 9b and R 10b As defined in embodiment A25 or A26.

[0643] A28. The antibody-drug conjugate according to embodiment A25, or a pharmaceutically acceptable salt or solvate thereof, wherein D has the structure shown in formula (D-3a) or (D-3b):

[0644] A29. The antibody-drug conjugate according to embodiment A25, or a pharmaceutically acceptable salt or solvate thereof, wherein D has the structure shown in formula (D-4a) or (D-4b):

[0645] A30. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of embodiments A23-A29, wherein -L- has the following structure -Z-L1-L2-L3-

[0646] in

[0647] Z is selected from wherein m is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7 or 8;

[0648] L1 is selected from the group consisting of: wherein n1 is independently an integer selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0649] L2 is a peptide residue consisting of 2-8 amino acids; and

[0650] L3 is selected from: where 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.

[0651] A31. The antibody-drug conjugate according to embodiment A30, or a pharmaceutically acceptable salt or solvate thereof,

[0652] Wherein L2 is a peptide residue consisting of 2, 3, 4, 5, 6 or 7 amino acids.

[0653] A32. The antibody-drug conjugate according to embodiment A31, or a pharmaceutically acceptable salt or solvate thereof,

[0654] Among them, Z is selected from Wherein m is 1, 2, 3, 4, 5, 6, 7 or 8;

[0655] L1 is selected from the group consisting of: wherein n1 is independently an integer selected from 0-12; and

[0656] L3 is selected from: where 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.

[0657] A33. The antibody-drug conjugate according to embodiment A31, or a pharmaceutically acceptable salt or solvate thereof,

[0658] Z is selected from

[0659] L1 is selected from absent or

[0660] L2 is selected from -Val-Ala-, -Gly-Gly-Phe-Gly-, -Val-Cit-, and -Glu-Val-Cit-; and

[0661] L3 is selected from:

[0662] A34. The antibody-drug conjugate according to embodiment A31, or a pharmaceutically acceptable salt or solvate thereof,

[0663] Wherein -Z-L1-L2-L3- is selected from the following structures

[0664] A35. The antibody-drug conjugate according to any one of embodiments A23-A34, or a pharmaceutically acceptable salt or solvate thereof, having an average DAR value of 2-10, such as 4-8.

[0665] A36. The antibody-drug conjugate according to embodiment A23, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is selected from

[0666] wherein Ab and p are as defined in Embodiment A23,

[0667] Preferably, Ab is a double-chain antibody, wherein:

[0668] (a) the antibody is in a symmetrical two-chain form, and wherein the polypeptide chain of the antibody comprises the amino acid sequence of SEQ ID NO: 53; or

[0669] (b) the antibody is in an asymmetric two-chain form, and wherein the antibody comprises a first and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 61 / 62, 69 / 70, or 71 / 72, respectively;

[0670] Preferably, the antibody-drug conjugate has an average DAR of 2-10 or 4-8.

[0671] A37. A pharmaceutical composition comprising the antibody of any one of embodiments A1-A17 or the immunoconjugate or immunofusion of embodiments A22-A36 and a pharmaceutically acceptable carrier.

[0672] A38. Use of the antibody of any one of embodiments A1 to A17 or the immunoconjugate or immunofusion of embodiments A22 to A36 as a medicament or for the preparation of a medicament.

[0673] A39. The use of embodiment A38, wherein the medicament is for treating and / or preventing cancer in an individual, wherein the cancer is a Trop2 and Nectin-4 double-positive solid tumor, preferably the tumor has at least 40% of cells showing high-level double-positive expression of Trop2 and Nectin-4.

[0674] In one aspect, the present invention also provides the following Group B embodiments:

[0675] B1. A multispecific antibody that binds to Trop2 and Nectin-4, wherein the antibody comprises at least one antigen-binding domain that specifically binds to Trop2 and at least one antigen-binding domain that specifically binds to Nectin-4.

[0676] B2. The multispecific antibody of embodiment B1, wherein the antibody has one or more characteristics selected from the group consisting of:

[0677] (a) the Trop2 and Nectin-4 antigen binding domains are respectively VHH domains;

[0678] (b) Antigen binding affinity K of the Trop2 and Nectin-4 antigen binding domains D The values ​​are 50x10 -8 M to 0.5x10 -8 M;

[0679] (c) the valency ratio of the Trop2 antigen-binding domain to the Nectin-4 antigen-binding domain in the antibody is 1:1 or 1:2; and

[0680] (d) The antibody is a trivalent or tetravalent bispecific antibody.

[0681] B3. The multispecific antibody of embodiment B1-B2, wherein: the antigen binding domain that specifically binds to Trop2 is a VHH domain, and

[0682] wherein the VHH domain comprises a CDR1, CDR2, and CDR3 sequence selected from the amino acid sequences of SEQ ID NOs: 5, 9, 13, 29, and 30;

[0683] More preferably, the CDR1, CDR2 and CDR3 sequences are:

[0684] (i) comprising or consisting of the amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively;

[0685] (ii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively;

[0686] (iii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 14, 15 and 16, respectively; or

[0687] (iv) an amino acid sequence comprising or consisting of SEQ ID NOs: 31, 32, and 33, respectively;

[0688] Still preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 5, 9, 13, 29 and 30, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity with said amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions,

[0689] Most preferably, the VHH domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 29 and 30, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 29 and 30.

[0690] B4. The multispecific antibody of embodiments B1-B3, wherein: the antigen binding domain that specifically binds to nectin-4 is a VHH domain, and

[0691] wherein the VHH domain comprises a CDR1, CDR2, and CDR3 sequence selected from the amino acid sequences of SEQ ID NOs: 17, 21, 25, 34, 35, and 38;

[0692] More preferably, the CDR1, CDR2 and CDR3 sequences are:

[0693] (i) comprising or consisting of the amino acid sequences of SEQ ID NOs: 18, 19 and 20, respectively;

[0694] (ii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 22, 23, and 24, respectively;

[0695] (iii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 26, 27, and 28, respectively;

[0696] (iv) an amino acid sequence comprising or consisting of SEQ ID NOs: 18, 36, and 20, respectively; or

[0697] (v) an amino acid sequence comprising or consisting of SEQ ID NOs: 18, 37, and 20, respectively;

[0698] Still preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35 and 38, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity with said amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions,

[0699] Most preferably, the VHH domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 21, 25, 34, 35 and 38, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 21, 25, 34, 35 and 38.

[0700] B5. The multispecific antibody of embodiments B1-B4, wherein the antibody further comprises:

[0701] (a) a half-life prolonging domain, preferably, the half-life prolonging domain is selected from a serum albumin binding peptide and an immunoglobulin Fc region; and / or

[0702] (b) a linker, preferably, the linker is 5-15 amino acids in length, more preferably, the linker comprises the amino acid sequence (G4S) n , where n = 1, 2 or 3.

[0703] B6. The multispecific antibody of embodiments B1-B5, 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.

[0704] B7. The multispecific antibody of embodiments B1-B6, wherein the antibody comprises two identical polypeptide chains, wherein the polypeptide chains comprise from N-terminus to C-terminus: (VHH A ) n1 -(VHHB ) n2 -HLE-(VHH A ) n3 -(VHH B ) n4，

[0705] wherein n1, n2, n3 and n4 are independently selected from integers of 0, 1 or 2, and preferably n3 and n4 are 0, and n1 and n2 are both 1;

[0706] Among them VHH A and VHH B wherein the VHH domains binding to antigen A and antigen B, respectively, wherein 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; wherein the symbol "-" indicates connection via a linker or direct connection.

[0707] B8. The multispecific antibody of embodiment B7, wherein the polypeptide chain comprises from N-terminus to C-terminus: VHH A -VHH B -HLE, and VHH A Nectin4 binding domain and VHH B Represents the Trop2 binding domain.

[0708] B9. The multispecific antibody of embodiments B1-B6, wherein the antibody comprises two different polypeptide chains, wherein:

[0709] The first polypeptide chain comprises from N-terminus to C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE-(VHH A ) n3 -(VHH B ) n4，

[0710] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH B ) m1 -(VHH A ) m2 -HLE-(VHH B ) m3 -(VHH A ) m4，

[0711] wherein n1, n2, n3 and n4 and m1, m2, m3 and m4 are independently selected from an integer of 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, further preferably m1=0 and m2=1 or m2=0 and m1=1;

[0712] Among them VHH A and VHH B wherein the VHH domains respectively bind to antigen A and antigen B, wherein 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 comprises a knob-into-hole mutation; wherein the symbol "-" indicates connection via a linker or direct connection.

[0713] B10. The multispecific antibody of embodiment B9, wherein:

[0714] The first polypeptide chain comprises from N-terminus to C-terminus: VHH A -VHH B -HLE ，

[0715] The second polypeptide chain comprises from N-terminus to C-terminus: VHH A -HLE ；

[0716] And among them VHH A Nectin4 binding domain and VHH B Represents the Trop2 binding domain.

[0717] B11. The multispecific antibody of embodiments B7-B11, wherein the immunoglobulin Fc region is an Fc region from IgG, for example, an Fc region from human IgG1 or IgG4, and preferably the Fc region comprises a mutation that reduces or eliminates the binding of the Fc region to FcγR.

[0718] B12. The multispecific antibody of embodiments B1-B6, wherein the antibody comprises a single polypeptide chain, wherein the polypeptide chain comprises from N-terminus to C-terminus: (VHH A ) n1 -(VHH B ) n2 -(HLE) n3 -(VHH A ) n4 -(VHH B ) n5 -(HLE) n6，

[0719] wherein n1, n2, n3, n4, n5 and n6 are independently selected from integers of 0, 1 or 2, and preferably n1+n4=1 or 2, n2+n5=1 or 2, and n3+n6=0 or 1;

[0720] Among them VHH A and VHH B wherein the VHH domains bind to antigen A and antigen B, respectively, wherein 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 via a linker or direct connection,

[0721] Preferably, the polypeptide chain comprises from N-terminus to C-terminus:

[0722] (i) VHH A -VHH B ,

[0723] (ii) VHH A -VHH B -HLE,

[0724] (iii) VHH B -VHH A -VHH B ,

[0725] (iv) VHH A -VHH B -VHH B ,

[0726] (v)VHH A -HLE-VHH B -VHH B ,or

[0727] (vi)VHH A -VHH A -HLE-VHH B -VHH B , wherein preferably A represents Trop2 and B represents Nectin4,

[0728] Still 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.

[0729] B13. The multispecific antibody of embodiments B1-B12, wherein:

[0730] (a) the antigen-binding domain that specifically binds to Nectin4 comprises the amino acid sequence of SEQ ID NO: 25 and the antigen-binding domain that specifically binds to Trop2 comprises the amino acid sequence of SEQ ID NO: 13;

[0731] (b) the antigen-binding domain that specifically binds to Nectin4 comprises the amino acid sequence of SEQ ID NO: 21 and the antigen-binding domain that specifically binds to Trop2 comprises the amino acid sequence of SEQ ID NO: 9;

[0732] (c) the antigen-binding domain that specifically binds to Nectin4 comprises the amino acid sequence of SEQ ID NO: 25 and the antigen-binding domain that specifically binds to Trop2 comprises the amino acid sequence of SEQ ID NO: 9;

[0733] (d) the antigen-binding domain that specifically binds to Nectin4 comprises the amino acid sequence of SEQ ID NO: 35 and the antigen-binding domain that specifically binds to Trop2 comprises an amino acid sequence selected from SEQ ID NOs: 29 and 30; or

[0734] (e) the antigen-binding domain that specifically binds to Nectin4 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 and 38, and the antigen-binding domain that specifically binds to Trop2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 and 30.

