Targeting Trop2 antibody coupling medicine as well as preparation method and application thereof

Abstract

The invention relates to a Trop2-targeting antibody coupling drug as well as a preparation method and application thereof, in particular to a nano antibody specifically bound with Trop2 or an antigen binding fragment thereof, a polypeptide construct thereof, an antibody coupling drug, a composition and medical application. The antibody coupling drug has excellent affinity to Trop2 protein, shows good targeting and inhibitory activity to Trop2 positive tumor cells, and is good in safety.

Description

Technical Field

[0001] The present invention relates to the field of targeted therapy, and in particular to an antibody-drug conjugate targeting Trop2, and a preparation method and use thereof. Background Art

[0002] Antibody-drug conjugates (ADCs) are a new type of biotherapeutic drug that is made by coupling a monoclonal antibody and a small molecule cytotoxic drug through a linker. Compared with traditional cytotoxic drugs, ADCs have the advantages of strong targeting and few toxic side effects, and show good therapeutic potential in clinical practice. Among them, the antibody part precisely delivers small molecule cytotoxic drugs to the tumor site by binding to the targeted antigen on the surface of tumor cells, thereby achieving a tumor-specific killing effect, which is one of the core factors affecting the efficacy of ADCs.

[0003] Trop2 (trophoblast cell surface antigen 2) is a transmembrane protein that is highly expressed in a variety of tumors, including breast cancer, pancreatic cancer, colon cancer, and ovarian cancer. It can promote tumor cell proliferation, invasion, metastasis, and spread. Its high expression is closely related to the shortened survival and poor prognosis of tumor patients. In April 2020, the FDA approved the first antibody-drug conjugate, Sacituzumab Govitecan (Trodelvy), for the treatment of triple-negative breast cancer patients. It is also the world's first approved antibody-drug conjugate targeting Trop2. Trodelvy is composed of a humanized IgG1 monoclonal antibody of Trop2 connected to the active metabolite SN-38 of the chemotherapy drug irinotecan. It can deliver chemotherapy drugs into tumor cells by binding to the Trop2 protein on the surface of tumor cells. For patients with metastatic triple-negative breast cancer, clinical results showed that the progression-free survival and overall survival of the Trodelvy group were significantly longer than those of the single-drug chemotherapy group, but adverse reactions such as bone marrow suppression and diarrhea occurred to varying degrees. Preclinical data showed that even though Trodelvy's DAR value was as high as 7.6, in Trop2-positive human gastric cancer xenografts (NCI-N87), a dose of 25 mg / kg twice a week for two weeks could only delay tumor growth, but could not eliminate it. Therefore, it is necessary to explore more effective, lower-dose, and less-frequently administered drugs targeting Trop2.

[0004] Traditional antibody-based targeted cancer therapy is one of the most promising ways to successfully treat cancer. However, due to the large size, low stability, slow clearance and high immunogenicity of antibodies, the full potential of antibodies is severely limited, so the development of new and successful cancer treatment strategies remains challenging. In 1993, the Hamers team of the Free University of Brussels in Belgium first isolated and observed a naturally occurring small-sized antibody of only about 15kDa from camel serum. Compared with traditional antibodies, nanoantibodies are functional antibodies that naturally lack light chains and only contain variable domains of heavy chains (VHH). It retains complete antigen binding ability and exhibits higher affinity and stability, low immunogenicity, good water solubility, and strong tissue penetration. The excellence of nanoantibodies breaks the application bottleneck of traditional antibodies due to their large size and complex structure, bringing new breakthroughs to the wider application of antibody drugs. Summary of the invention

