CN121591908A

CN121591908A - Trispecific antibody, antibody coupling drug, and preparation method and application thereof

Info

Publication number: CN121591908A
Application number: CN202411178345.6A
Authority: CN
Inventors: 罗文新; 刘雪; 陈远志; 刘涵; 许林; 袁娜; 宁文静; 唐济贤; 任云龙; 夏宁邵
Original assignee: Xiamen University
Current assignee: Xiamen University
Priority date: 2024-08-26
Filing date: 2024-08-26
Publication date: 2026-03-03

Abstract

The application 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 application provides trispecific antibodies comprising anti-EpCAM nanobodies, anti-cMet nanobodies, and anti-Trop 2 nanobodies. The application also provides a drug conjugate constructed based on the nano-antibody and the trispecific nano-antibody.

Description

Trispecific antibody, antibody coupling drug, and preparation method and application thereof

Technical Field

The invention relates to the field of biotechnology medicaments, in particular to a trispecific antibody-coupled medicament targeting EpCAM, cMet and Trop2, and a preparation method and application thereof.

Background

The Antibody Drug Conjugate (ADC) is a novel targeted chemotherapeutic Drug formed by coupling a monoclonal Antibody (Antibody) and a small molecular cytotoxicity Drug (Drug) through a Linker (Linker), has the strong killing effect of traditional small molecular chemotherapy and the tumor targeting of the Antibody Drug, and is one of the research and development hot spots of anticancer drugs in recent years.

EpCAM, known as EPITHELIAL CELL adhesion molecule (epithelial cell adhesion molecule), also known as CD326, belonging to the GA733 family of proteins, is a type I transmembrane glycoprotein, widely expressed on cancer cell membranes, in particular squamous cell carcinoma and adenocarcinoma. Is involved in the processes of tumor stem cells, cell proliferation, metabolism, angiogenesis, epithelial to mesenchymal transition (EMT), metastasis, chemotherapy/radiation resistance, and immunomodulation. During tumor development, EPCAM can cross-talk with a number of key signaling pathways such as Wnt/β -catenin, transforming growth factor- β/SMAD, epex/EGFR, PI3K/Akt/mTor, and P53, and the like, inducing biological changes in tumor cells. Oportuzumab monatox developed by Sesen Bio is the fastest-growing EpCAM ADC, which is in clinical stage III, and is formed by coupling a recombinant humanized anti-EpCAM antibody scFv with pseudomonas exotoxin a. Month 8 of 2021, FDA refuses approval Oportuzumab monatox for the treatment of high risk non-myogenic invasive bladder cancer (NMIBC) that is non-responsive to BCG. Currently Sesen Bio has voluntarily suspended further development of Oportuzumab monatox in the united states for the non-muscle invasive bladder cancer (NMIBC) indication.

CMet, collectively known as cell-MESENCHYMAL EPITHELIAL transition factor (cell-mesenchymal epithelial transforming factor), is a member of the receptor tyrosine kinase family. Aberrant activation of cMet signaling has been reported in various types of cancers, for reasons including protein overexpression, gene amplification or rearrangement, transcriptional regulation, and stimulation of autocrine or paracrine ligands. Studies have shown that CMet signaling pathways can enhance proliferation, survival, movement and migration, scattering, epithelial-to-mesenchymal transition (EMT), angiogenesis, invasion, and metastatic spread of tumor cells. No cMet-targeted ADCs are currently marketed worldwide. The fastest-growing one is ibovine Telisotuzumab Vedotin, currently in clinical stage III, where the anti-cMet monoclonal antibody ABT-700 is linked to the tubulin inhibitor MMAE via a cleavable linker, dar=3.1. On day 1 and 4 of 2022, the FDA awards Telisotuzumab Vedotin breakthrough therapy approval (BTD) for non-small cell lung cancer (NSCLC). The rest of cMet ADC development is still in an early clinical stage, and layout enterprises include forever, rong Chang biology, regeneration element, gift, etc.

Trop2 is called as trophblast Cell-Surface Antigen 2 (human Trophoblast Surface glycoprotein Antigen 2), is a transmembrane protein, is highly expressed in breast cancer, pancreatic cancer, colon cancer, ovarian cancer and other tumors, can promote the processes of proliferation, invasion, metastasis, diffusion and the like of tumor cells, and has high expression which is closely related to the reduction of the survival time and poor prognosis of tumor patients. Antibody therapeutic strategies such as monoclonal antibody drugs, antibody-conjugated drugs, bispecific antibodies, and the like, with Trop2 as a target are under development. The first Trop 2-targeting antibody-conjugated drug Sacituzumab Govitecan (Trodelvy) was approved by the FDA in month 4 of 2020, which was an antibody-conjugated drug consisting of a monoclonal antibody to humanized IgG1 of Trop2 linked to the active metabolite SN-38 of the chemotherapeutic drug irinotecan. It is capable of delivering chemotherapeutic agents into the interior of tumor cells by binding to the tumor cell surface Trop2 protein.

Although the ADC curative effect is widely verified, the blocking factors such as large volume of monoclonal antibody, low penetration rate of solid tumor and the like still need to be solved. The nanometer antibody is a small-size antibody with the relative molecular mass of only about 15kDa, which is separated and screened from camelid serum, and only contains a heavy chain variable region (Variabledomain of HEAVY CHAIN of HEAVY CHAIN anti-body, VHH), and is about 1/10 of that of the traditional antibody, and the crystal structure is in the shape of a rugby with the diameter of about 2.5nm and the length of about 4.2 nm. The unique molecular structure ensures that the ADC has good tissue penetrability, and is expected to bring more excellent curative effect and safety to the ADC.

Trispecific antibody drug conjugates are a leading concept. Few enterprises in the current layout are in the preclinical research stage, and in theory, the trispecific antibody can target tumor cells more specifically, enhance the cytotoxicity, overcome the drug resistance and reduce the side effects. In addition, the trispecific antibody can promote the crosslinking action between targets and promote the endocytosis of the antibody-coupled drug, thereby achieving better treatment effect. There is currently no publicly reported trispecific antibody-coupled drug targeting EpCAM, cMet and Trop 2.

Disclosure of Invention

The application discloses an antibody targeting EpCAM, an antibody targeting cMet and an antibody targeting Trop2, in particular a trispecific antibody targeting EpCAM, cMet and Trop2 simultaneously, wherein the antibody part can be an modified antibody of an EpCAM, cMet and Trop2 nanometer antibody and is derived from alpaca. The application also discloses a site-directed conjugated trispecific antibody-coupled drug targeting EpCAM, cMet and Trop2 simultaneously, which consists of a targeting moiety, a cytotoxic drug and a linker. In some embodiments, the application provides an antibody conjugated drug by attaching the anti-mitotic agent monomethyl auristatin E (MMAE, a tubulin inhibitor) as a toxic payload to the site of site-directed mutation of an antibody via the lysosomally cleavable dipeptide valine-citrulline (Val-Cit). In some embodiments, the trispecific antibody-coupled drug disclosed by the application is Anti-EpCAM/cMet/Trop2-VC-MMAE (DAR (apprxeq) 4) can effectively inhibit growth of tumor-bearing mice of HT-29 colorectal cancer cell lines of EpCAM positive, cMet positive and Trop2 weak positive, and has high-efficiency Anti-tumor activity and good safety.

On the basis of the above, the technical scheme of the application relates to the following aspects.

1. Trispecific antibodies

In one aspect, the invention provides a trispecific antibody that specifically binds EpCAM, cMet and Trop2 comprising a first antigen binding domain specific for EpCAM, a second antigen binding domain specific for cMet and a third antigen binding domain specific for Trop 2.

EpCAM binding domains

The first antigen binding domain specific for EpCAM comprised by the trispecific antibodies of the invention may be in any antibody format.

In certain embodiments, the first antigen binding domain is selected from a nanobody, a full length antibody (e.g., igG antibody), or an antigen binding fragment thereof (e.g., scFv, fab, scFab).

In certain embodiments, the first antigen binding domain is a VHH and the second antigen binding domain is a VHH comprising the following CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2) and CDR3 (complementarity determining region 3) sequences:

(a) CDR1 having a sequence as shown in SEQ ID NO. 4 or a sequence having substitution, deletion or addition of one or several amino acids (for example substitution, deletion or addition of 1, 2 or 3 amino acids) as compared with the sequence shown in SEQ ID NO. 4;

(b) CDR2 having a sequence as shown in SEQ ID NO. 5 or a sequence having substitution, deletion or addition of one or several amino acids (for example substitution, deletion or addition of 1, 2 or 3 amino acids) as compared with the sequence shown in SEQ ID NO. 5, and

(C) CDR3, it has a sequence as shown in SEQ ID NO:6, or has substitution, deletion or addition of one or several amino acids (for example substitution, deletion or addition of 1,2 or 3 amino acids) compared with the sequence shown in SEQ ID NO: 6.

In some embodiments, 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.

In some embodiments, the substitution is a conservative substitution.

In some embodiments, the first antigen binding domain comprises a VHH sequence as shown in SEQ ID NO. 1 or a variant thereof, which 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 to the sequence from which it is derived, or has one or several amino acid substitutions, deletions or additions (e.g. 1,2, 3, 4 or 5 amino acid substitutions, deletions or additions) as compared thereto, preferably the substitutions are conservative substitutions.

In some embodiments, the first antigen binding domain comprises a VHH sequence as set forth in SEQ ID NO. 1.

C-Met binding domain

The second antigen binding domain specific for c-Met comprised by the trispecific antibodies of the invention may be in any antibody form.

In certain embodiments, the second antigen binding domain is selected from a nanobody, a full length antibody (e.g., igG antibody), or an antigen binding fragment thereof (e.g., scFv, fab, scFab).

In certain embodiments, the second antigen binding domain is a VHH, and the second antigen binding domain is a VHH, comprising the following CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2) and CDR3 (complementarity determining region 3) sequences:

(a) CDR1 having a sequence as shown in SEQ ID NO. 7 or a sequence having substitution, deletion or addition of one or several amino acids (for example substitution, deletion or addition of 1, 2 or 3 amino acids) as compared with the sequence shown in SEQ ID NO. 7;

(b) CDR2 having a sequence as shown in SEQ ID NO. 8 or a sequence having substitution, deletion or addition of one or several amino acids (for example substitution, deletion or addition of 1, 2 or 3 amino acids) as compared with the sequence shown in SEQ ID NO. 8, and

(C) CDR3, it has a sequence as shown in SEQ ID NO:9, or has substitution, deletion or addition of one or several amino acids (for example substitution, deletion or addition of 1,2 or 3 amino acids) compared with the sequence shown in SEQ ID NO: 9.

In some embodiments, the second antigen binding domain 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.

In some embodiments, the second antigen binding domain comprises a VHH sequence as shown in SEQ ID NO. 2 or a variant thereof, which 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 to the sequence from which it is derived, or has one or several amino acid substitutions, deletions or additions (e.g. 1,2, 3, 4 or 5 amino acid substitutions, deletions or additions) as compared thereto, preferably the substitutions are conservative substitutions.

In some embodiments, the second antigen binding domain comprises a VHH sequence as set forth in SEQ ID NO. 2.