[0735] B14. The multispecific antibody of embodiments B1-B6, wherein the antibody is in a two-chain form, wherein:

[0736] (a) the antibody is in a symmetrical two-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 that is at least 95%, 96%, 97%, 98% or 99% identical thereto, preferably comprises the amino acid sequence of SEQ ID NO: 53; or

[0737] (b) the antibody is in an asymmetric two-chain form, and wherein the antibody comprises a first and a second polypeptide chain selected from the group consisting of:

[0738] (i) a first and a second polypeptide chain comprising SEQ ID NOs: 55 and 56, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0739] (ii) a first and a second polypeptide chain comprising SEQ ID NOs: 57 and 58, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0740] (iii) a first and a second polypeptide chain comprising SEQ ID NOs: 59 and 60, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0741] (iv) a first and a second polypeptide chain comprising SEQ ID NOs: 61 and 62, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0742] (v) a first and a second polypeptide chain comprising SEQ ID NOs: 63 and 64, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0743] (vi) a first and a second polypeptide chain comprising SEQ ID NOs: 65 and 66, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0744] (vii) a first and a second polypeptide chain comprising SEQ ID NOs: 67 and 68, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0745] (viii) a first and a second polypeptide chain comprising SEQ ID NOs: 69 and 70, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0746] (ix) a first and a second polypeptide chain comprising SEQ ID NOs: 71 and 72, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0747] (x) a first and a second polypeptide chain comprising SEQ ID NOs: 73 and 74, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto, and

[0748] (xi) a first and a second polypeptide chain comprising SEQ ID NOs: 75 and 76, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0749] Preferably, the first and second polypeptide chains comprise the amino acid sequences of SEQ ID NOs: 61 / 62, 69 / 70 or 71 / 72, respectively.

[0750] B15. The multispecific antibody of embodiments B1-B6, wherein the antibody is in single-chain form, wherein the polypeptide chain of the antibody comprises an amino acid sequence selected from SEQ ID NOs: 41-52 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical thereto.

[0751] B16. An anti-Trop2 antibody comprising a VHH domain that specifically binds to Trop2, and

[0752] wherein the VHH domain comprises a CDR1, CDR2, and CDR3 sequence selected from the amino acid sequences of SEQ ID NOs: 5, 9, 13, 29, and 30;

[0753] More preferably, the CDR1, CDR2 and CDR3 sequences are:

[0754] (i) comprising or consisting of the amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively;

[0755] (ii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively;

[0756] (iii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 14, 15 and 16, respectively; or

[0757] (iv) an amino acid sequence comprising or consisting of SEQ ID NOs: 31, 32, and 33, respectively;

[0758] Still preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 5, 9, 13, 29 and 30, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity with said amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions,

[0759] Most preferably, the VHH domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 29 and 30, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 29 and 30.

[0760] B17. An anti-Necti-4 antibody comprising a VHH domain that specifically binds to Nectin-4, and

[0761] wherein the VHH domain comprises a CDR1, CDR2, and CDR3 sequence selected from the amino acid sequences of SEQ ID NOs: 17, 21, 25, 34, 35, and 38;

[0762] Preferably, the CDR1, CDR2 and CDR3 sequences are:

[0763] (i) comprising or consisting of the amino acid sequences of SEQ ID NOs: 18, 19 and 20, respectively;

[0764] (ii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 22, 23, and 24, respectively;

[0765] (iii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 26, 27, and 28, respectively;

[0766] (iv) an amino acid sequence comprising or consisting of SEQ ID NOs: 18, 36, and 20, respectively; or

[0767] (v) an amino acid sequence comprising or consisting of SEQ ID NOs: 18, 37, and 20, respectively;

[0768] Still preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs: 17, 21, 25, 34, 35 and 38, or an amino acid sequence having at least 85%, 90%, 95% or 99% identity with said amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions,

[0769] Most preferably, the VHH domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 21, 25, 34, 35 and 38, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 21, 25, 34, 35 and 38.

[0770] B18. A polynucleotide encoding the antibody of any one of embodiments B1 to B17.

[0771] B19. A vector, preferably an expression vector, comprising the polynucleotide of embodiment B18.

[0772] B20. A host cell comprising the polynucleotide of embodiment B18 or the vector of embodiment B19, eg, a mammalian cell.

[0773] B21. A method for producing the antibody of any one of embodiments B1-B17, comprising:

[0774] The antibody is produced by culturing host cells containing proteins encoding the polypeptide chains under conditions suitable for expression of the polypeptide chains; and assembling the polypeptide chains under conditions suitable for assembly of the polypeptide chains into the antibody.

[0775] B22. An immunoconjugate or immunofusion comprising the antibody of any one of embodiments B1-B17.

[0776] B23. The immunoconjugate according to embodiment B22, which is an antibody-drug conjugate represented by formula (I):

[0777] Ab-(LD) p (I)

[0778] or a pharmaceutically acceptable salt or solvate thereof,

[0779] in:

[0780] Ab is the antibody of any one of embodiments B1-B17;

[0781] L is the connector;

[0782] D is a drug, preferably an anti-tumor compound; and

[0783] p is an integer selected from 1 to 16, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0784] B24. The antibody-drug conjugate according to embodiment B23, 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 exitecan, Dxd or MMAE.

[0785] B25. The antibody-drug conjugate according to embodiment B23, or a pharmaceutically acceptable salt or solvate thereof, wherein D has the structure represented by formula (D-1a) or formula (D-1b):

[0786] where R 1a Selected from H and C1-C6 alkyl;

[0787] R 2a Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and-SR 5a ;

[0788] R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ;and

[0789] R 4a and R 5a Independently selected from H and C1-C4 alkyl;

[0790] or

[0791] where R 1b 、R 2b 、R 3b 、R 4b 、R 5b and R 8b Each independently selected from C 1-8 Alkyl; for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl;

[0792] R 6b and R 7b Each independently selected from C 1-8 alkoxy, such as methoxy, ethoxy or propoxy;

[0793] R 9b Selected from C 1-8 alkyl and COOH; for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl; and

[0794] R 10b Selected from OH and H.

[0795] B26. The antibody-drug conjugate according to embodiment B25, or a pharmaceutically acceptable salt or solvate thereof, wherein

[0796] R 1a H; R 2a is a C1-C6 alkyl group; R 3a is halogen, preferably -F; R 4a is a C1-C4 alkyl group, preferably an ethyl group;

[0797] R 1b 、R 4b and R 8b Each independently selected from C 1-2 Alkyl; preferably methyl;

[0798] R 2b 、R 3b and R 5b Each independently selected from C 3-4 alkyl;

[0799] R 6b and R 7b Each independently selected from C 1-2 alkoxy; and

[0800] R 9b Selected from C 1-4 Alkyl and R 10b OH; or R 9b is COOH and R 10b For H.

[0801] B27. The antibody-drug conjugate according to embodiment B25, or a pharmaceutically acceptable salt or solvate thereof, wherein D has a structure represented by formula (D-2a) or formula (D-2b):

[0802] where R 1a 、R 2a 、R 3a and R 4a As defined in embodiment B25 or B26;

[0803] where R 1b 、R 2b 、R 3b 、R 4b 、R 5b 、R 6b 、R 7b 、R 8b 、R 9b and R 10b As defined in Embodiment B25 or B26.

[0804] B28. The antibody-drug conjugate according to embodiment B25, or a pharmaceutically acceptable salt or solvate thereof, wherein D has the structure shown in formula (D-3a) or (D-3b):

[0805] B29. The antibody-drug conjugate according to embodiment B25, or a pharmaceutically acceptable salt or solvate thereof, wherein D has the structure shown in formula (D-4a) or (D-4b):

[0806] B30. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of embodiments B23-B29, wherein -L- has the following structure -Z-L1-L2-L3-

[0807] in

[0808] Z is selected from wherein m is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7 or 8;

[0809] L1 is selected from the group consisting of: wherein n1 is independently an integer selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0810] L2 is a peptide residue consisting of 2-8 amino acids; and

[0811] L3 is selected from: where R1c 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.

[0812] B31. The antibody-drug conjugate according to embodiment B30, or a pharmaceutically acceptable salt or solvate thereof,

[0813] Wherein L2 is a peptide residue consisting of 2, 3, 4, 5, 6 or 7 amino acids.

[0814] B32. The antibody-drug conjugate according to embodiment B31, or a pharmaceutically acceptable salt or solvate thereof,

[0815] Among them, Z is selected from Wherein m is 1, 2, 3, 4, 5, 6, 7 or 8;

[0816] L1 is selected from the group consisting of: wherein n1 is independently an integer selected from 0-12; and

[0817] L3 is selected from: where 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.

[0818] B33. The antibody-drug conjugate according to embodiment B31, or a pharmaceutically acceptable salt or solvate thereof,

[0819] Z is selected from

[0820] L1 is selected from absent or

[0821] L2 is selected from -Val-Ala-, -Gly-Gly-Phe-Gly-, -Val-Cit-, and -Glu-Val-Cit-; and

[0822] L3 is selected from:

[0823] B34. The antibody-drug conjugate according to embodiment B31, or a pharmaceutically acceptable salt or solvate thereof,

[0824] Wherein -Z-L1-L2-L3- is selected from the following structures

[0825] B35. The antibody-drug conjugate according to any one of embodiments B23-B34, or a pharmaceutically acceptable salt or solvate thereof, having an average DAR value of 2-10, such as 4-8.

[0826] B36. The antibody-drug conjugate according to embodiment B23, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is selected from

[0827] wherein Ab and p are as defined in Embodiment B23,

[0828] Preferably, Ab is a double-chain antibody, wherein:

[0829] (a) the antibody is in a symmetrical two-chain form, and wherein the polypeptide chain of the antibody comprises the amino acid sequence of SEQ ID NO: 53; or

[0830] (b) the antibody is in an asymmetric two-chain form, and wherein the antibody comprises a first and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 61 / 62, 69 / 70 or 71 / 72, respectively:

[0831] Preferably, the antibody-drug conjugate has an average DAR of 2-10 or 4-8.

[0832] B37. A pharmaceutical composition comprising the antibody of any one of embodiments B1-B17 or the immunoconjugate or immunofusion of embodiments B22-B36 and a pharmaceutically acceptable carrier.

[0833] B38. Use of the antibody of any one of embodiments B1 to B17 or the immunoconjugate or immunofusion of embodiments B22 to B36 as a medicament or for the preparation of a medicament.

[0834] B39. The use of embodiment B38, wherein the medicament is for treating and / or preventing cancer in an individual, wherein the cancer is a Trop2 and Nectin-4 double-positive solid tumor, preferably the tumor has at least 40% cells showing high-level double-positive expression of Trop2 and Nectin-4.

[0835] In one aspect, the present invention also provides the following Group C embodiments:

[0836] C1. A multispecific antibody that binds to Trop2 and Nectin4, wherein the antibody comprises at least one antigen-binding domain that specifically binds to Trop2 and at least one antigen-binding domain that specifically binds to Nectin4.

