Targeting Trop2 / EpCAM bispecific antibody, antibody coupling drug, and preparation method and application thereof

Abstract

The invention relates to a bispecific antibody targeting Trop2 and EpCAM at the same time, an antibody coupling drug and a preparation method and application of the bispecific antibody and the antibody coupling drug. Specifically, the invention provides an anti-EpCAM nano antibody, and also provides a bispecific antibody comprising the anti-EpCAM nano antibody and an anti-Trop2 nano antibody. The invention also provides a drug conjugate constructed based on the nano antibody and the bispecific nano antibody.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, specifically to a bispecific antibody that simultaneously targets Trop2 and EpCAM, an antibody-drug conjugate, its preparation method, and its application. Background Technology

[0002] Antibody-drug conjugates (ADCs) are a highly effective and precise anti-tumor treatment strategy, often referred to as "magic bullets." They consist of antibodies, cytotoxic payloads, and chemical linkers, enabling precise targeted killing of tumor cells by binding to the antibody-antigen pair on the cell surface.

[0003] EpCAM, short for Epithelial Cell Adhesion Molecule, also known as CD326, TACSTD1, GA733-2, KSA, and CO17-1A, is a conserved type I transmembrane glycoprotein with a size of 35 kDa. EpCAM is highly expressed in various tumors of epithelial cell origin, particularly in gastrointestinal malignancies, while its expression is low in normal tissues. During tumor development, EpCAM crosstalks with many key signaling pathways, such as Wnt / β-catenin, transforming growth factor-β / SMAD, Epex / EGFR, PI3K / Akt / mTor, and p53, inducing biological changes in tumor cells. EpCAM is involved in processes such as tumor stem cell development, cell proliferation, metabolism, angiogenesis, epithelial-to-mesenchymal transition (EMT), metastasis, chemotherapy / radiation resistance, and immune regulation. Oportuzumab monatox, developed by Sesen Bio, is the most advanced EpCAM ADC, currently in Phase III clinical trials. It is composed of a recombinant humanized anti-EpCAM antibody scFv conjugated to Pseudomonas exotoxin A. In July 2020, Qilu Pharmaceutical entered into a licensing agreement with Sesen Bio, obtaining exclusive rights to develop and commercialize Oportuzumab monatox in Greater China. In February 2021, the FDA accepted its Biologics License Application (BLA) for Oportuzumab monatox for the treatment of high-risk non-muscle-invasive bladder cancer unresponsive to BCG.

[0004] Trop2, short for Trophoblast Cell-Surface Antigen 2, is a transmembrane protein highly expressed in various tumors, including breast cancer, pancreatic cancer, colon cancer, and ovarian cancer. It promotes tumor cell proliferation, invasion, metastasis, and spread, and its high expression is closely related to shortened survival and poor prognosis in cancer patients. Antibody therapy strategies targeting Trop2, such as monoclonal antibodies, antibody-drug conjugates (ADCs), and bispecific antibodies, are currently under development. In April 2020, the FDA approved the first Trop2-targeting ADC, Sacituzumab Govitecan (Trodelvy), which is an antibody-drug conjugate composed of a humanized Trop2 IgG1 monoclonal antibody linked to the active metabolite SN-38 of the chemotherapy drug irinotecan. It delivers chemotherapy drugs into tumor cells by binding to the Trop2 protein on the surface of tumor cells. Although ADC drugs targeting Trop-2 have shown excellent anti-tumor activity in a variety of solid tumors, they still face many challenges in clinical application, including tumor targeting, complex pharmacokinetic characteristics, insufficient effective load release, and side effects.

[0005] Although the efficacy of antibody-drug conjugates (ADCs) has been widely validated, obstacles such as large monoclonal antibody size and low penetration rate into solid tumors still need to be addressed. Antibodies naturally lacking light chains in camels and cartilaginous fish are known as heavy-chain antibodies (HCAbs), initially discovered in camel-dwelling animals (such as camels and alpacas). Traditional antibodies consist of two heavy chains and two light chains, while heavy-chain antibodies consist only of the heavy chain, composed of a single variable region, one hinge region, and two constant regions (CH2 and CH3). Their antigen-binding sites, known as the "VHH regions" or nanobodies, are functional units that can independently bind antigens. The superior properties of heavy-chain antibodies overcome the application bottlenecks caused by the large size and complex structure of traditional antibodies, and hold promise for bringing better efficacy and safety to ADCs.

[0006] Bispecific antibody-drug conjugates (ADCs) are a relatively new concept. Bispecific antibodies can promote the synergistic internalization of two targets, enabling more specific targeting of tumor cells and overcoming drug resistance. Currently, the development of bispecific antibody-drug conjugates is still in its early stages, with the most advanced being in Phase III clinical trials. JSKN003, a novel antibody-drug conjugate (ADC) targeting two HER2 epitopes developed by KNJ Biopharma, is undergoing a Phase III clinical trial in HER2-low-expressing, unresectable, and / or metastatic breast cancer. BL-B01D1, a first-in-class EGFR*HER3 bispecific antibody-drug conjugate developed by Baili Tianheng, has initiated a Phase III clinical trial for the treatment of nasopharyngeal carcinoma. Currently, there are no publicly reported bispecific antibody-drug conjugates targeting Trop2 and EpCAM. Developing a bispecific antibody-drug conjugate that simultaneously targets Trop2 and EpCAM would enhance the tumor targeting of Trop2 and EpCAM bispecific antibody-drug conjugates and reduce the toxicity of antibody-drug conjugates. Summary of the Invention

[0007] This application discloses nanobodies targeting EpCAM, exhibiting excellent affinity for the EpCAM protein. It also discloses the combination of antibodies targeting Trop2 and EpCAM, and specifically discloses bispecific antibodies simultaneously targeting Trop2 and EpCAM, wherein the antibody portion can be a modified antibody derived from alpacas and modified from Trop2 and EpCAM nanobodies. This application further discloses site-conjugated bispecific antibody-drug conjugates targeting Trop2 and EpCAM, comprising a targeting portion, a cytotoxic drug, and a linker. In some embodiments, this application uses the antimitotic agent monomethylorlistatine E (MMAE, a microtubule inhibitor) as a toxic payload, linking it to the antibody's site-specific mutation site via a lysosomal cleavable dipeptide valine-citrulline (Val-Cit), thereby obtaining the antibody-drug conjugate. The bispecific antibody-drug conjugates disclosed in this application exhibit good binding activity to both Trop2 and EpCAM proteins. In vivo, Anti-Trop2 / EpCAM-VC-MMAE effectively inhibited tumor growth in tumor-bearing mice with high / low expression of Trop2 / EpCAM, demonstrating highly efficient anti-tumor activity and good safety.

[0008] Based on the above, the technical solution of this application involves the following aspects.

[0009] 1. Nanobodies

[0010] This application utilizes phage display and screening techniques to isolate alpaca antibodies against EpCAM, which can specifically bind to EpCAM-positive cells. In some embodiments, the isolated alpaca antibodies exhibit good binding affinity to the EpCAM antigen.

[0011] In one aspect, this application provides a nanobody or antigen-binding fragment thereof that specifically binds to EpCAM, comprising the following CDR1 (complementarity-determining region 1), CDR2 (complementarity-determining region 2), and CDR3 (complementarity-determining region 3) sequences:

[0012] (a) CDR1, having: the sequence shown in SEQ ID NO:3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:3;

[0013] (b) CDR2, having: the sequence shown in SEQ ID NO:5, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence shown in SEQ ID NO:5; and

[0014] (c) CDR3, having: the sequence shown in SEQ ID NO:7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:7.

[0015] In some implementations, the substitution is a conservative substitution.

[0016] In some embodiments, the nanobody or its antigen-binding fragment comprises: CDR1 as shown in SEQ ID NO:3, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:5.

[0017] In some embodiments, the nanobody or its antigen-binding fragment further comprises the following FR1 (framework region 1), FR2 (framework region 2), FR3 (framework region 3), and FR4 (framework region 4) sequences:

[0018] (a)FR1, having: the sequence shown in SEQ ID NO:2, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:2;

[0019] (b) FR2, having: the sequence shown in SEQ ID NO:4, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence shown in SEQ ID NO:4; and

[0020] (c)FR3, having: the sequence shown in SEQ ID NO:6, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:6;

[0021] (d)FR4, which has: the sequence shown in SEQ ID NO:8, or the sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:8.

[0022] In some implementations, the substitution is a conservative substitution.

[0023] In some embodiments, the nanobody or its antigen-binding fragment comprises: FR1 as shown in SEQ ID NO:2, FR2 as shown in SEQ ID NO:4, FR3 as shown in SEQ ID NO:6, and FR4 as shown in SEQ ID NO:8.

[0024] In some embodiments, the amino acid sequence of the present invention is defined using the Kabat, IMGT, Chothia, or Abm numbering system.

[0025] In some embodiments, the nanobody comprises an amino acid sequence selected from the following:

[0026] (i) A sequence as shown in SEQ ID NO:1;

[0027] (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:1; or

[0028] (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:1.

[0029] In some implementations, the substitution is a conservative substitution.

[0030] 2. Peptide construct

[0031] This application further provides a polypeptide construct obtained by fusing the nanobody with Fc.

[0032] Specifically, in one aspect, this application provides a polypeptide construct that specifically binds to EpCAM, comprising the nanobody or antigen-binding fragment thereof described in any of the preceding claims, and an immunoglobulin Fc domain.

[0033] In some embodiments, the immunoglobulin Fc domain is directly or via a peptide linker linked to the N-terminus or C-terminus of the nanobody or its antigen-binding fragment.

[0034] In some embodiments, the immunoglobulin Fc domain is directly or via a peptide linker linked to the C-terminus of the nanobody or its antigen-binding fragment.

[0035] In some embodiments, the immunoglobulin Fc domain is a human Fc domain or a human-derived Fc domain. In some embodiments, the immunoglobulin Fc domain is selected from IgG1 isotype, IgG2 isotype, IgG3 isotype, and IgG4 isotype.

[0036] In some embodiments, the immunoglobulin Fc domain comprises the sequence shown in SEQ ID NO: 9, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with it, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) with it.

[0037] In some embodiments, the immunoglobulin Fc domain is mutated. This mutation can provide a binding site for therapeutic agents (e.g., cytotoxic drugs). In some embodiments, the immunoglobulin Fc domain contains mutations at the S239 and / or K290 positions, such mutations including, for example, S239C and / or K290C.

[0038] In some implementations, the immunoglobulin Fc domain is numbered according to the Kabat EU index.

[0039] In some embodiments, the polypeptide construct contains or is composed of an amino acid sequence as shown in SEQ ID NO:10.

[0040] In some embodiments, the polypeptide construct is a dimer, such as a homodimer or a heterodimer.

[0041] In some embodiments, the polypeptide construct comprises, but is not limited to, one or more of single-domain antibodies, single-chain antibodies, antibody Fabs, full-length antibody proteins, antigen-binding fragments, bispecific antibodies, multispecific antibodies, bivalent / multivalent single-domain antibodies, bivalent / multivalent single-chain antibodies, and bivalent / multivalent antibody Fabs.

[0042] 3. Bispecific antibodies

[0043] In one aspect, the present invention provides a bispecific antibody that specifically binds to Trop2 and EpCAM, comprising a first antigen-binding domain specific to Trop2 and a second antigen-binding domain specific to EpCAM.

[0044] Trop2 binding domain

[0045] The first antigen-binding domain specific to Trop2 contained in the bispecific antibody of the present invention can be any antibody form.

[0046] In some embodiments, the first antigen-binding domain is selected from nanobodies, full-length antibodies (e.g., IgG antibodies) or their antigen-binding fragments (e.g., scFv, Fab, scFab).

[0047] In some embodiments, the first antigen-binding domain is VHH, which includes the following CDR1 (complementarity-determining region 1), CDR2 (complementarity-determining region 2), and CDR3 (complementarity-determining region 3) sequences:

[0048] (a) CDR1, having: the sequence shown in SEQ ID NO:12, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:12;

[0049] (b) CDR2, having: the sequence shown in SEQ ID NO:13, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence shown in SEQ ID NO:13; and

[0050] (c) CDR3, having: the sequence shown in SEQ ID NO:14, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:14;

[0051] In some embodiments, the first antigen-binding domain includes: CDR1 as shown in SEQ ID NO:12, CDR2 as shown in SEQ ID NO:13, and CDR3 as shown in SEQ ID NO:14.

[0052] In some embodiments, the first antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:11 or a variant thereof; the variant having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity with the sequence from which it is derived, or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the original sequence; preferably, the substitutions are conservative substitutions.

[0053] In some embodiments, the first antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:11.

[0054] EpCAM combined structural domain

[0055] The bispecific antibody of the present invention contains a second antigen-binding domain specific to EpCAM, which can be in any antibody form.

[0056] In some embodiments, the second antigen-binding domain is selected from nanobodies, full-length antibodies (e.g., IgG antibodies) or their antigen-binding fragments (e.g., scFv, Fab, scFab).

[0057] In some embodiments, the second antigen-binding domain is VHH; it comprises the following CDR1 (complementarity-determining region 1), CDR2 (complementarity-determining region 2), and CDR3 (complementarity-determining region 3) sequences:

[0058] (a) CDR1, having: the sequence shown in SEQ ID NO:3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:3;

[0059] (b) CDR2, having: the sequence shown in SEQ ID NO:5, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence shown in SEQ ID NO:5; and

[0060] (c) CDR3, having: the sequence shown in SEQ ID NO:7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:7.

[0061] In some embodiments, the second antigen-binding domain includes: CDR1 as shown in SEQ ID NO:3, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:7.

[0062] In some embodiments, the second antigen-binding domain further comprises the following FR1 (framework region 1), FR2 (framework region 2), FR3 (framework region 3), and FR4 (framework region 4) sequences:

[0063] (a)FR1, having: the sequence shown in SEQ ID NO:2, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:2;

[0064] (b) FR2, having: the sequence shown in SEQ ID NO:4, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence shown in SEQ ID NO:4; and

[0065] (c)FR3, having: the sequence shown in SEQ ID NO:6, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:6;

[0066] (d)FR4, which has: the sequence shown in SEQ ID NO:8, or the sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:8.

[0067] In some implementations, the substitution is a conservative substitution.

[0068] In some embodiments, the second antigen-binding domain includes FR1 as shown in SEQ ID NO:2, FR2 as shown in SEQ ID NO:4, FR3 as shown in SEQ ID NO:6, and FR4 as shown in SEQ ID NO:8.

[0069] In some embodiments, the second antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:1 or a variant thereof; the variant having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity with the sequence from which it is derived, or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the original sequence; preferably, the substitutions are conservative substitutions.

[0070] In some embodiments, the second antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:1.

[0071] The amino acid sequences described in this application can be defined using the Kabat, IMGT, Chothia, or Abm numbering systems.

[0072] Structure of bispecific antibodies

[0073] Those skilled in the art will understand that any known bispecific antibody structure can be used in this invention. As an example, the following bispecific nanobody is provided, wherein both the first antigen-binding domain and the second antigen-binding domain are VHH.

