Tri-specific antibody, antibody coupling medicine as well as preparation method and application of antibody coupling medicine

Abstract

The invention relates to a trispecific antibody and an antibody coupling drug which simultaneously target EpCAM, cMet and Trop2, and a preparation method and application thereof. In particular, the present invention provides a trispecific antibody comprising an anti-EpCAM nanobody, an anti-cMet nanobody, and an anti-Trop2 nanobody. The invention also provides a drug conjugate constructed on the basis of the nano antibody and the trispecific nano antibody.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, specifically to trispecific antibody-drug conjugates targeting EpCAM, cMet, and Trop2, their preparation methods, and applications. Background Technology

[0002] Antibody-drug conjugates (ADCs) are a new type of targeted chemotherapy drug composed of monoclonal antibodies and small molecule cytotoxic drugs linked by a linker. They combine the powerful killing effect of traditional small molecule chemotherapy with the tumor-targeting properties of antibody drugs, and have become one of the hot topics in anticancer drug research and development in recent years.

[0003] EpCAM, short for Epithelial Cell Adhesion Molecule, also known as CD326, belongs to the GA733 protein family. It is a type I transmembrane glycoprotein widely expressed on cancer cell membranes, especially squamous cell carcinoma and adenocarcinoma. It participates in processes such as tumor stem cell development, cell proliferation, metabolism, angiogenesis, epithelial-to-mesenchymal transition (EMT), metastasis, chemotherapy / radiation resistance, and immune regulation. 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. 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 August 2021, the FDA refused to approve Oportuzumab monatox for the treatment of high-risk non-muscle-invasive bladder cancer (NMIBC) that is unresponsive to BCG. Sesen Bio has since voluntarily suspended further development of Oportuzumab monatox for the NMIBC indication in the United States.

[0004] cMet, short for cellular-mesenchymal epithelial transition factor, is a member of the receptor tyrosine kinase family. Aberrant activation of cMet signaling has been reported in various types of cancer, caused by factors including protein overexpression, gene amplification or rearrangement, transcriptional regulation, and stimulation by autocrine or paracrine ligands. Studies have shown that the CMet signaling pathway can enhance tumor cell proliferation, survival, motility and migration, scattering, epithelial-mesenchymal transition (EMT), angiogenesis, invasion, and metastasis. Currently, there are no cMet-targeting ADCs on the market globally. The most advanced is AbbVie's Telisotuzumab Vedotin, currently in Phase III clinical trials. It uses a cleavable linker to connect the anti-cMet monoclonal antibody ABT-700 to the microtubule inhibitor MMAE, with a DAR of 3.1. On January 4, 2022, the FDA granted Telisotuzumab Vedotin Breakthrough Therapy Designation (BTD) for non-small cell lung cancer (NSCLC). Other cMet ADCs are still in the early clinical stages of development, with companies involved including Hengrui, Rongchang Bio, Regeneron, and Eli Lilly.

[0005] 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.

[0006] Although the efficacy of ADCs has been widely validated, obstacles such as the large size of monoclonal antibodies and their low penetration rate into solid tumors still need to be addressed. Nanobodies, isolated and screened from camel serum, are small antibodies with a relative molecular mass of only about 15 kDa. They contain only the variable domain of heavy chain antibody (VHH), about 1 / 10 the size of traditional antibodies, and have a rugby ball-shaped crystal structure with a diameter of approximately 2.5 nm and a length of approximately 4.2 nm. This unique molecular structure gives them excellent tissue penetration, promising to bring better efficacy and safety to ADCs.

[0007] Trispecific antibody-drug conjugates (ADCs) are a cutting-edge concept. Currently, very few companies are involved in this field, and most are in the preclinical research stage. Theoretically, trispecific antibodies can target tumor cells more specifically, enhance cytotoxicity, overcome drug resistance, and reduce side effects. Furthermore, trispecific antibodies can promote cross-linking between targets, facilitating the internalization of antibody-drug conjugates, thereby achieving better therapeutic effects. Currently, there are no publicly reported trispecific antibody-drug conjugates targeting EpCAM, cMet, and Trop2. Summary of the Invention

[0008] This application discloses the combined use of antibodies targeting EpCAM, cMet, and Trop2, and particularly discloses a trispecific antibody simultaneously targeting EpCAM, cMet, and Trop2. The antibody portion can be a modified antibody derived from alpaca nanobodies targeting EpCAM, cMet, and Trop2. This application also discloses a site-conjugated trispecific antibody-drug conjugate simultaneously targeting EpCAM, cMet, and Trop2, consisting of a targeting portion, a cytotoxic drug, and a linker. In some embodiments, this application uses the antimitotic agent monomethylolpropamine 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. In some embodiments, the trispecific antibody-drug conjugate Anti-EpCAM / cMet / Trop2-VC-MMAE (DAR≈4) disclosed in this application can effectively inhibit the growth of tumors in HT-29 colorectal cancer cell line-bearing mice that are EpCAM-positive, cMet-positive, and Trop2-weakly positive, exhibiting highly efficient antitumor activity and good safety.

[0009] Based on the above, the technical solution of this application involves the following aspects.

[0010] 1. Trispecific antibodies

[0011] In one aspect, the present invention provides a trispecific antibody that specifically binds to EpCAM, cMet, and Trop2, comprising a first antigen-binding domain specific to EpCAM, a second antigen-binding domain specific to cMet, and a third antigen-binding domain specific to Trop2.

[0012] EpCAM combined structural domain

[0013] The first antigen-binding domain specific to EpCAM contained in the trispecific antibody of the present invention can be any antibody form.

[0014] 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).

[0015] In some embodiments, the first antigen-binding domain is VHH, and 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:

[0016] (a) CDR1, 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;

[0017] (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

[0018] (c) CDR3, 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.

[0019] In some embodiments, the first antigen-binding domain includes: CDR1 as shown in SEQ ID NO:4, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:6.

[0020] In some implementations, the substitution is a conservative substitution.

[0021] In some embodiments, the first 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.

[0022] In some embodiments, the first antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:1.

[0023] c-Met binding domain

[0024] The second antigen-binding domain specific to c-Met contained in the trispecific antibody of the present invention can be any antibody form.

[0025] 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).

[0026] 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:

[0027] (a) CDR1, 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;

[0028] (b) CDR2, having: the sequence shown in SEQ ID NO:8, 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:8; and

[0029] (c) CDR3, having: the sequence shown in SEQ ID NO:9, 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:9.

[0030] In some embodiments, the second antigen-binding domain includes: CDR1 as shown in SEQ ID NO:7, CDR2 as shown in SEQ ID NO:8, and CDR3 as shown in SEQ ID NO:9.

[0031] In some embodiments, the second antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:2 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.

[0032] In some embodiments, the second antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:2.

[0033] Trop2 binding domain

[0034] The third antigen-binding domain specific to Trop2 contained in the trispecific antibody of the present invention can be any antibody form.