[0837] C2. The multispecific antibody of embodiment C1, wherein the antibody has one or more characteristics selected from the group consisting of:

[0838] (a) the Trop2 and Nectin4 antigen binding domains are VHH domains respectively;

[0839] (b) Antigen binding affinity K of the Trop2 and Nectin4 antigen binding domains D The values ​​are 50x10 -8 M to 0.5x10 -8 M;

[0840] (c) the valency ratio of the Trop2 antigen-binding domain to the Nectin4 antigen-binding domain in the antibody is 1:1 or 1:2; and

[0841] (d) the antibody is a trivalent or tetravalent bispecific antibody; and

[0842] Preferably, the antibody further has one or more characteristics selected from the following: (e) synergistic binding on target tumor cells expressing human Nectin4 and Trop2; (f) synergistic endocytosis; and (g) improved tumor cell targeting distribution compared to single-target drugs of Nectin4 and Trop2.

[0843] C3. The multispecific antibody of any one of embodiments C1-C2, wherein: the antigen binding domain that specifically binds to Trop2 is a VHH domain, and

[0844] wherein the VHH domain comprises a CDR1, CDR2, and CDR3 sequence selected from the amino acid sequences of SEQ ID NOs: 5, 9, 13, 29, 30, 98, and 99;

[0845] More preferably, the CDR1, CDR2 and CDR3 sequences are:

[0846] (i) comprising or consisting of the amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively;

[0847] (ii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively;

[0848] (iii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 14, 15 and 16, respectively; or

[0849] (iv) an amino acid sequence comprising or consisting of SEQ ID NOs: 31, 32, and 33, respectively;

[0850] Still 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 with said amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions,

[0851] Most preferably, the VHH domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 29, 30, 98 and 99, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 29, 30, 98 and 99.

[0852] C4. The multispecific antibody of any one of embodiments C1-C3, wherein: the antigen binding domain that specifically binds to Nectin4 is a VHH domain, and

[0853] wherein the VHH domain comprises a CDR1, CDR2, and CDR3 sequence selected from the amino acid sequences of SEQ ID NOs: 17, 21, 25, 34, 35, 38, and 100-106;

[0854] More preferably, the CDR1, CDR2 and CDR3 sequences are:

[0855] (i) comprising or consisting of the amino acid sequences of SEQ ID NOs: 18, 19 and 20, respectively;

[0856] (ii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 22, 23, and 24, respectively;

[0857] (iii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 26, 27, and 28, respectively;

[0858] (iv) an amino acid sequence comprising or consisting of SEQ ID NOs: 18, 36, and 20, respectively; or

[0859] (v) an amino acid sequence comprising or consisting of SEQ ID NOs: 18, 37, and 20, respectively;

[0860] Still 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 with said amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions,

[0861] Most preferably, the VHH domain comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 21, 25, 34, 35, 38 and 100-106.

[0862] C5. The multispecific antibody of any one of embodiments C1-C4, wherein the antibody further comprises:

[0863] (a) a half-life prolonging domain, preferably, the half-life prolonging domain is selected from a serum albumin binding peptide and an immunoglobulin Fc region; and / or

[0864] (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.

[0865] C6. The multispecific antibody of any one of embodiments C1-C5, 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.

[0866] C7. The multispecific antibody of any one of embodiments C1-C6, wherein the antibody comprises two identical polypeptide chains, wherein the polypeptide chains comprise, from N-terminus to C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE-(VHH A ) n3 -(VHH B ) n4，

[0867] wherein n1, n2, n3 and n4 are independently selected from integers of 0, 1 or 2, and preferably n3 and n4 are 0, and n1 and n2 are both 1;

[0868] Among them VHH A and VHH B wherein the VHH domains binding to antigen A and antigen B, respectively, wherein 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; wherein the symbol "-" indicates connection via a peptide linker or direct connection.

[0869] C8. The multispecific antibody of embodiment C7, wherein the polypeptide chain comprises from N-terminus to C-terminus: VHH A-VHH B -HLE, and VHH A Nectin4 binding domain and VHH B Represents the Trop2 binding domain.

[0870] C9. The multispecific antibody of any one of embodiments C1-C6, wherein the antibody comprises two different polypeptide chains, wherein:

[0871] The first polypeptide chain comprises from N-terminus to C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE-(VHH A ) n3 -(VHH B ) n4，

[0872] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH B ) m1 -(VHH A ) m2 -HLE-(VHH B ) m3 -(VHH A ) m4，

[0873] wherein n1, n2, n3 and n4 and m1, m2, m3 and m4 are independently selected from an integer of 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, further preferably m1=0 and m2=1, or m2=0 and m1=1;

[0874] Among them VHH A and VHH B wherein the VHH domains respectively bind to antigen A and antigen B, wherein 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 comprises a knob-into-hole mutation; wherein the symbol "-" indicates connection via a peptide linker or direct connection.

[0875] C10. The multispecific antibody of embodiment C9, wherein:

[0876] The first polypeptide chain comprises from N-terminus to C-terminus: VHH A -VHH B -HLE ，

[0877] The second polypeptide chain comprises from N-terminus to C-terminus: VHHA -HLE ；

[0878] And among them VHH A Nectin4 binding domain and VHH B Represents the Trop2 binding domain.

[0879] C11. The multispecific antibody of any one of embodiments C7-C10, wherein the immunoglobulin Fc region is an Fc region from IgG, for example, an Fc region from human IgG1 or IgG4, and preferably the Fc region comprises a mutation that reduces or eliminates the binding of the Fc region to FcγR.

[0880] C12. The multispecific antibody of any one of embodiments C1-C6, wherein the antibody comprises a single polypeptide chain, wherein the polypeptide chain comprises, from N-terminus to C-terminus: (VHH A ) n1 -(VHH B ) n2 -(HLE) n3 -(VHH A ) n4 -(VHH B ) n5 -(HLE) n6，

[0881] wherein n1, n2, n3, n4, n5 and n6 are independently selected from integers of 0, 1 or 2, and preferably n1+n4=1 or 2, n2+n5=1 or 2, and n3+n6=0 or 1;

[0882] Among them VHH A and VHH B wherein the VHH domains bind to antigen A and antigen B, respectively, wherein 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 via a peptide linker or direct connection,

[0883] Preferably, the polypeptide chain comprises from N-terminus to C-terminus:

[0884] (i) VHH A -VHH B ,

[0885] (ii) VHH A -VHH B -HLE,

[0886] (iii) VHH B -VHH A -VHHB ,

[0887] (iv) VHH A -VHH B -VHH B ,

[0888] (v)VHH A -HLE-VHH B -VHH B ,or

[0889] (vi)VHH A -VHH A -HLE-VHH B -VHH B , wherein preferably A represents Trop2 and B represents Nectin4,

[0890] Still 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.

[0891] C13. The multispecific antibody of any one of embodiments C1-C12, wherein:

[0892] (a) the antigen-binding domain that specifically binds to Nectin4 comprises the amino acid sequence of SEQ ID NO: 25 and the antigen-binding domain that specifically binds to Trop2 comprises the amino acid sequence of SEQ ID NO: 13;

[0893] (b) the antigen-binding domain that specifically binds to Nectin4 comprises the amino acid sequence of SEQ ID NO: 21 and the antigen-binding domain that specifically binds to Trop2 comprises the amino acid sequence of SEQ ID NO: 9;

[0894] (c) the antigen-binding domain that specifically binds to Nectin4 comprises the amino acid sequence of SEQ ID NO: 25 and the antigen-binding domain that specifically binds to Trop2 comprises the amino acid sequence of SEQ ID NO: 9;

[0895] (d) the antigen-binding domain that specifically binds to Nectin4 comprises the amino acid sequence of SEQ ID NO: 35 and the antigen-binding domain that specifically binds to Trop2 comprises an amino acid sequence selected from SEQ ID NOs: 29 and 30; or

[0896] (e) the antigen-binding domain that specifically binds to Nectin4 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 and 38, and the antigen-binding domain that specifically binds to Trop2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 and 30.

[0897] C14. The multispecific antibody of embodiments C1-C6, wherein the antibody is in a two-chain form, wherein:

[0898] (a) the antibody is in a symmetrical two-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 that is at least 95%, 96%, 97%, 98% or 99% identical thereto, preferably comprises the amino acid sequence of SEQ ID NO: 53; or

[0899] (b) the antibody is in an asymmetric two-chain form, and wherein the antibody comprises a first and a second polypeptide chain selected from the group consisting of:

[0900] (i) a first and a second polypeptide chain comprising SEQ ID NOs: 55 and 56, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0901] (ii) a first and a second polypeptide chain comprising SEQ ID NOs: 57 and 58, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0902] (iii) a first and a second polypeptide chain comprising SEQ ID NOs: 59 and 60, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0903] (iv) a first and a second polypeptide chain comprising SEQ ID NOs: 61 and 62, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0904] (v) a first and a second polypeptide chain comprising SEQ ID NOs: 63 and 64, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0905] (vi) a first and a second polypeptide chain comprising SEQ ID NOs: 65 and 66, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0906] (vii) a first and a second polypeptide chain comprising SEQ ID NOs: 67 and 68, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0907] (viii) a first and a second polypeptide chain comprising SEQ ID NOs: 69 and 70, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0908] (ix) a first and a second polypeptide chain comprising SEQ ID NOs: 71 and 72, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0909] (x) a first and a second polypeptide chain comprising SEQ ID NOs: 73 and 74, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto, and

[0910] (xi) a first and a second polypeptide chain comprising SEQ ID NOs: 75 and 76, respectively, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical thereto,

[0911] Preferably, the first and second polypeptide chains comprise the amino acid sequences of SEQ ID NOs: 61 / 62, 69 / 70 or 71 / 72, respectively.

[0912] C15. The multispecific antibody of embodiments C1-C6, wherein the antibody is in a single-chain form, wherein the polypeptide chain of the antibody comprises an amino acid sequence selected from SEQ ID NOs:41-52 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical thereto.

[0913] C16. A VHH domain that specifically binds to Trop2, and

[0914] wherein the VHH domain comprises a CDR1, CDR2, and CDR3 sequence selected from the amino acid sequences of SEQ ID NOs: 5, 9, 13, 29, 30, 98, and 99;

[0915] More preferably, the CDR1, CDR2 and CDR3 sequences are:

[0916] (i) comprising or consisting of the amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively;

[0917] (ii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively;

[0918] (iii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 14, 15 and 16, respectively; or

[0919] (iv) an amino acid sequence comprising or consisting of SEQ ID NOs: 31, 32, and 33, respectively;

[0920] Still 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 with said amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions,

[0921] Most preferably, the VHH domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 29, 30, 98 and 99, or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 9, 13, 29, 30, 98 and 99.

[0922] C17. A VHH domain that specifically binds to Nectin4, and

[0923] wherein the VHH domain comprises a CDR1, CDR2, and CDR3 sequence selected from the amino acid sequences of SEQ ID NOs: 17, 21, 25, 34, 35, 38, and 100-106;

[0924] Preferably, the CDR1, CDR2 and CDR3 sequences are:

[0925] (i) comprising or consisting of the amino acid sequences of SEQ ID NOs: 18, 19 and 20, respectively;

[0926] (ii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 22, 23, and 24, respectively;

[0927] (iii) comprising or consisting of the amino acid sequences of SEQ ID NOs: 26, 27, and 28, respectively;

[0928] (iv) an amino acid sequence comprising or consisting of SEQ ID NOs: 18, 36, and 20, respectively; or

[0929] (v) an amino acid sequence comprising or consisting of SEQ ID NOs: 18, 37, and 20, respectively;

[0930] Still 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 with said amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions,

[0931] Most preferably, the VHH domain comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 21, 25, 34, 35, 38 and 100-106.