[0074] In some embodiments, the first antigen-binding domain and the second antigen-binding domain are optionally connected by a connector.

[0075] In some embodiments, the first antigen-binding domain is optionally connected to the N-terminus or C-terminus of the second antigen-binding domain via a connector.

[0076] In some embodiments, the first antigen-binding domain is optionally connected to the N-terminus of the second antigen-binding domain via a connector.

[0077] In some embodiments, the linker is a peptide linker (e.g., a rigid peptide linker or a flexible peptide linker); in some embodiments, the peptide linker consists of 10 to 20 amino acid residues (e.g., 10-19, 10-18, 10-17, 10-16, 10-15).

[0078] In some embodiments, the connector is a peptide connector containing one or more glycines and / or one or more serines.

[0079] In some embodiments, the peptide linker is (G4S)n, where n is an integer not less than 0, such as 1, 2, 3, or 4. In some embodiments, the peptide linker is (GGGGS)3.

[0080] In some embodiments, the bispecific nanobody further comprises an immunoglobulin Fc domain.

[0081] In some embodiments, the immunoglobulin Fc domain is optionally linked to the N-terminus and / or C-terminus (e.g., C-terminus) of the first or second antigen-binding domain via a peptide linker.

[0082] In some embodiments, the immunoglobulin Fc domain is optionally linked to the C-terminus of the first antigen-binding domain via a peptide linker. See exemplary structures. Figure 8 Antibody ② in the sample.

[0083] In some embodiments, the immunoglobulin Fc domain is optionally linked to the C-terminus of the second antigen-binding domain via a peptide linker. See exemplary structures. Figure 8 Antibody ① in the sample.

[0084] In some embodiments, the immunoglobulin Fc domain is optionally linked to the C-terminus of the first antigen-binding domain via a peptide linker, and optionally to the N-terminus of the second antigen-binding domain via a peptide linker. See exemplary structures. Figure 8 Antibody ③ in the sample.

[0085] In some embodiments, the immunoglobulin Fc domain is optionally linked to the C-terminus of the second antigen-binding domain via a peptide linker, and optionally to the N-terminus of the first antigen-binding domain via a peptide linker. See exemplary structures. Figure 8 Antibody ④ in the sample.

[0086] In some embodiments, the bispecific antibody comprises, from N-terminus to C-terminus, a first antigen-binding domain, a peptide linker, a second antigen-binding domain, and an immunoglobulin Fc domain. See exemplary structures below. Figure 8 Antibody ① in the sample.

[0087] In some embodiments, the bispecific antibody comprises, from N-terminus to C-terminus, the second antigen-binding domain, the peptide linker, the first antigen-binding domain, and the immunoglobulin Fc domain. See exemplary structures below. Figure 8 Antibody ② in the middle;

[0088] In some embodiments, the bispecific antibody comprises, from N-terminus to C-terminus, the following in sequence: a first antigen-binding domain, an immunoglobulin Fc domain, a peptide linker, and a second antigen-binding domain. See exemplary structures below. Figure 8 Antibody ③ in the sample.

[0089] In some embodiments, the bispecific antibody comprises, from N-terminus to C-terminus, the second antigen-binding domain, the immunoglobulin Fc domain, the peptide linker, and the first antigen-binding domain. See exemplary structures below. Figure 8 Antibody ④ in the sample.

[0090] In some embodiments, the immunoglobulin Fc domain is the Fc domain of IgG (e.g., the Fc domain of IgG1, IgG2, IgG3, or IgG4).

[0091] In some embodiments, the immunoglobulin Fc domain comprises a sequence as shown in SEQ ID NO:9, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to it, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to it.

[0092] In some embodiments, the immunoglobulin Fc domain is mutated. This mutation can provide a binding site for therapeutic agents (e.g., cytotoxic drugs). In some embodiments, the immunoglobulin Fc domain contains mutations at the S239 and / or K290 positions, such mutations including, for example, S239C and / or K290C.

[0093] In some implementations, the immunoglobulin Fc domain is numbered according to the Kabat EU index.

[0094] In some embodiments, the bispecific nanobody comprises a sequence as shown in SEQ ID NO: 17, 18, 19, or 20. The sequences shown herein do not contain an amino acid (such as methionine (Met)) encoded by a start codon (such as ATG) at their N-terminus. Those skilled in the art will understand that during the preparation of proteins through genetic engineering, the first molecule of the resulting polypeptide chain is often an amino acid (such as Met) encoded by the start codon due to the effect of the start codon. The bispecific antibody of this invention encompasses not only amino acid sequences that do not contain an amino acid (such as Met) encoded by a start codon at their N-terminus, but also amino acid sequences that do contain an amino acid (such as Met) encoded by a start codon at their N-terminus. Therefore, sequences that further contain an amino acid (such as Met) encoded by a start codon at the N-terminus of the aforementioned amino acid sequences are also within the scope of protection of this invention.

[0095] In some embodiments, the bispecific nanobody comprises a variant of the sequence shown in SEQ ID NO:17, 18, 19, or 20, the variant differing from SEQ ID NO:17, 18, 19, or 20 only in one or more (e.g., up to 20, 15, 10, or 5 conserved substitutions of amino acid residues) conserved substitutions, or having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the antibody from which it is derived or its antigen-binding fragment, and substantially retaining the biological function of the bispecific nanobody from which it is derived (e.g., specific binding to Trop2 and EpCAM, neutralizing the biological activity of Trop2 and EpCAM). For example, in some embodiments, the variant, compared to the bispecific nanobody from which it is derived, may be truncated at the N-terminus or C-terminus of the first antigen-binding domain and / or the second antigen-binding domain to contain only a portion of FR1 and / or FR4, or to lack one or both of those backbone regions, as long as it substantially maintains antigen binding and specificity.

[0096] In some embodiments, the bispecific antibody is a dimer, such as a homodimer or a heterodimer.

[0097] In some embodiments, the bispecific antibody is at a concentration of less than approximately 10 -5 M, for example, less than approximately 10 -6 M, 10 - 7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D Combined with Trop2;

[0098] In some embodiments, the bispecific antibody is at a concentration of less than approximately 10 -5 M, for example, less than approximately 10 -6 M, 10 - 7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D Combined with EpCAM.

[0099] The bispecific antibody of the present invention can be a monovalent or bivalent antibody having any antibody structure.

[0100] 4. Multispecific antibodies

[0101] On the other hand, this application provides a multispecific antibody comprising the bispecific antibody of the present invention. To generate the multispecific antibody, the bispecific antibody of the present invention can be linked (e.g., by chemical conjugation, gene fusion, non-covalent association, or other means) to one or more other binding molecules (e.g., additional antibodies, antibody fragments, peptides, or binding mimics).

[0102] In some implementations, the multispecific antibody specifically binds to Trop2 and EpCAM, and additionally specifically binds to one or more other targets.

[0103] In some embodiments, the multispecific antibody further comprises at least one third antibody having a third binding specificity against a third target.

[0104] 5. Nucleic acids, vectors, host cells, and expression methods

[0105] In another aspect, this application provides an isolated nucleic acid molecule comprising a polynucleotide sequence encoding the nanobody or its antigen-binding fragment, a polypeptide construct, a bispecific antibody, or a multispecific antibody. The nucleic acid can be obtained using methods known in the art, such as isolation from a phage display library, a yeast display library, immunization of animals, immortalized cells (e.g., mouse B cell hybridoma cells, EBV-mediated immortalized B cells), or chemical synthesis. The nucleic acid molecule can be codon-optimized for the host cell used for expression.

[0106] In another aspect, this application provides a vector containing the said nucleic acid molecule.

[0107] In some embodiments, the nucleic acid molecule is prepared as a recombinant nucleic acid. In some embodiments, the nucleic acid molecule is cloned into an expression vector. The expression vector may further contain additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The recombinant nucleic acid containing the nucleic acid can be prepared using techniques well known in the art, such as chemical synthesis, DNA recombination techniques (e.g., polymerase chain reaction (PCR) techniques), etc. (see Sambrook, J., EFFritsch, and T. Maniatis. (1989). Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The expression vector may also contain a polynucleotide sequence encoding a polypeptide or protein that facilitates the detection and / or separation of the expressed antibody or antigen-binding fragment. Such polypeptides or proteins may include, but are not limited to, affinity tags (e.g., biotin, polyhistidine tags (His6), or glutathione S-transferase (GSH) tags), polypeptides containing protease cleavage sites, and reporter proteins (e.g., fluorescent proteins). The nucleic acid molecule may be present in one or more vectors. In some embodiments, the expression vector is a DNA plasmid, such as a DNA plasmid for expression in bacterial, yeast, or mammalian cells. In other embodiments, the expression vector is a viral vector. In still other embodiments, the expression vector is a bacteriophage vector or a phage particle vector.

[0108] In another aspect, this application provides a host cell comprising at least one nucleic acid or vector as described above. In some embodiments, the host cell is used to express the nanobody or its antigen-binding fragment, polypeptide construct, bispecific antibody, or multispecific antibody. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, such as *Escherichia coli*), eukaryotic cells (e.g., yeast, insect cells, mammalian cells). Bacteria (e.g., *Escherichia coli* BL21(DE3)) are particularly advantageous for expressing smaller antigen-binding fragments. Suitable mammalian host cells for antibody expression include, but are not limited to, myeloma cells, HeLa cells, HEK cells (e.g., HEK 293 cells), Chinese hamster ovary (CHO) cells, and other mammalian cells suitable for antibody expression.

[0109] 6. Antibody Preparation

[0110] The antibodies of the present invention can be prepared by various methods known in the art, such as phage surface display technology and genetic engineering recombination technology. For example, DNA molecules encoding the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification, the obtained DNA molecules can be inserted into an expression vector, and then transfected into host cells. The transfected host cells can then be cultured under specific conditions to express the antibodies of the present invention. The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules.

[0111] In some implementations, the method includes the following steps:

[0112] (1) Transform host cells using at least one of the nucleic acids or expression vectors described herein;

[0113] (2) Culture the transformed host cells under suitable conditions to allow the expression of the nucleic acid or expression vector, and

[0114] (3) Isolate and purify the nanobody or its antigen-binding fragment, polypeptide construct, bispecific antibody or multispecific antibody from the host cell or culture medium.

[0115] In some embodiments, the host cell also contains a chaperone plasmid, which can help improve the solubility, stability, and / or folding of the antibody or antibody fragment. Techniques for isolating and purifying antibodies from host cells are well known to those skilled in the art.

[0116] 7. Conjugates

[0117] On the other hand, this application also provides conjugates comprising the nanobody or antigen-binding fragment of the present invention, a polypeptide construct, a bispecific antibody or a multispecific antibody, and a conjugated portion.

[0118] In some implementations, the targeting portion is optionally coupled to the coupling portion via a connector.

[0119] In some embodiments, the conjugation portion is selected from protein tags. Such protein tags are well known in the art, and examples include, but are not limited to, His, Flag, GST, MBP, HA, Myc, GFP, or biotin, and those skilled in the art know how to select appropriate protein tags (e.g., purification tags, detection tags, or tracer tags) according to the desired purpose. In some exemplary embodiments, the C-terminus of the bispecific antibody of the present invention is linked to a purification tag.

[0120] In some embodiments, the coupling portion is selected from detectable markers, such as enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), or biotin. The detectable markers described in this invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances such as acridinium esters), magnetic beads (e.g., The labeling includes thermometric markers such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.), and biotin for binding avidin (e.g., streptavidin) modified with the aforementioned markers. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable markers described above can be linked to the bispecific antibodies of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0121] In some embodiments, the coupling portion is selected from therapeutic agents, such as cytotoxic drugs.

[0122] In some embodiments, the coupling moiety is selected from other bioactive peptides.

[0123] 8. Antibody-drug conjugates (ADCs)

[0124] This application further provides an antibody-drug conjugate (ADC) comprising:

[0125] The targeting portion is selected from any of the nanobodies or their antigen-binding fragments, polypeptide constructs, bispecific antibodies or multispecific antibodies described above;

[0126] Cytotoxic drugs section; and

[0127] Connector for connecting the target portion and the cytotoxic drug portion.

[0128] Cytotoxic drugs can be conjugated to disulfide bonds on antibodies, or site-specific and stable conjugation can be achieved using engineered cysteine-mutated conjugate technology. Mutating a specific amino acid site on an antibody to cysteine ​​and reacting it with a drug-linker allows for site-specific conjugation, yielding highly uniform conjugates and improving the therapeutic index of ADC drugs.

[0129] In some embodiments, the targeting portion is connected to the linker via a thiol group on a cysteine ​​residue.

[0130] In some embodiments, the targeting portion is linked to the linker via a thiol group on a cysteine ​​residue of VHH.

[0131] In some embodiments, the targeting portion is connected to the linker via a thiol group on a cysteine ​​residue of a reduced disulfide bond in the hinge region.

[0132] In some implementations, the targeting portion is connected to the linker via a thiol group on a cysteine ​​residue in the Fc domain.

[0133] In some embodiments, the targeting portion is connected to the linker via a thiol group on a cysteine ​​residue at position 239 and / or 290 of the Fc domain.

[0134] In some embodiments, the cytotoxic drug is selected from microtubule inhibitors and DNA damage drugs.

[0135] In some embodiments, the microtubule inhibitor is selected from olritamine compounds (e.g., MMAE, MMAF), maytansine compounds (e.g., maytansine, maytanol, DM1, DM4), taxanes (e.g., taxol, docetaxel, carbazitaxel), vinblastines (e.g., vincristine), eribulin, and colchicine.

[0136] In some embodiments, the DNA damaging agent is selected from DNA alkylating agents (cazithromycin γ1l, N-acetyl-γ1I cazithromycin, atrazomycin, PBD, ducamycin), DNA topoisomerase inhibitors (e.g., camptothecin compounds (specifically camptothecin, SN-38, Dxd, irinotecan, belotetan, topotecan, PNU-159682), doxorubicin, daunorubicin, etoposide, mitoxantrone), and muscarinic acid.

[0137] In some embodiments, the cytotoxic drug is MMAE.

[0138] In some implementations, the connector may be a cuttable or non-cuttable connector.

[0139] In some embodiments, the cleavable linker is selected from protease-sensitive, pH-sensitive, and glutathione-sensitive linkers.

[0140] In some embodiments, the linker is selected from MC (6-maleimide hexanoyl), MCC (maleimide methylcyclohexane-1-carboxylate), MP (maleimide propionyl), Val-Cit (valine-citrulline), Val-Ala (valine-alanine), Ala-Phe (alanine-phenylalanine), PAB (p-aminobenzyloxycarbonyl), SPP (5-(succinimide)-4-(pyridine-2- (N-succinimide-1-yl)valerate, 6-(2,5-dioxopyrrolidone-1-yl)-4-(pyridin-2-ylthio)hexanoate, 6-(2,5-dioxopyrrolidone-1-yl)-5-methyl-4-(pyridin-2-ylthio)hexanoate, SMCC (N-succinimide-4-(N-maleimide-methyl)cyclohexane-1-carboxylate) or SIAB (N-succinimide-(4-iodo-acetyl)aminobenzoate) and any combination thereof.