[0005] The present application discloses a site-specific conjugated antibody-drug conjugate targeting Trop2, which is composed of a targeting moiety, a cytotoxic drug and a linker. In some embodiments, the antibody-drug conjugate has Figure 1 The structure shown, wherein the mAb is a nano-antibody Nano-C modified antibody, derived from alpacas, with the anti-mitotic agent monomethyl oleostatin E (MMAE, a tubulin inhibitor) as a toxic payload, and it is connected to the site-directed mutation site of the antibody through the lysosomal cleavable dipeptide valine-citrulline (Val-Cit), thereby obtaining an antibody-drug conjugate. In some embodiments, the antibody-drug conjugates C2-VC-MMAE (DAR≈2) and C3-VC-MMAE (DAR≈4) disclosed in the present application show nanomolar affinity and binding activity for Trop2 protein, and have strong tumor tissue penetration ability in the tumor sphere model, which is better than traditional monoclonal antibodies. In vivo, C2-VC-MMAE and C3-VC-MMAE have good targeting at the tumor site of the tumor-bearing mouse model, can effectively inhibit the growth of mouse tumors, show efficient anti-tumor activity, and have good safety. On the basis of the above, the technical solution of the present application involves the following aspects.

[0006] 1. Nanobodies

[0007] Based on phage display technology and screening technology, alpaca antibodies against Trop2 are isolated and obtained, which can specifically bind to Trop2 positive cells. In some embodiments, the isolated alpaca antibody "Nano-C" (SEQ ID NO: 1) has a KD value of nM level for Trop2 antigen.

[0008] In one aspect, the present application provides a nanobody or an antigen-binding fragment thereof that specifically binds to Trop2, comprising the following CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 2) and CDR3 (complementarity determining region 3) sequences:

[0009] (a) CDR1 having: a sequence as shown in SEQ ID NO: 2, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence as shown in SEQ ID NO: 2;

[0010] (b) CDR2 having: a sequence as shown in SEQ ID NO: 3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence as shown in SEQ ID NO: 3; and

[0011] (c) CDR3 having: a sequence as shown in SEQ ID NO: 4, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence shown in SEQ ID NO: 4.

[0012] In some embodiments, the substitutions are conservative substitutions.

[0013] In some embodiments, the Nanobody or antigen-binding fragment thereof comprises: a CDR1 as shown in SEQ ID NO:2, a CDR2 as shown in SEQ ID NO:3, and a CDR3 as shown in SEQ ID NO:4.

[0014] In some embodiments, the CDR sequences are defined using the Kabat, IMGT, Chothia, or Abm numbering systems.

[0015] In some embodiments, the Nanobody comprises an amino acid sequence selected from the group consisting of:

[0016] (i) the sequence shown in SEQ ID NO: 1;

[0017] (ii) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in SEQ ID NO: 1; or

[0018] (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence shown in SEQ ID NO:1.

[0019] In some embodiments, the substitutions are conservative substitutions.

[0020] 2. Peptide constructs

[0021] The present application further provides a polypeptide construct obtained by fusion of the nanobody with Fc.

[0022] Specifically, in another aspect, the present application provides a polypeptide construct that specifically binds to Trop2, which comprises the Nanobody or antigen-binding fragment thereof as described in any of the foregoing, and an immunoglobulin Fc domain.

[0023] In some embodiments, the immunoglobulin Fc domain is linked directly or via a peptide linker to the N-terminus or C-terminus of the Nanobody or antigen-binding fragment thereof.

[0024] In some embodiments, the immunoglobulin Fc domain is linked to the C-terminus of the Nanobody or antigen-binding fragment thereof directly or via a peptide linker.

[0025] In some embodiments, the immunoglobulin Fc domain is a human Fc domain or is derived from a human Fc domain. In some embodiments, the immunoglobulin Fc domain is selected from the group consisting of IgG1 isotype, IgG2 isotype, IgG3 isotype and IgG4 isotype.

[0026] In some embodiments, the immunoglobulin Fc domain comprises the sequence shown in SEQ ID NO: 5, 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.

[0027] In some embodiments, the immunoglobulin Fc domain is mutated. The mutation can 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 the position of S239 and / or K290, and the mutation includes, for example, S239C and / or K290C.

[0028] In some embodiments, the immunoglobulin Fc domain is numbered according to the Kabat EU index.