[0932] C18. An antibody comprising the VHH domain that specifically binds to Trop2 according to embodiment C16 and / or the VHH domain that specifically binds to Nectin4 according to embodiment C17.

[0933] C19. An antigen binding molecule comprising the antibody of embodiment C18.

[0934] C20. A polynucleotide encoding the antibody of any one of embodiments C1-C15, the VHH domain of embodiment C16 or C17, the antibody of embodiment C18, or the antigen binding molecule of embodiment C19.

[0935] C21. A vector, preferably an expression vector, comprising the polynucleotide of embodiment C20.

[0936] C22. A host cell comprising the polynucleotide of embodiment C20 or the vector of embodiment C21, eg, a mammalian cell.

[0937] C23. A method for producing the antibody of any one of embodiments C1-C15 or C18, the method comprising:

[0938] The antibody is produced by culturing host cells containing proteins encoding the polypeptide chains under conditions suitable for expression of the polypeptide chains; and assembling the polypeptide chains under conditions suitable for assembly of the polypeptide chains into the antibody.

[0939] C24. An immunoconjugate or immunofusion comprising the antibody of any one of embodiments C1-C15 or C18.

[0940] 25. Antibody-drug conjugate represented by formula (I):

[0941] Ab-(LD) p (I)

[0942] or a pharmaceutically acceptable salt or solvate thereof,

[0943] in:

[0944] Ab is the antibody of any one of embodiments C1-C15 or C18 or the antigen binding molecule of embodiment C19;

[0945] L is a linker;

[0946] D is a drug, preferably an anti-tumor compound; and

[0947] p is an integer selected from 1 to 16, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0948] C26. The antibody-drug conjugate according to embodiment C25, 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 exitecan, Dxd or MMAE.

[0949] C27. The antibody-drug conjugate according to embodiment C25, or a pharmaceutically acceptable salt or solvate thereof, wherein D has the structure shown in Formula (D-1a) or Formula (D-1b):

[0950] where R 1a Selected from H and C1-C6 alkyl;

[0951] R 2a Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and-SR 5a ;

[0952] R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ;and

[0953] R 4a and R 5a Independently selected from H and C1-C4 alkyl;

[0954] or

[0955] where R 1b 、R 2b 、R 3b 、R 4b 、R 5b and R 8b Each independently selected from C 1-8Alkyl; for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl;

[0956] R 6b and R 7b Each independently selected from C 1-8 alkoxy, such as methoxy, ethoxy or propoxy;

[0957] R 9b Selected from C 1-8 alkyl and COOH; for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl; and

[0958] R 10b Selected from OH and H.

[0959] C28. The antibody-drug conjugate according to embodiment C27, or a pharmaceutically acceptable salt or solvate thereof, wherein

[0960] R 1a H; R 2a is a C1-C6 alkyl group; R 3a is halogen, preferably -F; R 4a is a C1-C4 alkyl group, preferably an ethyl group;

[0961] R 1b 、R 4b and R 8b Each independently selected from C 1-2 Alkyl; preferably methyl;

[0962] R 2b 、R 3b and R 5b Each independently selected from C 3-4 alkyl;

[0963] R 6b and R 7b Each independently selected from C 1-2 alkoxy; and

[0964] R 9b Selected from C 1-4 Alkyl and R 10b OH; or R 9b is COOH and R 10b For H.

[0965] C29. The antibody-drug conjugate according to embodiment C27, or a pharmaceutically acceptable salt or solvate thereof, wherein D has a structure represented by formula (D-2a) or formula (D-2b):

[0966] where R 1a 、R2a 、R 3a and R 4a As defined in embodiment C27 or C28;

[0967] where R 1b 、R 2b 、R 3b 、R 4b 、R 5b 、R 6b 、R 7b 、R 8b 、R 9b and R 10b As defined in Embodiment C27 or C28.

[0968] C30. The antibody-drug conjugate according to embodiment C27, or a pharmaceutically acceptable salt or solvate thereof, wherein D has the structure shown in formula (D-3a) or (D-3b):

[0969] C31. The antibody-drug conjugate according to embodiment C27, or a pharmaceutically acceptable salt or solvate thereof, wherein D has the structure shown in formula (D-4a) or (D-4b):

[0970] C32. The antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to any one of embodiments C25-C31, wherein -L- has the following structure -Z-L1-L2-L3-

[0971] in

[0972] Z is selected from wherein m is independently an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7 or 8;

[0973] L1 is selected from the group consisting of: wherein n1 and m1 are each independently an integer selected from 0-20, for example an integer selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0974] L2 is an amino acid residue or a peptide residue consisting of 2-8 amino acids; and

[0975] L3 is selected from: wherein X is selected from -NH-, -O- and -S-; R 1c Each independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl-, C 1-8Alkoxy, halogen, nitro and cyano; Su is each independently selected from pentose, pentose uronic acid, hexose and hexose uronic 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;.

[0976] C33. The antibody-drug conjugate according to embodiment C32, or a pharmaceutically acceptable salt or solvate thereof,

[0977] Among them, Z is selected from Wherein m is 1, 2, 3, 4, 5, 6, 7 or 8;

[0978] Preferably, Z is selected from

[0979] C34. The antibody-drug conjugate according to any one of embodiments C32-C33, or a pharmaceutically acceptable salt or solvate thereof,

[0980] wherein L1 is selected from the group consisting of: wherein n1 is independently an integer selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0981] Preferably, L1 is absent or

[0982] C35. The antibody-drug conjugate according to any one of embodiments C32-C34, or a pharmaceutically acceptable salt or solvate thereof,

[0983] wherein L2 is an amino acid residue or a peptide residue consisting 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 acid is optionally replaced by one or more C 1-6 Alkyl substitution;

[0984] 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);

[0985] Still more preferably, L2 is selected from -Ala-, -Val-, -Gly-, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Val-Cit- and -Glu-Val-Cit-.

[0986] C36. The antibody-drug conjugate according to any one of embodiments C32-C35, or a pharmaceutically acceptable salt or solvate thereof,

[0987] Wherein, L3 is selected from:

[0988] where R 1c 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;

[0989] Preferably, L3 is selected from:

[0990] The variables are defined above.

[0991] C37. The antibody-drug conjugate according to any one of embodiments C32-C36, or a pharmaceutically acceptable salt or solvate thereof,

[0992] Wherein, Su is each independently:

[0993] More preferably, Su are each independently

[0994] Still more preferably, Su are each independently

[0995] C38. The antibody-drug conjugate according to any one of embodiments C32-C36, or a pharmaceutically acceptable salt or solvate thereof,

[0996] Wherein, L3 is selected from:

[0997] Preferably, L3 is selected from:

[0998] C39. The antibody-drug conjugate according to embodiment C35, or a pharmaceutically acceptable salt or solvate thereof,

[0999] Z is selected from

[1000] L1 is selected from the group consisting of absence and

[1001] L2 is selected from -Gly-, -Val-Ala- and -Gly-Gly-Phe-Gly; and

[1002] L3 is selected from:

[1003] C40. The antibody-drug conjugate according to embodiment C35, or a pharmaceutically acceptable salt or solvate thereof,

[1004] Wherein -Z-L1-L2-L3- is selected from the following structures

[1005] C41. The antibody-drug conjugate according to any one of embodiments C25-C40, or a pharmaceutically acceptable salt or solvate thereof, having an average DAR value of 2-10, such as 4-8.

[1006] C42. The antibody-drug conjugate according to embodiment C25, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is selected from

[1007] wherein Ab and p are as defined in Embodiment C25,

[1008] Preferably, Ab is a diabody, wherein:

[1009] (a) the antibody is in a symmetrical two-chain form, and wherein the polypeptide chain of the antibody comprises the amino acid sequence of SEQ ID NO: 53; or

[1010] (b) the antibody is in an asymmetric two-chain form, and wherein the antibody comprises a first and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 61 / 62, 69 / 70 or 71 / 72, respectively:

[1011] Preferably, the antibody-drug conjugate has an average DAR of 2-10 or 4-8.

[1012] C43. A pharmaceutical composition comprising the antibody of any one of embodiments C1-C15 or C18, the antigen binding molecule of embodiment C19, or the immunoconjugate or immunofusion of embodiment C24, or the antibody-drug conjugate of any one of embodiments C25-C42, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.

[1013] C44. Use of the antibody of any one of embodiments C1-C15 or C18, the antigen-binding molecule of embodiment C19, or the immunoconjugate or immunofusion of embodiment C24, or the antibody-drug conjugate of any one of embodiments C25-C42, or a pharmaceutically acceptable salt or solvate thereof, as a medicament or for the preparation of a medicament.

[1014] C45. The use of embodiment C44, 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); bile duct cancer (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.

[1015] Any or all features described above and throughout this application may 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 and should not be construed as constituting any limitation. Example

[1016] Materials and methods

[1017] Reference antibodies and their preparation

[1018] In this example, the reference antibody Datopotamab (also known as V-BMK1) and the reference antibody Enfortumab (also known as V-BMK2) were used.

[1019] The "reference antibody Datopotamab" is an anti-Trop2 antibody constructed based on the heavy and light chain variable region amino acid sequences of the monospecific, bivalent antibody Datopotamab (FDA UNII: BA3HQU7PO9) published by NCATS Inxight Drugs. In the context of the reference antibody Datopotamab, unless otherwise explicitly stated, the reference antibody will have a Fab antigen-binding domain and a human IgG1 heavy chain constant region and a kappa light chain.

[1020] The "reference antibody Enfortumab" is an anti-nectin 4 antibody constructed based on the heavy and light chain variable region amino acid sequences of the monospecific, bivalent antibody Enfortumab (FDA UNII: U1HUE4W970) published by NCATS Inxight Drugs. In the context of the reference antibody Enfortumab, unless otherwise explicitly stated, the reference antibody will have a Fab antigen-binding domain and a human IgG1 heavy chain constant region and a kappa light chain.

[1021] The sequences of the heavy chain variable region and light chain variable region of two reference antibodies are shown in SEQ ID NOs: 1-4, respectively.

[1022] The reference antibody was prepared as follows. The reference antibody coding sequence was synthesized by GenWeiZhi (Shanghai, China), cloned into the pcDNA 3.4 expression vector, and transfected into Expi293F cells. The culture supernatant of the transfected cells was collected, and the antibody was separated and purified using a protein A column. The concentration of the purified antibody was measured using a NanoDrop; the protein purity was determined by SDS-PAGE and analytical HPLC-SEC, and then stored at -80°C until use.

[1023] Antigens and their preparation

[1024] The extracellular sequence information (Gln31-Tyr 274) of human Trop2 (UniProt_P09758-1) and cynomolgus macaque Trop2 (XP_005543292.2) and the extracellular sequence information (Gly 32-Ser 349) of human Nectin4 (UniProt_Q96NY8-1) and cynomolgus macaque Nectin4 (XP_005541277.1) were retrieved from the database. A His tag was added to the C-terminus of each of these proteins. After optimization based on human codon usage, the genes were synthesized and subcloned into the pcDNA3.4 vector. After verification by Sanger sequencing, the plasmids were extracted and used for future use. The constructed eukaryotic expression vectors for these proteins were transiently transfected into Expi293F cells. The protein expression supernatant was collected from the transfected cell culture and purified using a nickel column. The protein purity was determined by SDS-PAGE and was >95%. The activities of the prepared human Trop2-His and human Nectin4-His recombinant antigen proteins were confirmed by ELISA using the aforementioned reference anti-Trop2 antibody (Datopotamab) and reference anti-Nectin 4 antibody (Enfortumab).