[0035] In some embodiments, the third antigen-binding domain is selected from nanobodies, full-length antibodies (e.g., IgG antibodies) or their antigen-binding fragments (e.g., scFv, Fab, scFab).

[0036] In some embodiments, the third antigen-binding domain is VHH, which comprises the following CDR1 (complementarity-determining region 1), CDR2 (complementarity-determining region 2), and CDR3 (complementarity-determining region 3) sequences:

[0037] (a) CDR1, having: the sequence shown in SEQ ID NO:10, 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:10;

[0038] (b) CDR2, having: the sequence shown in SEQ ID NO:11, 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:11; and

[0039] (c) CDR3, 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;

[0040] In some embodiments, the third antigen-binding domain includes: CDR1 as shown in SEQ ID NO:10, CDR2 as shown in SEQ ID NO:11, and CDR3 as shown in SEQ ID NO:12.

[0041] In some embodiments, the third antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:3 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.

[0042] In some embodiments, the triantigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:3.

[0043] The amino acid sequences described in this application can be defined using the Kabat, IMGT, Chothia, or Abm numbering systems.

[0044] Structure of trispecific antibodies

[0045] Those skilled in the art will understand that any known trispecific antibody structure can be used in this invention. As an example, the following trispecific nanobody is provided, wherein the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain are all VHH.

[0046] In some implementations, the trispecific nanobody sequentially comprises a first antigen-binding domain, a second antigen-binding domain, and a third antigen-binding domain.

[0047] In some embodiments, the trispecific nanobody further comprises an immunoglobulin Fc domain.

[0048] In some embodiments, the immunoglobulin Fc domain is located between the first antigen-binding domain and the second antigen-binding domain, or after the first antigen-binding domain, or between the second antigen-binding domain and the third antigen-binding domain.

[0049] In some embodiments, the immunoglobulin Fc domain is optionally linked to the N-terminus of the first antigen-binding domain, the second antigen-binding domain, or the third antigen-binding domain via a peptide linker.

[0050] In some embodiments, the immunoglobulin Fc domain is optionally linked to the C-terminus of the first antigen-binding domain, the second antigen-binding domain, or the third antigen-binding domain via a peptide linker.

[0051] In some embodiments, the trispecific nanobody comprises, from N-terminus to C-terminus, a second antigen-binding domain, a third antigen-binding domain, a first antigen-binding domain, and an immunoglobulin Fc domain. See exemplary structures below. Figure 1 T1 in the middle.

[0052] In some embodiments, the trispecific nanobody comprises, from N-terminus to C-terminus, a first antigen-binding domain, a third antigen-binding domain, an immunoglobulin Fc domain, and a second antigen-binding domain. See exemplary structures for details. Figure 1 T3 in the middle.

[0053] In some embodiments, the trispecific nanobody comprises, from N-terminus to C-terminus, a first antigen-binding domain, an immunoglobulin Fc domain, a second antigen-binding domain, and a third antigen-binding domain. See exemplary structures for details. Figure 1 T6 in the middle.

[0054] In some embodiments, the immunoglobulin Fc domain is the Fc domain of IgG (e.g., the Fc domain of IgG1, IgG2, IgG3, or IgG4).

[0055] In some embodiments, the immunoglobulin Fc domain comprises a sequence as shown in SEQ ID NO:13, 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.

[0056] 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.

[0057] In some implementations, the immunoglobulin Fc domain is numbered according to the Kabat EU index.

[0058] In the trispecific antibody of the present invention, the peptide linker can be a rigid peptide linker or a flexible peptide linker.

[0059] In some embodiments, the peptide linker consists of 10 to 20 (e.g., 10-19, 10-18, 10-17, 10-16, 10-15) amino acid residues.

[0060] In some embodiments, the connector is a peptide connector containing one or more glycines and / or one or more serines.

[0061] 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.

[0062] In some embodiments, the trispecific nanobody comprises a sequence as shown in SEQ ID NO: 17, 18, or 19. The sequence shown herein does not contain an amino acid (such as methionine (Met)) encoded by a start codon (such as ATG) at its N-terminus. Those skilled in the art will understand that during the preparation of proteins through genetic engineering, the first digit 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 trispecific antibody of the present 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 the present invention.

[0063] In some embodiments, the bispecific nanobody comprises a variant of the sequence shown in SEQ ID NO:17, 18, or 19, the variant differing from SEQ ID NO:17, 18, or 19 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 trispecific nanobody from which it is derived (e.g., specifically binding to EpCAM, cMet, and Trop2, neutralizing the biological activity of EpCAM, cMet, and Trop2). For example, in some embodiments, the variant may be truncated at the N-terminus or C-terminus of the first antigen-binding domain, the second antigen-binding domain, and / or the third antigen-binding domain, so that it contains only a portion of FR1 and / or FR4, or lacks one or both of those backbone regions, as long as it substantially maintains antigen binding and specificity.

[0064] In some implementations, the trispecific antibody is a dimer, such as a homodimer or a heterodimer.

[0065] In some embodiments, the trispecific 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.

[0066] In some embodiments, the trispecific 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 c-Met.

[0067] In some embodiments, the trispecific 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.

[0068] The trispecific antibody of the present invention can be a monovalent or bivalent antibody having any antibody structure.

[0069] 2. Multispecific antibodies

[0070] On the other hand, this application provides a multispecific antibody comprising the trispecific antibody of the present invention. To generate the multispecific antibody, the trispecific 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).

[0071] In some implementations, the multispecific antibody specifically binds to EpCAM, cMet, and Trop2, and additionally specifically binds to one or more other targets.

[0072] 3. Nucleic acids, vectors, host cells, and expression methods

[0073] In another aspect, this application provides an isolated nucleic acid molecule comprising a polynucleotide sequence encoding the trispecific or 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.

[0074] In another aspect, this application provides a vector containing the said nucleic acid molecule.

[0075] 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.

[0076] 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 trispecific 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.

[0077] 4. Antibody preparation

[0078] 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.

[0079] In some implementations, the method includes the following steps:

[0080] (1) Transform host cells using at least one of the nucleic acids or expression vectors described herein;

[0081] (2) Culture the transformed host cells under suitable conditions to allow the expression of the nucleic acid or expression vector, and

[0082] (3) Isolate and purify the trispecific antibody or multispecific antibody from the host cell or culture medium.

[0083] 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.

[0084] 5. Conjugates

[0085] On the other hand, this application also provides conjugates comprising the trispecific or multispecific antibody of the present invention and the conjugated portion.

[0086] In some implementations, the targeting portion is optionally coupled to the coupling portion via a connector.

[0087] 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 trispecific antibody of the present invention is linked to a purification tag.

[0088] 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 trispecific antibodies of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0089] In some embodiments, the coupling portion is selected from therapeutic agents, such as cytotoxic drugs.

[0090] In some embodiments, the coupling moiety is selected from other bioactive peptides.

[0091] 6. Antibody-drug conjugates (ADCs)

[0092] This application further provides an antibody-drug conjugate (ADC) comprising:

[0093] The target portion is selected from any of the trispecific or multispecific antibodies described above;

[0094] Cytotoxic drugs section; and

[0095] Connector for connecting the target portion and the cytotoxic drug portion.