Trop2 binding domains

The third antigen binding domain specific for Trop2 comprised by the trispecific antibodies of the invention may be in any antibody format.

In certain embodiments, the third antigen binding domain is selected from a nanobody, a full length antibody (e.g., igG antibody), or an antigen binding fragment thereof (e.g., scFv, fab, scFab).

In certain embodiments, the third antigen binding domain is a VHH comprising the following CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2) and CDR3 (complementarity determining region 3) sequences:

(a) CDR1 having a sequence as shown in SEQ ID NO. 10 or a sequence having substitution, deletion or addition of one or several amino acids (for example substitution, deletion or addition of 1, 2 or 3 amino acids) as compared with the sequence shown in SEQ ID NO. 10;

(b) CDR2 having a sequence as shown in SEQ ID NO. 11 or a sequence having substitution, deletion or addition of one or several amino acids (for example substitution, deletion or addition of 1, 2 or 3 amino acids) as compared with the sequence shown in SEQ ID NO. 11, and

(C) CDR3 having a sequence as shown in SEQ ID NO. 12 or a sequence having substitution, deletion or addition of one or several amino acids (for example substitution, deletion or addition of 1,2 or 3 amino acids) as compared with the sequence shown in SEQ ID NO. 12;

In some embodiments, the third 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.

In some embodiments, the third antigen binding domain comprises a VHH sequence as shown in SEQ ID NO. 3 or a variant thereof, which 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 to the sequence from which it is derived, or has one or several amino acid substitutions, deletions or additions (e.g. 1,2, 3, 4 or 5 amino acid substitutions, deletions or additions) as compared thereto, preferably the substitutions are conservative substitutions.

In some embodiments, the tri-antigen binding domain comprises a VHH sequence as set forth in SEQ ID NO. 3.

The amino acid sequences of the application may be defined using the Kabat, IMGT, chothia or Abm numbering system.

Structure of trispecific antibodies

It will be appreciated by those skilled in the art that all of the trispecific antibody structural forms known in the art may be used in the present invention. As an example, the following trispecific nanobodies are provided, wherein the first, second and third antigen-binding domains are VHH.

In certain embodiments, the trispecific nanobody comprises, in order, a first antigen-binding domain, a second antigen-binding domain, and a third antigen-binding domain.

In certain embodiments, the trispecific nanobody further comprises an immunoglobulin Fc domain.

In certain 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.

In certain embodiments, the immunoglobulin Fc domain is linked to the N-terminus of the first, second, or third antigen binding domain, optionally through a peptide linker.

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

In certain embodiments, the trispecific nanobody comprises, in order 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. See T1 in fig. 1 for an exemplary structure.

In certain embodiments, the trispecific nanobody comprises, in order 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. See T3 in fig. 1 for an exemplary structure.

In certain embodiments, the trispecific nanobody comprises, in order 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. See T6 in fig. 1 for an exemplary structure.

In certain embodiments, the immunoglobulin Fc domain is an Fc domain of IgG (e.g., an Fc domain of IgG1, igG2, igG3, or IgG 4).

In certain embodiments, the immunoglobulin Fc domain comprises a sequence as set forth 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 thereto, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared thereto.

In some embodiments, the immunoglobulin Fc domain is mutated. The mutation may provide a site for binding to a therapeutic agent (e.g., a cytotoxic drug). In some embodiments, the immunoglobulin Fc domain comprises a mutation at position S239 and/or K290, e.g., comprising S239C and/or K290C.

In some embodiments, the immunoglobulin Fc domains are numbered according to the Kabat EU index.

In the trispecific antibody of the present invention, the peptide linker may be a rigid peptide linker or a flexible peptide linker.

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

In certain embodiments, the linker is a peptide linker comprising one or more glycine and/or one or more serine.

In certain embodiments, the peptide linker is (G4S) n, n being an integer no less than 0, e.g., 1,2, 3, or 4. In certain embodiments, the peptide linker is (GGGGS) ₃.

In certain embodiments, the trispecific nanobody comprises the sequence shown as SEQ ID NO. 17, 18 or 19. The sequences shown herein do not contain an amino acid (e.g., methionine (Met)) encoded by an initiation codon (e.g., ATG) at their N-terminus. Those skilled in the art understand that in the preparation of proteins by genetic engineering, the first position of the resulting polypeptide chain is often the amino acid encoded by the start codon (e.g., met) due to the action of the start codon. The trispecific antibody of the present invention encompasses not only an amino acid sequence which does not comprise an amino acid (e.g., met) encoded by the initiation codon at its N-terminus, but also an amino acid sequence which comprises an amino acid (e.g., met) encoded by the initiation codon at its N-terminus. Thus, sequences further comprising an amino acid (e.g., met) encoded by the start codon at the N-terminus of the amino acid sequence described above are also within the scope of the present invention.

In certain embodiments, the bispecific nanobody comprises a variant of the sequence shown in SEQ ID NO 17, 18, or 19 that differs from SEQ ID NO 17, 18, or 19 only by conservative substitutions of one or more (e.g., conservative substitutions of up to 20, up to 15, up to 10, or up to 5 amino acids), or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the antibody or antigen binding fragment thereof from which it is derived, and substantially retains the biological function of the trispecific nanobody from which it is derived (e.g., specifically binds EpCAM, cMet, and Trop2, neutralizes the biological activity of EpCAM, cMet, and Trop 2). For example, in some embodiments, the variant may be truncated at the N-or C-terminus of the first, second, and/or third antigen binding domains as compared to the trispecific nanobody from which it is derived so that it comprises only a portion of FR1 and/or FR4, or lacks one or both of those framework regions, so long as it substantially retains antigen binding and specificity.

In some embodiments, the trispecific antibody is a dimer, such as a homodimer or a heterodimer.

In some embodiments, the trispecific antibody binds EpCAM with a K _D of less than about 10 ^-5 M, for example less than about 10 ^-6M、10^- ⁷M、10^-8M、10^-9 M or 10 ^-10 M or less.

In some embodiments, the trispecific antibody binds to c-Met with a K _D of less than about 10 ^-5 M, e.g., less than about 10 ^-6M、10^- ⁷M、10^-8M、10^-9 M or 10 ^-10 M or less.

In some embodiments, the trispecific antibody binds Trop2 with a K _D of less than about 10 ^-5 M, for example less than about 10 ^-6M、10^- ⁷M、10^-8M、10^-9 M or 10 ^-10 M or less.

The trispecific antibodies of the invention may be monovalent or bivalent antibodies having any antibody structure.

2. Multispecific antibodies

In another aspect, the application provides a multispecific antibody comprising a trispecific antibody of the application. To produce the multispecific antibodies, a trispecific antibody of the application may be linked (e.g., by chemical coupling, 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).

In certain embodiments, the multispecific antibody specifically binds EpCAM, cMet, and Trop2, and additionally specifically binds one or more other targets.

3. Nucleic acids, vectors, host cells and expression methods

In another aspect, the application provides an isolated nucleic acid molecule comprising a polynucleotide sequence encoding the trispecific antibody or multispecific antibody. The nucleic acid may be obtained using methods known in the art, for example, isolated from phage display libraries, yeast display libraries, immunized animals, immortalized cells (e.g., mouse B cell hybridoma cells, EBV-mediated immortalized B cells), or chemical synthesis. The nucleic acid molecule may be codon optimized for the host cell used for expression.

In another aspect, the application provides a vector comprising the nucleic acid molecule.

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 comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The recombinant nucleic acids comprising the nucleic acids may 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.,E.F.Fritsch,and T.Maniatis.(1989).Molecular cloning:a laboratory manual,2nd ed.Cold Spring Harbor Laboratory,Cold Spring Harbor,N.Y.). expression vectors may also comprise polynucleotide sequences encoding polypeptides or proteins, such polypeptides or proteins may include, but are not limited to, affinity tags (e.g., biotin, polyhistidine tag (His ₆) or glutathione S-transferase (GSH) tags), polypeptides comprising protease cleavage sites, and reporter proteins (e.g., fluorescent proteins) the nucleic acid molecules may be present in one or more vectors in some embodiments, in other embodiments, the expression vector is a viral vector, in other embodiments, the expression vector is a phage vector or a phagemid vector.

In another aspect, the 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 antibody or multispecific antibody. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, e.g., E.coli), eukaryotic cells (e.g., yeast, insect cells, mammalian cells). Bacteria such as E.coli BL21 (DE 3) are particularly advantageous for expressing smaller antigen binding fragments. Mammalian host cells suitable 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 expression of antibodies.

4. Preparation of antibodies

The antibodies of the invention may be prepared by various methods known in the art, for example, by phage surface display techniques, genetic engineering recombinant techniques. For example, a DNA molecule encoding an antibody of the invention is obtained by chemical synthesis or PCR amplification, the resulting DNA molecule is inserted into an expression vector, and the host cell is transfected. The transfected host cells are then cultured under specific conditions and express the antibodies of the invention. The antigen binding fragments of the invention may be obtained by hydrolysis of the intact antibody molecule.

In some embodiments, the method comprises the steps of:

(1) Transforming a host cell with at least one nucleic acid or expression vector described herein;

(2) Culturing the transformed host cell under suitable conditions to allow expression of the nucleic acid or expression vector, and

(3) Isolating and purifying the trispecific antibody or the multispecific antibody from the host cell or the culture medium.

In some embodiments, the host cell further comprises a chaperone plasmid, which may help to 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.

5. Conjugate(s)

In another aspect, the application also provides a conjugate comprising a trispecific antibody or multispecific antibody of the application and a coupling moiety.

In certain embodiments, the targeting moiety is conjugated to the coupling moiety, optionally through a linker.

In certain embodiments, the coupling moiety is selected from the group consisting of a protein tag (protein tag). Such protein tags are well known in the art, examples of which include, but are not limited to His, flag, GST, MBP, HA, myc, GFP or biotin, and it is known to those skilled in the art how to select an appropriate protein tag (e.g., a purification tag, a detection tag, or a tracer tag) depending on the intended purpose. In certain exemplary embodiments, the C-terminus of the trispecific antibodies of the invention is linked to a purification tag.

In certain embodiments, the coupling moiety is selected from a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin. The detectable label according to the present invention may be any substance that is detectable by fluorescence, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, examples of which include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-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 acridine esters), magnetic beads (e.g.,) A calorimetric label such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to the label-modified avidin (e.g., streptavidin) described above. In certain embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In certain embodiments, a detectable label as described above may be attached to a trispecific antibody of the invention by linkers (linker) of different lengths to reduce potential steric hindrance.

In certain embodiments, the coupling moiety is selected from the group consisting of therapeutic agents, such as cytotoxic drugs.

In certain embodiments, the coupling moiety is selected from the group consisting of additional biologically active polypeptides.

6. Antibody conjugated medicine (ADC)

The application further provides an antibody conjugated drug (ADC) comprising:

a targeting moiety selected from the group consisting of a trispecific antibody or a multispecific antibody of any one of the preceding claims;

Cytotoxic drug moiety, and

A linker for linking the targeting moiety and the cytotoxic drug moiety.