[1025] Engineered cell lines expressing recombinant antigens and their preparation

[1026] A CHO-K1 engineered cell line expressing human Trop2 and a CHO-K1 engineered cell line expressing human Nectin4 were prepared as follows.

[1027] The full-length genes for human Trop2 and human Nectin4 were inserted into the lentiviral expression vector pLVX-puro, respectively. Lentiviruses were packaged using 293T cells and then infected into CHO-K1 cells. Cells were cultured in a selective medium containing 8 μg / ml puromycin to generate polyclonal cell lines. Stable CHO-K1-human Trop2 and CHO-K1-human Nectin4 cell lines expressing high levels of Trop2 and Nectin4 were selected by limiting dilution. FACS analysis using the reference antibodies Datopotamab and Enfortumab demonstrated >90% antigen expression positivity in the stable cell lines.

[1028] ELISA binding assay

[1029] Dilute the antigen to 0.5-1.0μg / ml with PBS, coat the plate with 100μl / well, and incubate at 2-8℃ overnight or at 37℃ for 2h. Wash the plate three times with 0.05% PBST on a plate washer, add 250μl / well blocking solution (1% BSA or 8% skim milk powder in PBS), and let it stand at room temperature for more than 1h to block. Dilute the sample to be tested with blocking solution to the appropriate concentration. Wash the plate three times with 0.05% PBST on a plate washer, add 100μl / well of the diluted sample, and let it stand at room temperature for about 1h. Wash the plate three times with 0.05% PBST on a plate washer, add 100μl / well of the secondary antibody-HRP diluted to the target dilution multiple, and let it stand at room temperature for about 0.5 or 1h. Wash the plate three times with 0.05% PBST on a plate washer, add 100μl / well of TMB, observe the color change, and immediately add 100μl / well of stop solution when the color reaches the appropriate level. Read OD450-OD650.

[1030] FACS binding assay

[1031] Target cells were cultured at 1-5 × 10 5 Plate a 96-well plate at a density of 100 cells and centrifuge at 300g for 5 minutes at 4°C. Add the sample to be tested diluted to the appropriate concentration and incubate at 2-8°C for about 1 hour. Centrifuge at 4°C, remove the supernatant, wash twice with 200μl / well FACS buffer (1% BSA or 2% FBS in PBS), and centrifuge at 4°C. Add 100μl / well of flow cytometry secondary antibody diluted to the target dilution multiple, resuspend the cells, and incubate at 2-8°C in the dark for about 0.5h or 1h. After washing twice with 200μl / well FACS buffer, resuspend the cells in 100μl / well FACS buffer and detect on the machine. Measure the MFI of the cells using a flow cytometer (Beckman Coulter).

[1032] Endocytosis assay

[1033] Target cells were cultured at 1-5 × 10 5 Plate cells and add the test sample diluted to the appropriate concentration. Incubate at 2-8°C for approximately 0.5 h to allow the test sample to bind to the cells. Centrifuge at 800 x g for 3 min at 4°C, remove the supernatant, and wash the cells 2-3 times with 200 μl / well of pre-chilled FACS buffer to remove excess unbound test sample. Resuspend the cells in 100 μl / well of pre-chilled FACS buffer. Divide the cells equally into two groups and incubate them at 4°C and 37°C for 4 h, respectively. After incubation, immediately add ice-cold FACS buffer to terminate the endocytosis assay. Centrifuge at 800 x g for 3 min at 4°C, and wash the cells 2-3 times with 200 μl / well of pre-chilled FACS buffer. Immediately add 100 μl / well of the secondary flow cytometry antibody diluted to the desired dilution, resuspend the cells, and incubate at 2-8°C in the dark for approximately 30 min to 1 h. After washing the cells 2-3 times with 200 μL / well FACS buffer, resuspend the cells in 100 μL / well FACS buffer and analyze them on a flow cytometer. The MFI of the cells was determined using a flow cytometer (Beckman Coulter). The internalization level of the cell surface-bound antibody was calculated using the following formula:

[1034] Endocytosis = MFI of sample incubated at 4°C - MFI of sample incubated at 37°C.

[1035] Internalization rate (%) = 100% - (MFI of the sample incubated at 37°C / MFI of the sample incubated at 4°C) × 100%.

[1036] SPR assay

[1037] The binding affinity between antibodies and antigens is detected by surface plasmon resonance (SPR) technology.

[1038] Immobilized Antibody Detection: 10 μg / mL of antibody was immobilized on a Protein A chip. Diluted antigen was injected at a flow rate of 30 μL / min, with an association time of 120 seconds and a dissociation time of 200 seconds. After each dissociation step, 10 mM glycine (pH 2.0) was used to regenerate the chip. Data were analyzed using a 1:1 binding model.

[1039] Immobilized Antigen Detection Antibody: Antigen was immobilized on a CM5 chip at a concentration of 10 μg / mL. Diluted antibody was injected at a flow rate of 30 μL / min, with an association time of 120 seconds and a dissociation time of 200 seconds. After each dissociation, the antibody was regenerated using 10 mM glycine (pH 2.0). Data were analyzed using a 1:1 binding model.

[1040] SEC-HPLC

[1041] Appropriate amounts of protein samples were loaded onto a TSK-gel G3000SWxL column (Tosoh Corporation) or a Zenix-CSEC-300 column (Sepax Technologies) at ambient column temperature. An Agilent 1260 HPLC system was used with a mobile phase consisting of 0.05 M sodium phosphate, 0.3 M sodium chloride, pH 6.8 ± 0.1, and the sample was isocratically eluted at a flow rate of 0.8 mL / min for 20 minutes. The eluted protein was detected by UV absorbance at 280 nm.

[1042] CEX-HPLC

[1043] Load an appropriate amount of protein sample onto the ProPac column at ambient temperature. TM The samples were eluted over a 70-minute gradient using an Agilent 1260 HPLC system with a WCX-10 BioLC column (Thermo Scientific). The mobile phase A consisted of 2-methylpiperazine, imidazole, and Tris (pH 5.0 ± 0.1) and a mobile phase B consisting of 100 mM sodium chloride, 2-methylpiperazine, imidazole, and Tris (pH 10.8 ± 0.1). The eluted protein was detected by UV absorbance at 280 nm.

[1044] HIC-HPLC

[1045] Load an appropriate amount of protein sample onto the MAbPac at ambient column temperature. TM The sample was eluted on a HIC-Butyl column (Thermo SCIENTIFIC) using an Agilent 1260 HPLC system with a gradient elution of mobile phases A and B at a flow rate of 0.8 mL / min over 35 minutes. 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 by UV absorbance at 280 nm.

[1046] Thermal stability determination by DSF

[1047] Based on micro-differential scanning fluorescence (nanoDSF) technology, with a temperature range of 25-95°C and a temperature change rate of 1°C / min., the denaturation temperature (Tm and Tonset) of the antibody protein and the onset temperature of protein aggregation (Tagg) are accurately measured by the 280-450nm fluorescence spectrum and the changes in the 266nm or 473nm laser scattering light intensity to evaluate the thermal stability of the antibody protein.

[1048] Example 1.1 VHH screening

[1049] Alpaca immunization and magnetic sorting technology were used to screen candidate VHH sequences that bind to Trop2 and Nectin4 through preliminary property characterization. In brief, the recombinant antigen proteins human Trop2-His and human Nectin4-His prepared above were used to immunize alpacas (Alpaca), respectively, with an immunization interval of 14 days. Starting from the second immunization, peripheral blood was collected seven days after each immunization, and the immune serum titer was monitored by ELISA binding experiment. After the serum immune titer reached the standard for blood collection and library construction, peripheral blood was collected from the immunized alpacas and peripheral blood mononuclear cells (PBMC) were isolated. Total RNA was extracted from PBMCs, and PrimeScript was used to use RNA as a template. TM II 1st Strand cDNA Synthesis Kit (Takara) was used for reverse transcription to prepare cDNA. Using cDNA as a template, the first round of PCR amplification produced nucleic acid fragments of conventional IgG (containing VH) and pure heavy chain IgG (containing VHH) lacking the CH1 domain. The two types of nucleic acids were separated on an agarose gel, and the encoding nucleic acid containing VHH was extracted and purified before a second round of PCR amplification. The VHH fragments were separated and purified on the gel. The recovered VHH gene fragments were mixed with the linearized yeast display vector pDisplay and co-transfected into yeast competent cells by electroporation to produce a yeast display library that displays VHH antibodies on the yeast cell surface. Yeast cells that bind to the target antigen were enriched from the constructed library by magnetic sorting using streptavidin magnetic beads that had been incubated with the target antigen and thus bound to the antigen.

[1050] The yeast culture obtained after magnetic bead sorting is coated on SDCAA plates, and single clones are selected for culture. After 48 hours of induction, the monoclonal cell cultures are incubated with biotin-antigen and then PE-streptavidin. After incubation, flow cytometry (FACS) is performed to identify positive monoclonal yeast cells that bind to the target antigen. Genomic DNA is extracted from the cultures of the positive yeast cell clones and used for PCR amplification of the antibody sequence and sequencing.

[1051] Based on the sequencing results, candidate VHH sequences with significant sequence differences were selected and fused to the human IgG1-Fc sequence at the C-terminus. These sequences were then ligated into the expression vector pcDNA3.4. After verification by sequencing, the vectors were transiently transfected into HEK-293F cells (hereinafter referred to as "293F cells"). The culture supernatant was collected and the binding and endocytosis properties of the expressed antibodies were characterized. The anti-Trop2 VHH and anti-Nectin4 VHH antibodies listed in Table 1 were ultimately screened and obtained.

[1052] Table 1. VHH antibodies and their variable region sequences

[1053] Example 1.2. In vitro characterization of candidate VHHs

[1054] Candidate VHH-Fc antibody expression and purification

[1055] The gene encoding the VHH antibody sequence was synthesized and inserted into the expression vector pcDNA3.4, fused to the hIgG1 Fc sequence (SEQ ID NO: 85) at its C-terminus. The constructed expression vector was transiently transfected into 293F cells. After 7 days of continuous culture of the transfected cells, the culture supernatant was collected and filtered through a 0.45 μm filter. The filtrate was transferred to a sterile centrifuge tube, and the antibody was purified using a Protein A column. The purity of the antibody product was determined by SEC-HPLC.

[1056] ELISA antigen binding property detection

[1057] Binding of candidate VHH-Fc antibodies to human and monkey antigens was tested using ELISA binding assays. For the Trop2 ELISA binding assay, ELISA plates were coated with either human Trop2-his or cynomolgus monkey Trop2-his antigens, and assays were performed using an anti-hFc-HRP secondary antibody (Abcam, CAT#ab97225). For the Nectin4 ELISA binding assay, plates were coated with either human Nectin4-his or cynomolgus monkey Nectin4-his antigens, and assays were performed using an anti-hFc-HRP secondary antibody (Abcam, CAT#ab97225). The ELISA results are shown in Table 2 below.

[1058] Table 2. Anti-Trop2 VHH-Fc binding ELISA test results

[1059] As shown in the table above, in the Trop2 ELISA binding assay, all candidates showed good binding to human and monkey Trop2 antigens, and the EC50 ratios of all candidates for monkey and human Trop2 antigens were between 0.7 and 0.8, indicating that the candidates had similar affinities to human and monkey Trop2 antigens.