[0096] 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.

[0097] In some embodiments, the targeting portion is connected to the linker via a thiol group on a cysteine ​​residue.

[0098] In some embodiments, the targeting portion is linked to the linker via a thiol group on a cysteine ​​residue of VHH.

[0099] 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.

[0100] In some implementations, the targeting portion is connected to the linker via a thiol group on a cysteine ​​residue in the Fc domain.

[0101] 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.

[0102] In some embodiments, the cytotoxic drug is selected from microtubule inhibitors and DNA damage drugs.

[0103] 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.

[0104] 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.

[0105] In some embodiments, the cytotoxic drug is MMAE.

[0106] In some implementations, the connector may be a cuttable or non-cuttable connector.

[0107] In some embodiments, the cleavable linker is selected from protease-sensitive, pH-sensitive, and glutathione-sensitive linkers.

[0108] 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.

[0109] In some implementations, the connector is MC-Val-Cit-PAB.

[0110] 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:

[0111]

[0112] In some implementations, the antibody-drug conjugate is selected from:

[0113]

[0114] Where x = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0115] Ab is any of the trispecific antibodies described above.

[0116] In some implementation schemes, the antibody-drug conjugate is:

[0117]

[0118] Where x = 1, 2, 3, 4, 5 or 6;

[0119] Ab contains or is composed of an amino acid sequence as shown in SEQ ID NO:17, 18 or 19.

[0120] 7. Composition

[0121] 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.

[0122] 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...

[0123] In another aspect, this application provides a composition comprising or consisting of one or more of the following antibody-drug conjugates:

[0124]

[0125] Where x = 1, 2, 3, 4, 5, 6, 7 or 8;

[0126] Ab contains or is composed of an amino acid sequence as shown in SEQ ID NO:17, 18 or 19.

[0127] 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.

[0128] 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.

[0129] 8. Antibody composition

[0130] This application also provides an antibody composition comprising a first antibody that specifically binds to EpCAM, a second antibody that specifically binds to c-Met, and a third antibody that specifically binds to Trop2.

[0131] Antibodies that specifically bind to EpCAM

[0132] The first antibody that specifically binds to EpCAM 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, bi / multivalent single-domain antibodies, bi / multivalent single-chain antibodies, and bi / multivalent antibody Fabs.

[0133] In some embodiments, the first 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.

[0134] In some embodiments, the nanobody that specifically binds to EpCAM 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:

[0135] (a) CDR1, 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;

[0136] (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

[0137] (c) CDR3, 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.

[0138] In some embodiments, the nanobody that specifically binds to EpCAM or its antigen-binding fragment comprises: CDR1 as shown in SEQ ID NO:4, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:6.

[0139] In some embodiments, the nanobody that specifically binds to EpCAM or its antigen-binding fragment comprises a VHH sequence as shown in SEQ ID NO:1 or a variant thereof; the variant has 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 has 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.

[0140] In some embodiments, the first antibody comprises a VHH sequence as shown in SEQ ID NO:1.

[0141] Antibodies that specifically bind to c-Met

[0142] The second antibody specifically binding c-Met 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.

[0143] In some embodiments, the second antibody is a nanobody that specifically binds to c-Met or its antigen-binding fragment, or a polypeptide construct comprising the nanobody or its antigen-binding fragment.

[0144] In some embodiments, the nanobody that specifically binds to c-Met 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:

[0145] (a) CDR1, 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;

[0146] (b) CDR2, having: the sequence shown in SEQ ID NO:8, 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:8; and

[0147] (c) CDR3, having: the sequence shown in SEQ ID NO:9, 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:9.

[0148] In some embodiments, the nanobody that specifically binds to c-Met or its antigen-binding fragment comprises: CDR1 as shown in SEQ ID NO:7, CDR2 as shown in SEQ ID NO:8, and CDR3 as shown in SEQ ID NO:9.

[0149] In some embodiments, the nanobody that specifically binds to c-Met or its antigen-binding fragment comprises a VHH sequence as shown in SEQ ID NO:2 or a variant thereof; the variant has 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 has 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.

[0150] In some embodiments, the nanobody that specifically binds to c-Met or its antigen-binding fragment comprises a VHH sequence as shown in SEQ ID NO:2.

[0151] Antibodies that specifically bind to Trop2

[0152] The antibody composition of the present invention may contain a third antibody that specifically binds to Trop2, which may be in any form of antibody, 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.

[0153] In some embodiments, the third antibody is a nanobody that specifically binds to Trop2 or its antigen-binding fragment, or a polypeptide construct containing the nanobody or its antigen-binding fragment.

[0154] 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:

[0155] (a) CDR1, having: the sequence shown in SEQ ID NO:10, 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:10;

[0156] (b) CDR2, having: the sequence shown in SEQ ID NO:11, 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:11; and

[0157] (c) CDR3, 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.

[0158] In some embodiments, the nanobody that specifically binds to Trop2 or its antigen-binding fragment comprises: CDR1 as shown in SEQ ID NO:10, CDR2 as shown in SEQ ID NO:11, and CDR3 as shown in SEQ ID NO:12.

[0159] In some embodiments, the Trop2-specific nanobody or its antigen-binding fragment comprises a VHH sequence as shown in SEQ ID NO:3 or a variant thereof; the variant has 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 has one or more amino acid substitutions, deletions, or additions compared to the original sequence; preferably, the substitutions are conservative substitutions.

[0160] In some embodiments, the first antibody comprises a VHH sequence as shown in SEQ ID NO:3.

[0161] peptide constructs

[0162] In some embodiments, the first antibody is a polypeptide construct comprising a nanobody that specifically binds to EpCAM or an antigen-binding fragment thereof. In some embodiments, the second antibody is a polypeptide construct comprising a nanobody that specifically binds to -Met or an antigen-binding fragment thereof. In some embodiments, the third antibody is a polypeptide construct comprising a nanobody that specifically binds to Trop2 or an antigen-binding fragment thereof.

[0163] In some embodiments, the polypeptide construct includes an immunoglobulin Fc domain.

[0164] 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.

[0165] 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.

[0166] In some embodiments, the immunoglobulin Fc domain comprises the sequence shown in SEQ ID NO: 13, 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.

[0167] 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.

[0168] In some embodiments, the polypeptide construct contains or is composed of an amino acid sequence as shown in SEQ ID NO:14, 15 or 16.

[0169] In some implementations, the polypeptide construct is a dimer.

[0170] 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.

[0171] 9. Pharmaceutical Composition

[0172] In another aspect, this application provides a pharmaceutical composition comprising any of the trispecific antibodies, multispecific antibodies, nucleic acid molecules, carriers, host cells, conjugates, antibody-drug conjugates, compositions, or antibody compositions described in any of the preceding claims, and optionally a carrier or excipient.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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).

[0182] 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.

[0183] In some implementations, suitable diluents include water, glycerol, methanol, ethanol, and other biocompatible diluents.