[0141] In some implementations, the connector is MC-Val-Cit-PAB.

[0142] In some embodiments, each peptide chain of the polypeptide construct is linked to 0, 1, 2, 3, 4, or 5 of the following structures via VHH, cysteine ​​residues in the reduced disulfide bond of the hinge region, or cysteine ​​residues in the Fc domain:

[0143]

[0144] In some implementations, the antibody-drug conjugate is selected from:

[0145]

[0146] Where x = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0147] Ab is any of the polypeptide constructs or bispecific antibodies described above.

[0148] In some implementation schemes, the antibody-drug conjugate is:

[0149]

[0150] Where x = 1, 2, 3, 4, 5 or 6;

[0151] Ab contains or is composed of an amino acid sequence as shown in SEQ ID NO:10, 17, 18, 19 or 20.

[0152] 9. Composition

[0153] In another aspect, this application provides a composition comprising or consisting of one or more of the antibody-drug conjugates described in any of the preceding claims.

[0154] In some embodiments, the DAR value of the composition is 1-10, for example 1-1.5, 1-2, 1-2.5, 1-3, 1-3.5, 1-4, 1-4.5, 1-5, 1-5.5, 1-6, 1-6.5, 1-7, 1-7.5, 1-8, 1-8.5, 1-9, 1-9.5, 1-10, 1.5-2, 1.5-2.5, 1.5-3, 1.5-3.5, 1.5-4, 1.5-4.5, 1.5-5, 1.5-5.5, 1.5-6, 1.5-6.5, 1.5-7, 1.5-7.5, 1.5-8, 1.5-8.5, 1.5-9, 1.5-9.5, 1.5-10, 2-2.5, 2-3, 2- 3.5, 2-4, 2-4.5, 2-5, 2-5.5, 2-6, 2-6.5, 2-7, 2-7.5, 2-8, 2-8.5, 2-9, 2-9.5, 2-10, 2.5-3, 2.5-3.5, 2.5-4, 2.5-4.5, 2.5-5, 2.5-5.5, 2.5-6, 2.5-6.5 2.5-7, 2.5-7.5, 2.5-8, 2.5-8.5, 2.5-9, 2.5-9.5, 2.5-10, 3-3.5, 3-4, 3-4.5, 3-5, 3-5.5, 3-6, 3-6.5, 3-7, 3-7.5, 3-8, 3-8.5, 3-9, 3-9.5, 3-10, 3.5-4 3.5-4.5, 3.5-5, 3.5-5.5, 3.5-6, 3.5-6.5, 3.5-7, 3.5-7.5, 3.5-8, 3.5-8.5, 3.5-9, 3.5-9.5, 3.5-10, 4-4.5, 4-5, 4-5.5, 4-6, 4.5-5, 4.5-5.5, 4.5- 6, 4.5-6.5, 4.5-7, 4.5-7.5, 4.5-8, 4.5-8.5, 4.5-9, 4.5-9.5, 4.5-10, 5-5.5, 5-6, 5-6.5, 5-7, 5-7.5, 5-8, 5-8.5, 5-9, 5-9.5, 5-10, 5.5-6, 5.5-6.5, 5 .5-7, 5.5-7.5, 5.5-8, 5.5-8.5, 5.5-9, 5.5-9.5, 5.5-10, 6-6.5, 6-7, 6-7.5, 6-8, 6-8.5, 6-9, 6-9.5, 6-10, 6.5-7, 6.5-7.5, 6.5-8, 6.5-8.5, 6.5-9, 6. 5-9.5, 6.5-10, 7-7.5, 7-8, 7-8.5, 7-9, 7-9.5, 7-10, 7.5-8, 7.5-8.5, 7.5-9, 7.5-9.5, 7.5-10, 8-8.5, 8-9, 8-9.5, 8-10, 8.5-9, 8.5-9.5, 8.5-10, 9-9.5, 9-10, 9.5-10...

[0155] In another aspect, this application provides a composition comprising or consisting of one or more of the following antibody-drug conjugates:

[0156]

[0157] Where x = 1, 2, 3, 4, 5, 6, 7 or 8;

[0158] Ab contains or is composed of an amino acid sequence as shown in SEQ ID NO:10, 17, 18, 19 or 20.

[0159] In some embodiments, the DAR value of the composition is 1-8, for example 1-1.5, 1-2, 1-2.5, 1-3, 1-3.5, 1-4, 1-4.5, 1-5, 1-5.5, 1-6, 1-6.5, 1-7, 1-7.5, 1-8, 1.5-2, 1.5-2.5, 1.5-3, 1.5-3.5, 1.5-4, 1.5-4.5, 1.5-5, 1.5-5.5, 1.5-6, 1. 5-6.5, 1.5-7, 1.5-7.5, 1.5-8, 2-2.5, 2-3, 2-3.5, 2-4, 2-4.5, 2-5, 2-5.5, 2-6, 2-6.5, 2-7, 2-7.5, 2-8, 2.5-3, 2.5-3.5, 2.5-4, 2.5-4.5, 2.5-5, 2.5-5.5, 2.5-6, 2.5-6.5, 2.5-7, 2.5-7.5, 2.5-8 3-3.5, 3-4, 3-4.5, 3-5, 3-5.5, 3-6, 3-6.5, 3-7, 3-7.5, 3-8, 3.5-4, 3.5-4.5, 3.5-5, 3.5-5.5, 3.5-6, 3.5-6.5, 3.5-7, 3.5-7.5, 3.5-8, 4-4.5, 4-5, 4-5.5, 4-6, 4-6.5, 4-7, 4-7.5, 4-8, 4.5-5, 4 0.5-5.5, 4.5-6, 4.5-6.5, 4.5-7, 4.5-7.5, 4.5-8, 5-5.5, 5-6, 5-6.5, 5-7, 5-7.5, 5-8, 5.5-6, 5.5-6.5, 5.5-7, 5.5-7.5, 5.5-8, 6-6.5, 6-7, 6-7.5, 6-8, 6.5-7, 6.5-7.5, 6.5-8, 7-7.5, 7-8 or 7.5-8.

[0160] In some embodiments, the composition has a DAR value of 1-5, for example 3.5-4.5, or even 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4 or 4.5.

[0161] 10. Antibody composition

[0162] This application also provides an antibody composition comprising a first antibody that specifically binds to Trop2 and a second antibody that specifically binds to EpCAM.

[0163] Antibodies that specifically bind to Trop2

[0164] The first antibody that specifically binds to Trop2 in the antibody composition of the present invention can be any antibody form, including but not limited to one or more of single-domain antibodies, single-chain antibodies, antibody Fabs, full-length antibody proteins, antigen-binding fragments, bispecific antibodies, multispecific antibodies, bivalent / multivalent single-domain antibodies, bivalent / multivalent single-chain antibodies, and bivalent / multivalent antibody Fabs.

[0165] In some embodiments, the first antibody is a nanobody that specifically binds to Trop2 or an antigen-binding fragment thereof, or a polypeptide construct comprising the nanobody or an antigen-binding fragment thereof.

[0166] In some embodiments, the nanobody that specifically binds to Trop2 or its antigen-binding fragment comprises the following CDR1 (complementarity-determining region 1), CDR2 (complementarity-determining region 2), and CDR3 (complementarity-determining region 3) sequences:

[0167] (a) CDR1, having: the sequence shown in SEQ ID NO:12, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:12;

[0168] (b) CDR2, having: the sequence shown in SEQ ID NO:13, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence shown in SEQ ID NO:13; and

[0169] (c) CDR3, having: the sequence shown in SEQ ID NO:14, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:14.

[0170] In some embodiments, the nanobody that specifically binds to Trop2 or its antigen-binding fragment comprises: CDR1 as shown in SEQ ID NO:12, CDR2 as shown in SEQ ID NO:13, and CDR3 as shown in SEQ ID NO:14.

[0171] In some embodiments, the nanobody that specifically binds to Trop2 or its antigen-binding fragment comprises a VHH sequence as shown in SEQ ID NO:11 or a variant thereof; the variant has at least 80% sequence identity with the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions compared to the original sequence; preferably, the substitution is a conservative substitution.

[0172] In some embodiments, the first antibody comprises a VHH sequence as shown in SEQ ID NO:11.

[0173] Antibodies that specifically bind to EpCAM

[0174] The antibody composition of the present invention may contain a second antibody that specifically binds to EpCAM, which may be in any antibody form, including but not limited to one or more of the following: single-domain antibody, single-chain antibody, antibody Fab, full-length antibody protein, antigen-binding fragment, bispecific antibody, multispecific antibody, bi / multivalent single-domain antibody, bi / multivalent single-chain antibody, and bi / multivalent antibody Fab.

[0175] In some embodiments, the second antibody is a nanobody that specifically binds to EpCAM or an antigen-binding fragment thereof, or a polypeptide construct comprising the nanobody or an antigen-binding fragment thereof.

[0176] In some embodiments, the nanobody or antigen-binding fragment that specifically binds to EpCAM is the nanobody or antigen-binding fragment described in aspect 1 above.

[0177] peptide constructs

[0178] In some embodiments, the first antibody is a polypeptide construct comprising a nanobody that specifically binds to Trop2 or an antigen-binding fragment thereof. In some embodiments, the second antibody is a polypeptide construct comprising a nanobody that specifically binds to EpCAM or an antigen-binding fragment thereof.

[0179] In some embodiments, the polypeptide construct includes an immunoglobulin Fc domain.

[0180] In some embodiments, the immunoglobulin Fc domain is directly or via a peptide linker linked to the N-terminus or C-terminus of the nanobody or its antigen-binding fragment.

[0181] In some embodiments, the immunoglobulin Fc domain is directly or via a peptide linker linked to the C-terminus of the nanobody or its antigen-binding fragment.

[0182] In some embodiments, the immunoglobulin Fc domain comprises the sequence shown in SEQ ID NO: 9, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with it, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) with it.

[0183] In some embodiments, the immunoglobulin Fc domain may or may not contain mutations at the S239 and / or K290 positions, such mutations as S239C and / or K290C. In some embodiments, the immunoglobulin Fc domain is numbered according to the Kabat EU index.

[0184] In some embodiments, the polypeptide construct contains or is composed of an amino acid sequence as shown in SEQ ID NO:15 or 16.

[0185] In some implementations, the polypeptide construct is a dimer.

[0186] In some embodiments, the polypeptide construct comprises, but is not limited to, one or more of single-domain antibodies, single-chain antibodies, antibody Fabs, full-length antibody proteins, antigen-binding fragments, bispecific antibodies, multispecific antibodies, bivalent / multivalent single-domain antibodies, bivalent / multivalent single-chain antibodies, and bivalent / multivalent antibody Fabs.

[0187] 11. Pharmaceutical Composition

[0188] In another aspect, this application provides a pharmaceutical composition comprising any of the nanobodies or antigen-binding fragments thereof described above, a polypeptide construct comprising the nanobodies or antigen-binding fragments thereof, the bispecific antibody, multispecific antibody, nucleic acid molecule, carrier, host cell, conjugate, antibody-drug conjugate, composition or antibody composition, and optionally a carrier or excipient.

[0189] The excipient may be one described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986). Non-limiting examples of suitable excipients include buffers, preservatives, binders, lubricants, disintegrants, chelating agents, surfactants, flavoring agents, sweeteners, and coloring agents.

[0190] In some embodiments, suitable buffers include calcium bicarbonate, sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium gluconate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide, and other calcium salts or combinations thereof.

[0191] In some embodiments, suitable preservatives include antioxidants such as α-tocopherol and ascorbate, and antimicrobial agents such as parabens, chlorobutanol, and phenol. Antioxidants may further include EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, para-aminobenzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol, and N-acetylcysteine, etc.

[0192] In some implementations, suitable binders include starches such as potato starch, corn starch, and wheat starch; sugars such as sucrose, glucose, dextrose, lactose, and maltodextrin; natural and synthetic gums; gelatin; cellulose derivatives such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, methyl cellulose, and ethyl cellulose; polyvinylpyrrolidone (polyvinylpyrrolidone); polyethylene glycol (PEG); waxes; calcium carbonate; calcium phosphate; alcohols such as sorbitol, xylitol, mannitol, and water, or combinations thereof.

[0193] In some implementations, suitable lubricants include metal stearate salts (such as magnesium stearate, calcium stearate, aluminum stearate), fatty acid esters (such as sodium stearoyl fumarate), fatty acids (such as stearic acid), fatty alcohols, glyceryl betaine, mineral oil, paraffin wax, hydrogenated vegetable oil, leucine, polyethylene glycol (PEG), metal dodecyl sulfate salts (such as sodium dodecyl sulfate, magnesium dodecyl sulfate), sodium chloride, sodium benzoate, sodium acetate, and talc, or combinations thereof.

[0194] In some embodiments, the disintegrant may be a non-effervescent disintegrant. Suitable non-effervescent disintegrants include starches such as corn starch, potato starch, pregelatinized and modified starches, sweeteners, clays such as bentonite, microcrystalline cellulose, alginate, sodium glycolate starch, gums such as agar, guar gum, locust bean gum, ark sylvestris gum, pectin, and tragacanth gum. In some embodiments, the disintegrant may be an effervescent disintegrant. Suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

[0195] In some implementations, suitable flavoring agents may be selected from cinnamon oil; wintergreen oil; peppermint oil; clover oil; hay oil; fennel oil; eucalyptus oil; vanilla; citrus oils such as lemon oil, orange oil, grape and grapefruit oil; and fruit flavorings, including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple and apricot flavorings.

[0196] In some implementations, suitable sweeteners include glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts, such as sodium salts; dipeptide sweeteners, such as aspartame; dihydrochalcone compounds, glycyrrhizin; stevia (stevioside); chlorinated derivatives of sucrose, such as sucralose; and sugar alcohols, such as sorbitol, mannitol, xylitol, etc.

[0197] In some implementations, suitable colorants include food, pharmaceutical and cosmetic pigments (FD&C), pharmaceutical and cosmetic pigments (D&C), and external pharmaceutical and cosmetic pigments (Ext.D&C).

[0198] In some embodiments, suitable chelating agents include ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA); disodium, trisodium, tetrasodium, dipotassium, tripotassium, dilithium, and diammonium salts of EDTA; barium, calcium, cobalt, copper, dysprosium, europium, iron, indium, lanthanum, magnesium, manganese, nickel, samarium, strontium, or zinc chelates of EDTA; and trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid. Monohydrate; N,N-bis(2-hydroxyethyl)glycine; 1,3-diamino-2-hydroxypropane-N,N,N′,N′-tetraacetic acid; 1,3-diaminopropane-N,N,N′,N′-tetraacetic acid; ethylenediamine-N,N′-diacetic acid; ethylenediamine-N,N′-dipropionic acid dihydrochloride; ethylenediamine-N,N′-bis(methylenephosphonic acid) hemihydrate; N-(2-hydroxyethyl)ethylenediamine-N N,N′,N′-triacetic acid; ethylenediamine-N,N,N′,N′-tetra(methylenephosphonic acid); O,O′-bis(2-aminoethyl)ethylene glycol-N,N,N′,N′-tetraacetic acid; N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid; 1,6-hexanediamine-N,N,N′,N′-tetraacetic acid; N-(2-hydroxyethyl)iminodiacetic acid; iminodiacetic acid; 1,2- Diaminopropane-N,N,N′,N′-tetraacetic acid; hypozinogenyltriacetic acid; hypozinogenyltripropionic acid; trisodium salt of hypozinogenyltris(methylene phosphate); 7,19,30-trioxa-1,4,10,13,16,22,27,33-octaazabicyclo[11,11,11]tripentadecanehexahydrobromide; or triethylenetetramine-N,N,N′,N″,N″′,N″′-hexaacetic acid, etc.