The cytotoxic drug is conjugated to disulfide bonds on the antibody, or site-specifically and stably conjugated to the antibody by engineered mutant cysteine conjugation techniques. The amino acid at the specific site of the antibody is mutated into cysteine, and the cysteine reacts with the drug-connector, so that the site-specific coupling can be realized, the conjugate with high uniformity can be obtained, and the therapeutic index of the ADC drug can be improved.

In some embodiments, the targeting moiety is linked to the linker through a thiol group on a cysteine residue.

In some embodiments, the targeting moiety is linked to the linker through a sulfhydryl group on a cysteine residue on a VHH.

In some embodiments, the targeting moiety is linked to the linker through a sulfhydryl group on a cysteine residue on a reduced disulfide bond in the hinge region.

In some embodiments, the targeting moiety is linked to the linker through a thiol group on a cysteine residue of the Fc domain.

In some embodiments, the targeting moiety is linked to the linker through a thiol group on a cysteine residue at position 239 and/or 290 of the Fc domain.

In some embodiments, the cytotoxic drug is selected from the group consisting of a tubulin inhibitor and a DNA damaging drug.

In some embodiments, the tubulin inhibitor is selected from the group consisting of orestatin compounds (e.g., MMAE, MMAF), maytansinoids (e.g., maytansine, maytansinol, DM1, DM 4), taxanes (e.g., paclitaxel Taxol, docetaxel, cabazitaxel Carbazitaxel), vinca alkaloids (e.g., vinblastine, vincristine), eribulin, and colchicine.

In some embodiments, the DNA damaging agent is selected from the group consisting of DNA alkylating agents (calicheamicin γ1l, N-acetyl- γ1i calicheamicin, aflatoxin, PBD, du Kamei), DNA topoisomerase inhibitors (e.g., camptothecins, SN-38, dxd, irinotecan, belotecan, topotecan, PNU-159582), doxorubicin, daunorubicin, etoposide, mitoxantrone), and amanitine.

In some embodiments, the cytotoxic drug is MMAE.

In some embodiments, the linker is a cleavable or non-cleavable linker.

In some embodiments, the cleavable linker is selected from the group consisting of protease-sensitive, pH-sensitive, and glutathione-sensitive linkers.

In some embodiments, the linker is selected from the group consisting of MC (6-maleimidocaproyl), MCC (maleimidomethyl cyclohexane-1-carboxylate), MP (maleimidopropionyl), val-Cit (valine-citrulline), val-Ala (valine-alanine), ala-Phe (alanine-phenylalanine), PAB (p-aminobenzyloxycarbonyl), SPP (5- (succinimidyl) -4- (pyridin-2-ylsulfanyl) pentanoate), 6- (2, 5-dioxopyrrolidin-1-yl) -4- (pyridin-2-ylsulfanyl) hexanoate, 6- (2, 5-dioxopyrrolidin-1-yl) -5-methyl-4- (pyridin-2-ylsulfanyl) hexanoate, SMCC (N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate), or SIAB (N-succinimidyl (4-iodo-acetyl) aminobenzoate), and any combinations thereof.

In some embodiments, the linker is MC-Val-Cit-PAB.

In some embodiments, each peptide chain of the polypeptide construct is linked to 0, 1, 2,3, 4 or 5 of the following structures by VHH, cysteine residues in disulfide bonds after hinge region reduction, or cysteine residues of Fc domains:

in some embodiments, the antibody-conjugated drug is selected from the group consisting of:

wherein x=1, 2,3,4, 5, 6, 7, 8, 9 or 10;

ab is a trispecific antibody according to any of the preceding claims.

In some embodiments, the antibody-conjugated drug is:

wherein x=1, 2,3, 4, 5 or 6;

ab contains or consists of the amino acid sequence shown in SEQ ID NO 17, 18 or 19.

7. Composition and method for producing the same

In another aspect, the application provides a composition comprising or consisting of one or more of the antibody-conjugated drugs of any of the preceding claims.

In some embodiments, the DAR value of the composition is from 1 to 10, e.g 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.

In another aspect, the application provides a composition comprising or consisting of one or more of the following antibody-conjugated drugs:

Wherein x=1, 2, 3, 4, 5, 6, 7 or 8;

In some embodiments, the DAR value of the composition is from 1 to 8, such as 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、4.5-5.5、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 to 8.

In some embodiments, the DAR value of the composition is from 1 to 5, such as from 3.5 to 4.5, and still more such as 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, or 4.5.

8. Antibody compositions

The application also provides an antibody composition comprising a first antibody that specifically binds EpCAM, a second antibody that specifically binds c-Met, and a third antibody that specifically binds Trop 2.

Antibodies that specifically bind EpCAM

The first antibody that specifically binds EpCAM comprised in the antibody compositions of the present invention may be in any antibody format, including, but not limited to, one or more of single domain antibodies, single chain antibodies, antibody fabs, full length antibodies, antigen binding fragments, bispecific antibodies, multispecific antibodies, di/multivalent single domain antibodies, di/multivalent single chain antibodies, and di/multivalent antibody fabs.

In some embodiments, the first antibody is a nanobody or antigen-binding fragment thereof that specifically binds EpCAM, or a polypeptide construct comprising the nanobody or antigen-binding fragment thereof.

In certain embodiments, the nanobody or antigen-binding fragment thereof that specifically binds EpCAM comprises the following CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2), and CDR3 (complementarity determining region 3) sequences:

In some embodiments, the nanobody or antigen-binding fragment thereof that specifically binds to EpCAM 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.

In some embodiments, the nanobody or antigen-binding fragment thereof that specifically binds EpCAM comprises a sequence comprising a VHH sequence as set forth in SEQ ID NO:1, or a variant thereof, said 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 compared to the sequence from which it is derived, or having one or several amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1,2, 3,4, or 5 amino acids) compared thereto, preferably, said substitutions are conservative substitutions.

In some embodiments, the first antibody comprises a VHH sequence as set forth in SEQ ID NO. 1.

Antibodies that specifically bind c-Met

The secondary antibody that specifically binds c-Met comprised in the antibody compositions of the present invention may be in any antibody format, including, but not limited to, one or more of single domain antibodies, single chain antibodies, antibody fabs, full length antibodies, antigen binding fragments, bispecific antibodies, multispecific antibodies, di/multivalent single domain antibodies, di/multivalent single chain antibodies, and di/multivalent antibody fabs.

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

In certain embodiments, the nanobody or antigen-binding fragment thereof that specifically binds c-Met comprises the following CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2), and CDR3 (complementarity determining region 3) sequences:

In some embodiments, the nanobody or antigen-binding fragment thereof that specifically binds c-Met 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.

In some embodiments, the nanobody or antigen-binding fragment thereof that specifically binds c-Met comprises a VHH sequence as shown in SEQ ID NO.2 or a variant thereof, which 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 compared to the sequence from which it is derived, or has one or several amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2, 3, 4, or 5 amino acids) compared thereto, preferably the substitutions are conservative substitutions.

In some embodiments, the nanobody or antigen-binding fragment thereof that specifically binds c-Met comprises a VHH sequence as set forth in SEQ ID NO. 2.

Antibodies that specifically bind Trop2

The third antibody that specifically binds Trop2 comprised in the antibody compositions of the invention may be in any antibody format, including, but not limited to, one or more of single domain antibodies, single chain antibodies, antibody Fab, full length antibodies, antigen binding fragments, bispecific antibodies, multispecific antibodies, di/multivalent single domain antibodies, di/multivalent single chain antibodies, and di/multivalent antibody Fab.

In some embodiments, the third antibody is a nanobody or antigen-binding fragment thereof that specifically binds Trop2, or a polypeptide construct comprising the nanobody or antigen-binding fragment thereof.

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

(C) CDR3, it has a sequence as shown in SEQ ID NO:12, or has one or several amino acid substitutions, deletions or additions (for example 1, 2 or 3 amino acid substitutions, deletions or additions) compared with the sequence shown in SEQ ID NO: 12.

In some embodiments, the nanobody or antigen-binding fragment thereof that specifically binds Trop2 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.

In some embodiments, the nanobody or antigen-binding fragment thereof that specifically binds Trop2 comprises a VHH sequence as set forth in SEQ ID NO:3 or variant thereof, which 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 to the sequence from which it is derived, or one or several amino acid substitutions, deletions, or additions thereto, preferably the substitutions are conservative substitutions.

In some embodiments, the first antibody comprises a VHH sequence as set forth in SEQ ID NO. 3.

Polypeptide constructs

In some embodiments, the first antibody is a polypeptide construct comprising a nanobody or antigen-binding fragment thereof that specifically binds EpCAM. In some embodiments, the second antibody is a polypeptide construct comprising a nanobody or antigen-binding fragment thereof that specifically binds to-Met. In some embodiments, the third antibody is a polypeptide construct comprising a nanobody or antigen-binding fragment thereof that specifically binds Trop 2.

In some embodiments, the polypeptide construct comprises an immunoglobulin Fc domain.

In some embodiments, the immunoglobulin Fc domain is linked directly or through a peptide linker to the N-terminus or C-terminus of the nanobody or antigen-binding fragment thereof.

In some embodiments, the immunoglobulin Fc domain is linked directly or through a peptide linker to the C-terminus of the nanobody or antigen-binding fragment thereof.

In some embodiments, the immunoglobulin Fc domain comprises a sequence as set forth 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 thereto, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared thereto.

In some embodiments, the immunoglobulin Fc domain comprises or does not comprise a mutation at the S239C and/or K290C position, e.g., comprising S239C and/or K290C. In some embodiments, the immunoglobulin Fc domains are numbered according to the Kabat EU index.

In some embodiments, the polypeptide construct comprises or consists of the amino acid sequence set forth in SEQ ID NO. 14, 15 or 16.

In some embodiments, the polypeptide construct is a dimer.

In some embodiments, the polypeptide constructs comprise, but are not limited to, one or more of single domain antibodies, single chain antibodies, antibody Fab, antibody full length proteins, antigen binding fragments, bispecific antibodies, multispecific antibodies, bi/multivalent single domain antibodies, bi/multivalent single chain antibodies, and bi/multivalent antibody Fab.

9. Pharmaceutical composition

In another aspect, the application provides a pharmaceutical composition comprising a trispecific antibody, multispecific antibody, nucleic acid molecule, vector, host cell, conjugate, antibody-coupled drug, composition or antibody composition of any one of the preceding claims, and optionally a carrier or excipient.

The excipient may be an excipient as described in Handbook of Pharmaceutical Excipients, american Pharmaceutical Association (1986). Non-limiting examples of suitable excipients include buffers, preservatives, binders, lubricants, disintegrants, chelating agents, surfactants, flavouring agents, sweetening agents, colouring agents.

In some embodiments, suitable buffering agents include calcium 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, and the like, or combinations thereof.

In some embodiments, suitable preservatives include antioxidants, such as alpha-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, N-acetyl cysteine, and the like.

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

In some embodiments, suitable lubricants include metal salts of stearic acid (e.g., magnesium stearate, calcium stearate, aluminum stearate), fatty acid esters (e.g., sodium stearyl fumarate), fatty acids (e.g., stearic acid), fatty alcohols, glyceryl behenate, mineral oils, waxes, hydrogenated vegetable oils, leucine, polyethylene glycol (PEG), metal salts of lauryl sulfate (e.g., sodium lauryl sulfate, magnesium lauryl sulfate), sodium chloride, sodium benzoate, sodium acetate, and talc, or combinations thereof.