[0184] 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.

[0185] 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.

[0186] 10. Pharmaceutical Uses

[0187] In one aspect, this application provides the use of the trispecific 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 a subject of a disease related to EpCAM, c-Met, and / or Trop2.

[0188] In another aspect, this application provides for the prevention and / or treatment of diseases associated with EpCAM, c-Met, and / or Trop2, including administering an effective amount of the trispecific 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.

[0189] In some embodiments, the disease associated with EpCAM, c-Met, and / or Trop2 is a tumor, such as a tumor that is positive for EpCAM, c-Met, and / or Trop2. 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.

[0190] In some implementations, the subject is a mammal, such as a human;

[0191] In some embodiments, the trispecific 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.

[0192] 11. Purpose of Testing

[0193] On the other hand, the use of the trispecific antibodies, conjugates or antibody compositions of the present invention in the preparation of diagnostic reagents for detecting the presence or level of EpCAM, c-Met and / or Trop2 in a sample or for diagnosing whether a subject has a disease related to EpCAM, c-Met and / or Trop2 is provided.

[0194] In some embodiments, the conjugate used to prepare the detection reagent comprises the trispecific antibody of the present invention and a detectable label.

[0195] In some implementations, the trispecific antibody used to prepare the detection reagent is labeled with a detectable tag.

[0196] In some embodiments, the trispecific 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 trispecific antibody of the present invention.

[0197] On the other hand, this application provides a method for detecting the presence or content of EpCAM, c-Met and / or Trop2 in a sample, which includes using the trispecific antibody, conjugate or antibody composition of the present invention.

[0198] In some embodiments, the method is an immunological assay, such as immunoblotting, enzyme immunoassay (e.g., ELISA), chemiluminescent immunoassay, fluorescence immunoassay, or radioimmunoassay.

[0199] In some embodiments, the conjugate used in the method comprises the trispecific antibody of the present invention and a detectable label.

[0200] In some implementations, the trispecific antibody used in the method carries a detectable label.

[0201] In some embodiments, the trispecific 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 trispecific antibody of the present invention.

[0202] In some implementations, the method includes the following steps:

[0203] (1) Contact the sample with the trispecific antibody, conjugate or antibody composition of the present invention;

[0204] (2) Detect the formation of a complex between the trispecific antibody, conjugate or antibody composition and the antigen or detect the amount of the complex.

[0205] The formation of the complex indicates the presence of an antigen or cells expressing an antigen;

[0206] The antigen is selected from EpCAM, c-Met, and Trop2.

[0207] 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).

[0208] In some embodiments, the method is used to diagnose whether a subject has a disease associated with EpCAM, c-Met, and / or Trop2. In such embodiments, the method may further include the step of comparing the levels of EpCAM, c-Met, and / or Trop2 in a sample from the subject with reference values. The reference values ​​may be the levels of EpCAM, c-Met, and / or Trop2 in a sample from a subject known not to have a disease associated with EpCAM, c-Met, and / or Trop2 (e.g., a healthy control) (also referred to as a “negative reference value”). For example, if the levels of EpCAM, c-Met, and / or Trop2 in a sample from the subject are elevated relative to a negative reference value, it indicates that the subject has a disease associated with EpCAM, c-Met, and / or Trop2.

[0209] 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.

[0210] In some embodiments, the c-MET-related diseases are characterized by elevated c-MET expression and / or excessive c-MET activity. In some embodiments, the c-MET-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.

[0211] 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.

[0212] 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.

[0213] Terminology Definition

[0214] 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.

[0215] 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.

[0216] 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.

[0217] As used in this article, the term "c-Met" stands for cellular-mesenchymal epithelial transition factor, a member of the receptor tyrosine kinase family. c-Met is a proto-oncogene located on chromosome 7q21eq31. Initially translated into a single-stranded precursor protein in vivo, it undergoes post-translational modification to become a three-dimensional structure linked by disulfide bonds. The mature c-Met molecule consists of a 50 kDa extracellular α-chain and a 140 kDa transmembrane β-chain. The amino acid sequence can be found, for example, in NCBI Gene ID: NP_000236.2.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] As used herein, the term "trispecific antibody" refers to an antibody that has binding specificity to three different antigens (or epitopes). A trispecific antibody comprises three antigen-binding domains with binding specificity to different antigens (or epitopes), thereby enabling it to bind to three different binding sites and / or target molecules. In some cases, the different antigen-binding domains are linked by peptide linkers. The term "trispecific nanobody" refers to a trispecific antibody formed by three nanobodies.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 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:

[0226] 1) Alanine (A), Serine (S), Threonine (T);

[0227] 2) Aspartic acid (D), glutamic acid (E);

[0228] 3) Asparagine (N), glutamine (Q);

[0229] 4) Arginine (R), Lysine (K);

[0230] 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and

[0231] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0232] 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).

[0233] 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.

[0234] 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.

[0235] 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).

[0236] 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.

[0237] 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.

[0238] 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.

[0239] 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.

[0240] As used in this article, the term "contact" refers to direct physical association; it includes both solid and liquid forms.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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.

[0245] Beneficial effects of the invention

[0246] This application provides formulations for the combined use of antibodies targeting EpCAM, cMet, and Trop2. Specifically, it provides trispecific antibodies simultaneously targeting EpCAM, cMet, and Trop2, as well as antibody-drug conjugates (ADCs), compositions containing these, and their uses. The ADCs exhibit good binding activity to EpCAM, cMet, and Trop2 proteins, demonstrating good targeting and inhibitory activity against EpCAM-positive, cMet-positive, and / or Trop2-positive tumors, with a good safety profile. Attached Figure Description

[0247] Figure 1 Three representative structures of the three-antibody are shown as examples.

[0248] Figure 2 The band sizes of Anti-cMet / Trop2 / EpCAM TsAb (T1), Anti-EpCAM / Trop2 / cMet TsAb (T3), and Anti-EpCAM / cMet / Trop2 TsAb (T6) identified by SDS-PAGE are shown (1 for non-reduction and 2 for reduction).

[0249] Figure 3 An example of the structure of an ADC (Anti-EpCAM / cMet / Trop2(T6)-VC-MMAE) is shown.

[0250] Figure 4 The DAR value of Anti-EpCAM / cMet / Trop2(T6)-VC-MMAE was detected by hydrophobic interaction chromatography (HIC).

[0251] Figure 5 The results show the detection results of the binding activity of Anti-cMet / Trop2 / EpCAM TsAb (T1), Anti-EpCAM / Trop2 / cMet TsAb (T3), and Anti-EpCAM / cMet / Trop2 TsAb (T6) to the antigen EpCAM using ELISA.

[0252] Figure 6 The results show the detection results of the binding activity of Anti-cMet / Trop2 / EpCAM TsAb (T1), Anti-EpCAM / Trop2 / cMet TsAb (T3), and Anti-EpCAM / cMet / Trop2 TsAb (T6) to the antigen cMet using the ELISA method.