[0199] In some implementations, suitable diluents include water, glycerol, methanol, ethanol, and other biocompatible diluents.

[0200] In some embodiments, suitable surfactants include polysorbate, sodium lauryl sulfate, sodium stearoyl fumarate, polyoxyethylene alkyl ethers, dehydrated sorbitan fatty acid esters, polyethylene glycol (PEG), polyoxyethylene castor oil derivatives, glycol esters of fatty acids, glycerides of fatty acids, or combinations thereof.

[0201] The pharmaceutical compositions described herein can be formulated into various dosage forms and administered via a variety of different routes, such as oral, rectal, or parenteral administration. The term "parenteral" as used herein can include intra-arterial, intracardiac, intraventricular, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrasheathal, intravenous, intravitreal, epidural, subcutaneous, inhalation, transdermal, transmucosal, sublingual, buccal, and topical (including epidermal, dermal, enema, eye drops, ear drops, intranasal, and vaginal) administration. In some exemplary embodiments, the route of administration may be by injection, such as intramuscular, intravenous, subcutaneous, or intraperitoneal injection. Oral formulations may include capsules, tablets, pouches, pills, sugar tablets, lozenges, powders, and granules, etc.

[0202] 12. Pharmaceutical Uses

[0203] In one aspect, this application provides the use of the nanobody described herein or an antigen-binding fragment thereof, or a polypeptide construct comprising the nanobody or an antigen-binding fragment thereof, for the preparation of a medicament for the prevention and / or treatment of EpCAM-related diseases in subjects.

[0204] In one aspect, this application provides prevention and / or treatment of diseases associated with EpCAM, including administering to a subject in need an effective amount of the nanobody described herein or an antigen-binding fragment thereof, or a polypeptide construct comprising the nanobody or an antigen-binding fragment thereof.

[0205] In some embodiments, the EpCAM-related disease is a tumor, such as an EpCAM-positive tumor. In some embodiments, the tumor is selected from colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), lung cancer, oral squamous cell carcinoma, ovarian cancer (e.g., epithelial ovarian cancer), cervical cancer, bladder cancer, prostate cancer, pancreatic cancer, liver cancer, and retinoblastoma.

[0206] In some implementations, the subject is a mammal, such as a human.

[0207] In some embodiments, the nanobody or its antigen-binding fragment, peptide construct or pharmaceutical composition may be used alone or in combination with other pharmaceutically active agents.

[0208] In another aspect, this application provides the use of the bispecific antibodies, multispecific antibodies, nucleic acid molecules, vectors, host cells, conjugates, antibody-drug conjugates, compositions, antibody compositions, or pharmaceutical compositions described herein for the preparation of a medicament for the prevention and / or treatment in subjects of diseases related to Trop2 and / or EpCAM.

[0209] In another aspect, this application provides for the prevention and / or treatment of diseases related to Trop2 and / or EpCAM, including administering an effective amount of the bispecific antibody, multispecific antibody, nucleic acid molecule, vector, host cell, conjugate, antibody-drug conjugate, composition, antibody composition, or pharmaceutical composition described herein to a subject in need of such treatment.

[0210] In some embodiments, the Trop2-related and / or EpCAM-related disease is a tumor, such as a Trop2 and / or EpCAM-positive tumor. In some embodiments, the tumor is selected from colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), lung cancer, oral squamous cell carcinoma, ovarian cancer (e.g., epithelial ovarian cancer), cervical cancer, bladder cancer, prostate cancer, pancreatic cancer, liver cancer, and retinoblastoma.

[0211] In some implementations, the subject is a mammal, such as a human;

[0212] In some embodiments, the bispecific antibody, multispecific antibody, nucleic acid molecule, vector, host cell, conjugate, antibody-drug conjugate, composition, antibody composition, or pharmaceutical composition may be used alone or in combination with other pharmaceutically active agents.

[0213] 13. Purpose of Testing

[0214] In one aspect, this application provides the use of the nanobody or antigen-binding fragment thereof of the present invention, or a polypeptide construct comprising the nanobody or antigen-binding fragment thereof, in the preparation of a diagnostic reagent for detecting the presence or level of EpCAM in a sample or for diagnosing whether a subject has a disease related to EpCAM.

[0215] In some embodiments, the nanobodies or antigen-binding fragments thereof, or peptide constructs used to prepare the detection reagents, are labeled with detectable tags.

[0216] In some embodiments, the nanobodies or antigen-binding fragments thereof, or peptide constructs used to prepare the detection reagents do not contain a detectable label. In such embodiments, the detection reagents may further comprise other reagents (such as a second antibody) capable of detecting the nanobodies or antigen-binding fragments thereof, or peptide constructs of the present invention.

[0217] In one aspect, this application provides a method for detecting the presence or level of EpCAM in a sample, comprising using the nanobody or antigen-binding fragment thereof of the present invention, or a polypeptide construct comprising said nanobody or antigen-binding fragment thereof.

[0218] In some embodiments, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0219] In some embodiments, the nanobody or its antigen-binding fragment or peptide construct used in the method is labeled with a detectable tag.

[0220] In some embodiments, the nanobodies or their antigen-binding fragments or peptide constructs used in the method do not carry a detectable label. Therefore, the method may also include using other reagents (such as a second antibody) carrying a detectable label to detect the nanobodies or their antigen-binding fragments or peptide constructs of the present invention.

[0221] In some implementations, the method includes the following steps:

[0222] (1) Contact the sample with the nanobody or its antigen-binding fragment or polypeptide construct of the present invention;

[0223] (2) Detect the formation of a complex between the nanobody or its antigen-binding fragment or polypeptide construct and the antigen, or detect the amount of the complex.

[0224] The formation of the complex indicates the presence of an antigen or cells expressing an antigen;

[0225] The antigen is EpCAM.

[0226] The method can be used for diagnostic purposes or non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient).

[0227] In some embodiments, the method is used to diagnose whether a subject has a disease related to EpCAM. In such embodiments, the method may further include the step of comparing the amount of EpCAM in a sample from the subject with a reference value. The reference value may be the level of EpCAM in a sample from a subject known not to have a disease related to EpCAM (e.g., a healthy control) (also referred to as a "negative reference value"). For example, if the amount of EpCAM in a sample from the subject is elevated relative to a negative reference value, it indicates that the subject has a disease related to EpCAM.

[0228] In some embodiments, the EpCAM-related diseases are characterized by elevated EpCAM expression and / or excessive EpCAM activity. In some embodiments, the EpCAM-related diseases are tumors, including but not limited to colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), lung cancer, oral squamous cell carcinoma, ovarian cancer (e.g., epithelial ovarian cancer), cervical cancer, bladder cancer, prostate cancer, pancreatic cancer, liver cancer, and retinoblastoma.

[0229] In some embodiments, the sample may be selected from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissue (e.g., surgically removed tumor tissue, biopsy sections, or fine-needle aspiration tissue), histological preparations, etc.

[0230] On the other hand, the use of the bispecific antibodies, conjugates or antibody compositions of the present invention in the preparation of diagnostic reagents for detecting the presence or level of Trop2 and / or EpCAM in a sample or for diagnosing whether a subject has a disease related to Trop2 and / or EpCAM.

[0231] In some embodiments, the conjugate used to prepare the detection reagent comprises the bispecific antibody of the present invention and a detectable label.

[0232] In some implementations, the bispecific antibody used to prepare the detection reagent is labeled with a detectable tag.

[0233] In some embodiments, the bispecific antibody used to prepare the detection reagent does not carry a detectable label. In such embodiments, the detection reagent may further comprise other reagents (such as a second antibody) capable of detecting the bispecific antibody of the present invention.

[0234] On the other hand, this application provides a method for detecting the presence or content of Trop2 and / or EpCAM in a sample, which includes using the bispecific antibody, conjugate, or antibody composition of the present invention.

[0235] In some embodiments, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0236] In some embodiments, the conjugate used in the method comprises the bispecific antibody of the present invention and a detectable label.

[0237] In some embodiments, the bispecific antibody used in the method is labeled with a detectable tag.

[0238] In some embodiments, the bispecific antibody used in the method does not carry a detectable label. Therefore, the method may also include using other reagents (such as a second antibody) carrying a detectable label to detect the bispecific antibody of the present invention.

[0239] In some implementations, the method includes the following steps:

[0240] (1) Contact the sample with the bispecific antibody, conjugate or antibody composition of the present invention;

[0241] (2) Detect the formation of a complex between the bispecific antibody, conjugate or antibody composition and the antigen or detect the amount of the complex.

[0242] The formation of the complex indicates the presence of an antigen or cells expressing an antigen;

[0243] The antigen is selected from Trop2 or EpCAM.

[0244] The method can be used for diagnostic purposes or non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient).

[0245] In some embodiments, the method is used to diagnose whether a subject has a disease related to Trop2 and / or EpCAM. In such embodiments, the method may further include the step of comparing the amount of Trop2 and / or EpCAM in a sample from the subject with a reference value. The reference value may be the level of Trop2 and / or EpCAM in a sample from a subject known not to have a disease related to Trop2 and / or EpCAM (e.g., a healthy control) (also referred to as a "negative reference value"). For example, if the amount of Trop2 and / or EpCAM in a sample from the subject is elevated relative to a negative reference value, it indicates that the subject has a disease related to Trop2 and / or EpCAM.

[0246] In some embodiments, the Trop2-related diseases are characterized by elevated Trop2 expression and / or excessive Trop2 activity. In some embodiments, the Trop2-related diseases are tumors, including but not limited to colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), lung cancer, oral squamous cell carcinoma, ovarian cancer (e.g., epithelial ovarian cancer), cervical cancer, and bladder cancer.

[0247] In some embodiments, the EpCAM-related diseases are characterized by elevated EpCAM expression and / or excessive EpCAM activity. In some embodiments, the EpCAM-related diseases are tumors, including but not limited to colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), lung cancer, oral squamous cell carcinoma, ovarian cancer (e.g., epithelial ovarian cancer), cervical cancer, bladder cancer, prostate cancer, pancreatic cancer, liver cancer, and retinoblastoma.

[0248] In some embodiments, the sample may be selected from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissue (e.g., surgically removed tumor tissue, biopsy sections, or fine-needle aspiration tissue), histological preparations, etc.

[0249] Terminology Definition

[0250] In this document, unless otherwise stated, scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the procedures described herein, such as molecular genetics, nucleic acid chemistry, cell culture, biochemistry, and cell biology, are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0251] As used herein, the singular forms of “a” and “an” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, the terms “including,” “comprising,” “having,” “containing,” or variations thereof are open-ended, not exclusive or exhaustive.

[0252] As used herein, the term "Trop2" stands for Trophoblast Cell-Surface Antigen 2, a transmembrane protein. Trop-2 is encoded by the TACSTD2 gene located on chromosome 1 and consists of 323 amino acids. The Trop-2 protein structure includes a hydrophobic leader peptide, an extracellular domain, a transmembrane domain, and a cytoplasmic tail. The sequence of Trop2 is well known to those skilled in the art, and the amino acid sequence can be found, for example, in NCBI Gene ID: NP_002344.2.

[0253] As used herein, the term "EpCAM" stands for Epithelial Cell Adhesion Molecule, also known as CD326, TACSTD1, GA733-2, KSA, CO17-1A, etc. It is a conserved type I transmembrane glycoprotein with a size of 35 kDa. The gene encoding human EpCAM is located on chromosome 2. Human EpCAM is a polypeptide composed of 314 amino acids, consisting of a hydrophobic leader peptide, a large extracellular domain (N-terminus) of 242 amino acids, a single transmembrane domain of 23 amino acids, and a short cytoplasmic domain (C-terminus) of 26 amino acids. The amino acid sequence of EpCAM can be found in NCBI Gene ID: NP_002345.2. As used herein, the terms "antibody" and "monoclonal antibody" refer to immunoglobulin molecules that are typically composed of two pairs of polypeptide chains (each pair having one light chain (LC) and one heavy chain (HC)). Each chain has a variable region, called the heavy chain variable region (VH) and the light chain variable region (VL). Together, VH and VL are responsible for binding to the antigen recognized by the antibody. Mammalian immunoglobulins have five main heavy chain classes (or isotypes), which determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW, and IgNAR. IgY is a primary antibody produced by birds and reptiles, functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.

[0254] The variable region of an antibody comprises a framework region (FR) and hypervariable regions (HVR), referred to as the complementarity-determining region (CDR). The CDR is primarily responsible for binding to epitopes of the antigen. VH and VL consist of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The allocation of amino acids in each region or domain can follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0255] As used herein, the terms "nanobody," "single-domain antibody," "VHH antibody," or "camel antibody" refer to an antibody that lacks additional antibody domains and possesses a single domain (variable region) capable of specifically binding to an antigen or epitope. The term "nanobody" has the meaning commonly understood by those skilled in the art as an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), typically derived from the variable region of a heavy chain antibody (e.g., a camel antibody or a shark antibody). Typically, a nanobody consists of four framework regions and three complementarity-determining regions, having a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N-terminus or C-terminus to contain only a portion of FR1 and / or FR4, or to lack one or both of those framework regions, as long as they substantially maintain antigen binding and specificity. Nanobodies are also called single-domain antibodies (sdAbs), and the two terms are used interchangeably.

[0256] As used herein, the term "bispecific antibody" refers to an antibody that has binding specificity to two different antigens (or epitopes). A bispecific antibody comprises two antigen-binding domains with binding specificity to different antigens (or epitopes), thereby enabling it to bind to two different binding sites and / or target molecules. In some cases, the different antigen-binding domains are linked by peptide linkers. The term "bispecific nanobody" refers to a bispecific antibody formed by two nanobodies.

[0257] The term "multispecific antibody" refers to an antibody that has binding specificity against at least two (e.g., two, three, or four) different antigens (or epitopes). A multispecific antibody comprises multiple antigen-binding domains that have binding specificity against different antigens (or epitopes), thereby enabling it to bind to at least two different binding sites and / or target molecules. In some cases, the individual antigen-binding domains are linked by peptide linkers.

[0258] As used herein, the term "Fc region" or "Fc domain" refers to a portion of the heavy chain constant region containing CH2 and CH3. In some embodiments, the Fc region includes a hinge, CH2, and CH3. In some embodiments, when the Fc region includes a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0259] As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of lymphocytes or by cells transfected with the coding sequence of a single antibody. Monoclonal antibodies can be produced by methods known to those skilled in the art. Monoclonal antibodies include humanized monoclonal antibodies.