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, alginates, sodium starch glycolate, gums such as agar, guar gum, locust bean gum, karaya gum, pectin (pecitin) 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.

In some embodiments, suitable flavoring agents may be selected from cinnamon oil, oil of wintergreen, 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 essences including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple and apricot essences.

In some embodiments, 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 the sodium salt, dipeptide sweeteners such as aspartame, dihydrochalcone compounds, glycyrrhizin, stevia (Stevia Rebaudiana) (stevioside), chlorinated derivatives of sucrose such as sucralose, and sugar alcohols such as sorbitol, mannitol, xylitol (sylitol), and the like.

In some embodiments, suitable colorants include food, drug and cosmetic pigments (FD & C), drug and cosmetic pigments (D & C), and external drug and cosmetic pigments (ext.d & C).

In some embodiments, suitable chelating agents include ethylenediamine-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 chelate of EDTA, 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 ' -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 ' -triacetic acid, ethylenediamine-N, N, N ', N ' -tetra (methylenephosphonic acid), O ' -bis (2-aminoethyl) ethylene glycol-N, N ', N ' -tetraacetic acid, N, N-bis (2-hydroxybenzyl) ethylenediamine-N, N ' -diacetic acid, ethylenediamine-N, N ' -dipropylenediamine-6, N ' -diaminopropane-2, N ' -diacetic acid, n, N ', N ' -tetraacetic acid, nitrilotriacetic acid, trisodium salt of nitrilotris (methylene phosphoric acid), 7,19,30-trioxa-1,4,10,13,16,22,27,33-octaazabicyclo [11,11,11] cyclopentadecane hexahydrobromide, or triethylenetetramine-N, N, N ', N ' ', N ' ' ', N ' ' ' -hexaacetic acid, and the like.

In some embodiments, suitable diluents include water, glycerol, methanol, ethanol, and other biocompatible diluents.

In some embodiments, suitable surfactants include polysorbates, sodium lauryl sulfate, sodium stearyl fumarate, polyoxyethylene alkyl ethers, sorbitan fatty acid esters, polyethylene glycols (PEG), polyoxyethylene castor oil derivatives, sugar esters of fatty acids, glycerides of fatty acids, combinations thereof, and the like.

The pharmaceutical compositions described herein may be formulated in a variety of dosage forms and administered by a variety of different means, such as oral, rectal or parenteral. The term "parenteral" as used herein may include intraarterial, intracardiac, intracerebroventricular, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravenous, intravitreal, epidural, subcutaneous, inhalation, transdermal, transmucosal, sublingual, buccal and topical (including epidermis, dermis, enema, eye drops, ear drops, intranasal, vaginal) administration. In some exemplary embodiments, the route of administration may be by injection, such as intramuscular, intravenous, subcutaneous, or intraperitoneal injection. Formulations for oral use may include capsules, tablets, caplets, pills, dragees, lozenges, powders, granules and the like.

10. Pharmaceutical use

In one aspect, the application provides the use of a trispecific antibody, multispecific antibody, nucleic acid molecule, vector, host cell, conjugate, antibody-coupled medicament, composition, antibody composition or pharmaceutical composition described herein for the preparation of a medicament for the prevention and/or treatment of a disease associated with EpCAM, c-Met and/or Trop2 in a subject.

In another aspect, the application provides a method of preventing and/or treating a disease associated with EpCAM, c-Met, and/or Trop2, comprising administering to a subject in need thereof an effective amount of a trispecific antibody, multispecific antibody, nucleic acid molecule, vector, host cell, conjugate, antibody-coupled drug, composition, antibody composition, or pharmaceutical composition described herein.

In some embodiments, the disease associated with EpCAM, c-Met, and/or Trop2 is a tumor, e.g., a tumor that is positive with EpCAM, c-Met, and/or Trop 2. 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 carcinoma, ovarian cancer (e.g., ovarian epithelial cancer), cervical and bladder cancer, prostate cancer, pancreatic cancer, liver cancer, and retinoblastoma.

In some embodiments, the subject is a mammal, e.g., a human;

In some embodiments, the trispecific antibody, multispecific antibody, nucleic acid molecule, vector, host cell, conjugate, antibody-coupled drug, composition, antibody composition, or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent.

11. Detection use

In another aspect, there is provided the use of a trispecific antibody, conjugate or antibody composition of the invention in the preparation of a detection reagent 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 associated with EpCAM, c-Met and/or Trop 2.

In certain embodiments, the conjugates used to prepare the detection reagents comprise a trispecific antibody of the invention and a detectable label.

In certain embodiments, the trispecific antibodies used to prepare the detection reagents bear a detectable label.

In certain embodiments, the trispecific antibodies used to prepare the detection reagents are free of detectable labels. In such embodiments, the detection reagent may further comprise other reagents (e.g., a secondary antibody) capable of detecting the trispecific antibodies of the invention.

In another aspect, the application provides a method of detecting the presence or amount of EpCAM, c-Met and/or Trop2 in a sample comprising the use of a trispecific antibody, conjugate or antibody composition of the application.

In certain embodiments, the method is an immunological assay, such as an immunoblot, an enzyme immunoassay (e.g., ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

In certain embodiments, the conjugates used in the methods comprise a trispecific antibody of the invention and a detectable label.

In certain embodiments, the trispecific antibodies used in the methods bear a detectable label.

In certain embodiments, the trispecific antibodies used in the methods do not bear a detectable label. Thus, the method may further comprise detecting the trispecific antibodies of the invention using other reagents (e.g., secondary antibodies) bearing a detectable label.

In certain embodiments, the method comprises the steps of:

(1) Contacting the sample with a trispecific antibody, conjugate or antibody composition of the invention;

(2) Detecting the formation of a complex between the trispecific antibody, conjugate or antibody composition and an antigen or detecting the amount of said complex.

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

wherein the antigen is selected from EpCAM, c-Met and Trop2.

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

In certain embodiments, the methods are used to diagnose whether a subject has a disease associated with EpCAM, c-Met, and/or Trop 2. In such embodiments, the method may further comprise the step of comparing the amount of EpCAM, c-Met, and/or Trop2 in the sample from the subject to a reference value. The reference value may be the level of EpCAM, c-Met, and/or Trop2 in a sample from a subject (e.g., a healthy control) known to be free of diseases associated with EpCAM, with c-Met, and/or with Trop2 (also referred to as a "negative reference value"). For example, if the amount of EpCAM, c-Met, and/or Trop2 in a sample from the subject is increased relative to a negative reference value, the subject is indicated to have a disease associated with EpCAM, c-Met, and/or Trop 2.

In certain embodiments, the EpCAM-related disease is characterized by elevated EpCAM expression and/or excessive EpCAM activity. In certain embodiments, the EpCAM-related disease is a tumor, including, but not limited to, colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (e.g., triple negative breast cancer), lung cancer, oral squamous carcinoma, ovarian cancer (e.g., ovarian epithelial cancer), cervical and bladder cancer, prostate cancer, pancreatic cancer, liver cancer, and retinoblastoma.

In certain embodiments, the disease associated with c-MET is characterized by increased c-MET expression and/or excessive c-MET activity. In certain embodiments, the disease associated with c-MET is a tumor, including but not limited to colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), lung cancer, oral squamous carcinoma, ovarian cancer (e.g., ovarian epithelial cancer), cervical cancer, and bladder cancer.

In certain embodiments, the Trop 2-related disorder is characterized by increased Trop2 expression and/or excessive Trop2 activity. In certain embodiments, the Trop 2-related disease is a tumor, including but not limited to colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), lung cancer, oral squamous carcinoma, ovarian cancer (e.g., ovarian epithelial cancer), cervical cancer, and bladder cancer.

In certain embodiments, the sample may be selected from urine, blood, serum, plasma, saliva, ascites fluid, circulating cells, circulating tumor cells, non-tissue associated cells (i.e., free cells), tissue (e.g., surgically resected tumor tissue, biopsy or fine needle aspirate tissue), histological preparations, and the like.

Definition of terms

In this document, unless otherwise indicated, scientific and technical terms used have the meanings commonly understood by one of ordinary skill in the art. Moreover, the procedures of molecular genetics, nucleic acid chemistry, cell culture, biochemistry, cell biology and the like as used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

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

As used herein, the term "EpCAM", collectively referred to as EPITHELIAL CELL adhesion molecule (epithelial cell adhesion molecule), also known as CD326, TACSTD, GA733-2, ksa, co17-1A, etc., is a conserved type I transmembrane glycoprotein of size 35kDa. The gene encoding human EpCAM is located on chromosome 2, human EpCAM is a polypeptide consisting of 314 amino acids, consisting of a hydrophobic leader peptide, a large extracellular domain of 242 amino acids (N-terminus), a single transmembrane domain of 23 amino acids and a short cytoplasmic domain of 26 amino acids (C-terminus). 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 generally 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 (variable region) known as the heavy chain variable region (VH) and the light chain variable region (VL), respectively. VH and VL together are responsible for binding to the antigen recognized by the antibody. Mammalian immunoglobulins have five major heavy chain classes (or isotypes) that determine the functional activities of the antibody molecule, igM, igD, igG, igA and IgE. Antibody isoforms not found in mammals include IgX, igY, igW and IgNAR. IgY is a primary antibody produced by birds and reptiles and is functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, whereas IgX antibodies are present in amphibians.

As used herein, the term "c-Met" is fully referred to as cell-MESENCHYMAL EPITHELIAL transition factor (interstitial epithelial transforming factor), a member of the receptor tyrosine kinase family. c-Met is a protooncogene located on chromosome 7 and having the band 7q21eq31, and is initially translated in vivo into a single-chain precursor protein, which is post-translationally modified to have a three-dimensional structure linked by disulfide bonds. The mature c-MET molecule consists of an extracellular alpha chain with a molecular weight of 50kDa and a transmembrane beta chain of 140 kDa. The amino acid sequence can be found, for example, in NCBI Gene ID NP-000236.2.

As used herein, the term "Trop2" is commonly referred to as trophblast Cell-Surface Antigen 2 (human feeder Cell Surface glycoprotein Antigen 2), a transmembrane protein. Trop-2 is encoded by TACSTD 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 the person skilled in the art, wherein the amino acid sequence can be found, for example, in NCBI Gene ID: NP-002344.2.

The antibody variable region comprises a Framework Region (FR) and a hypervariable region (hypervariable regions, HVR), referred to as the "complementarity determining region (complementaritydeterminingregion, CDR)". The CDRs are primarily responsible for binding to epitopes of the antigen. VH and VL are composed of 3 CDRs and 4 FRs arranged from amino-terminus to carboxyl-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The allocation of amino acids in each region or domain may follow the definition of Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991)), or Chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883.

As used herein, the terms "nanobody", "single-domain antibody", "VHH antibody" or "camelid antibody" refer to an antibody that does not have additional antibody domains present and has a single domain (variable region) capable of specifically binding an antigen or an epitope of an antigen. The term "nanobody" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment consisting of a single monomer variable antibody domain (e.g., a single heavy chain variable region), typically derived from a variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, nanobodies consist of 4 framework regions and 3 complementarity determining regions, having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Nanobodies may be truncated at the N-or C-terminus such that they comprise only a portion of FR1 and/or FR4, or lack one or both of those framework regions, so long as they substantially retain antigen binding and specificity. Nanobodies are also known as single-domain antibodies (sdabs), which are used interchangeably.