[0253] Figure 7 The results show the detection results of the binding activity of Anti-cMet / Trop2 / EpCAM TsAb(T1), Anti-EpCAM / Trop2 / cMet TsAb(T3), and Anti-EpCAM / cMet / Trop2 TsAb(T6) to antigen Trop2 using ELISA.

[0254] Figure 8 The binding ability of Anti-EpCAM / cMet / Trop2 TsAb(T6) and the parental antibody in the pancreatic cancer cell line BxPC3 was demonstrated by flow cytometry.

[0255] Figure 9 The internalization ability of Anti-EpCAM / cMet / Trop2 TsAb(T6) and parental antibodies in the pancreatic cancer cell line BxPC3 was demonstrated by flow cytometry.

[0256] Figure 10 The binding ability of Anti-EpCAM / cMet / Trop2 TsAb(T6) and parental antibodies in the colorectal cancer cell line HT-29 was demonstrated by flow cytometry.

[0257] Figure 11 The internalization ability of Anti-EpCAM / cMet / Trop2 TsAb(T6) and parental antibodies in the colorectal cancer cell line HT-29 was demonstrated by flow cytometry.

[0258] Figure 12 The internalization capacity of Anti-cMet / Trop2 / EpCAM TsAb (T1), Anti-EpCAM / Trop2 / cMet TsAb (T3), and Anti-EpCAM / cMet / Trop2 TsAb (T6) in the pancreatic cancer cell line BxPC3 was demonstrated by immunofluorescence assay.

[0259] Figure 13 The internalization ability of Anti-cMet / Trop2 / EpCAM TsAb (T1), Anti-EpCAM / Trop2 / cMet TsAb (T3), Anti-EpCAM / cMet / Trop2 TsAb (T6) and parental antibodies in the colorectal cancer cell line HT-29 was demonstrated by immunofluorescence assay.

[0260] Figure 14 The internalization ability of Anti-cMet / Trop2 / EpCAM TsAb (T1), Anti-EpCAM / Trop2 / cMet TsAb (T3), Anti-EpCAM / cMet / Trop2 TsAb (T6) and parental antibodies in SKOV3 ovarian cancer cells was demonstrated by immunofluorescence assay.

[0261] Figure 15 The study demonstrated the tumor-suppressive effect of single-injection administration of Anti-EpCAM / cMet / Trop2(T6)-VC-MMAE on the BxPC3 tumor-bearing mouse pancreatic cancer cell line with high expression of EpCAM / cMet / Trop2.

[0262] Figure 16 The results of single-injection administration of Anti-EpCAM / cMet / Trop2(T6)-VC-MMAE showed the changes in body weight in BxPC3 tumor-bearing mice with pancreatic cancer cell lines that are highly expressed in EpCAM / cMet / Trop2.

[0263] Figure 17 The study demonstrated the tumor-suppressive effect of single-injection Anti-EpCAM / cMet / Trop2(T6)-VC-MMAE on HT-29 tumor-bearing mice with EpCAM-high / cMet-high / Trop2-low expression colorectal cancer cells.

[0264] Figure 18 The study showed the changes in body weight in HT-29 tumor-bearing mice with EpCAM-high / cMet-high / Trop2-low expression colorectal cancer cells after a single injection of Anti-EpCAM / cMet / Trop2(T6)-VC-MMAE.

[0265] Figure 19 The study demonstrated the tumor-suppressive effect of single-injection of Anti-EpCAM / Trop2 / cMet / (T3)-VC-MMAE on HT-29 tumor-bearing mice with EpCAM-high / cMet-high / Trop2-low expression colorectal cancer cells.

[0266] Figure 20 The results show the changes in body weight in HT-29 tumor-bearing mice with EpCAM-high / cMet-high / Trop2-low expression colorectal cancer cells after single-injection administration of Anti-EpCAM / Trop2 / cMet / (T3)-VC-MMAE.

[0267] Sequence information

[0268] The sequence information involved in this invention is described in the table below:

[0269]

[0270]

[0271]

[0272] Detailed Implementation

[0273] 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.

[0274] Example 1: Site-directed modification and design of EpCAM, cMet, and Trop2 parental peptide constructs 1.1 In the early stages of the laboratory, nanobodies targeting EpCAM, cMet and Trop2 were screened from the alpaca antibody library based on phage display and screening techniques. The amino acid sequences are shown in Table 1.

[0275] Table 1. Amino acid sequence of parental VHH

[0276]

[0277]

[0278] 1.2 The screened Anti-EpCAM, Anti-cMet, and Anti-Trop2 nanobodies were tandemly expressed with the Fc segments CH2 and CH3 (SEQ ID NO:13) of traditional antibodies 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, named "Anti-EpCAM Ab", "Anti-cMet Ab", and "Anti-Trop2 Ab", respectively. See Table 2.

[0279] Table 2. Amino acid sequences (with Fc tags) of “Anti-EpCAM Ab”, “Anti-cMet Ab”, and “Anti-Trop2 Ab”.

[0280]

[0281]

[0282] 1.3: Construction of a trispecific antibody with a "3+0" structure

[0283] Anti-cMet VHH, Anti-Trop2 VHH, Anti-EpCAM VHH and the Fc segments CH2 and CH3 (SEQ ID NO:13) of the 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 "3+0" structure Anti-cMet / Trop2 / EpCAM polypeptide construct, named "Anti-cMet / Trop2 / EpCAM TsAb(T1)", as shown in Table 3.

[0284] Table 3. Amino acid sequence of “Anti-cMet / Trop2 / EpCAM TsAb(T1)” (with Fc tag)

[0285]

[0286]

[0287] 1.4: Construction of a trispecific antibody with a "2+1" structure

[0288] Anti-EpCAM VHH, Anti-Trop2 VHH, the Fc fragments CH2 and CH3 of the traditional antibody (SEQ ID NO:13), and Anti-cMet VHH 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 "2+1" structure Anti-EpCAM / Trop2 / cMet polypeptide construct, named "Anti-EpCAM / Trop2 / cMet TsAb(T3)", as shown in Table 4.

[0289] Table 4. Amino acid sequence of “Anti-EpCAM / Trop2 / cMet TsAb(T3)” (with Fc tag)

[0290]

[0291]

[0292] 1.5 Anti-EpCAM VHH, the Fc fragments CH2 and CH3 (SEQ ID NO:13) of the traditional antibody, Anti-cMet VHH, and Anti-Trop2 VHH 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 "1+2" ​​structure Anti-EpCAM / cMet / Trop2 polypeptide construct, named "Anti-EpCAM / cMet / Trop2 TsAb(T6)", as shown in Table 5.