[1060] As also shown in the table above, in the Nectin4 ELISA binding test, all candidates showed good binding to human and monkey Nectin4 antigens, and the EC50 ratios of all candidates for binding to monkey and human Nectin4 were 0.8-0.9, indicating that the candidates had similar affinities to human and monkey Nectin4 antigens.

[1061] Endocytic property detection

[1062] Based on preliminary FACS binding experiments, three concentrations of 100 nM, 10 nM, and 2 nM were selected as the concentrations for endocytosis assay. The candidate anti-Trop2 VHH-Fc antibodies and anti-Nectin4 VHH-Fc antibodies were tested for endocytosis on tumor cells NCI-N87 and BT474. 5 The VHH-Fc antibody to be tested was added to each well of target cells at a concentration of 100 nM, 10 nM, or 2 nM and incubated at 4°C for 0.5 h. The cells were then divided into two groups and incubated at 4°C and 37°C for 4 h, respectively. After the incubation, anti-hIgG-Fc-PE (1:500 dilution) was used to incubate at 4°C in the dark for 30 min. The cells were fixed with 1% paraformaldehyde (PFA) and analyzed by FACS.

[1063] As shown in Figures 1A and 1B, for the candidate anti-Trop2 VHH-Fc antibodies tested, the trends in the endocytosis assay results were essentially consistent across the three concentrations tested. Candidates A04 and G08 exhibited better binding to target cells and non-inferior endocytosis compared to V-BMK1, demonstrating robust endocytosis in NCI-87 cells (Trop2) with high Trop2 expression at a concentration of 2 nM. hi ) and BT474 cells with low Trop2 expression level (Trop2 low The binding and endocytosis of H03 to target cells were slightly inferior to those of V-BMK1. At a concentration of 2 nM, H03 could be internalized in NCI-87 cells (Trop2 hi ) endocytosis rate was 54% and BT474 cells with low expression level of Trop2 (Trop2 low ) had an endocytosis rate of 38%.

[1064] As shown in Figures 2A and 2B, the trends in endocytosis assay results for the candidate anti-Nectin4 VHH-Fc antibodies tested were largely consistent across the three concentrations tested. All candidates, F9-Fc, F12-Fc, and D9-Fc, were able to bind to target cells, but the levels of target binding and endocytosis rates varied significantly among the candidates.

[1065] Epitope binning

[1066] The epitope grouping of candidate anti-VHH-Fc antibodies and related reference antibodies (Datopotamab or Enfortumab) was detected by ELISA binding assay.

[1067] In the ELISA binding assay, the plate was coated with the analyte antibody diluted to 1.0 μg / ml. The antigen (Trop2-his or Nectin4-his) was diluted to 300 ng / ml in blocking buffer, and the competitor antibody was diluted to 30 μg / ml in blocking buffer. Equal volumes of antigen and competitor were mixed and incubated at room temperature for 1 hour. An equal volume of the antigen and blocking buffer mixture was used as a control for the competition experiment to calculate the inhibitory rate of the competitor against the analyte. 100 μl of the prepared mixture was added to the ELISA plate at 1 well and incubated at room temperature for 1 hour. Anti-His antibody-HRP (Sino Biological / 105327-MM02T-H) was used as a secondary antibody. After incubation at room temperature for 1 hour, the plate was detected.

[1068] As shown by the ELISA results in Table 3 , the anti-Trop2 candidates A04-Fc, G08-Fc, and H0-Fc did not bind to the same epitope as Datopotamab.

[1069] As shown in the ELISA results in Table 4, the anti-nectin4 antibody F09-Fc had weak signal inhibition on the binding of enfortumab to the nectin4 antigen, indicating that its binding epitope may be adjacent to enfortumab; while the anti-nectin4 antibodies D9-Fc and F12-Fc were grouped with different epitopes from enfortumab.

[1070] Table 3 Anti-Trop2 epitope competition

[1071] Table 4 Anti-Nectin4 epitope competition

[1072] FACS antigen binding property detection

[1073] The binding of the anti-Trop2 VHH-Fc candidate antibodies A04-Fc, G08-Fc, and H03-Fc to target cells was tested using a FACS binding assay. The assay was performed under the following conditions: NCI-N87 target cells (2×10 5 3 wells) + VHH-Fc or reference antibody (375 nM, 6× dilution, 4°C for 1 hour) + anti-hIgG Fc-PE secondary antibody (1:500, 4°C for 0.5 hour). The FACS binding results are shown in Figure 3. All three candidate antibodies exhibited good target cell binding properties.

[1074] The binding of the anti-nectin4 VHH-Fc candidate antibodies D9-Fc, F09-Fc, and F12-Fc to target cells was tested using a FACS binding assay. 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 (375nM, 6× dilution, 4℃1h) + anti-hIgG Fc-PE secondary antibody (1:500, 4℃0.5h); or BT474 target cells (2×10 5 4 wells) + F09-Fc or reference antibody (350 nM, 5× dilution, 4°C for 1 hour) + anti-hIgG Fc-PE secondary antibody (1:500, 4°C for 1 hour). FACS binding results showed (Figure 4(A) and (B)) that all three candidate antibodies exhibited good target cell binding properties.

[1075] Example 1.3. Sequence Optimization and Characterization of VHH Components

[1076] Sequence optimization of anti-Trop2 VHH constructs

[1077] The anti-Trop2 VHH sequence A04 was selected and subjected to sequence optimization.

[1078] The original VHH sequence was humanized using the "best match method". The amino acid sequence of the VHH framework region was compared and analyzed using the human germline V gene database to select the best germline sequence. The VHH CDR sequence was used to replace the best matching human CDR sequence to generate a humanized VHH sequence. Multiple residues in the framework region were backmutated and post-translational modification (PTM) was removed. The humanized sequence was reverse translated and sent to Jin Weizhi (Shanghai, China) for gene synthesis. It was then constructed into the pcDNA 3.4 expression vector and humanized VHHs were expressed in the form of human IgG4 half body by fusion of the human IgG4 sequence (SEQ ID NO: 87) at the C-terminus, thereby obtaining a VHH antibody protein.

[1079] The VHH sequences of the PTM-removed and humanized antibodies of A04 are shown in Table 5 below, where the CDR sequences defined according to the AbM protocol are underlined.

[1080] Table 5. Anti-Trop2 VHH sequences

[1081] Table 6 below shows the expression and purification results of the A04 humanized antibody expressed in the form of VHH-hIgG4 half antibody.

[1082] Table 6. A04.m1 humanized antibody expression and purification results

[1083] The obtained antibodies A04.m1-hIgG4, A04m1.m10-hIgG4, A04m1.m11-hIgG4 and A04.m1.m12-hIgG4 were detected by FACS. The binding of the two antibodies to the target cells NCI-N87 was similar to that of the parent antibody (Table 7). The FACS binding test conditions were as follows: target cells (1×10 5 / well) + sample (200 nM, 5X dilution, incubation at 4°C for 1 h) + anti-Fc-PE (1:500, incubation at 4°C for 0.5 h).

[1084] Table 7. Binding activity of A04 humanized antibody to NCI-N87

[1085] SPR assay was performed to verify the human-monkey cross-reactivity of the selected A04 humanized antibody (Table 8).

[1086] Table 8. Binding affinity kinetics of A04 humanized antibody to human and cynomolgus monkey Trop2 antigen

[1087] The physicochemical properties of the VHH half antibodies prepared from 293F host cells as described above were analyzed using HIC-HPLC and DSF assays. The results are shown in Table 9 below.

[1088] Table 9. Physical and chemical properties of A04 humanized antibody DA data

[1089] Sequence optimization of anti-nectin 4 VHH constructs

[1090] The anti-Nectin4 VHH sequence F09 (hereinafter referred to as "F09") was selected and sequence optimized.

[1091] The original VHH sequence was humanized using the "best match method." The amino acid sequences of the VHH framework regions were aligned and analyzed using a human germline V gene database to select the best germline sequence. The best-matched human CDR sequence was replaced with the VHH CDR sequence to generate the humanized VHH sequence. Multiple residues in the framework region were backmutated and post-translational modifications (PTMs) were removed. The humanized sequence was reverse translated and sent to GeneWeizhi (Shanghai, China) for gene synthesis. The humanized VHHs were then constructed into a pcDNA 3.4 expression vector and expressed as a C-terminal His-tag fusion. The antibody was purified using a nickel column, and the product purity was determined by SEC-HPLC to obtain the VHH-His antibody protein.

[1092] 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.

[1093] Table 10 Humanized and optimized VHH sequences

[1094] Table 11 below shows the expression and purification results of the F09 optimized antibody expressed in the VHH-his format.

[1095] Table 11. F09 optimized antibody expression and purification results

[1096] The obtained antibodies were subjected to FACS binding assays and showed that the obtained antibodies had similar antigen binding affinity to the parent antibody F09. The binding affinity kinetics of the obtained antibodies to human and cynomolgus monkey Nectin4 antigens were examined by SPR assays, which showed comparable binding affinity to the parent antibody and similar human-monkey cross-reactivity (Table 12).

[1097] Table 12. Binding affinity kinetics of optimized F09 antibodies to human and cynomolgus monkey Nectin4 antigens

[1098] After converting F09.m2m5 into an Fc bivalent format, F09.m2m5-Fc still showed similar binding to F09-Fc when binding to target cells BT474. The FACS assay used the following conditions: BT474 target cells (2×10 5 / well) + antibody sample (350nM, 5× dilution, 4°C for 1h) + anti-hIgG Fc-PE secondary antibody (1:500, 4°C for 1h).

[1099] Based on the above data, F09.m2m5 was selected, and the immunogenicity of the humanized VHH sequence was analyzed using WeMol software. Mutated residues were introduced into F09.m2m5 to obtain the de-immunogenic sequences F09.m2m5-2 and F09.m2m5-4.

[1100] Table 13. F09.m2m5 de-immunized VHH sequences

[1101] Cross-reactivity SPR and FACS assays were performed on the deimmunized F09.m2m5 sample. The results of the SPR assay are shown in Table 14. The results of the FACS assay are shown in Figure 5. The sample maintained cross-reactivity with human Nectin4 and cyno Nectin4 engineered cell lines, and did not cross-react with CHO-K1 cells. The physicochemical properties of F09.m2m5.his, F09.m2m5-2.his, and F09.m2m5-4.his were analyzed 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.

[1102] Table 14. Binding affinity kinetics of F09.m2m5 deimmunized samples to human and cynomolgus monkey Nectin4 antigens

[1103] Table 15. Physicochemical properties of F09.m2m5.his and its de-immunized antibodies

[1104] Example 1.4 Generation of multispecific anti-Trop2 / Nectin4 antibody molecules

[1105] This example describes the design and construction of the structure and expression vector of an exemplary anti-Trop2 / Nectin4 bispecific antibody (BsAb).

[1106] Multispecific antibody molecule design

[1107] The following bispecific antibody molecule constructs were designed:

[1108] A. Single chain form, from N-terminus to C-terminus contains: (VHH A ) n1 -(VHH B ) n2 -(HLE) n3 -(VHH A ) n4 -(VHH B ) n5 -(HLE) n6，

[1109] wherein n1, n2, n3, n4, n5 and n6 are independently selected from integers of 0, 1 or 2; wherein VHH A and VHH B 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 "-" indicates connection via a peptide linker or direct connection, preferably a peptide linker of 5-15 amino acids in length,

[1110] Preferably, VHH Trop2 Domain structure and VHH Nectin4 The ratio of the domains is 1:1 or 1:2,

[1111] Preferably, VHH Trop2 The domains are 1-2, and VHH Nectin4 There are 1-2 domains;

[1112] Optionally, there are 0 or 1 HLE domains;

[1113] For example, the single-chain form of Table 16 below:

[1114] Table 16. Anti-Trop2 and Nectin4 single chain antibody formats

[1115] For single-chain antibodies, in order to facilitate subsequent purification, a purification tag (such as a His tag) can be optionally added to the C-terminus of the antibody molecule, optionally through a small peptide linker (such as AAA) or by direct connection.