As used herein, the term "trispecific antibody" refers to an antibody that has binding specificity for three different antigens (or epitopes). A trispecific antibody comprises three antigen-binding domains with binding specificity for different antigens (or epitopes) so as to be able to bind to three different binding sites and/or target molecules. In some cases, the different antigen binding domains are linked by a peptide linker. The term "trispecific nanobody" refers to a trispecific antibody formed from three nanobodies.

The term "multispecific antibody" refers to an antibody that has binding specificity for at least two (e.g., two, three, or four) different antigens (or epitopes). A multispecific antibody comprises a plurality of antigen-binding domains having binding specificities for different antigens (or epitopes) so as to be capable of binding at least two different binding sites and/or target molecules. In some cases, the individual antigen binding domains are linked by a peptide linker.

As used herein, the term "Fc region" or "Fc domain" refers to the portion of the heavy chain constant region that comprises CH2 and CH3. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. In some embodiments, the hinge mediates dimerization between two Fc-containing polypeptides when the Fc region comprises a hinge. The Fc region may be any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, igG2, igG3, or IgG4.

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.

As used herein, the term "conservative variant" refers to a protein that contains conservative amino acid substitutions that do not substantially affect or reduce the affinity of the protein. For example, a nanobody or polypeptide construct that specifically binds EpCAM may include up to 1, up to 2, up to 5, up to 10, or up to 15 conservative substitutions and specifically binds a polypeptide of EpCAM. Conservative amino acid substitutions of functionally similar amino acids are well known to those of ordinary skill in the art. The following six groups are considered examples of amino acids that are conservative substitutions for one another:

1) Alanine (a), serine (S), threonine (T);

2) Aspartic acid (D), glutamic acid (E);

3) Asparagine (N), glutamine (Q);

4) Arginine (R), lysine (K);

5) Isoleucine (I), leucine (L), methionine (M), valine (V), and

6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

As used herein, amino acid residues are abbreviated as 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).

As used herein, the term "identity" refers to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (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), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be achieved using, for example, the method of Needleman et al (1970) J.mol.biol.48:443-453, which may be conveniently performed by a computer program such as the Align program (DNAstar, inc.). The percent 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)) which has been integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI biol.48:444-453 (1970)) algorithms that have been incorporated into the GAP program of the GCG software package (available on www.gcg.com) using the Blossum 62 matrix or PAM250 matrix and the GAP weights (GAP WEIGHT) of 16, 14, 12, 10, 8, 6 or 4 and the length weights of 1, 2, 3, 4, 5 or 6.

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 a reaction between an antibody and an antigen to which it is directed. The binding affinity between two molecules can be described by the K _D value. The K _D value refers to the dissociation constant obtained from the ratio of kd (the rate of dissociation of a particular binding molecule-target molecule interaction; also known as koff) to ka (the rate of association of a particular binding molecule-target molecule interaction; also known as kon), or kd/ka expressed as molar concentration (M). The smaller the K _D value, the more tightly the two molecules bind and the higher the affinity. In certain embodiments, an antibody that specifically binds (or has specificity for) an antigen refers to an antibody that binds the antigen with less than about 10 ^-5 M, such as less than about 10 ^-6M、10^-7M、10^-8M、10^-9 M or 10 ^-10 M or less K _D. The K _D value can be determined by methods well known in the art, for example, using Surface Plasmon Resonance (SPR) in a BIACORE instrument.

As used herein, the terms "polynucleotide," "nucleic acid," and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, which may generally include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

As used herein, the term "isolated" refers to a substance (e.g., a nucleic acid molecule or polypeptide) that is separated from the source or environment in which it is present, i.e., that does not substantially comprise any other components.

As used herein, the term "vector" is a medium used to introduce an exogenous nucleic acid into a host cell, which is amplified or expressed when the vector is transformed into an appropriate host cell. Vectors typically remain episomal, but may be designed to integrate a gene or portion thereof into the chromosome of the genome. In this context, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), and the like.

As used herein, the term "expression vector" refers to a vector capable of expressing DNA operably linked to regulatory sequences (e.g., promoters, ribosome binding sites) capable of effecting expression of the DNA. Regulatory sequences may comprise promoter and terminator sequences, and optionally may comprise origins of replication, selectable markers, enhancers, polyadenylation signals, and the like. The expression vector may be a plasmid, phage vector, recombinant virus or other vector that when introduced into an appropriate host cell results in expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic and/or prokaryotic cells as well as expression vectors that remain episomal or are integrated into the genome of the host cell.

As used herein, the term "host cell" is a cell that is used to receive, maintain, replicate, or amplify a vector. Host cells may also be used to express polypeptides encoded by nucleic acids or vectors. The host cell may be a eukaryotic cell or a prokaryotic cell.

As used herein, the term "contacting" refers to direct physical association, including both solid and liquid forms.

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 a desired targeted 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 the intended target cell.

As used herein, the terms "subject," "patient," or "individual" include mammals and non-mammals. The mammal may be any member of the class of mammals including, but not limited to, humans, non-human primates such as chimpanzees, apes or other monkey animals, farm animals such as cattle, horses, sheep, goats, pigs, domestic animals such as rabbits, dogs (or dogs), cats, laboratory animals including rodents such as rats, mice and guinea pigs, and the like. Non-mammals may include birds, fish, and the like. In some embodiments, the subject may be a mammal. In some embodiments, the subject may be a human. In some cases, the person may be an adult. In some cases, the person may be a child. In some cases, the person may be 0-17 years old. In some cases, the person may be 18-130 years old. In some cases, the subject may be a male. In some cases, the subject may be a female. In some cases, the subject is diagnosed with or suspected of having a disease. In some cases, the disease is cancer. The subject may be a patient or an individual. In some cases, a subject, patient, or individual may be used interchangeably.

As used herein, the terms "treat," "treating," "ameliorating" or "alleviating" include alleviating or alleviating a symptom of a disease, inhibiting a disease, e.g., arresting the development of a disease, alleviating a disease, causing a disease to resolve, alleviating a symptom caused by a disease, or stopping a symptom of a disease. The terms "treat," "treating," "ameliorating" or "alleviating" may further include obtaining a therapeutic benefit. Therapeutic benefit may refer to eradication of the disease being treated. In addition, therapeutic benefits may also be realized by eradicating one or more of the physiological symptoms associated with the disorder being treated, such that the subject obtains an observable improvement, although in some embodiments, the subject may still be afflicted with the underlying disorder.

As used herein, the terms "effective amount," "therapeutically effective amount" refer to a sufficient amount of the administered drug that will at least partially alleviate the symptoms of the disease being treated. The dosing regimen may be adjusted to provide the best desired response. For example, a single bolus may be administered, or several divided doses may be administered over time, or the dose may be proportionally reduced or increased depending on the treatment situation. It is noted that the dosage value may vary with the type and severity of the condition to be alleviated, and may include single or multiple doses. It is further understood that for any particular individual, the particular dosage regimen will be adjusted over time according to the individual's needs and the pharmaceutical instructions or the physician's expertise. Generally, an effective dose is about 0.0001 to about 50mg, for example about 0.01 to about 10 mg/kg/day per kg body weight per day (single or divided administration). For a 70kg human, this amounts to about 0.007 mg/day to about 3500 mg/day, for example about 0.7 mg/day to about 700 mg/day. In some cases, dosage levels not higher than the lower limit of the aforementioned range may be sufficient, while in other cases larger doses may still be employed without causing any adverse side effects, provided that the larger dose is first divided into several smaller doses for administration throughout the day.

Advantageous effects of the invention

The application provides a scheme of combining an antibody targeting EpCAM, an antibody targeting cMet and an antibody targeting Trop2, and particularly provides a trispecific antibody and an antibody coupling medicament which simultaneously target EpCAM, cMet and Trop2, and a composition and application containing the same. The antibody coupling drug can have better binding activity to EpCAM, cMet and Trop2 proteins, has better targeting and inhibiting activity to EpCAM positive, cMet positive and/or Trop2 positive tumors, and has good safety.

Drawings

Fig. 1 exemplarily shows three representative structures of the three antibodies.

FIG. 2 shows the identification of the band sizes of Anti-cMet/Trop2/EpCAM TsAb (T1), anti-EpCAM/Trop2/cMet TsAb (T3), anti-EpCAM/cMet/Trop2 TsAb (T6) by SDS-PAGE (1 is non-reducing, 2 is reducing).

Fig. 3 shows schematically the structure of an ADC (Anti-EpCAM/cMet/Trop 2 (T6) -VC-MMAE).

FIG. 4 shows DAR values for detection of Anti-EpCAM/cMet/Trop2 (T6) -VC-MMAE by Hydrophobic Interaction Chromatography (HIC).

FIG. 5 shows the results of ELISA assays for binding activity of Anti-cMet/Trop2/EpCAM TsAb (T1), anti-EpCAM/Trop2/cMet TsAb (T3), anti-EpCAM/cMet/Trop2 TsAb (T6) to antigen EpCAM.

FIG. 6 shows the results of ELISA assays for binding activity of Anti-cMet/Trop2/EpCAM TsAb (T1), anti-EpCAM/Trop2/cMet TsAb (T3), anti-EpCAM/cMet/Trop2 TsAb (T6) to antigen cMet.

FIG. 7 shows the results of ELISA assays for binding activity of Anti-cMet/Trop2/EpCAM TsAb (T1), anti-EpCAM/Trop2/cMet TsAb (T3), anti-EpCAM/cMet/Trop2 TsAb (T6) to antigen Trop 2.

FIG. 8 shows the binding capacity of Anti-EpCAM/cMet/Trop2 TsAb (T6) and parent antibodies in pancreatic cancer cell line BxPC3 using flow cytometry.

FIG. 9 shows the ability of Anti-EpCAM/cMet/Trop2 TsAb (T6) and parent antibodies to internalize in pancreatic cancer cell line BxPC3 using flow cytometry.

FIG. 10 shows the binding capacity of Anti-EpCAM/cMet/Trop2 TsAb (T6) and parent antibodies in colorectal cancer cell line HT-29 detected by flow cytometry.

FIG. 11 shows the ability of Anti-EpCAM/cMet/Trop2 TsAb (T6) and parent antibodies to internalize in colorectal cancer cell line HT-29 using flow cytometry.

FIG. 12 shows the ability to detect internalization of Anti-cMet/Trop2/EpCAM TsAb (T1), anti-EpCAM/Trop2/cMet TsAb (T3), anti-EpCAM/cMet/Trop2 TsAb (T6) in pancreatic cancer cell line BxPC3 by immunofluorescence.

FIG. 13 shows the ability of immunofluorescence to detect internalization of Anti-cMet/Trop2/EpCAM TsAb (T1), anti-EpCAM/Trop2/cMet TsAb (T3), anti-EpCAM/cMet/Trop2 TsAb (T6) and parent antibodies in HT-29 cells of colorectal cancer cell line.