[0293] Table 5. Amino acid sequence of “Anti-EpCAM / cMet / Trop2 TsAb(T6)” (with Fc tag)

[0294]

[0295]

[0296] Example 2: Anti-EpCAMAb, Anti-cMetAb, Anti-Trop2Ab, Anti-cMet / Trop2 / EpCAM Instantaneous tables of TsAb(T1), Anti-EpCAM / Trop2 / cMetTsAb(T3), and Anti-EpCAM / cMet / Trop2TsAb(T6) Affinity chromatography purification

[0297] 2.1 Plasmid Acquisition: Anti-EpCAM Ab, Anti-cMet Ab, Anti-Trop2 Ab, Anti-cMet / Trop2 / EpCAM TsAb (T1), Anti-EpCAM / Trop2 / cMet TsAb (T3), and Anti-EpCAM / cMet / Trop2 TsAb (T6) 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 s, and then cultured overnight at 37°C in ampicillin-resistant LB agar plates. Single colonies were picked and amplified in ampicillin-resistant LB agar at 37°C, 220 rpm for 15 h, and then plasmids were extracted using an endotoxin-free plasmid extraction kit.

[0298] 2.2 Antibody Expression: Expi-293F cells were transiently transfected with a single plasmid to express Anti-EpCAM Ab, Anti-cMet Ab, Anti-Trop2 Ab, Anti-cMet / Trop2 / EpCAM TsAb (T1), Anti-EpCAM / Trop2 / cMet TsAb (T3), and Anti-EpCAM / cMet / Trop2 TsAb (T6). The prepared cell density was 4 × 10⁻⁶ cells / year. 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.

[0299] 2.3 Antibody Purification: Collect the cell expression supernatants of Anti-EpCAM Ab, Anti-cMet Ab, Anti-Trop2 Ab, Anti-cMet / Trop2 / EpCAM TsAb (T1), Anti-EpCAM / Trop2 / cMet TsAb (T3), and Anti-EpCAM / cMet / Trop2TsAb (T6), and centrifuge at 10,000 rpm for 25 min. Filter the cell supernatant through a 0.22 μm filter for later use.

[0300] 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.

[0301] Figure 2 The figure shows the band sizes of antibodies identified by SDS-PAGE. As shown in the figure, under non-reducing conditions, the band sizes of EpCAM, cMet, and Trop2 nanobodies are around 80–90 kDa, while the band sizes of Anti-cMet / Trop2 / EpCAM TsAb (T1), Anti-EpCAM / Trop2 / cMet TsAb (T3), and Anti-EpCAM / cMet / Trop2 TsAb (T6) are around 140–160 kDa. Under reducing conditions, the band sizes of T1, T3, and T6 antibodies are around 80 kDa, which is consistent with the theoretical values. Furthermore, the bands are clean and uniform, indicating high antibody purity.

[0302] Example 3: Preparation of Anti-EpCAM / cMet / Trop2(T6)-VC-MMAE

[0303] Using the antimitotic agent monomethyl orlistatine E (MMAE, a microtubule inhibitor) as a toxic payload, it is linked to the site-specific mutation site of a T6 antibody via a lysosomal cleavable MC-Val-Cit-PAB (maleimide hexanoyl-valine-citrulline-p-aminobenzoyloxycarboxyl) linker, thereby obtaining an antibody-drug conjugate (e.g., Figure 3 (As shown). The detailed experimental steps are as follows:

[0304] (1) Add 0.5M EDTA to the antibody reaction system to make its working concentration reach 5mM;

[0305] (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).

[0306] (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.

[0307] (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 / cMet / Trop2 TsAb(T6) 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-EpCAM / cMet / Trop2(T6)-VC-MMAE was obtained.

[0308] The parental antibody-drug conjugates Anti-EpCAM-VC-MMAE, Anti-cMet-VC-MMAE, and Anti-Trop2-VC-MMAE were obtained using the same reaction system.

[0309] Example 4: Identification of Anti-EpCAM / cMet / Trop2(T6)-VC-MMAE by Hydrophobic Interaction Chromatography (HIC) Average DAR value

[0310] (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);

[0311] (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 8 As shown, Anti-EpCAM / cMet / Trop2(T6)-VC-MMAE has a DAR≈4 at pH=7.5, which is consistent with the theoretical value.

[0312] Example 5: ELISA detection of Anti-cMet / Trop2 / EpCAMTsAb(T1), Anti-EpCAM / Trop2 / The binding activity of cMet TsAb (T3) and Anti-EpCAM / cMet / Trop2TsAb (T6) with antigens EpCAM, cMet, and Trop2.

[0313] Antigens EpCAM, cMet, and Trop2 (purchased from Sino Biological) were 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-EpCAM Ab, Anti-cMet Ab, Anti-Trop2 Ab, Anti-cMet / Trop2 / EpCAMTsAb (T1), Anti-EpCAM / Trop2 / cMet TsAb (T3), Anti-EpCAM / cMet / Trop2 TsAb (T6), and Negative control were diluted to a series of different concentrations and added to 96-well plates pre-coated with EpCAM / cMet / 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°C for 30 min, washed 5 times with PBST, and 100 μL of substrate chromogenic solution A / B (1:1 mixture) was added to each well. After chromogenic development for about 10 min, a blue gradient change was observed. Then, 50 μL of stop solution was added to each well, and the color turned yellow. The absorbance was measured at wavelengths of 450 nm and 630 nm.

[0314] The results showed that Anti-EpCAM Ab, Anti-cMet / Trop2 / EpCAM TsAb (T1), Anti-EpCAM / Trop2 / cMet TsAb (T3), and Anti-EpCAM / cMet / Trop2 TsAb (T6) bound to the EpCAM antigen in a concentration-dependent manner; Anti-cMet Ab, T1, T3, and T6 bound to the cMet antigen in a concentration-dependent manner; and Anti-Trop2 Ab, T1, T3, and T6 bound to the Trop2 antigen in a concentration-dependent manner. EC50 50 All were at the nM / L level (as shown in Table 6). However, the binding activity of the T3 and T6 trispecific antibodies was significantly lower than that of the parent antibody. Overall, T1 showed the best binding activity. The negative control did not show a specific binding trend (e.g., Figure 5 , Figure 6 , Figure 7 (As shown).

[0315] Table 6: EC5 values ​​of trispecific antibodies and parental antibodies 50 value

[0316]

[0317] Example 6: Identification of the binding and internalization effects of Anti-EpCAM / cMet / Trop2TsAb(T6) 6.1 Flow cytometry analysis of the binding and internalization capacity of Anti-EpCAM / cMet / Trop2 TsAb(T6) in BxPC3 and HT-29 cells

[0318] The pancreatic cancer cell line BxPC3, which highly expresses EpCAM / cMet / Trop2, and the colorectal cancer cell line HT-29, which expresses EpCAM / cMet / Trop2 low, were selected. BxPC3 and HT-29 (2×10⁻⁶ cells / 3.5%) were then used as the basis for further analysis. 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 (Goatanti-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 flow cytometry (LSRFortessa X-20; BD). Figure 8 , Figure 9 , Figure 10 , Figure 11 As shown, the binding rate of Anti-EpCAM / cMet / Trop2 TsAb(T6) in the BxPC3 cell line was significantly higher than that of the parental antibody, and the binding rate in the HT-29 cell line was also better than that of the parental antibody. After 3 hours of incubation, the internalization value of Anti-EpCAM / cMet / Trop2 TsAb(T6) in the BxPC3 cell line was more than 300% of that of the parental antibody, and the internalization value in the HT-29 cell line was also better than that of the parental antibody.