[1116] B. Symmetrical double-chain form: composed of two identical polypeptide chains, each chain from N-terminus to 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 an integer of 0, 1 or 2;

[1117] Among them VHH A and VHH Bwherein the VHH domains respectively 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, in particular a human IgG1 or human IgG4 Fc region; wherein the symbol "-" represents connection via a peptide linker or direct connection, preferably a peptide linker of 5-15 amino acids in length,

[1118] Preferably, VHH Trop2 Domain structure and VHH Nectin4 The ratio of the domains is 1:1 or 1:2,

[1119] Preferably, VHH Trop2 The domains are 1-2, and VHH Nectin4 There are 1-2 domains;

[1120] More preferably, each chain comprises from N-terminus to C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE, wherein n1 and n2 are independently selected from an integer of 1 or 2, more preferably n1 and n2 are 1, such as the symmetrical double-chain form of Table 17 below:

[1121] Table 17. Symmetrical Diabody Formats Against Trop2 and Nectin4

[1122] Due to dimerization of the immunoglobulin Fc region, the first and second polypeptide chains can associate to form a homodimer, thereby generating a two-chain form of the multispecific binding molecule.

[1123] C. Asymmetric double-chain form: composed of two different polypeptide chains,

[1124] The first polypeptide chain comprises from N-terminus to C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE-(VHH A ) n3 -(VHH B ) n4，

[1125] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH B ) m1 -(VHH A ) m2 -HLE-(VHH B ) m3 -(VHH A ) m4，

[1126] wherein n1, n2, n3 and n4 and m1, m2, m3 and m4 are independently selected from an integer of 0, 1 or 2;

[1127] Among them VHH A and VHH B wherein the VHH domains respectively 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, in particular a human IgG1 or IgG4 Fc region; wherein the symbol "-" represents connection via a peptide linker or direct connection, preferably a peptide linker of 5-15 amino acids in length,

[1128] Preferably, VHH Trop2 Domain structure and VHH Nectin4 The ratio of the domains is 1:1 or 1:2,

[1129] Preferably, VHH Trop2 The domains are 1-2, and VHH Nectin4 There are 1-2 domains;

[1130] More preferably,

[1131] The first polypeptide chain comprises from N-terminus to C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE ，

[1132] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH B ) m1 -(VHH A ) m2 -HLE ，

[1133] wherein n1, n2 and m1, m2 are independently selected from integers of 0, 1 or 2, but n1 and n2 cannot be 0 at the same time, m1 and m2 cannot be 0 at the same time, and the sum of n1 and m2 and the sum of n2 and m1 must be greater than or equal to 1;

[1134] More preferably, wherein:

[1135] The first polypeptide chain comprises from N-terminus to C-terminus: (VHH A ) n1 -(VHH B ) n2 -HLE ，

[1136] The second polypeptide chain comprises from N-terminus to C-terminus: (VHHB ) m1 -HLE ； Or (VHH A ) m2 -HLE ，

[1137] wherein n1, m1 and m2 are independently selected from an integer of 1 or 2, and n2 is an integer of 0 or 1;

[1138] More preferably,

[1139] The first polypeptide chain comprises from N-terminus to C-terminus: (VHH A )-HLE ，

[1140] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH B )-HLE ；

[1141] or,

[1142] The first polypeptide chain comprises from N-terminus to C-terminus: (VHH A )-(VHH B )-HLE ，

[1143] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH A )-HLE ；

[1144] or,

[1145] The first polypeptide chain comprises from N-terminus to C-terminus: (VHH A )-(VHH A )-HLE ，

[1146] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH B )-HLE ；

[1147] or,

[1148] The first polypeptide chain comprises from N-terminus to C-terminus: (VHH A )-(VHH B )-HLE ，

[1149] The second polypeptide chain comprises from N-terminus to C-terminus: (VHH B )-HLE ；

[1150] For example, the double-stranded form of Table 18 below:

[1151] Table 18. Anti-Trop2 and Nectin4 asymmetric diabody formats

[1152] Due to the dimerization of the immunoglobulin Fc region, the first and second polypeptide chains can associate to form a heterodimer, thereby generating a two-chain multispecific binding molecule. Preferably, to promote heterodimerization of the first and second polypeptide chains, a knob-into-hole mutation can be introduced into the Fc region of the first and second polypeptide chains, such as a (T366W / T366S, L368A, Y407V) mutation or a (T366Y / Y407T) mutation.

[1153] Construction of single-chain multispecific antibody molecules

[1154] Exemplary single-chain bispecific constructs as shown below in Tables 19 and 20 were constructed, and their corresponding amino acid sequences are provided in the Sequence Listing.

[1155] In Table 19, non-humanized VHH sequences were used as building blocks, where VHH Trop2 The 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; the serum albumin binding peptide as the half-life extension domain (HLE) is from the VHH domain VHH SA , more specifically from VHH SA Sequence MSA21 (hereinafter referred to as HLE1, SEQ ID NO: 39); the symbol “-” indicates that the two VHH domains are connected by a peptide linker.

[1156] Table 19. Single-chain multispecific antibodies *Peptide linker = G4S

[1157] In Table 20, humanized VHH sequences were used as building blocks, where VHH Trop2 The amino acid sequence is from A04.m1 (hereinafter referred to as S1), and its amino acid sequence is shown in SEQ ID NO: 29; VHH Nectin4The 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 , 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.

[1158] Table 20. Single-chain multispecific antibodies *Peptide linker = (G4S)2

[1159] The single-chain antibodies listed in Tables 19 and 20 were constructed into the pcDNA 3.4 expression vector with a 6xHis tag added to the C-terminus. The cells were transfected into Expi293F cells and cultured for 3 days. The culture supernatant of the transfected cells was collected and the expressed antibodies were purified using a nickel column. Antibody concentrations were measured using a NanoDrop ELISA. Protein purity was determined by SDS-PAGE and analytical HPLC-SEC, and the protein was then stored at -80°C.

[1160] Construction of multi-chain multispecific antibody molecules

[1161] Constructs are shown below in Tables 21 and 22 as exemplary multi-chain forms of bispecific antibodies, the corresponding amino acid sequences of which are provided in the Sequence Listing.

[1162] In Table 21, non-humanized VHH sequences were used as building blocks, where VHH Trop2 The 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; the Fc region of the immune protein serving as the half-life extension domain (HLE) is from human IgG, specifically an hIgG1 Fc sequence comprising a knob mutation and a 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.

[1163] Table 21. Multi-chain multispecific antibodies in Fc dimer form Note: The two VHH domains are connected by a peptide linker G4S, and the VHH domain and Fc domain are directly connected.

[1164] In Table 22, humanized VHH sequences were used as building blocks, where VHH Trop2 The amino acid sequence is from A04.m1 (hereinafter referred to as S1) or A04.m1m12 (hereinafter referred to as S2), and the amino acid sequence is shown in SEQ ID NO: 29 or SEQ ID NO: 30, respectively; VHH Nectin4 The amino acid sequence is from F09.m2m5 (hereinafter referred to as F1) or F09.m2m5-2 (hereinafter referred to as F2), and its amino acid sequence is shown in SEQ ID NO: 34 and SEQ ID NO: 38, respectively; the immune protein Fc region serving as the half-life extension domain (HLE) is from human IgG, specifically an hIgG1 sequence containing or not containing a knob mutation and a 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. Table 22 also shows the parental anti-Nectin4 antibody and parental anti-Trop2 antibody as controls, wherein parental anti-Nectin4 contains VHH Nectin4 Domain (F2), amino acid sequence as shown in SEQ ID NO: 109; Parental anti-Trop2 comprises VHH trop2 Domain (S2) and VHH targeting rotavirus RV domain (from DOI: 10.1053 / j.gastro.2013.06.053), the first and second chain amino acid sequences are shown in SEQ ID NOs: 107 and 108, respectively.

[1165] Table 22. Multi-chain multispecific antibodies in Fc dimer form *The two VHH domains are connected by a peptide linker (G4S)2, and the VHH domain and Fc domain are directly connected or connected through G4S.

[1166] 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, and V-hu29-Fc are also referred to as V-hu21, V-hu22, V-hu23, V-hu24, V-hu25, V-hu26, V-hu27, V-hu28, and V-hu29, respectively.

[1167] The first / second polypeptide chain of the double-chain antibody in the form of an Fc dimer shown in Tables 21 and 22 was constructed into a pcDNA 3.4 expression vector (if the antibody is a symmetrical structure, one vector is generated; if the antibody is an asymmetric structure, two vectors containing the first and second polypeptide chain encoding genes are generated, respectively), and transfected into HEK293F cells. The cells were cultured for 3 days, and the culture supernatant of the transfected cells was collected and loaded onto a Protein A column (MabSelect PrismA, Cytiva) for purification. The antibody was eluted with acetic acid-sodium acetate solution (pH 3.5) and then immediately neutralized with 2M Tris. The antibody concentration was measured using Nano Drop. The protein purity was determined by SDS-PAGE and analytical HPLC-SEC, and then stored at -80°C.

[1168] Control bispecific antibody construction

[1169] Control bispecific antibodies V-11 and V-12 were constructed using variable region amino acid sequences from the reference antibodies datopotamab and enfortumab. Antibody V-11 consists of the Fab portion of datopotamab linked to the Fc region at the C-terminus and the scFv portion of enfortumab linked to the Fc region at 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 linked to the Fc region at the C-terminus and the Fab portion of enfortumab linked to the Fc region at 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. Control antibodies V-11 and V-12 were produced and purified using essentially the same method as described above. Antibody concentration was measured using NanoDrop. Protein purity was determined by SDS-PAGE and analytical HPLC-SEC, and then stored at -80°C.

[1170] Example 1.5 Characterization of multispecific anti-Trop2 / Nectin4 antibody molecules

[1171] Analysis of target antigen expression levels on tumor cells

[1172] Using the reference antibodies Datopotamab and Enfortumab, FACS binding assay was performed to detect the expression of Trop2 antigen and Nectin4 antigen on various target tumor cells.

[1173] The test results are shown in Figure 6. In the tested tumor cell line MKN45, both antigens were expressed at very low levels, making it an antigen-negative cell. Among the other tested tumors, except for BT474 cells, which had very low levels of Trop2 expression, all other tumor cells showed high Trop2 expression, which was significantly higher than the expression level of Nectin4. Comparison of Nectin4 expression levels among different tumors showed that HT1197 and MDA-MB-231 cells had very low Nectin4 expression, while NCI-N87, SK-BR-3, and BT474 cells had relatively moderate levels of Nectin4 expression, while the remaining tumors had relatively high levels of Nectin4 expression.

[1174] Nonspecific binding analysis

[1175] The binding of the constructed and prepared multispecific antibody molecules to the antigen homologs was tested by ELISA binding assay. The ELISA binding assay used the following conditions: antigen Trop1 / 2 or Nectin1 / 2 / 3 / 4 (1 μg / ml, overnight on the plate at 2-8°C) + test antibody sample (200 nM, 6× dilution, 1 hour at room temperature) + Anti-Fc-HRP secondary antibody (1:5000, 1 hour at room temperature).