FIG. 14 shows the ability of immunofluorescence to detect internalization of Anti-cMet/Trop2/EpCAM TsAb (T1), anti-EpCAM/Trop2/cMet TsAb (T3), anti-EpCAM/cMet/Trop2 TsAb (T6) and parent antibodies in ovarian cancer SKOV3 cells.

FIG. 15 shows the tumor inhibiting effect of Anti-EpCAM/cMet/Trop2 (T6) -VC-MMAE single needle administration on EpCAM high-expression/cMet high-expression/Trop 2 high-expression pancreatic cancer cell line BxPC3 tumor-bearing mice.

FIG. 16 shows the results of body weight change of Anti-EpCAM/cMet/Trop2 (T6) -VC-MMAE single needle administration on EpCAM high-expression/cMet high-expression/Trop 2 high-expression pancreatic cancer cell line BxPC3 tumor-bearing mice.

FIG. 17 shows the tumor inhibiting effect of Anti-EpCAM/cMet/Trop2 (T6) -VC-MMAE single needle administration on HT-29 tumor bearing mice of EpCAM high expression/cMet high expression/Trop 2 low expression colorectal cancer cell line.

FIG. 18 shows the results of body weight change in HT-29 tumor-bearing mice on the EpCAM high/cMet high/Trop 2 low expression colorectal cancer cell line by single needle administration of Anti-EpCAM/cMet/Trop2 (T6) -VC-MMAE.

FIG. 19 shows the tumor inhibiting effect of Anti-EpCAM/Trop 2/cMet/(T3) -VC-MMAE single needle administration on HT-29 tumor bearing mice of EpCAM high expression/cMet high expression/Trop 2 low expression colorectal cancer cell line.

FIG. 20 shows the results of body weight change of Anti-EpCAM/Trop 2/cMet/(T3) -VC-MMAE single needle administration to HT-29 tumor-bearing mice of the EpCAM high/cMet high/Trop 2 low expressing colorectal cancer cell line.

Sequence information

The information of the sequences to which the invention relates is described in the following table:

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1 site-directed engineering and design of EpCAM, cMet and Trop2 parental polypeptide constructs nanobodies targeting EpCAM, cMet and Trop2 were screened from alpaca antibody libraries based on phage display technology and screening technology in the early laboratory, and the amino acid sequences are shown in table 1.

TABLE 1 amino acid sequence of parental VHH

1.2 The screened Anti-EpCAM, anti-cMet and Anti-Trop2 nanobody are expressed in series with the traditional antibody Fc segments CH2 and CH3 (SEQ ID NO: 13), the half life is prolonged, PTT5-H is used as an expression vector, serine 239 is mutated into cysteine, lysine 290 is mutated into cysteine, and the amino acids are named as "Anti-EpCAM Ab", "Anti-cMet Ab" and "Anti-Trop2 Ab", respectively. As shown in table 2.

Table 2 amino acid sequences of "Anti-EpCAM Ab", "Anti-cMet Ab" and "Anti-Trop2 Ab" (with Fc tag)

1.3 Construction of a trispecific antibody of the "3+0" Structure

The Anti-cMet VHH, anti-Trop2 VHH, anti-EpCAM VHH and the traditional antibody Fc segments CH2 and CH3 (SEQ ID NO: 13) are expressed in series, PTT5-H is used as an expression vector, serine at position 239 is mutated into cysteine, and lysine at position 290 is mutated into cysteine, so that an Anti-cMet/Trop2/EpCAM polypeptide construct with a '3+0' structure is obtained, which is named as 'Anti-cMet/Trop 2/EpCAM TsAb (T1)', and shown in Table 3.

The amino acid sequence of "Anti-cMet/Trop2/EpCAM TsAb (T1)" (with Fc tag)

1.4 Construction of a trispecific antibody of the "2+1" Structure

The Anti-EpCAM VHH, the Anti-Trop2 VHH, the Fc fragments CH2, CH3 (SEQ ID NO: 13) of the conventional antibody and the Anti-cMet VHH were expressed in tandem, the expression vector was PTT5-H mutated from serine 239 to cysteine and then mutated from lysine 290 to cysteine, and the Anti-EpCAM/Trop2/cMet polypeptide construct with a "2+1" structure, named "Anti-EpCAM/Trop2/cMet TsAb (T3)", was obtained as shown in Table 4.

Table 4. Amino acid sequence of "Anti-EpCAM/Trop2/cMet TsAb (T3)" (with Fc tag)

1.5 Tandem expression of Anti-EpCAM VHH, conventional antibody Fc fragments CH2, CH3 (SEQ ID NO: 13), anti-cMet VHH and Anti-Trop2 VHH, the expression vector was PTT5-H mutated to cysteine at serine 239 and then lysine at 290 to cysteine, resulting in an Anti-EpCAM/cMet/Trop2 polypeptide construct of "1+2" structure, designated "Anti-EpCAM/cMet/Trop2 TsAb (T6)", as shown in Table 5.

Table 5 amino acid sequence of "Anti-EpCAM/cMet/Trop2 TsAb (T6)" (with Fc tag)

Example 2：Anti-EpCAMAb、Anti-cMetAb、Anti-Trop2Ab,Anti-cMet/Trop2/EpCAM TsAb(T1)、Anti-EpCAM/Trop2/cMetTsAb(T3)、Anti-EpCAM/cMet/Trop2TsAb(T6) transient expression and affinity chromatography purification

2.1 Harvesting plasmid 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) plasmid was synthesized by general biosystems (Anhui) Inc. The plasmid was introduced into DH-5α E.coli competent cells (Shenzhen Kangbody) by rapid transformation, and cultured overnight at 37℃in an ampicillin-resistant LB plate after heat shock for 90 s. The monoclonal colonies were picked up and grown in ampicillin-resistant LB medium for 15h at 37℃at 220rpm, followed by plasmid extraction using endotoxin-free plasmid extraction kit.

2.2 Antibody expression Expi-293F cells were prepared at a density of 4X 10 ⁶/mL, a viability of 95% and a volume of 200mL by transient transfection of Expi-293F cells with elemental particles to express 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).. 0.5mg of each antibody single plasmid was filtered through 0.22 μm and added to 5mL of CD05 medium. Simultaneously, 2mgPEI was added to 5mL of the CD05 medium, immediately vortexed for 8s, and allowed to stand for 2min. The 7mlPEI mixture was added to the plasmid mixture, immediately vortexed for 8s, and allowed to stand for 8min. Finally, the former mixture was added dropwise to 200mL of the cell liquid with a pipette, and the mixture was gently mixed while adding. Culturing in a constant temperature shaking table at 5% CO ₂ and 37 deg.C, taking out the culture medium after 4 hr, supplementing 200MLFREESTYLE culture medium, and culturing in a constant temperature shaking table for 7-8 days.

2.3 Purification of cell expression supernatant from antibody collection 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), centrifuged at 10000rpm for 25min. The cell supernatant was filtered 0.22 μm for use.

The AKTA purifying instrument is operated by setting the flow rate of 8mL/min and the maximum pressure of 0.3MPa by software, washing the instrument pipeline fully by using B solution (100 mM citric acid monohydrate), reducing the flow rate to 2mL/min, loading a protein A medium-pressure chromatographic column, balancing protein A medium to a base line level by using 95% A solution (200 mM disodium hydrogen phosphate) at the flow rate of 8mL/min for stabilization, carrying out sampling at the flow rate of 8mL/min, raising and maintaining the visible UV value to a certain level, wherein the peak is a penetrating peak, balancing by using 95% A solution after the sampling, reducing the peak to the base line level for stabilization, eluting by using 70% B solution, wherein the visible peak is firstly risen and then reduced to the base line, eluting the target protein during the peak forming process, collecting eluent during the process, flushing the pipeline by using 100% B solution, filling the pipeline and the protein A column by using 20% ethanol, and removing the column, and preserving at 4 ℃. Eluted proteins were dialyzed in 20mM PBS via dialysis bag, dialyzed at 4℃for 24h. The antibody concentration was measured by a microplate reader or BCA, and if the antibody concentration was <0.5mg/mL, it was concentrated by passing through a 10Kd concentration tube (Millipore) at 3000rpm for 10 min. The antibodies were kept at-20 ℃ for later use after sub-packaging.

FIG. 2 shows the identification of the band size of antibodies by SDS-PAGE. As shown in the figure, under the non-reduction condition, the stripe sizes of the EpCAM, cMet and Trop2 nano-antibodies are about 80-90 kDa, the stripe sizes of the Anti-cMet/Trop2/EpCAM TsAb (T1), the Anti-EpCAM/Trop2/cMet TsAb (T3) and the Anti-EpCAM/cMet/Trop2 TsAb (T6) are about 140-160 kDa, and under the reduction condition, the stripe sizes of the T1, T3 and T6 antibodies are about 80kDa, are consistent with the theoretical value, and the stripe is clean and single, which indicates that the antibody purity is high.

Example 3 preparation of antibody-conjugated drug Anti-EpCAM/cMet/Trop2 (T6) -VC-MMAE

An antibody-conjugated drug was obtained by ligating an anti-mitotic agent monomethyl auristatin E (MMAE, a tubulin inhibitor) as a toxic load to the site-directed mutation site of the T6 antibody via a lysosomally cleavable MC-Val-Cit-PAB (maleimidocaproyl-valine-citrulline-p-aminobenzoyloxy carboxyl) linker (shown in FIG. 3). The experimental details were as follows:

(1) Adding 0.5M EDTA into the antibody reaction system to make the working concentration reach 5mM;

(2) Reduction by adding 10eq Tris (2-carboxyethyl) phosphine hydrochloride (TCEP; BEYOTIME) to the reaction system, incubating at 37℃for 2h, exposing disulfide bonds on the engineered antibody and cysteine-activated thiol groups on the mutation sites, displacing the buffer by ultrafiltration of the concentrate tube (10 Kd; millipore);

(3) Oxidation, namely adding excessive Dehydroascorbic acid (DHAA) (50 eq), incubating for 3 hours at room temperature, re-oxidatively connecting the reduced disulfide bonds, only exposing the engineered cysteine sites, and using an ultrafiltration concentration tube to perform buffer replacement to remove the oxidant DHAA;

(4) Coupling, namely dissolving MC-vc-PAB-MMAE (MCE, CAS No.: 646502-53-6) in DMSO with storage concentration of 10mM, adding 10% of the total reaction system, mixing by vortex, incubating at 4 ℃, adding 6eq MC-vc-PAB-MMAE into the reaction system of the Anti-EpCAM/cMet/Trop2 TsAb (T6) engineering antibody, incubating at 4 ℃ for 4 hours, and removing excessive small molecules, cysteine, DMSO and other impurities by an ultrafiltration concentration tube. Obtaining the antibody coupling drug Anti-EpCAM/cMet/Trop2 (T6) -VC-MMAE.

The parent antibody coupling drugs Anti-EpCAM-VC-MMAE, anti-cMet-VC-MMAE and Anti-Trop2-VC-MMAE are obtained by the same reaction system.