[0319] 6.2 Immunofluorescence assay was used to detect the internalization capacity of Anti-cMet / Trop2 / EpCAM TsAb (T1), Anti-EpCAM / Trop2 / cMetTsAb (T3), and Anti-EpCAM / cMet / Trop2 TsAb (T6) in BxPC3, HT-29, and SKOV3 cells.

[0320] On day 1, HT-29 colorectal cancer cell line, BxPC3 pancreatic cancer cell line, and SKOV3 ovarian cancer cell line were cultured overnight at 10,000 cells / well. On day 2, primary antibodies (Anti-EpCAM Ab, Anti-cMet Ab, Anti-Trop2 Ab, Anti-cMet / Trop2 / EpCAM TsAb (T1), Anti-EpCAM / Trop2 / cMet TsAb (T3), Anti-EpCAM / cMet / Trop2 TsAb (T6), and a negative control) were incubated at 3-hour and 1-hour intervals. The dilution buffer was 2% FBSDMEM, and the final antibody concentration was 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 addition: Secondary antibody (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody), 1:2000 dilution, incubated at 4℃ for 30 min. Cell nucleus staining: Wash three times with PBS, add 100 μL of 1:2000 DAPI diluted by inverting, and incubate at room temperature for 10 min. Image acquisition using a high-content cell screening imaging system (Opera Phenix).

[0321] like Figure 12 , Figure 13 , Figure 14 As shown, under uniform fluorescence intensity conditions, T1, T3, and T6 all exhibited significant internalization effects in the BxPC3 cell line. In the HT-29 and SKOV3 cell lines, the internalization effects of Anti-cMet / Trop2 / EpCAM TsAb (T1), Anti-EpCAM / Trop2 / cMet TsAb (T3), and Anti-EpCAM / cMet / Trop2 TsAb (T6) were significantly better than those of Anti-Trop2 Ab, Anti-EpCAM Ab, and Anti-cMet Ab.

[0322] Example 7: Anti-EpCAM / cMet / Trop2(T6)-VC-MMAE in BxPC3 pancreatic cancer cell line tumor-bearing mice The single-needle tumor-suppressing effect

[0323] 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. 3Tumors were uniformly grouped according to size, with the following groups: Anti-EpCAM-VC-MMAE 2.5μM, Anti-cMet-VC-MMAE 2.5μM, Anti-Trop2-VC-MMAE 2.5μM, Anti-EpCAM / cMet / Trop2(T6)-VC-MMAE 2.5μM, and PBS. A single tail vein injection was administered, and tumor volume and body weight were monitored every 3–4 days.

[0324] 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 15 , Figure 16 As shown: At a dose of 2.5 μM, Anti-Trop2-VC-MMAE 2.5 μM and Anti-EpCAM / cMet / Trop2(T6)-VC-MMAE 2.5 μM can completely clear 100 mm. 3 The growth of BxPC3 tumors was inhibited, while Anti-EpCAM-VC-MMAE 2.5μM and Anti-cMet-VC-MMAE 2.5μM only inhibited tumor growth, showing a significant difference compared to Anti-EpCAM / cMet / Trop2(T6)-VC-MMAE. Meanwhile, no decrease in body weight or other abnormalities were observed in any of the groups of animals.

[0325] Example 8: Anti-EpCAM / cMet / Trop2(T6)-VC-MMAE in HT-29 colorectal cancer-bearing mice Single-needle tumor suppression effect

[0326] 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: Anti-Trop2-VC-MMAE 5μM, Anti-EpCAM / cMet / Trop2(T6)-VC-MMAE 5μM, and PBS. The tumors were administered via a single tail vein injection, and tumor volume and body weight were monitored every 3–4 days.

[0327] 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 17 , Figure 18As shown: At a dose of 5 μM, Anti-EpCAM / cMet / Trop2(T6)-VC-MMAE can completely remove 100 mm. 3 The growth of HT-29 tumors was inhibited, while Anti-Trop2-VC-MMAE 5μM only inhibited tumor growth, showing a significant difference compared to Anti-EpCAM / cMet / Trop2(T6)-VC-MMAE. Simultaneously, no weight loss or other abnormalities were observed.

[0328] Example 9: Anti-tumor effect of Anti-EpCAM / Trop2 / cMet(T3)-VC-MMAE in animals

[0329] 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 group receiving Anti-EpCAM / Trop2 / cMet(T3)-VC-MMAE 5μM and PBS. A single tail vein injection was administered, and tumor volume and body weight were monitored every 3–4 days.

[0330] 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 19 , Figure 20 As shown: At a dose of 5 μM, Anti-EpCAM / Trop2 / cMet(T3)-VC-MMAE 5 μM can completely clear 100 mm. 3 The HT-29 tumor grew without any decrease in the animal's weight or other abnormalities.

[0331] 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 trispecific antibody that specifically binds to EpCAM, cMet, and Trop2, comprising a first antigen-binding domain specific to EpCAM, a second antigen-binding domain specific to cMet, and a third antigen-binding domain specific to Trop2.

2. The trispecific antibody of claim 1, 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: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; (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: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; Preferably, the first antigen-binding domain comprises: CDR1 as shown in SEQ ID NO:4, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:6; Preferably, the first 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.

3. The trispecific antibody of claim 1 or 2, 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: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; (b) CDR2, having: the sequence shown in SEQ ID NO:8, 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:8; and (c) CDR3, having: the sequence shown in SEQ ID NO:9, 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:9; 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:2 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.

4. The trispecific antibody of any one of claims 1-3, wherein the third 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:10, 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:10; (b) CDR2, having: the sequence shown in SEQ ID NO:11, 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:11; and (c) CDR3, 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; Preferably, the second antigen-binding domain comprises: CDR1 as shown in SEQ ID NO:10, CDR2 as shown in SEQ ID NO:11, and CDR3 as shown in SEQ ID NO:12; Preferably, the second antigen-binding domain comprises a VHH sequence as shown in SEQ ID NO:3 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.

5. The trispecific antibody according to any one of claims 1-4, wherein, The trispecific antibody is a trispecific nanobody, which sequentially comprises a first antigen-binding domain, a second antigen-binding domain, and a third antigen-binding domain. Preferably, the trispecific nanobody further comprises an immunoglobulin Fc domain; Preferably, the immunoglobulin Fc domain is located between the first antigen-binding domain and the second antigen-binding domain, or after the first antigen-binding domain, or between the second antigen-binding domain and the third antigen-binding domain. Preferably, the immunoglobulin Fc domain is optionally linked to the N-terminus of the first antigen-binding domain, the second antigen-binding domain, or the third 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, the second antigen-binding domain, or the third antigen-binding domain via a peptide linker. Preferably, the trispecific nanobody comprises, from the N-terminus to the C-terminus, a second antigen-binding domain, a third antigen-binding domain, a first antigen-binding domain, and an immunoglobulin Fc domain. Preferably, the trispecific nanobody comprises, from the N-terminus to the C-terminus, a first antigen-binding domain, a third antigen-binding domain, an immunoglobulin Fc domain, and a second antigen-binding domain. Preferably, the trispecific nanobody comprises, from the N-terminus to the C-terminus, a first antigen-binding domain, an immunoglobulin Fc domain, a second antigen-binding domain, and a third antigen-binding domain.