[0260] As used herein, the term "conservative variant" refers to a protein containing a conserved amino acid substitution that substantially does not affect or reduce the protein's affinity. For example, nanobodies or peptide constructs that specifically bind EpCAM may include up to one, two, five, ten, or fifteen conserved substitutions and specifically bind EpCAM to the peptide. Conserved amino acid substitutions of functionally similar amino acids are well known to those skilled in the art. The following six groups are considered examples of amino acids that are conserved substitutions for each other:

[0261] 1) Alanine (A), Serine (S), Threonine (T);

[0262] 2) Aspartic acid (D), glutamic acid (E);

[0263] 3) Asparagine (N), glutamine (Q);

[0264] 4) Arginine (R), Lysine (K);

[0265] 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and

[0266] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0267] As used herein, the amino acid residue abbreviations are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0268] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoIBiol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0269] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules (i.e., a binding molecule and a target molecule), such as the reaction between an antibody and its target antigen. The binding affinity between two molecules can be measured using Kx. D Value description. K D The value refers to the dissociation constant obtained by the ratio of kd (the dissociation rate of a specific binding molecule-target molecule interaction; also known as koff) to ka (the association rate of a specific binding molecule-target molecule interaction; also known as kon), or kd / ka expressed as molar concentration (M). DThe smaller the value, the tighter the binding between the two molecules, and the higher the affinity. In some embodiments, an antibody that specifically binds to a certain antigen (or an antibody that is specific to a certain antigen) refers to an antibody with a binding affinity of less than approximately 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D Bind to the antigen. K D The value can be determined by methods well known in the art, such as using surface plasmon resonance (SPR) in a BIACORE instrument.

[0270] As used herein, the terms “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” refer to an oligomer or polymer containing at least two linked nucleotides or nucleotide derivatives, which may typically include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0271] As used herein, the term "isolated" means that a substance (such as a nucleic acid molecule or polypeptide) is isolated from its source or environment in which it exists, i.e., it does not contain any other components.

[0272] As used herein, the term "vector" is a medium used to introduce exogenous nucleic acids into host cells, whereby the exogenous nucleic acid is amplified or expressed when the vector is transformed into a suitable host cell. Vectors typically remain free, but can be designed to integrate genes or portions thereof into chromosomes of the genome. In this paper, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, granules, phage vectors, phage particles, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), etc.

[0273] As used herein, the term "expression vector" refers to a vector capable of expressing DNA operatively linked to a regulatory sequence (such as a promoter, ribosome binding site) that can influence DNA expression. The regulatory sequence may include promoter and terminator sequences and optionally may include origin of replication, selection markers, enhancers, polyadenylation signals, etc. The expression vector may be a plasmid, phage vector, recombinant virus, or other vector that, when introduced into a suitable host cell, results in the expression of clonal DNA. Suitable expression vectors are well known to those skilled in the art and include reproducible expression vectors in eukaryotic and / or prokaryotic cells, as well as expression vectors that remain free or are integrated into the host cell genome.

[0274] As used herein, the term "host cell" is a cell used to receive, maintain, replicate, or amplify a vector. Host cells can also be used to express nucleic acids or polypeptides encoded by the vector. Host cells can be eukaryotic or prokaryotic cells.

[0275] As used in this article, the term "contact" refers to direct physical association; it includes both solid and liquid forms.

[0276] As used herein, the term "cytotoxic drug" refers to any drug or compound capable of killing cells. "Cytotoxicity" refers to the toxicity of a molecule to its intended target cell, not to the cells of the rest of the organism. In contrast, the term "toxicity" refers to the toxicity of a molecule to cells other than its intended target cell.

[0277] As used herein, the terms “subject,” “patient,” or “individual” include both mammals and non-mammals. Mammals can be any member of the class Mammalia, including but not limited to humans; non-human primates such as chimpanzees, apes, or other monkeys; livestock such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs (or canines), and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs; and so on. Non-mammals can include birds, fish, etc. In some embodiments, the subject can be a mammal. In some embodiments, the subject can be a human. In some cases, the human can be an adult. In some cases, the human can be a child. In some cases, the human can be 0-17 years old. In some cases, the human can be 18-130 years old. In some cases, the subject can be male. In some cases, the subject can be female. In some cases, the subject has been diagnosed with or is suspected of having a disease. In some cases, the disease is cancer. The subject can be a patient or an individual. In some cases, subject, patient, or individual may be used interchangeably.

[0278] As used herein, the terms “treatment,” “management,” “improvement,” or “relief” include alleviating or reducing the symptoms of a disease, suppressing the disease (e.g., preventing its progression), alleviating the disease, causing the disease to regress, relieving symptoms caused by the disease, or stopping the symptoms of the disease. The terms “treatment,” “management,” “improvement,” or “relief” may further include obtaining a therapeutic benefit. A therapeutic benefit may refer to the eradication of the treated disease. Additionally, a therapeutic benefit may also be achieved by eradicating one or more physiological symptoms associated with the treated disease, resulting in an observable improvement in the subject, although in some implementations the subject may still suffer from the underlying disease.

[0279] As used herein, the terms "effective dose" and "therapeutic effective dose" refer to an adequate amount of medication administered that will at least partially relieve the symptoms of the disease being treated. Dosing regimens can be adjusted to provide the optimal desired response. For example, a single bolus injection may be administered, or several fractions may be given over time, or the dose may be reduced or increased proportionally depending on treatment progress. It should be noted that dose values ​​can vary depending on the type and severity of the disease to be alleviated, and may include single or multiple doses. To further understand, for any given individual, the specific dosing regimen should be adjusted over time based on individual needs and the drug's instructions or the professional judgment of a clinician. Generally, the effective dose is approximately 0.0001 to approximately 50 mg per kg of body weight per day, for example, approximately 0.01 to approximately 10 mg / kg / day (single or fractionated doses). For a 70 kg person, this would total approximately 0.007 mg / day to approximately 3500 mg / day, for example, approximately 0.7 mg / day to approximately 700 mg / day. In some cases, a dose level not exceeding the lower limit of the aforementioned range may be sufficient, while in other cases, a larger dose may still be used without causing any harmful side effects, provided that the larger dose is first divided into several smaller doses for administration throughout the day.

[0280] Beneficial effects of the invention

[0281] This application provides a combination of an antibody targeting Trop2 and an antibody targeting EpCAM, and specifically provides a bispecific antibody that simultaneously targets Trop2 and EpCAM, as well as antibody-drug conjugates, compositions containing them, and their uses. The antibody-drug conjugates exhibit good binding activity to both Trop2 and EpCAM proteins, demonstrate good targeting and inhibitory activity against Trop2 and EpCAM double-positive or single-positive tumors, and have good safety profiles. Attached Figure Description

[0282] Figure 1 The purity analysis results of P1 nanobodies are shown (reducing gel and non-reducing gel).

[0283] Figure 2 The structure of an anti-EpCAM antibody-drug conjugate (P1-VC-MMAE) is shown as an example.

[0284] Figure 3 The results show the detection results of the binding activity of P1, P1 ADC and antigen EpCAM by ELISA.

[0285] Figure 4 The results of detecting the DAR value of P1-VC-MMAE using hydrophobic interaction chromatography (HIC) are shown.

[0286] Figure 5The internalization rate of the P1 antibody in three cell lines was shown by flow cytometry.

[0287] Figure 6 The binding and internalization of the P1 antibody in three cell lines were shown as verified by immunofluorescence.

[0288] Figure 7 The in vivo targeting of the P1 antibody was demonstrated.

[0289] Figure 8 The structures of four different bispecific antibodies targeting Trop2 / EpCAM are shown.

[0290] Figure 9 The band size of ①Anti-Trop2 / EpCAM BsAb was shown by SDS-PAGE identification.

[0291] Figure 10 The structure of an anti-Trop2 / EpCAM bispecific antibody-drug conjugate (Anti-Trop2 / EpCAM-VC-MMAE) is shown as an example.

[0292] Figure 11 The results demonstrate the ELISA method for detecting the binding activity of Anti-Trop2 Ab, Anti-EpCAM Ab, and ①Anti-Trop2 / EpCAMBsAb with antigen Trop2.

[0293] Figure 12 The results demonstrate the ELISA method for detecting the binding activity of Anti-Trop2 Ab, Anti-EpCAM Ab, and ①Anti-Trop2 / EpCAMBsAb with the antigen EpCAM.

[0294] Figure 13 The results of the detection of the DAR value of ①Anti-Trop2 / EpCAM-VC-MMAE by hydrophobic interaction chromatography (HIC) are shown.

[0295] Figure 14 The results of flow cytometry analysis of the binding and internalization abilities of ①Anti-Trop2 / EpCAM BsAb and parental antibodies in the pancreatic cancer cell line BxPC3 are shown.

[0296] Figure 15 The results of flow cytometry analysis of the binding and internalization abilities of ①Anti-Trop2 / EpCAM BsAb and parental antibodies in the colorectal cancer cell line HT-29 are shown.

[0297] Figure 16The in vitro killing effect of ① Anti-Trop2 / EpCAM-VC-MMAE was demonstrated.

[0298] Figure 17A and Figure 17B The results show the tumor-suppressing effects and weight changes of BxPC3 tumor-bearing mice with high Trop2 / EpCAM-VC-MMAE and high EpCAM-VC-MMAE single-dose administration, respectively.

[0299] Figure 18A and Figure 18B The results show the tumor-suppressing effects and weight changes of HT-29 tumor-bearing mice with low Trop2 expression / high EpCAM expression colorectal cancer cells after a single dose of Anti-Trop2 / EpCAM-VC-MMAE and Anti-Trop2-VC-MMAE.

[0300] Sequence information

[0301] Information about the sequences involved in this invention is described in the table below:

[0302]

[0303]

[0304] Detailed Implementation

[0305] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0306] Example 1: Site-directed modification and binding activity of EpCAM nanobodies

[0307] 1.1 In the early stages of the laboratory, a nanobody specifically targeting EpCAM was screened from an alpaca antibody library based on phage display and screening techniques. The amino acid sequence is shown in Table 1 SEQ ID NO:1.

[0308] The method described by KR Abhinandan et al. (Abhinandan KR, Martin AC. Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains. Mol Immunol. 2008 Aug; 45(14):3832-9. doi:10.1016 / j.molimm.2008.05.022.) and IMGT (THE INTERNATIONAL IMMUNOGENETICS INFORMATION) were used. The dual strategy determined the CDR sequence of VHH, as shown in Table 1.

[0309] Table 1. Description of the EpCAM nanobody sequence (hereinafter referred to as P1).

[0310]

[0311]

[0312] 1.2 The selected VHH was expressed in tandem with the Fc segments CH2 and CH3 (SEQ ID NO:9) of the traditional antibody to extend the half-life. The expression vector used was PTT5-H, with serine at position 239 mutated to cysteine ​​and lysine at position 290 mutated to cysteine, named "P1", as shown in Table 2.

[0313] Table 2. Amino acid sequence of nanobody P1 (with Fc tag)

[0314]

[0315] Example 2: Transient expression and affinity chromatography purification of P1

[0316] 2.1 Plasmid Acquisition: The Anti-EpCAM Ab plasmid was synthesized by General Biosystems (Anhui) Co., Ltd. The plasmid was rapidly transferred into DH-5α Escherichia coli competent cells (Shenzhen Kangti), heat-shocked for 90 seconds, and then cultured overnight at 37°C in ampicillin-resistant LB agar. Single colonies were picked and amplified in ampicillin-resistant LB agar at 37°C, 220 rpm for 15 hours. The plasmid was then extracted using an endotoxin-free plasmid extraction kit.

[0317] 2.2 Antibody Expression: Expi-293F cells were transiently transfected with a single plasmid to express Anti-EpCAM Ab. The cell density was 4 × 10⁻⁶ cells / cells. 6200 mL of Expi-293F cells with a viability of 95% were cultured. 0.5 mg of the antibody plasmid was filtered through a 0.22 μm filter and added to 5 mL of CD05 medium. Simultaneously, 2 mg of PEI was added to 5 mL of CD05 medium, vortexed for 8 seconds, and incubated for 2 minutes. Then, 7 mL of the PEI mixture was added to the plasmid mixture, vortexed for 8 seconds, and incubated for 8 minutes. Finally, the mixture was pipetted dropwise into 200 mL of cell culture medium, gently mixing as it was added. The cells were cultured in a 5% CO2, 37°C shaker for 4 hours. After 4 hours, 200 mL of freestyle medium was added, and the cells were cultured again in a shaker for 7-8 days for expression.

[0318] 2.3 Antibody Purification: Collect the cell expression supernatant of Anti-EpCAM Ab and centrifuge at 10,000 rpm for 25 min. Filter the cell supernatant through a 0.22 μm filter for later use.

[0319] AKTA purification instrument operation: Set the software flow rate to 8 mL / min and the maximum pressure to 0.3 MPa. First, thoroughly flush the instrument tubing with solution B (100 mM citric acid monohydrate), reduce the flow rate to 2 mL / min, load the protein A medium-pressure chromatography column, and equilibrate the protein A medium with 95% solution A (200 mM disodium hydrogen phosphate dodecahydrate) at a flow rate of 8 mL / min until the baseline level stabilizes, which takes about 15 min. Load the sample at a flow rate of 8 mL / min. The UV value will rise and remain at a certain level; this peak is the breakthrough peak. After loading the sample, equilibrate again with 95% solution A. The peak value will drop to the baseline level and stabilize. Elute with 70% solution B. During this process, the peak value will first rise and then drop to the baseline. The formation of the elution peak is the elution of the target protein. Collect the eluent from this process. Rinse the tubing for contaminating proteins with 100% solution B, then fill the tubing and protein A column with 20% ethanol. Remove the column and store it at 4°C. The eluted proteins were dialyzed into 20 mM PBS via a dialysis bag at 4°C for 24 h. Antibody concentration was measured using a microplate reader or BCA. If the antibody concentration was <0.5 mg / mL, it was concentrated using a 10 kDa Millipore concentrator at 3000 rpm for 10 min. The antibodies were aliquoted and stored at -20°C for later use.

[0320] Figure 1 The purity analysis results of P1 nanobodies are shown (reducing gel and non-reducing gel). As shown in the figure: under non-reducing conditions, the band size of P1 nanobodies is about 80 kDa, while under reducing conditions, the band size of P1 nanobodies is about 45 kDa, which is consistent with the theoretical value. Moreover, the bands are clean and uniform, indicating that the purity of P1 nanobodies is high.