Example 4 Hydrophobic Interaction Chromatography (HIC) identification of average DAR values for Anti-EpCAM/cMet/Trop2 (T6) -VC-MMAE

(1) HIC-UPLC (waters) analysis was performed using a silica gel based HPLC column (4.6X100 mm,3.5 μm, agilent) to determine the drug-antibody ratio (DAR);

(2) The antibodies and their conjugates were eluted by a linear gradient for 40min from buffer a (1.5M ammonium sulfate, 50mM sodium phosphate) to buffer B (80% sodium phosphate, 20% isopropanol), ph=7.5, 0.5ml/min,25 ℃. As shown in fig. 8, anti-EpCAM/cMet/Trop2 (T6) -VC-MMAE at ph=7.5, dar≡4, in agreement with the theoretical value.

EXAMPLE 5 ELISA assay for binding Activity of Anti-cMet/Trop2/EpCAMTsAb (T1), anti-EpCAM/Trop2/cMet TsAb (T3), anti-EpCAM/cMet/Trop2TsAb (T6) to antigens EpCAM, cMet and Trop2

The antigens EpCAM, cMet and Trop2 ((purchased from Sino Biological) were diluted to 1. Mu.g/mL with PBS, plated on 96-well plates, washed 5 times with PBST after overnight incubation at 4℃and blocked overnight with 2% BSA, the blocking solution was blotted off, washed 5 times with PBST, dried 24h 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)、Negative control was diluted to a range of different concentrations, added to 96-well plates which had been coated with the antigen EpCAM/cMet/Trop2, incubated 1h at 37℃and washed 5 times with PBST, then added with a secondary antibody Goat Anti-Human IgG (HRP) (1:2000 dilution), incubated 30min at 37℃and washed 5 times with PBST, mixed 1:1 with substrate chromogenic A/B solution, 100. Mu.L/well, developed for about 10min, exhibited a blue gradient change, then added with 50. Mu.L/Kong Zhongzhi solution, the color was changed to yellow, and absorbance values were measured at 450nm/630 nm.

The results show that ,Anti-EpCAM Ab、Anti-cMet/Trop2/EpCAM TsAb(T1)、Anti-EpCAM/Trop2/cMet TsAb(T3)、Anti-EpCAM/cMet/Trop2 TsAb(T6) binds to antigen EpCAM in a concentration-dependent manner, anti-cMet Ab, T1, T3, T6 binds to antigen cMet in a concentration-dependent manner, anti-Trop2 Ab, T1, T3, T6 binds to antigen Trop2 in a concentration-dependent manner, and EC ₅₀ is at nM/L level (as shown in Table 6), but the binding activity of the trispecific antibodies of both T3 and T6 forms is obviously lower than that of the parent antibody, and in combination, the binding activity of T1 is optimal, and the Negative control does not show a specific binding trend (as shown in FIGS. 5,6 and 7).

TABLE 6 EC ₅₀ values for trispecific antibodies and parent antibodies

EXAMPLE 6 identification of binding and internalization Effect of Anti-EpCAM/cMet/Trop2TsAb (T6) 6.1 flow cytometry detection of binding and internalization Capacity of Anti-EpCAM/cMet/Trop2TsAb (T6) in BxPC3, HT-29 cells

Pancreatic cancer cell line BxPC3 with high EpCAM expression/cMet expression/Trop 2 expression and colorectal cancer cell line HT-29 with high EpCAM expression/cMet expression/Trop 2 expression are selected. BxPC3, HT-29 (2X 10 ⁵/well) and antibody (10. Mu.g/mL) were resuspended in pre-chilled PBS, and triplicate replicates were spun at 3min at 20℃at 1h,1200rpm, and two replicates were spun at 37℃for 30min and 3h,1200rpm,3min, and spun at 3h, respectively, with 2% FBS1640, the secondary antibody (Goat Anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, dylight ^TM 650; invitrogen) was diluted at 1:400, incubated at 4℃for 30min, and analyzed by analytical flow cytometry (LSRFortessaX-20; BD) as shown in FIGS. 8, 9, 10, and 11. The binding rate of Anti-EpCAM/cMet/Trop2 TsAb (T6) in BxPC3 cell line was significantly higher than that of the parent antibody, and the binding rate of Anti-Ep/cMet 2 (T6) in HT-29 cell line was also better than that of the parent antibody in the BxPC3 cell line after 3h, and the Anti-Ep/cMet 2 (T6) was also better than the parent antibody in the parent cell line at 300% of the same value in the HT-29 cell line.

6.2 Immunofluorescence assay for internalization ability of Anti-cMet/Trop2/EpCAM TsAb (T1), anti-EpCAM/Trop2/cMet TsAb (T3), anti-EpCAM/cMet/Trop2TsAb (T6) in BxPC3, HT-29, SKOV3 cells

The first day, colorectal cancer cell line HT-29, pancreatic cancer cell line BxPC3 and ovarian cancer cell line SKOV3,10000/well were plated overnight. The next day, primary antibodies were incubated for 3 hours and 1 hour periods, 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 negative controls, with 2% FBSDMEM as diluent and 100nM/L final antibody concentration. Fixing, namely, PBS is washed three times, and 100ul of 4% formaldehyde solution is added and kept stand for 20min. Permeation, namely, PBS is washed for 3 times, 100ul of immunofluorescence permeation liquid is added, and the mixture is kept stand for 5min. Blocking, PBS was washed 2 times, and 100. Mu.L of 2% BSA was added thereto, followed by standing for 30 minutes. Secondary antibody (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody) was added, diluted 1:2000 and incubated at 4℃for 30min. The cell nucleus is stained with PBS for 3 times, diluted with DAPI 1:2000, mixed upside down, added with 100 mu L and stood for 10min at normal temperature. The shots were loaded through a high content cell screening imaging analysis system (Opera Phenix).

As shown in FIGS. 12, 13 and 14, T1, T3 and T6 all had remarkable internalization effect in BxPC3 cell line under the condition of uniform fluorescence intensity. In HT-29 and SKOV3 cell lines, the internalization effects of Anti-cMet/Trop2/EpCAM TsAb (T1), anti-EpCAM/Trop2/cMet TsAb (T3), anti-EpCAM/cMet/Trop2 TsAb (T6) were significantly better than those of Anti-Trop2 Ab, anti-EpCAM Ab and Anti-cMet Ab.

Example 7 anti-EpCAM/cMet/Trop2 (T6) -VC-MMAE Single needle tumor inhibiting Effect in pancreatic cancer cell line BxPC3 tumor bearing mice

Nude mice (5-6 weeks old, female) were selected, bxPC3 was inoculated subcutaneously according to the number of 2X 10 ⁶ cells, and tumor volumes and body weights were monitored every 3-4 days after tumor volumes were about 100mm ³, and were uniformly grouped according to tumor sizes, each group was 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、PBS. single needle tail intravenous injection.

Tumor size was measured using calipers and volume calculated using the following formula v= (W ² x L)/2, where V = tumor volume, W = smaller vertical diameter, L = larger vertical diameter. When the tumor size reached 1500mm ³, mice were euthanized. As shown in FIG. 15 and FIG. 16, at a dose of 2.5. Mu.M, anti-Trop2-VC-MMAE 2.5. Mu.M and Anti-EpCAM/cMet/Trop2 (T6) -VC-MMAE 2.5. Mu.M can completely clear the growth of a 100mm ³ BxPC3 tumor, while Anti-EpCAM-VC-MMAE 2.5. Mu. M, anti-cMet-VC-MMAE 2.5. Mu.M can only inhibit tumor growth, with a significant difference from Anti-EpCAM/cMet/Trop2 (T6) -VC-MMAE. While no weight loss or other abnormalities occurred in the animals of each group.

Example 8 anti-EpCAM/cMet/Trop2 (T6) -VC-MMAE Single needle tumor inhibiting Effect in colorectal cancer HT-29 tumor bearing mice

Nude mice (5-6 week old, female) were selected and inoculated subcutaneously with HT-29 in a cell number of 2X 10 ⁶, and treated to a tumor volume of about 100mm ³, uniformly grouped according to tumor size, and grouped in Anti-Trop2-VC-MMAE 5. Mu. M, anti-EpCAM/cMet/Trop2 (T6) -VC-MMAE 5. Mu. M, PBS. The tumor volume and the body weight are monitored every 3-4 days by single needle tail intravenous injection.

Tumor size was measured using calipers and volume calculated using the following formula v= (W ² x L)/2, where V = tumor volume, W = smaller vertical diameter, L = larger vertical diameter. When the tumor size reached 1500mm ³, mice were euthanized. As shown in FIGS. 17 and 18, at a dose of 5. Mu.M, anti-EpCAM/cMet/Trop2 (T6) -VC-MMAE can completely eliminate the growth of 100mm ³ HT-29 tumor, while Anti-Trop2-VC-MMAE only can inhibit the growth of tumor at 5. Mu.M, which is significantly different from Anti-EpCAM/cMet/Trop2 (T6) -VC-MMAE. While no weight loss or other abnormalities occur.

EXAMPLE 9 anti-EpCAM/Trop2/cMet (T3) -VC-MMAE anti-tumor Effect in animals

Nude mice (5-6 week old, female) were selected and inoculated subcutaneously with HT-29 in a number of 2X 10 ⁶ cells, and the tumor volumes were uniformly grouped according to tumor size to about 100mm ³, with the group being Anti-EpCAM/Trop2/cMet (T3) -VC-MMAE 5. Mu. M, PBS. The tumor volume and the body weight are monitored every 3-4 days by single needle tail intravenous injection.

Tumor size was measured using calipers and volume calculated using the following formula v= (W ² x L)/2, where V = tumor volume, W = smaller vertical diameter, L = larger vertical diameter. When the tumor size reached 1500mm ³, mice were euthanized. As shown in FIGS. 19 and 20, at a dose of 5. Mu.M, anti-EpCAM/Trop2/cMet (T3) -VC-MMAE 5. Mu.M completely cleared the growth of 100mm ³ HT-29 tumors without loss of animal weight or other abnormalities.

While specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure and that such modifications would be within the scope 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 EpCAM, cMet and Trop2 comprising a first antigen binding domain specific for EpCAM, a second antigen binding domain specific for cMet and a third antigen binding domain specific for Trop 2.

2. The trispecific antibody of claim 1, wherein the first antigen-binding domain is a VHH comprising the following CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2) and CDR3 (complementarity determining region 3) sequences:

(C) CDR3 having a sequence as shown in SEQ ID NO. 6 or a sequence having substitution, deletion or addition of one or several amino acids (for example substitution, deletion or addition of 1,2 or 3 amino acids) as compared with 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 having at least 80% sequence identity compared to the sequence from which it is derived or one or several amino acid substitutions, deletions or additions compared to it, preferably the substitution is a conservative substitution.

3. The trispecific antibody of claim 1 or 2, which is a VHH comprising the following CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2) and CDR3 (complementarity determining region 3) sequences:

(C) CDR3 having a sequence as shown in SEQ ID NO. 9 or a sequence having substitution, deletion or addition of one or several amino acids (for example substitution, deletion or addition of 1,2 or 3 amino acids) as compared with 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 having at least 80% sequence identity compared to the sequence from which it is derived or one or several amino acid substitutions, deletions or additions compared to it, preferably the substitution is a conservative substitution.