6. The trispecific antibody according to any one of claims 1-5, wherein the immunoglobulin Fc domain is the Fc domain of IgG; Preferably, the immunoglobulin Fc domain comprises a sequence as shown in SEQ ID NO:13, or a sequence having at least 80% sequence identity with it, or a sequence having one or more amino acid substitutions, deletions or additions compared to it; Preferably, the Fc domain of the immunoglobulin is mutated; Preferably, the immunoglobulin Fc domain contains mutations at the S239 and / or K290 positions, such mutations as S239C and / or K290C; 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.

7. The trispecific antibody according to any one of claims 1-6, wherein the trispecific antibody has one or more of the following characteristics: (1) The trispecific antibody comprises a sequence as shown in SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19, or a variant comprising a sequence as shown in SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19, wherein the variant differs from SEQ ID NO:17, 18 or 19 only in the conserved substitution of one or more amino acid residues, or has at least 85% sequence identity with the antibody from which it is derived or its antigen-binding fragment, and substantially retains the biological function of the trispecific nanobody from which it is derived; (2) The trispecific antibody is a dimer; (3) The trispecific 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; (4) The trispecific 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 c-Met; (5) The trispecific 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; (5) The trispecific antibody is a monovalent or bivalent antibody with any antibody structure.

8. A multispecific antibody comprising the trispecific antibody according to any one of claims 1-7; Preferably, the multispecific antibody specifically binds to EpCAM, cMet, and Trop2, and additionally specifically binds to one or more other targets.

9. An isolated nucleic acid molecule encoding the trispecific antibody of any one of claims 1-7 or the multispecific antibody of claim 8.

10. A vector comprising the nucleic acid molecule of claim 9; preferably, the vector is a cloning vector or an expression vector.

11. A host cell comprising the nucleic acid molecule of claim 9 or the vector of claim 10.

12. A method for preparing a trispecific antibody according to any one of claims 1-7 or a multispecific antibody according to claim 8, comprising culturing a host cell according to claim 11 under conditions that allow protein expression, and recovering the nanobody or its antigen-binding fragment, trispecific antibody or multispecific antibody from the cultured host cell culture.

13. A conjugate comprising the trispecific antibody of any one of claims 1-7 or the multispecific antibody of claim 8, 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.

14. An antibody-drug conjugate (ADC) comprising: a targeting portion selected from the trispecific antibody of any one of claims 1-7 or the multispecific antibody of claim 8; Cytotoxic drugs section; and Connector for connecting the target portion and the cytotoxic drug portion.

15. The antibody-drug conjugate of claim 14, 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.

16. The antibody-drug conjugate of claim 14 or 15, 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.

17. 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.

18. The antibody-drug conjugate according to any one of claims 14-17, 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:

19. The antibody-drug conjugate according to any one of claims 14-18, wherein the conjugate is selected from: in, x = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Ab is the trispecific antibody as described in any one of claims 1-7.

20. The antibody-drug conjugate according to any one of claims 14-19, 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:17, 18 or 19.

21. A composition comprising or consisting of one or more antibody-drug conjugates according to any one of claims 14-20; Preferably, the composition has a DAR value of 1-10.

22. A composition comprising or consisting of one or more antibody-drug conjugates of claim 19; 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.

23. An antibody composition comprising a first antibody that specifically binds to EpCAM, a second antibody that specifically binds to c-Met, and a third antibody that specifically binds to Trop2.

24. The antibody composition of claim 23, wherein the first 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; Preferably, the nanobody that specifically binds to EpCAM 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: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; (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: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; Preferably, the nanobody that specifically binds to EpCAM or its antigen-binding fragment comprises: CDR1 as shown in SEQ ID NO:4, CDR2 as shown in SEQ ID NO:5, and CDR3 as shown in SEQ ID NO:6; Preferably, the nanobody that specifically binds to EpCAM or its antigen-binding fragment 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 conserved substitution.

25. The antibody composition of claim 23 or 24, wherein the second antibody is a nanobody that specifically binds to c-Met 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 c-Met 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: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; (b) CDR2, having: the sequence shown in SEQ ID NO:8, 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:8; and (c) CDR3, having: the sequence shown in SEQ ID NO:9, 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:9; Preferably, the nanobody that specifically binds to c-Met or its antigen-binding fragment comprises: CDR1 as shown in SEQ ID NO:7, CDR2 as shown in SEQ ID NO:8, and CDR3 as shown in SEQ ID NO:9; Preferably, the nanobody that specifically binds to c-Met or its antigen-binding fragment comprises a VHH sequence as shown in SEQ ID NO:2 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 conserved substitution.

26. The antibody composition of any one of claims 23-25, wherein the third 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:10, 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:10; (b) CDR2, having: the sequence shown in SEQ ID NO:11, 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:11; and (c) CDR3, 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; Preferably, the nanobody that specifically binds to Trop2 or its antigen-binding fragment comprises: CDR1 as shown in SEQ ID NO:10, CDR2 as shown in SEQ ID NO:11, and CDR3 as shown in SEQ ID NO:12; Preferably, the nanobody that specifically binds to Trop2 or its antigen-binding fragment comprises a VHH sequence as shown in SEQ ID NO:3 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 according to any one of claims 23-26, 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: 13, 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:14, 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, trispecific antibody, multispecific antibody, bivalent / multivalent single-domain antibody, bivalent / multivalent single-chain antibody, and bivalent / multivalent antibody Fab.

28. A pharmaceutical composition comprising the trispecific antibody of any one of claims 1-7, the multispecific antibody of claim 8, the nucleic acid molecule of claim 9, the vector of claim 10, the host cell of claim 11, the conjugate of claim 13, the antibody-drug conjugate of any one of claims 14-20, the composition of any one of claims 21-22, or the antibody composition of any one of claims 23-27, and optionally a carrier or excipient.

29. Use of the trispecific antibody of any one of claims 1-7, the multispecific antibody of claim 8, the nucleic acid molecule of claim 9, the vector of claim 10, the host cell of claim 11, the conjugate of claim 13, the antibody-drug conjugate of any one of claims 14-20, the composition of any one of claims 21-22, or the antibody composition of any one of claims 23-27, or the pharmaceutical composition of claim 28 for the preparation of a medicament for the prevention and / or treatment in a subject of a disease related to EpCAM, c-Met, and / or Trop2; Preferably, the disease associated with EpCAM, c-Met, and / or Trop2 is a tumor, such as an EpCAM, c-Met, and / or Trop2 positive tumor; Preferably, the tumors include 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 trispecific 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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