[1176] The results showed that the multispecific antibody molecules had no specific binding to homologs Trop1 and Nectin1 / 2 / 3. The results of nonspecific binding analysis for antibodies V-hu21-Fc and V-hu23-Fc are shown in Figure 7.

[1177] Tumor cell binding activity

[1178] Based on the above analysis of antigen expression on tumor cells, we selected cell lines NCI-N87 (Trop2 hi , Nectin4 med )、BT474(Trop2 low , Nectin4 hi ) and MDA-MB-468(Trop2 hi , Nectin4 hi ), the binding ability of the bispecific antibody of the present invention (abbreviated as "VBsAb") on different target cells was tested by FACS binding assay.

[1179] The single-chain antibody was tested using the following FACS binding conditions: target cells (1-2×10 5 / well) + sample (200nM, 3× dilution, 4℃1h) + anti-His-APC secondary antibody (Biolegend, 1:200, 4℃1h)

[1180] The Fc dimerized diabody was tested using the following FACS binding conditions: target cells (2 × 10 5 / well) + sample (200nM, 3× dilution, 4°C for 1h) + anti-human IgG Fc-PE secondary antibody (Thermo, 1:500, 4°C for 0.5h).

[1181] The binding of the non-humanized V BsAb of the present invention and the Fc-containing V BsAbs (V-10, V-13–V-18) on the NCI-N87 cell line, which highly expresses Trop2, is shown in Figures 8A-8B . The binding of the non-humanized V BsAb of the present invention and the Fc-containing V BsAbs (V-10, V-13–V-18) on the BT474 cell line, which has low Trop2 expression and relatively high Nectin4 expression, is shown in Figures 9A-9B .

[1182] In the tumor cells MDA-MB-468 and NCI-87 with high Trop2 expression, the symmetric humanized V BsAbs of the present invention (V-hu24-Fc and V-hu25-Fc) with two Trop2 binding sites and two Nectin4 binding sites had comparable high binding activity to the control antibody Datopotamab; the asymmetric humanized V BsAbs of the present invention (V-hu21-Fc, hu23-Fc, and hu28-Fc) with one Trop2 binding site and two Nectin4 binding sites showed comparable binding activity, all higher than the control antibody Enfortumab, and the difference from Enfortumab was more obvious in the target tumor cell NCI-87 with lower Nectin4 levels; in addition, the humanized V BsAb (V-hu29-Fc) with only one Trop2 binding site and one Nectin4 binding site showed low binding activity similar to that of Enfortumab ( Figures 10A and 10B ). In BT474 cells with low Trop2 expression and high Nectin4 expression, the humanized V BsAb of the present invention exhibited binding behavior similar to that of the control Enfortumab ( FIG10C ).

[1183] The humanized V-hu21-Fc in the form of an Fc dimer was selected and the binding activity of the antibody to various tumor cells was further tested by FACS binding assay. In the experiment, the anti-nectin4 monospecific bivalent antibody Parental anti-nectin4 was used as a control. The FACS binding assay conditions were as follows: target cells (2×10 5 / well) + sample (200 nM, 3× dilution, 4°C for 1 hour) + anti-human IgG Fc-PE secondary antibody (Thermo, 1:500, 4°C for 0.5 hours). The results are shown in Figure 11.

[1184] Humanized V-hu23-Fc in the form of an Fc dimer was selected, and the antibody's binding activity to HT1376 and NCI-H292 (human mucoepidermoid lung carcinoma, CBP60187, Nanjing Kebai Biotechnology Co., Ltd.) tumor cells was further tested by FACS binding assay. Parental anti-Trop2 and parental anti-Nectin4 were used as controls in the assay. The FACS binding assay conditions were as follows: target cells (3×10 5 / well) + sample (200 nM, 3× dilution, 4° C. 1 h) + anti-human IgG Fc-PE secondary antibody (Thermo, 1:500, 4° C. 0.5 h). The results are shown in FIG12 .

[1185] The results showed that V-21-Fc and V-23-Fc exhibited synergistic binding effects on cells with double positive targets compared with the parent control molecules.

[1186] Internalization assay

[1187] Using an endocytosis assay, the internalization ability of the bispecific antibody molecules of the present invention on different target cells, NCI-N87, BT-474, HT1376, MDA-MB-468 and NCI-H292 cells, was detected.

[1188] The endocytic ability of the V BsAbs of the present invention was tested using the aforementioned cell lines expressing different target antigens. As shown in Figures 13A-E, all tested V BsAbs exhibited good endocytic activity, and V-21-Fc and V-hu23-Fc showed synergistic endocytic effects on target double-positive tumor cells (HT1376 and NCI-H292) compared with their parental control molecules, parental anti-Trop2 and parental anti-Nectin4.

[1189] Binding kinetics SPR assay

[1190] The affinity of the bispecific antibody V-hu21 for human and cynomolgus macaque Trop2 antigens and Nectin4 antigens (Sino Biological) was determined by SPR. 10 μg / mL of antibody was captured on a Protein A chip for 15 seconds. Two-fold serial dilutions of antigen were then injected at a flow rate of 30 μL / min, with an association time of 120 seconds and a dissociation time of 200 seconds. After each dissociation step, the chip was regenerated with 10 mM glycine (pH 2.0). Data were analyzed using a 1:1 binding model.

[1191] The affinity results of the candidate molecule V-hu21-Fc for human and cynomolgus monkey Trop2 are shown in Table 23. The results showed that the candidate molecule V-hu21-Fc cross-reacted with cynomolgus monkey Trop2 and exhibited similar binding and dissociation rates with human and cynomolgus monkey Trop2 antigens.

[1192] Table 23. Affinity of V-hu21-Fc for human and cynomolgus monkey Trop2 antigens

[1193] The affinity results of the candidate molecule V-hu21-Fc for human and cynomolgus monkey Nectin4 are shown in Table 24. The results showed that the candidate molecule V-hu21-Fc cross-reacted with cynomolgus monkey Nectin4 and exhibited similar binding and dissociation rates with human Nectin4 antigen and cynomolgus monkey Nectin4 antigen.

[1194] Table 24. Affinity of V-hu21-Fc for human and cynomolgus monkey Nectin4 antigens

[1195] In addition, the affinity of the bispecific antibody molecule V-hu23-Fc for human and cynomolgus monkey Trop2 antigens or human and cynomolgus monkey Nectin4 antigens was also tested by SPR. The results are shown in Tables 25 and 26 below.

[1196] Table 25. Affinity of V-hu23-Fc for human and cynomolgus monkey Trop2 antigens

[1197] Table 26. Affinity of V-hu23-Fc for human and cynomolgus monkey Nectin4 antigens

[1198] Developability Assessment

[1199] Physicochemical properties of candidate molecules V-hu21, hu23, and hu24-Fc were analyzed using SEC-HPLC, CEX-HPLC, HIC-HPLC, and DLS. The samples exhibited development-ready physicochemical properties as shown in Table 27.

[1200] Table 27. Physicochemical properties of V-hu21, hu23, and hu24-Fc (DA data)

[1201] Example 2. Preparation and characterization of antibody-drug conjugates (ADCs)

[1202] The linker-payloads used in some embodiments of the present invention are known in the prior art and / or commercially available. When the drawn structure is inconsistent with the actual situation, the structure should be modified according to the actual situation.

[1203] Materials and methods

[1204] General Synthesis Method A

[1205] Place 5.45 mg / ml of an antibody (e.g., the corresponding bispecific antibody prepared according to the above antibody preparation example) in a 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 a shaker (x500 rpm) and react at 37°C for 2 hours. Add an additional 6 molar equivalents of TCEP (5 mM) to the mixture. Shake the reaction mixture on a shaker (500 rpm) at 37°C for an additional 2 hours. Then, ultrafiltration (MWCO 30 kd) is performed to remove TCEP, and the volume is filled up to the original volume with 20 mM His-HAc buffer. 6 molar equivalents of linker-payload (i.e., linker-payload) (5 mg / ml in DMA) were added dropwise to the fully reduced antibody, while the DMA ...

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 each a VHH domain; (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: it exhibits (e) cooperative binding, (f) cooperative 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.

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. 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 said antibody comprises two identical polypeptide chains, and wherein said 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 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.

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 a Nectin4 binding domain and VHH B represents a 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 comprises, 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, wherein 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 includes a knob-into-hole mutation; wherein the symbol "-" represents connection through 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 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 the VHH domains that bind to antigen A and antigen B, wherein A and B are different from each other and 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 the 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, 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 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 SEQ ID NOs: 59 and 60 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, (iv) A first and a second polypeptide chain each comprising SEQ ID NOs: 61 and 62 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, (v) A first and a second polypeptide chain each comprising SEQ ID NOs: 63 and 64 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, (vi) A first and a second polypeptide chain each comprising SEQ ID NOs: 65 and 66 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, (vii) A first and a second polypeptide chain each comprising SEQ ID NOs: 67 and 68 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, (viii) A first and a second polypeptide chain each comprising SEQ ID NOs: 69 and 70 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, (ix) A first and a second polypeptide chain each comprising SEQ ID NOs: 71 and 72 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, (x) A first and a second polypeptide chain each comprising SEQ ID NOs: 73 and 74 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto, and (xi) A first and a second polypeptide chain each comprising SEQ ID NOs: 75 and 76 or an amino acid sequence 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 chains of the antibody comprise 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 or consist of the amino acid sequences of SEQ ID NOs: 6, 7 and 8; (ii) Each comprise or consist of the amino acid sequences of SEQ ID NOs: 10, 11 and 12; (iii) Comprising or consisting of the amino acid sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively; or (iv) Comprising or consisting of the amino acid sequences set forth in 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 to 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 set forth in SEQ ID NOs: 18, 19, and 20, respectively; (ii) Comprising or consisting of the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively; (iii) Comprising or consisting of the amino acid sequences set forth in SEQ ID NOs: 26, 27, and 28, respectively; (iv) Comprising or consisting of the amino acid sequences set forth in SEQ ID NOs: 18, 36, and 20, respectively; or (v) Comprising or consisting of the amino acid sequences set forth in 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 to Trop2 according to claim 16 and / or the VHH domain that specifically binds to Nectin4 according to 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. 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 or a pharmaceutically acceptable salt or solvate thereof according to claim 25, wherein D has the structure shown 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 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 or a pharmaceutically acceptable salt or solvate thereof according to claim 27, wherein D has the 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 or a pharmaceutically acceptable salt or solvate thereof according to claim 27, wherein D has the structure shown in formula (D-3a) or (D-3b):

31. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 27, wherein D has the structure shown 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 where m is independently an integer selected from 1-10, for example 1, 2, 3, 4, 5, 6, 7 or 8; L1 is selected from non - existent, where n1 and m1 are each independently an integer selected from 0-20, for example an integer selected from 0-12, for example 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-existence, where n1 is independently an integer selected from 0-12, for example 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, L1 is absent or 35. The antibody-drug conjugate according to any one of claims 32-34 or a pharmaceutically acceptable salt or solvate thereof, 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); Still 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 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; Preferably, L3 is selected from: as defined for each variable above.

37. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 32-36, Among them, Each S is independently: More preferably, each of the Sus is independently Still 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, Among them, -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, having an average DAR value of 2-10, such as 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 wherein 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.

44. Use of 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 as a medicament or for the preparation of a medicament.

45. 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.

Images