[0321] Example 3: Preparation of antibody-drug conjugate P1-VC-MMAE

[0322] Using the antimitotic agent monomethylolpropamine E (MMAE, a microtubule inhibitor) as a toxic payload, it is linked to the antibody's site-directed mutagenic site via a lysosomal cleavable MC-Val-Cit-PAB (maleimide hexanoyl-valine-citrulline-p-aminobenzoyloxycarboxyl) linker, thereby obtaining antibody-drug conjugates (such as...). Figure 2 (As shown). The detailed experimental steps are as follows:

[0323] (1) Add 0.5M EDTA to the antibody reaction system to make its working concentration reach 5mM;

[0324] (2) Reduction: Add 10 eq of Tris(2-carboxyethyl)phosphine hydrochloride (TCEP; BEYOTIME) to the reaction system and incubate at 37°C for 2 h. The disulfide bonds on the engineered antibody and the cysteine-activated thiol groups on the mutation sites are exposed. Replace the buffer through an ultrafiltration concentrator (10Kd; Millipore).

[0325] (3) Oxidation: Add excess Dehydroascorbic acid (DHAA) (50 eq), incubate at room temperature for 3 h, and re-oxidize and link the reduced disulfide bonds, exposing only the engineered cysteine ​​sites. Use an ultrafiltration concentration tube to replace the buffer and remove the oxidant DHAA.

[0326] (4) Conjugation: MC-vc-PAB-MMAE (MCE, CAS No.: 646502-53-6) was dissolved in DMSO at a storage concentration of 10 mM. The added DMSO accounted for 10% of the total reaction system. The mixture was vortexed and incubated at 4°C. 6 eq of MC-vc-PAB-MMAE was added to the reaction system of the Anti-EpCAM Ab engineered antibody and incubated at 4°C for 4 h. Excess small molecules, cysteine, and impurities such as DMSO were removed using an ultrafiltration concentrator. The antibody-drug conjugate P1-VC-MMAE was obtained.

[0327] Example 4: ELISA detection of the binding activity of P1, P1 ADC and antigen EpCAM.

[0328] The antigen EpCAM (purchased from Sino Biological) was diluted to 1 μg / mL with PBS and plated into 96-well plates (100 μL per well). After incubation overnight at 4°C, the plates were washed five times with PBST, blocked overnight with 2% BSA, the blocking buffer was removed, and the plates were washed five times with PBST and dried for 24 h. P1, P1-VC-MMAE (P1 ADC), and the negative control (125s) were diluted to different concentrations and added to 96-well plates pre-coated with EpCAM antigen (100 μL per well). The plates were incubated at 37°C for 1 h and washed five times with PBST. Goat Anti-Human Antigen was then added. IgG (HRP) secondary antibody (1:2000 dilution) was incubated at 37°C for 30 min, washed 5 times with PBST, and 100 μg / well of substrate chromogenic solution A / B was added. After approximately 10 min of development, a blue gradient was observed. Then, 50 μL / well of stop solution was added, and the color turned yellow. Absorbance was measured at 450 nm and 630 nm. The results showed that P1 and P1-VC-MMAE bound to the antigen EpCAM in a concentration-dependent manner, and EC... 50 All were at the ng / mL level (as shown in Table 3), and the negative control did not show a specific binding trend at 125s (e.g. Figure 3 (As shown).

[0329] Table 3. EC5 of P1 and P1 ADC 50 Value (ELISA)

[0330]

[0331] Example 5: Hydrophobic interaction chromatography (HIC) for determining the drug-antibody conjugate ratio (DAR) of P1-VC-MMAE

[0332] (1) A silica-based HPLC column (4.6×100mm, 3.5μm, Agilent) was used for HIC-UPLC (waters) analysis to determine the drug-antibody ratio (DAR);

[0333] (2) Antibodies and their conjugates were eluted via a linear gradient for 40 min from buffer A (1.5 M ammonium sulfate, 50 mM sodium phosphate) to buffer B (80% sodium phosphate, 20% isopropanol), pH 7.5, 0.5 mL / min, 25 °C. Figure 4 As shown: P1-VC-MMAE has a DAR of 4 at pH = 7.5, which is consistent with the theoretical value.

[0334] Example 6: Identification of the internalization effect of P1 antibody

[0335] 6.1 Flow cytometry analysis of the internalization capacity of P1 antibody in HT-29, SKOV3, and BxPC3 cells

[0336] EpCAM-high expression colorectal cancer cell line HT-29, human ovarian adenocarcinoma cell line SKOV3, and human orthotopic pancreatic adenocarcinoma cell line BxPC3 were selected. BxPC3, SKOV3, and HT-29 (2×10⁻⁶ cells / year) were then used as subjects. 5 The antibody (10 μg / mL) was resuspended in pre-chilled PBS, and three replicates were prepared. The samples were incubated at 4°C for 1 hour, centrifuged at 1200 rpm for 3 minutes, and washed. Two replicates were taken and resuspended in 2% FBS 1640, incubated at 37°C for 30 minutes and 3 hours respectively, centrifuged at 1200 rpm for 3 minutes, and washed. The secondary antibody (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, DyLight) was then added. TM 650 (Invitrogen) 1:400 dilution, incubated at 4°C for 30 min, and analyzed by loading onto an analytical flow cytometer (LSRFortessaX-20; BD).

[0337] Figure 5 The internalization rate of the P1 antibody in three cell lines was shown by flow cytometry. Figure 5 As shown: In the HT-29 cell line, the internalization efficiency of P1 antibody after 3 hours of incubation was 89.4%; in the SKOV3 cell line, the internalization efficiency was 32.1%; and in the BxPC3 cell line, the internalization efficiency was 64.1%.

[0338] 6.2 Immunofluorescence verification of the internalization effect of P1 antibody

[0339] On day 1, HT-29 colorectal cancer cell line, SKOV3 human ovarian adenocarcinoma cell line, and BxPC3 pancreatic cancer cell line were cultured overnight at 10,000 wells. On day 2, primary antibody (P1) was incubated at two intervals, 3 hours and 1 hour, with a dilution of 2% FBSDMEM and a final antibody concentration of 100 nM / L. Fixation: Wash three times with PBS, add 100 μL of 4% formaldehyde solution, and incubate for 20 min. Permeabilization: Wash three times with PBS, add 100 μL of immunofluorescence permeabilization buffer, and incubate for 5 min. Blocking: Wash twice with PBS, add 100 μL of 2% BSA, and incubate for 30 min. Secondary antibody: Secondary antibody (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody), diluted 1:2000, incubated at 4°C for 30 min. Cell nucleus staining: Wash three times with PBS, dilute DAPI 1:2000, mix thoroughly by inverting, add 100 μL, and incubate at room temperature for 10 min. Imaging is performed using a high-content cell screening imaging system (Opera Phenix).

[0340] like Figure 6 As shown: In both the HT-29 and BxPC3 cell lines, P1 showed significant internalization, with the internalization effect being very significant within 1 hour. In the SKOV3 cell line, because the EpCAM expression level of SKOV3 was slightly lower than that of the HT-29 and BxPC3 cell lines, the internalization effect of P1 was weaker at the same fluorescence intensity, but significant antibody internalization was still visible.

[0341] Example 7: In vivo targeting of P1 antibody in BxPC3 tumor-bearing mouse model

[0342] The antibody was coupled with Cyanine 5.5 NHS ester (Luminex Life Science Solutions) at a ratio of 1:10, mixed by rotation at 4°C overnight, and then desalted using a Zeba column. TM Spin Desalting Columns,7K MWCO,0.5mL; Thermo Scientific TM Centrifugation was used to remove free luciferin. Nude mice (5-6 weeks old, female) were selected, and BxPC3 was administered at a rate of 2 × 10⁻⁶. 6 Cells were subcutaneously seeded until the tumor volume reached 100 mm². 3 Mice were grouped uniformly according to tumor size and administered 200 μg of antibody via tail vein injection. Small animal fluorescence in vivo imaging (CaliperIVIS Lumina II) was performed on days 1, 3, 5, and 7.

[0343] like Figure 7 As shown, P1 was enriched at tumor sites in tumor-bearing mice on day 1. Subsequently, due to metabolism in the mice, antibodies in other non-specific organs were metabolized, while antibodies at the tumor site remained significantly enriched on day 7. On day 7, the mice were dissected, and the heart, liver, spleen, lungs, kidneys, and tumors were removed. Fluorescence signals in each organ and tumor site were detected, revealing that the antibodies were mainly enriched in the liver and tumor sites, highlighting the potential for liver toxicity.

[0344] Example 8: Site-directed modification and design of Trop2 and EpCAM parental peptide constructs

[0345] 8.1 In the early stages, the laboratory used phage display and screening techniques to screen nanobodies targeting Trop2 and EpCAM specifically from an alpaca antibody library. The amino acid sequences are shown in Table 4, with the CDR sequence indicated by underline.

[0346] Table 4. Amino acid sequences of the parental VHH of Trop2 and EpCAM

[0347]

[0348]

[0349] 8.2 The screened Anti-Trop2 and Anti-EpCAM nanobodies were tandemly expressed with the Fc segments CH2 and CH3 (SEQ ID NO:9) of a conventional antibody to extend their half-life. The expression vector used was PTT5-H, with serine at position 239 mutated to cysteine ​​and lysine at position 290 mutated to cysteine ​​(underlined), and named "Anti-Trop2 Ab" and "Anti-EpCAM Ab" respectively. See Table 5.

[0350] Table 5. Amino acid sequences of Anti-Trop2 Ab and Anti-EpCAM Ab (with Fc tags)

[0351]

[0352]

[0353] 8.3 Anti-Trop2 VHH and Anti-EpCAM VHH, along with the Fc fragments CH2 and CH3 (SEQ ID NO: 9) of a traditional antibody, were expressed in tandem using the PTT5-H expression vector. Serine at position 239 was mutated to cysteine, and lysine at position 290 was mutated to cysteine ​​to obtain the Anti-Trop2 / EpCAM polypeptide construct, named “①Anti-Trop2 / EpCAM BsAb”, as shown in Table 6. Figure 8 As shown.

[0354] Table 6. ① Amino acid sequence of Anti-Trop2 / EpCAM BsAb (with Fc tag)

[0355]

[0356] Example 9: Transient expression of Anti-Trop2 Ab, Anti-EpCAM Ab, and Anti-Trop2 / EpCAM BsAb and Affinity chromatography purification

[0357] 9.1 Plasmid Acquisition: Anti-Trop2 Ab, Anti-EpCAM Ab, and Anti-Trop2 / EpCAM BsAb plasmids were synthesized by General Biosystems (Anhui) Co., Ltd. The plasmids were rapidly transferred into DH-5α Escherichia coli competent cells (Shenzhen Kangti), heat-shocked for 90 seconds, and then cultured overnight at 37°C in ampicillin-resistant LB agar. Single colonies were picked and amplified in ampicillin-resistant LB agar at 37°C, 220 rpm for 15 hours. Plasmids were then extracted using an endotoxin-free plasmid extraction kit.

[0358] 9.2 Antibody Expression: Expi-293F cells were transiently transfected with single plasmids to express Anti-Trop2 Ab, Anti-EpCAM Ab, and Anti-Trop2 / EpCAM BsAb. The prepared density was 4 × 10⁻⁶ cells / cells. 6 200 mL of Expi-293F cells with a viability of 95% were cultured. 0.5 mg of each antibody plasmid was filtered through a 0.22 μm filter and added to 5 mL of CD05 medium. Simultaneously, 2 mg of PEI was added to 5 mL of CD05 medium, vortexed for 8 seconds, and incubated for 2 minutes. Then, 7 mL of the PEI mixture was added to the plasmid mixture, vortexed for 8 seconds, and incubated for 8 minutes. Finally, the mixture was pipetted dropwise into 200 mL of cell culture medium, gently mixing as it was added. The cells were cultured in a 5% CO2, 37°C shaker for 4 hours. After 4 hours, 200 mL of freestyle medium was added, and the cells were cultured again in a shaker for 7-8 days for expression.

[0359] 9.3 Antibody Purification: Collect the cell expression supernatant of Anti-Trop2 Ab, Anti-EpCAM Ab, and Anti-Trop2 / EpCAM BsAb, and centrifuge at 10,000 rpm for 25 min. Filter the cell supernatant through a 0.22 μm filter for later use.

[0360] AKTA purification instrument operation: Set the software flow rate to 8 mL / min and the maximum pressure to 0.3 MPa. First, thoroughly flush the instrument tubing with solution B (100 mM citric acid monohydrate), reduce the flow rate to 2 mL / min, load the protein A medium-pressure chromatography column, and equilibrate the protein A medium with 95% solution A (200 mM disodium hydrogen phosphate dodecahydrate) at a flow rate of 8 mL / min until the baseline level stabilizes, which takes about 15 min. Load the sample at a flow rate of 8 mL / min. The UV value will rise and remain at a certain level; this peak is the breakthrough peak. After loading the sample, equilibrate again with 95% solution A. The peak value will drop to the baseline level and stabilize. Elute with 70% solution B. During this process, the peak value will first rise and then drop to the baseline. The formation of the elution peak is the elution of the target protein. Collect the eluent from this process. Rinse the tubing for contaminating proteins with 100% solution B, then fill the tubing and protein A column with 20% ethanol. Remove the column and store it at 4°C. The eluted proteins were dialyzed into 20 mM PBS via a dialysis bag at 4°C for 24 h. Antibody concentration was measured using a microplate reader or BCA. If the antibody concentration was <0.5 mg / mL, it was concentrated using a 10 kDa Millipore concentrator at 3000 rpm for 10 min. The antibodies were aliquoted and stored at -20°C for later use.

[0361] Figure 9The image shows the band size of ①Anti-Trop2 / EpCAM BsAb identified by SDS-PAGE. As shown in the figure, under non-reducing conditions, the band size of ①Anti-Trop2 / EpCAM BsAb is around 110–120 kDa, with a small amount of aggregates; under reducing conditions, the band size of ①Anti-Trop2 / EpCAM BsAb is around 60 kDa, which is consistent with the theoretical value.

[0362] Bispecific antibodies with different structures were prepared according to the methods in Examples 8 and 9, such as... Figure 8 As shown, they are respectively:

[0363] ②Anti EpCAM / Trop2 BsAb, from N-terminus to C-terminus, contains Anti-EpCAM VHH, Anti-Trop2VHH, and the traditional antibody Fc fragment CH2 and CH3;

[0364] ③Anti Trop2 / EpCAM BsAb, from N-terminus to C-terminus, contains Anti-Trop2 VHH, traditional antibody Fc segment CH2, CH3, and Anti-EpCAM VHH;

[0365] ④Anti EpCAM / Trop2 BsAb, from N-terminus to C-terminus, contains Anti-EpCAM VHH, traditional antibody Fc segment CH2, CH3, and Anti-Trop2 VHH.

[0366] The amino acid sequences are shown in Table 7.

[0367] Table 7. Amino acid sequences (with Fc tags) of ②Anti EpCAM / Trop2 BsAb, ③Anti Trop2 / EpCAM BsAb, and ④Anti EpCAM / Trop2 BsAb.