4. A trispecific antibody according to any of claims 1-3, which third antigen binding domain is a VHH comprising the following CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2) and CDR3 (complementarity determining region 3) sequences:

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 having at least 80% sequence identity compared to the sequence from which it is derived or one or several amino acid substitutions, deletions or additions compared to it, preferably the substitution is a conservative substitution.

5. The trispecific antibody of any one of claims 1-4, wherein the trispecific antibody is a trispecific nanobody comprising, in order, 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 and second antigen binding domains, or after the first antigen binding domain, or between the second and third antigen binding domains;

preferably, the immunoglobulin Fc domain is linked to the N-terminus of the first, second or third antigen binding domain, optionally via a peptide linker;

Preferably, the immunoglobulin Fc domain is linked to the C-terminus of the first, second or third antigen binding domain, optionally via a peptide linker;

preferably, the trispecific nanobody comprises, in order 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, in order 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, in order 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 of any one of claims 1-5, wherein the immunoglobulin Fc domain is an Fc domain of IgG;

Preferably, the immunoglobulin Fc domain comprises a sequence as set forth in SEQ ID No. 13, or a sequence having at least 80% sequence identity thereto, or a sequence having one or more amino acid substitutions, deletions or additions thereto;

Preferably, the immunoglobulin Fc domain is mutated;

Preferably, the immunoglobulin Fc domain comprises a mutation at position S239 and/or K290, e.g., comprising S239C and/or K290C;

preferably, the linker is a peptide linker (e.g., a rigid peptide linker or a flexible peptide linker);

preferably, the linker is a peptide linker comprising one or more glycine and/or one or more serine;

Preferably, the peptide linker is (G4S) n, n being an integer not less than 0, for example 1, 2, 3 or 4.

7. The trispecific antibody of any one of claims 1-6, which 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, which variant differs from SEQ ID NO. 17, 18 or 19 only by conservative substitution of one or more amino acid residues, or has at least 85% sequence identity to the antibody or antigen binding fragment thereof from which it is derived, 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 binds EpCAM with a K _D of less than about 10 ^-5 M, for example less than about 10 ^-6M、10^-7M、10^-8M、10^-9 M or 10 ^-10 M or less;

(4) The trispecific antibody binds to c-Met with a K _D of less than about 10 ^-5 M, e.g., less than about 10 ^-6M、10^-7M、10^-8M、10^-9 M or 10 ^-10 M or less;

(5) The trispecific antibody binds Trop2 with a K _D of less than about 10 ^-5 M, for example less than about 10 ^-6M、10^-7M、10^-8M、10^-9 M or 10 ^-10 M or less;

(5) The trispecific antibody is a monovalent or diabody having any antibody structure.

8. A multispecific antibody comprising the trispecific antibody of any one of claims 1-7;

preferably, the multispecific antibody specifically binds EpCAM, cMet and Trop2, and additionally specifically binds 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 of making the trispecific antibody of any one of claims 1-7 or the multispecific antibody of claim 8, comprising culturing the host cell of claim 11 under conditions that allow expression of the protein, and recovering the nanobody or antigen-binding fragment thereof, 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 coupling moiety;

Preferably, the coupling moiety is selected from a protein tag (e.g., a purification tag), a detectable label, e.g., an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance), or biotin, a therapeutic agent, e.g., a cytotoxic drug, or an additional biologically active polypeptide.

14. An antibody conjugated drug (ADC) comprising a targeting moiety selected from the trispecific antibody of any one of claims 1-7 or the multispecific antibody of claim 8;

Cytotoxic drug moiety, and

A linker for linking the targeting moiety and the cytotoxic drug moiety.

15. The antibody-conjugated drug of claim 14, wherein said targeting moiety is linked to said linker through a sulfhydryl group on a cysteine residue;

Preferably, the targeting moiety is linked to the linker through a thiol group on a cysteine residue of a VHH, fc domain or a thiol group exposed by a cysteine residue in a reduced disulfide bond in the hinge region;

Preferably, the targeting moiety is linked to the linker via a VHH, a sulfhydryl group on a cysteine residue in a disulfide bond or a cysteine residue at positions 239 and/or 290 of the Fc domain after reduction of the hinge region.

16. The antibody-conjugated drug of claim 14 or 15, wherein said cytotoxic drug is selected from the group consisting of a tubulin inhibitor and a DNA damaging drug;

Preferably, the tubulin inhibitor is selected from the group consisting of orestatin compounds (e.g., MMAE, MMAF), maytansinoids (e.g., maytansine, maytansinol, DM1, DM 4), taxanes (e.g., paclitaxel Taxol, docetaxel, cabazitaxel Carbazitaxel), vinca alkaloids (e.g., vinblastine, vincristine), eribulin, and colchicine;

Preferably, the DNA damaging agent is selected from the group consisting of DNA alkylating agents (calicheamicin γ1l, N-acetyl- γ1i calicheamicin, aflatoxin, PBD, du Kamei), DNA topoisomerase inhibitors (e.g. camptothecins, SN-38, dxd, irinotecan, belotecan, topotecan, PNU-159582), doxorubicin, daunorubicin, etoposide, mitoxantrone) and amanita;

Preferably, the cytotoxic drug is MMAE.

17. The antibody-conjugated drug of any of claims 16-18, wherein said 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 the group consisting of MC (6-maleimidocaproyl), MCC (maleimidomethyl cyclohexane-1-carboxylate), MP (maleimidopropionyl), val-Cit (valine-citrulline), val-Ala (valine-alanine), ala-Phe (alanine-phenylalanine), PAB (p-aminobenzyloxycarbonyl), SPP (5- (succinimidyl) -4- (pyridin-2-ylsulfanyl) pentanoate), 6- (2, 5-dioxopyrrolidin-1-yl) -4- (pyridin-2-ylsulfanyl) hexanoate, 6- (2, 5-dioxopyrrolidin-1-yl) -5-methyl-4- (pyridin-2-ylsulfanyl) hexanoate, SMCC (N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate) or SIAB (N-succinimidyl (4-iodo-acetyl) aminobenzoate), and any combination thereof;

preferably, the linker is MC-Val-Cit-PAB.

18. The antibody-conjugated drug of any of claims 14-17, wherein each peptide chain of the targeting moiety is linked to 0, 1,2, 3, 4 or 5 of the following structures by VHH, a cysteine residue in a disulfide bond after hinge region reduction, or a cysteine residue of an Fc domain:

19. the antibody-conjugated drug of any of claims 14-18, which is selected from the group consisting of:

wherein x=1, 2,3,4, 5, 6, 7, 8, 9 or 10;

ab is the trispecific antibody of any one of claims 1-7.

20. The antibody-conjugated drug of any of claims 14-19, which is:

wherein x=1, 2,3, 4, 5 or 6;

21. A composition comprising or consisting of one or more antibody-conjugated drugs of any one of claims 14-20;

Preferably, the composition has a DAR value of 1 to 10.

22. A composition comprising or consisting of one or more antibody-conjugated drugs of claim 19;

Preferably, the composition has a DAR value of 1-8;

More preferably, the DAR value of the composition is from 1 to 5, such as from 3.5 to 4.5, and still more such as 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 EpCAM, a second antibody that specifically binds c-Met, and a third antibody that specifically binds Trop 2.

24. The antibody composition of claim 23, wherein the first antibody is a nanobody or antigen-binding fragment thereof that specifically binds EpCAM, or a polypeptide construct comprising the nanobody or antigen-binding fragment thereof;

preferably, the nanobody or antigen-binding fragment thereof that specifically binds EpCAM comprises the following CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2) and CDR3 (complementarity determining region 3) sequences:

preferably, the nanobody or antigen-binding fragment thereof that specifically binds to EpCAM 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 or antigen-binding fragment thereof that specifically binds EpCAM comprises a VHH sequence as shown in SEQ ID No. 1 or a variant thereof, said variant having a sequence identity of at least 80% compared to the sequence from which it is derived, or having one or several amino acid substitutions, deletions or additions compared to it, preferably said substitutions are conservative substitutions.

25. The antibody composition of claim 23 or 24, wherein the second antibody is a nanobody or antigen-binding fragment thereof that specifically binds c-Met, or a polypeptide construct comprising said nanobody or antigen-binding fragment thereof;

Preferably, the nanobody or antigen-binding fragment thereof that specifically binds to c-Met comprises the following CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2) and CDR3 (complementarity determining region 3) sequences:

Preferably, the nanobody or antigen-binding fragment thereof that specifically binds to c-Met 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 or antigen-binding fragment thereof that specifically binds c-Met comprises a VHH sequence as shown in SEQ ID NO. 2 or a variant thereof, said variant having at least 80% sequence identity compared to the sequence from which it is derived or having one or several amino acid substitutions, deletions or additions compared to it, preferably said substitutions are conservative substitutions.

26. The antibody composition of any one of claims 23-25, wherein the third antibody is a nanobody or antigen-binding fragment thereof that specifically binds Trop2, or a polypeptide construct comprising the nanobody or antigen-binding fragment thereof;

Preferably, the nanobody or antigen-binding fragment thereof that specifically binds Trop2 comprises the following CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2) and CDR3 (complementarity determining region 3) sequences:

preferably, the nanobody or antigen-binding fragment thereof that specifically binds Trop2 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 or antigen-binding fragment thereof that specifically binds Trop2 comprises a VHH sequence as shown in SEQ ID No. 3 or a variant thereof, said variant having at least 80% sequence identity compared to the sequence from which it is derived or having one or several amino acid substitutions, deletions or additions compared to it, preferably said substitutions are conservative substitutions.

27. The antibody composition of any one of claims 23-26, wherein the polypeptide construct comprises an immunoglobulin Fc domain;

Preferably, the immunoglobulin Fc domain is linked directly or through a peptide linker to the N-terminus or C-terminus of the nanobody or antigen-binding fragment thereof; preferably, the immunoglobulin Fc domain is linked directly or through a peptide linker to the C-terminus of the nanobody or antigen-binding fragment thereof;

Preferably, the immunoglobulin Fc domain comprises a sequence as set forth 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 thereto, or a sequence having one or several amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) thereto;

Preferably, the immunoglobulin Fc domain comprises or does not comprise a mutation at position S239C and/or K290C, e.g. comprising S239C and/or K290C, preferably, the immunoglobulin Fc domain is numbered according to the Kabat EU index;

Preferably, the polypeptide construct comprises or consists of the amino acid sequence shown as 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 a single domain antibody, a single chain antibody, an antibody Fab, an antibody full length protein, an antigen binding fragment, a trispecific antibody, a multispecific antibody, a di/multivalent single domain antibody, a di/multivalent single chain antibody, and a di/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-conjugated drug 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-conjugated drug 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 preventing and/or treating a disease associated with EpCAM, c-Met and/or Trop2 in a subject;

Preferably, the disease associated with EpCAM, c-Met and/or Trop2 is a tumor, for example EpCAM, c-Met and/or Trop2 positive tumors;

Preferably, the tumor colorectal cancer, gastric cancer, pancreatic cancer, breast cancer (e.g., triple-negative breast cancer), lung cancer, oral squamous carcinoma, ovarian cancer (e.g., ovarian epithelial cancer), cervical and 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, vector, host cell, conjugate, antibody-coupled drug, composition, antibody composition or pharmaceutical composition is used alone or in combination with another pharmaceutically active agent.