[0368]

[0369]

[0370] Example 10: Preparation of Anti-Trop2 / EpCAM-VC-MMAE, an Antibody-Drug Conjugate

[0371] Using the antimitotic agent monomethylolpropamine E (MMAE, a microtubule inhibitor) as a toxic payload, it is linked to the antibody's site-directed mutagenic site via a lysosomal cleavable MC-Val-Cit-PAB (maleimide hexanoyl-valine-citrulline-p-aminobenzoyloxycarboxyl) linker, thereby obtaining antibody-drug conjugates (such as...). Figure 10 (As shown). The detailed experimental steps are as follows:

[0372] (1) Add 0.5M EDTA to the antibody reaction system to make its working concentration reach 5mM;

[0373] (2) Reduction: Add 10 eq of Tris(2-carboxyethyl)phosphine hydrochloride (TCEP; BEYOTIME) to the reaction system and incubate at 37°C for 2 h. The disulfide bonds on the engineered antibody and the cysteine-activated thiol groups on the mutation sites are exposed. Replace the buffer through an ultrafiltration concentrator (10Kd; Millipore).

[0374] (3) Oxidation: Add excess Dehydroascorbic acid (DHAA) (50 eq), incubate at room temperature for 3 h, and re-oxidize and link the reduced disulfide bonds, exposing only the engineered cysteine ​​sites. Use an ultrafiltration concentration tube to replace the buffer and remove the oxidant DHAA.

[0375] (4) Conjugation: MC-vc-PAB-MMAE (MCE, CAS No.: 646502-53-6) was dissolved in DMSO at a storage concentration of 10 mM. The added DMSO accounted for 10% of the total reaction system. The mixture was vortexed and incubated at 4°C. 6 eq of MC-vc-PAB-MMAE was added to the reaction system of the Anti-Trop2 / EpCAM BsAb engineered antibody and incubated at 4°C for 4 h. Excess small molecules, cysteine, and impurities such as DMSO were removed using an ultrafiltration concentrator. The antibody-drug conjugate Anti-Trop2 / EpCAM-VC-MMAE was obtained.

[0376] The parental antibody-drug conjugates Anti-Trop2-VC-MMAE and Anti-EpCAM-VC-MMAE were obtained using the same reaction system.

[0377] Example 11: ELISA detection of Anti-Trop2Ab, Anti-EpCAMAb, and ①Anti-Trop2 / EpCAMBsAb Binding activity with antigen Trop2

[0378] Trop2 antigen (purchased from Sino Biological) was diluted to 1 μg / mL with PBS and plated into 96-well plates (100 μL per well). After incubation overnight at 4°C, the plates were washed five times with PBST, blocked overnight with 2% BSA, the blocking buffer was removed, and the plates were washed five times with PBST and dried for 24 h. Anti-Trop2 Ab, Anti-EpCAM Ab, Anti-Trop2 / EpCAM BsAb, and Negative control were diluted to different concentrations and added to 96-well plates pre-coated with Trop2 antigen (100 μL per well). The plates were incubated at 37°C for 1 h and washed five times with PBST. Goat Anti-Human Antigen was then added. IgG (HRP) secondary antibody (1:2000 dilution) was incubated at 37℃ for 30 min, washed 5 times with PBST, and 100 μL of a 1:1 mixture of substrate chromogenic solution A / B was added to each well. After approximately 10 min of incubation, a blue gradient was observed. Then, 50 μL of stop solution was added to each well, and the color turned yellow. Absorbance was measured at 450 nm and 630 nm. The results showed that Anti-Trop2Ab and ①Anti-Trop2 / EpCAM BsAb bind to antigen Trop2 in a concentration-dependent manner, EC50... 50 All were at the ng / mL level (as shown in Table 8). Anti-EpCAM Ab and Negative control did not show a specific binding trend (e.g., ...). Figure 11 (As shown).

[0379] Table 8. EC5 values ​​of Anti-Trop2 Ab, Anti-EpCAM Ab, and Anti-Trop2 / EpCAM BsAb 50 Value (Trop2)

[0380]

[0381] Example 12: ELISA detection of Anti-Trop2Ab, Anti-EpCAMAb, and ①Anti-Trop2 / EpCAM BsAb Binding activity with antigen EpCAM

[0382] Antigen EpCAM (purchased from Sino Biological) was diluted to 1 μg / mL with PBS and plated into 96-well plates (100 μL per well). After incubation overnight at 4°C, the plates were washed five times with PBST, blocked overnight with 2% BSA, the blocking buffer was removed, and the plates were washed five times with PBST and dried for 24 h. Anti-Trop2 Ab, Anti-EpCAM Ab, Anti-Trop2 / EpCAM BsAb, and Negative control were diluted to different concentrations and added to 96-well plates pre-coated with EpCAM antigen (100 μL per well). The plates were incubated at 37°C for 1 h and washed five times with PBST. Goat Anti-Human Antigen was then added. IgG (HRP) secondary antibody (1:2000 dilution) was incubated at 37℃ for 30 min, washed 5 times with PBST, and 100 μL of a 1:1 mixture of substrate chromogenic solution A / B was added to each well. After approximately 10 min of incubation, a blue gradient was observed. Then, 50 μL of stop solution was added to each well, and the color turned yellow. Absorbance was measured at 450 nm and 630 nm. The results showed that Anti-EpCAMAb and Anti-Trop2 / EpCAM BsAb bound to the antigen EpCAM in a concentration-dependent manner, with EC50... 50 All were at the ng / mL level (as shown in Table 9). The Anti-Trop2 Ab and Negative control did not show a specific binding trend (e.g., ...). Figure 12 (As shown).

[0383] Table 9. EC5 values ​​of Anti-Trop2 Ab, Anti-EpCAM Ab, and ①Anti-Trop2 / EpCAM BsAb 50 Value (EpCAM)

[0384]

[0385] Example 13: Hydrophobic interaction chromatography (HIC) for the identification of the average value of ① Anti-Trop2 / EpCAM-VC-MMAE DAR value

[0386] (1) A silica-based HPLC column (4.6×100mm, 3.5μm, Agilent) was used for HIC-UPLC (waters) analysis to determine the drug-antibody ratio (DAR);

[0387] (2) Antibodies and their conjugates were eluted via a linear gradient for 40 min from buffer A (1.5 M ammonium sulfate, 50 mM sodium phosphate) to buffer B (80% sodium phosphate, 20% isopropanol), pH 7.5, 0.5 mL / min, 25 °C. Figure 13As shown: ① At pH=7.5, the DAR=4 for Anti-Trop2 / EpCAM-VC-MMAE, which is consistent with the theoretical value.

[0388] Example 14: ① Identification of the internalization effect of Anti-Trop2 / EpCAM BsAb

[0389] Flow cytometry was used to detect the internalization ability of bispecific antibodies in BxPC3 and HT-29 cells.

[0390] Pancreatic cancer cell line BxPC3, which highly expresses Trop2 and EpCAM, and colorectal cancer cell line HT-29, which lowly expresses Trop2 and highly expresses EpCAM, were selected. BxPC3 and HT-29 (2×10⁻⁶ cells / year) were then used as complementary cancer cells. 5 The antibody (10 μg / mL) was resuspended in pre-chilled PBS, and three replicates were prepared. The samples were incubated at 4°C for 1 hour, centrifuged at 1200 rpm for 3 minutes, and washed. Two replicates were taken and resuspended in 2% FBS 1640, incubated at 37°C for 30 minutes and 3 hours respectively, centrifuged at 1200 rpm for 3 minutes, and washed. The secondary antibody (Goat anti-Human IgG FcCross-Adsorbed Secondary Antibody, DyLight) was then added. TM 650 (Invitrogen) 1:400 dilution, incubated at 4°C for 30 min, and analyzed by loading onto an analytical flow cytometer (LSRFortessaX-20; BD).

[0391] like Figure 14 , Figure 15 As shown: ① The binding and internalization rates of Anti-Trop2 / EpCAM BsAb in the BxPC3 cell line were significantly higher than those of the parent antibody, and the binding rate in the HT-29 cell line was also better than that of the parent antibody.

[0392] Example 15: ① In vitro killing effect of Anti-Trop2 / EpCAMBsAb

[0393] Logarithmic growth phase BxPC3 cells were centrifuged, resuspended, and counted. 5000 cells / well were seeded in 96-well Bioland cell culture plates and incubated overnight at 37°C. Then, ADCs were diluted 2-fold (500 nM initially, 2% 1640 medium) in 20 serial dilutions, with three parallel curves for each ADC, 100 μL / well. After incubation at 37°C for 72 h, 10 μL of CCK8 reagent (MCE; cat. No: HY-K0301) was added to each well for an additional 1-2 h. The color reaction was observed, and the absorbance at 450 nm was measured using a microplate reader. A blank control group (no cells) and a negative control group (no drug treatment) were set up during the experiment. Cell viability was calculated using the following formula:

[0394] Cell killing rate = [(Ac-As) / (Ac-Ab)] × 100%

[0395] As: Absorbance of experimental wells (including cells, culture medium, CCK-8 solution and drug solution);

[0396] Ac: Absorbance of control wells (containing cells, culture medium, and CCK-8 solution, but excluding drugs);

[0397] Ab: Absorbance of blank wells (containing culture medium and CCK-8 solution, but excluding cells and drugs).

[0398] IC 50 Value computation was performed using GraphPad Prism software. Figure 16 As shown in Table 10, ① Anti-Trop2 / EpCAM-VC-MMAE and MMAE have similar ICs. 50 The values ​​are all in the pM / L range.

[0399] Table 10. IC 50 Value (CCK8)

[0400] <![CDATA[IC 50 (nM / L)]]> Anti-Trop2 / EpCAM-VC-MMAE 0.2027 MMAE 0.02323

[0401] Example 16: ① Single-needle tumor suppression effect of Anti-Trop2 / EpCAM-VC-MMAE

[0402] 16.1 Single-injection tumor-suppressing effect of Anti-Trop2 / EpCAM-VC-MMAE in TROP2-high / EpCAM-high pancreatic cancer BxPC3 tumor-bearing mice

[0403] Select nude mice (5-6 weeks old, female), and BxPC3 at a ratio of 2×10 6 Cells were subcutaneously seeded until the tumor volume reached 100 mm². 3 Tumors were uniformly grouped according to size, with the group receiving Anti-Trop2 / EpCAM-VC-MMAE 2.5μM or PBS. A single tail vein injection was administered, and tumor volume and body weight were monitored every 3–4 days.

[0404] The tumor size was measured using calipers, and its volume was calculated using the following formula: V = (W / W) 2 ×L) / 2, where V = tumor volume, W = smaller vertical diameter, and L = larger vertical diameter. When the tumor size reaches 1500 mm... 3 The mice were euthanized. Figure 17A As shown: At a dose of 2.5 μM, ① Anti-Trop2 / EpCAM-VC-MMAE can completely clear 100 mm 3 The growth of BxPC3 tumors was observed. Simultaneously, the animal did not experience weight loss or other abnormalities. Figure 17B ).

[0405] 16.2①Single-injection tumor-suppressing effect of Anti-Trop2 / EpCAM-VC-MMAE in HT-29 tumor-bearing mice with low TROP2 expression / high EpCAM expression colorectal cancer

[0406] Select nude mice (5-6 weeks old, female), and administer HT-29 at a rate of 2×10⁻⁶. 6 Cells were subcutaneously seeded until the tumor volume reached 100 mm². 3 Tumors were uniformly grouped according to size, with the following groups: ① Anti-Trop2 / EpCAM-VC-MMAE 5μM, PBS. A single tail vein injection was administered, and tumor volume and body weight were monitored every 3–4 days.

[0407] The tumor size was measured using calipers, and its volume was calculated using the following formula: V = (W / W) 2 ×L) / 2, where V = tumor volume, W = smaller vertical diameter, and L = larger vertical diameter. When the tumor size reaches 1500 mm... 3 The mice were euthanized. Figure 18A As shown: At a dose of 5 μM, Anti-Trop2 / EpCAM-VC-MMAE can completely clear 100 mm 3 The growth of HT-29 tumors was observed without any decrease in animal weight or other abnormalities. Figure 18B ).

[0408] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A nanobody or its antigen-binding fragment that specifically binds to EpCAM, comprising the following CDR1 (complementarity-determining region 1), CDR2 (complementarity-determining region 2), and CDR3 (complementarity-determining region 3) sequences: (a) CDR1, having: the sequence shown in SEQ ID NO:3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:3; (b) CDR2, having: the sequence shown in SEQ ID NO:5, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence shown in SEQ ID NO:5; and (c) CDR3, having: the sequence shown in SEQ ID NO:7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:7; Preferably, the substitution is a conservative substitution; Preferably, the nanobody or its antigen-binding fragment comprises: CDR1 as shown in SEQ ID NO:3, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:

7.

2. The nanobody of claim 1 or its antigen-binding fragment, wherein the nanobody comprises an amino acid sequence selected from the following: (i) A sequence as shown in SEQ ID NO:1; (ii) A sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence shown in SEQ ID NO:1; or (iii) A sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:1; Preferably, the substitution is a conservative substitution.

3. A polypeptide construct that specifically binds to EpCAM, comprising the nanobody or its antigen-binding fragment as described in claim 1 or 2; Preferably, the polypeptide construct contains an immunoglobulin Fc domain; Preferably, the immunoglobulin Fc domain is directly or via a peptide linker linked to the N-terminus or the C-terminus of the nanobody or its antigen-binding fragment; preferably, the immunoglobulin Fc domain is directly or via a peptide linker linked to the C-terminus of the nanobody or its antigen-binding fragment. Preferably, the immunoglobulin Fc domain comprises the sequence shown in SEQ ID NO: 9, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to it, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to it; Preferably, the immunoglobulin Fc domain may or may not contain mutations at the S239 and / or K290 positions, such mutations as S239C and / or K290C; preferably, the immunoglobulin Fc domain is numbered according to the Kabat EU index. Preferably, the polypeptide construct contains or is composed of the amino acid sequence shown in SEQ ID NO:10; Preferably, the polypeptide construct is a dimer; Preferably, the polypeptide construct comprises, but is not limited to, one or more of the following: single-domain antibody, single-chain antibody, antibody Fab, full-length antibody protein, antigen-binding fragment, bispecific antibody, multispecific antibody, bi / multivalent single-domain antibody, bi / multivalent single-chain antibody, and bi / multivalent antibody Fab.

4. A bispecific antibody that specifically binds to Trop2 and EpCAM, comprising a first antigen-binding domain specific to Trop2 and a second antigen-binding domain specific to EpCAM.

5. The bispecific antibody of claim 4, wherein the first antigen-binding domain is VHH, comprising the following CDR1 (complementarity-determining region 1), CDR2 (complementarity-determining region 2), and CDR3 (complementarity-determining region 3) sequences: (a) CDR1, having: the sequence shown in SEQ ID NO:12, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:12; (b) CDR2, having: the sequence shown in SEQ ID NO:13, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence shown in SEQ ID NO:13; and (c) CDR3, having: the sequence shown in SEQ ID NO:14, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:14; Preferably, the first antigen-binding domain comprises: CDR1 as shown in SEQ ID NO:12, CDR2 as shown in SEQ ID NO:13, and CDR3 as shown in SEQ ID NO:14; Preferably, the first antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:11 or a variant thereof; the variant has at least 80% sequence identity with the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions compared to the original sequence; preferably, the substitution is a conservative substitution.

6. The bispecific antibody of claim 4 or 5, wherein the second antigen-binding domain is VHH, comprising the following CDR1 (complementarity-determining region 1), CDR2 (complementarity-determining region 2), and CDR3 (complementarity-determining region 3) sequences: (a) CDR1, having: the sequence shown in SEQ ID NO:3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:3; (b) CDR2, having: the sequence shown in SEQ ID NO:5, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence shown in SEQ ID NO:5; and (c) CDR3, having: the sequence shown in SEQ ID NO:7, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:7; Preferably, the second antigen-binding domain comprises: CDR1 as shown in SEQ ID NO:3, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:7; Preferably, the second antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:1 or a variant thereof; the variant has at least 80% sequence identity with the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions compared to the original sequence; preferably, the substitution is a conservative substitution.

7. The bispecific antibody according to any one of claims 4-6, wherein, The first antigen-binding domain and the second antigen-binding domain are optionally connected by a connector; Preferably, the first antigen-binding domain is optionally connected to the N-terminus or C-terminus of the second antigen-binding domain via a connector; Preferably, the first antigen-binding domain is optionally connected to the N-terminus of the second antigen-binding domain via a connector; Preferably, the connector is a peptide connector (e.g., a rigid peptide connector or a flexible peptide connector); Preferably, the linker is a peptide linker containing one or more glycines and / or one or more serines; Preferably, the peptide linker is (G4S)n, where n is an integer not less than 0, such as 1, 2, 3 or 4.

8. The bispecific antibody according to any one of claims 4-7, further comprising an immunoglobulin Fc domain; Preferably, the immunoglobulin Fc domain is optionally linked to the N-terminus and / or C-terminus (e.g., C-terminus) of the first or second antigen-binding domain via a peptide linker; Preferably, the immunoglobulin Fc domain is optionally linked to the C-terminus of the first antigen-binding domain via a peptide linker; Preferably, the immunoglobulin Fc domain is optionally linked to the C-terminus of the second antigen-binding domain via a peptide linker; Preferably, the immunoglobulin Fc domain is optionally linked to the C-terminus of the first antigen-binding domain via a peptide linker, and optionally linked to the N-terminus of the second antigen-binding domain via a peptide linker; Preferably, the immunoglobulin Fc domain is optionally linked to the C-terminus of the second antigen-binding domain via a peptide linker, and optionally linked to the N-terminus of the first antigen-binding domain via a peptide linker; Preferably, the bispecific antibody comprises, from the N-terminus to the C-terminus, the following in sequence: (1) the first antigen-binding domain, the peptide linker, the second antigen-binding domain, and the immunoglobulin Fc domain; Preferably, the bispecific antibody comprises, from the N-terminus to the C-terminus, the second antigen-binding domain, the peptide linker, the first antigen-binding domain, and the immunoglobulin Fc domain. Preferably, the bispecific antibody comprises, from the N-terminus to the C-terminus, the first antigen-binding domain, the immunoglobulin Fc domain, the peptide linker, and the second antigen-binding domain. Preferably, the bispecific antibody comprises, from the N-terminus to the C-terminus, the second antigen-binding domain, the immunoglobulin Fc domain, the peptide linker, and the first antigen-binding domain. Preferably, the immunoglobulin Fc domain is the Fc domain of IgG (e.g., the Fc domain of IgG1, IgG2, IgG3 or IgG4). Preferably, the immunoglobulin Fc domain comprises a sequence as shown in SEQ ID NO:9, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to it, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to it; Preferably, the immunoglobulin Fc domain may or may not contain mutations at the S239 and / or K290 positions, such mutations including, for example, S239C and / or K290C.

9. The bispecific antibody according to any one of claims 4-8, wherein the bispecific antibody has one or more of the following characteristics: (1) The bispecific antibody comprises a sequence as shown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20; (2) The bispecific antibody is a dimer; (3) The bispecific antibody is in a concentration of less than approximately 10 -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D Combined with Trop2; (4) The bispecific antibody is in a concentration of less than approximately 10 -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D Combined with EpCAM. (5) The bispecific antibody is a monovalent or bivalent antibody with any antibody structure.

10. A multispecific antibody comprising the bispecific antibody according to any one of claims 4-9; Preferably, the multispecific antibody specifically binds to Trop2 and EpCAM, and additionally specifically binds to one or more other targets; Preferably, the multispecific antibody further comprises at least one third antibody having a third binding specificity against a third target.

11. An isolated nucleic acid molecule encoding the nanobody or its antigen-binding fragment as described in claim 1 or 2, the polypeptide construct as described in claim 3, the bispecific antibody as described in any one of claims 4-9, or the multispecific antibody as described in claim 10.

12. A vector comprising the nucleic acid molecule of claim 11; preferably, the vector is a cloning vector or an expression vector.

13. A host cell comprising the nucleic acid molecule of claim 11 or the vector of claim 12.

14. A method for preparing the nanobody or antigen-binding fragment thereof according to claim 1 or 2, the polypeptide construct according to claim 3, the bispecific antibody according to any one of claims 4-9, or the multispecific antibody according to claim 10, comprising culturing the host cell according to claim 10 under conditions that allow protein expression, and recovering the nanobody or antigen-binding fragment thereof, bispecific antibody, or multispecific antibody from the cultured host cell culture.

15. A conjugate comprising the nanobody or antigen-binding fragment of claim 1 or 2, the polypeptide construct of claim 3, the bispecific antibody of any one of claims 4-9 or the multispecific antibody of claim 10, and a conjugation portion; Preferably, the coupling portion is selected from protein tags, such as purification tags; detectable markers, such as enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), or biotin; therapeutic agents, such as cytotoxic drugs; or other bioactive peptides.

16. An antibody-drug conjugate (ADC) comprising: a targeting portion selected from the nanobody or antigen-binding fragment thereof as described in claim 1 or 2, the polypeptide construct as described in claim 3, the bispecific antibody as described in any one of claims 4-9, or the multispecific antibody as described in claim 10; Cytotoxic drugs section; and Connector for connecting the target portion and the cytotoxic drug portion.

17. The antibody-drug conjugate of claim 16, wherein the targeting portion is linked to the linker via a thiol group on a cysteine ​​residue; Preferably, the targeting portion is connected to the linker via a thiol group on a cysteine ​​residue in the VHH or Fc domain, or a thiol group exposed by a cysteine ​​residue in a reduced disulfide bond in the hinge region. Preferably, the targeting portion is connected to the linker via a thiol group on a cysteine ​​residue in the VHH, the cysteine ​​residue at position 239 and / or 290 of the Fc domain after the reduction of the hinge region.

18. The antibody-drug conjugate of claim 16 or 17, wherein the cytotoxic agent is selected from microtubule inhibitors and DNA damage agents; Preferably, the microtubule inhibitor is selected from olistatin compounds (e.g., MMAE, MMAF), maytansine compounds (e.g., maytansine, maytanol, DM1, DM4), taxanes (e.g., taxol, docetaxel, carbazitaxel), vinblastines (e.g., vincristine, vinblastine), eribulin, and colchicine; Preferably, the DNA damaging agent is selected from DNA alkylating agents (cazithromycin γ1l, N-acetyl-γ1I cazithromycin, atrazomycin, PBD, ducamycin), DNA topoisomerase inhibitors (e.g., camptothecin compounds (specifically camptothecin, SN-38, Dxd, irinotecan, belotetan, topotecan, PNU-159682), doxorubicin, daunorubicin, etoposide, mitoxantrone), and muscarine; Preferably, the cytotoxic drug is MMAE.

19. The antibody-drug conjugate according to any one of claims 16-18, wherein the linker is a cleavable or non-cleavable linker; Preferably, the cleavable linker is selected from protease-sensitive, pH-sensitive, and glutathione-sensitive linkers; Preferably, the linker is selected from MC (6-maleimide hexanoyl), MCC (maleimide methylcyclohexane-1-carboxylate), MP (maleimide propionyl), Val-Cit (valine-citrulline), Val-Ala (valine-alanine), Ala-Phe (alanine-phenylalanine), PAB (p-aminobenzyloxycarbonyl), SPP (5-(succinimide)-4-(pyridin-2-ylthio) 6-(2,5-dioxopyrrolidone-1-yl)-4-(pyridin-2-ylthio)hexanoate, 6-(2,5-dioxopyrrolidone-1-yl)-5-methyl-4-(pyridin-2-ylthio)hexanoate, SMCC (N-succinimide-4-(N-maleimide-methyl)cyclohexane-1-carboxylate) or SIAB (N-succinimide-(4-iodo-acetyl)aminobenzoate) and any combination thereof; Preferably, the connector is MC-Val-Cit-PAB.

20. The antibody-drug conjugate according to any one of claims 16-19, wherein each peptide chain of the targeting portion is linked to 0, 1, 2, 3, 4, or 5 of the following structures via VHH, cysteine ​​residues in the reduced disulfide bond of the hinge region, or cysteine ​​residues in the Fc domain:

21. The antibody-drug conjugate according to any one of claims 16-20, wherein the conjugate is selected from: in, x = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Ab is the polypeptide construct of claim 3 or the bispecific antibody of any one of claims 4-9.

22. The antibody-drug conjugate according to any one of claims 16-21, wherein: in, x = 1, 2, 3, 4, 5 or 6; Ab contains or is composed of an amino acid sequence as shown in SEQ ID NO:10, 17, 18, 19 or 20.

23. A composition comprising or consisting of one or more antibody-drug conjugates according to any one of claims 16-22; Preferably, the composition has a DAR value of 1-10.

24. A composition comprising or consisting of one or more antibody-drug conjugates of claim 22; Preferably, the DAR value of the composition is 1-8; More preferably, the composition has a DAR value of 1-5, for example 3.5-4.5, or even 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4 or 4.

5.

25. An antibody composition comprising a first antibody that specifically binds to Trop2 and a second antibody that specifically binds to EpCAM.

26. The antibody composition of claim 25, wherein the first antibody is a nanobody that specifically binds to Trop2 or an antigen-binding fragment thereof, or a polypeptide construct comprising the nanobody or an antigen-binding fragment thereof; Preferably, the nanobody that specifically binds to Trop2 or its antigen-binding fragment comprises the following CDR1 (complementarity-determining region 1), CDR2 (complementarity-determining region 2), and CDR3 (complementarity-determining region 3) sequences: (a) CDR1, having: the sequence shown in SEQ ID NO:12, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:12; (b) CDR2, having: the sequence shown in SEQ ID NO:13, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence shown in SEQ ID NO:13; and (c) CDR3, having: the sequence shown in SEQ ID NO:14, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO:14; Preferably, the nanobody that specifically binds to Trop2 or its antigen-binding fragment comprises: CDR1 as shown in SEQ ID NO:12, CDR2 as shown in SEQ ID NO:13, and CDR3 as shown in SEQ ID NO:14; Preferably, the nanobody that specifically binds to Trop2 or its antigen-binding fragment comprises a VHH sequence as shown in SEQ ID NO:11 or a variant thereof; the variant has at least 80% sequence identity with the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions compared to the original sequence; preferably, the substitution is a conservative substitution.

27. The antibody composition of claim 25 or 26, wherein the second antibody is a nanobody that specifically binds to EpCAM or an antigen-binding fragment thereof, or a polypeptide construct comprising said nanobody or an antigen-binding fragment thereof; Preferably, the nanobody that specifically binds to EpCAM or its antigen-binding fragment is as defined in claim 1 or 2.

28. The antibody composition according to any one of claims 25-27, wherein the polypeptide construct comprises an immunoglobulin Fc domain; Preferably, the immunoglobulin Fc domain is directly or via a peptide linker linked to the N-terminus or the C-terminus of the nanobody or its antigen-binding fragment; preferably, the immunoglobulin Fc domain is directly or via a peptide linker linked to the C-terminus of the nanobody or its antigen-binding fragment. Preferably, the immunoglobulin Fc domain comprises the sequence shown in SEQ ID NO: 9, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to it, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to it; Preferably, the immunoglobulin Fc domain may or may not contain mutations at the S239 and / or K290 positions, such mutations as S239C and / or K290C; preferably, the immunoglobulin Fc domain is numbered according to the Kabat EU index. Preferably, the polypeptide construct contains or is composed of an amino acid sequence as shown in SEQ ID NO:15 or 16; Preferably, the polypeptide construct is a dimer; Preferably, the polypeptide construct comprises, but is not limited to, one or more of the following: single-domain antibody, single-chain antibody, antibody Fab, full-length antibody protein, antigen-binding fragment, bispecific antibody, multispecific antibody, bi / multivalent single-domain antibody, bi / multivalent single-chain antibody, and bi / multivalent antibody Fab.

29. A pharmaceutical composition comprising the nanobody or antigen-binding fragment thereof as claimed in claim 1 or 2, the polypeptide construct of claim 3, the bispecific antibody of any one of claims 4-9, the multispecific antibody of claim 10, the nucleic acid molecule of claim 11, the carrier of claim 12, the host cell of claim 13, the conjugate of claim 15, the antibody-drug conjugate of any one of claims 16-22, the composition of any one of claims 23-24, or the antibody composition of any one of claims 25-28, and optionally a carrier or excipient.

30. Use of the nanobody of claim 1 or 2 or its antigen-binding fragment, or the polypeptide construct of claim 3, for the preparation of a medicament for the prevention and / or treatment of EpCAM-related diseases in subjects; Preferably, the EpCAM-related disease is a tumor, such as an EpCAM-positive tumor; Preferably, the tumor is selected from colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), lung cancer, oral squamous cell carcinoma, ovarian cancer (e.g., ovarian epithelial carcinoma), cervical cancer, bladder cancer, prostate cancer, pancreatic cancer, liver cancer, and retinoblastoma. Preferably, the subject is a mammal, such as a human; Preferably, the nanobody or its antigen-binding fragment, polypeptide construct or pharmaceutical composition is used alone or in combination with other pharmaceutically active agents.

31. Use of the bispecific antibody of any one of claims 4-9, the multispecific antibody of claim 10, the nucleic acid molecule of claim 11, the vector of claim 12, the host cell of claim 13, the conjugate of claim 15, the antibody-drug conjugate of any one of claims 16-22, the composition of any one of claims 23-24, the antibody composition of any one of claims 25-28, or the pharmaceutical composition of claim 29 for the preparation of a medicament for the prevention and / or treatment in a subject of diseases related to Trop2 and / or EpCAM; Preferably, the Trop2-related and / or EpCAM-related disease is a tumor, such as a Trop2 and / or EpCAM-positive tumor; Preferably, the tumor is selected from colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), lung cancer, oral squamous cell carcinoma, ovarian cancer (e.g., ovarian epithelial carcinoma), cervical cancer, bladder cancer, prostate cancer, pancreatic cancer, liver cancer, and retinoblastoma. Preferably, the subject is a mammal, such as a human; Preferably, the bispecific antibody, multispecific antibody, nucleic acid molecule, carrier, host cell, conjugate, antibody-drug conjugate, composition, antibody composition, or pharmaceutical composition is used alone or in combination with other pharmaceutically active agents.

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