[0029] In some embodiments, the polypeptide construct comprises or consists of the amino acid sequence shown in SEQ ID NO: 6, 7 or 8.

[0030] In some embodiments, the polypeptide construct is a dimer, such as a homodimer or a heterodimer.

[0031] In some embodiments, 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 bispecific antibody, a multispecific antibody, a bi / multivalent single domain antibody, a bi / multivalent single chain antibody, and a bi / multivalent antibody Fab.

[0032] 3. Nucleic Acids, Vectors, Host Cells and Expression Methods

[0033] In another aspect, the application provides a kind of isolated nucleic acid molecule, it comprises the polynucleotide sequence encoding the nano antibody or its antigen-binding fragment or the polypeptide construct. The nucleic acid can be obtained by methods known in the art, for example, isolated from phage display library, yeast display library, immune animal, immortalized cell (for example, mouse B cell hybridoma cell, EBV-mediated immortalized B cell) or chemical synthesis. The nucleic acid molecule can be codon optimized for the host cell used for expression.

[0034] In another aspect, the present application provides a vector comprising the nucleic acid molecule.

[0035] 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 acid comprising the nucleic acid may be prepared using techniques well known in the art, such as chemical synthesis, DNA recombination technology (e.g., polymerase chain reaction (PCR) technology), etc. (see Sambrook, J., EF Fritsch, and T. Maniatis. (1989). Molecular cloning: a laboratory manual, 2nded. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The expression vector may also comprise a polynucleotide sequence encoding a polypeptide or protein, which may facilitate detection and / or separation of the expressed antibody or antigen-binding fragment. Such polypeptides or proteins may include, but are not limited to, affinity tags (e.g., biotin, polyhistidine tags (His6) or glutathione S-transferase (GSH) tags), polypeptides comprising protease cleavage sites, and reporter proteins (e.g., fluorescent proteins). The nucleic acid molecule may be present in one or more vectors. In some embodiments, the expression vector is a DNA plasmid, such as a DNA plasmid for expression in bacteria, yeast or mammalian cells. In other embodiments, the expression vector is a viral vector. In other embodiments, the expression vector is a phage vector or a phagemid vector.

[0036] 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 nanobody or its antigen-binding fragment or the polypeptide construct. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, such as Escherichia coli), eukaryotic cells (e.g., yeast, insect cells, mammalian cells).

[0037] Bacteria (e.g., E. coli BL21 (DE3)) 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 expressing antibodies.

[0038] In another aspect, the present application provides a method for producing the Nanobody or its antigen-binding fragment or the polypeptide construct in a host cell, wherein the method comprises the following steps:

[0039] (1) transforming a host cell with at least one nucleic acid or expression vector described herein;

[0040] (2) culturing the transformed host cell under appropriate conditions to allow expression of the nucleic acid or expression vector, and

[0041] (3) isolating and purifying the antibody or antigen-binding fragment thereof or the polypeptide construct from host cells or culture medium.

[0042] In some embodiments, the host cell further comprises a chaperone plasmid, which can help improve the solubility, stability and / or folding of the antibody or antibody fragment.Techniques for isolating and purifying antibodies from host cells are well known to those skilled in the art.

[0043] 4. Antibody-drug conjugates (ADCs)

[0044] The present application further provides an antibody-drug conjugate (ADC), comprising:

[0045] A targeting moiety selected from any of the polypeptide constructs described above;

[0046] Cytotoxic drug moiety; and

[0047] A linker is used to connect the targeting part and the cytotoxic drug part.

[0048] Cytotoxic drugs are coupled to disulfide bonds on antibodies, or site-specific and stable coupling is achieved with antibodies through engineered mutant cysteine ​​coupling technology. Mutating the amino acid at a specific site of the antibody to cysteine ​​and reacting with the drug-linker can achieve site-specific coupling, obtain a highly uniform conjugate, and improve the therapeutic index of ADC drugs.

[0049] In some embodiments, the polypeptide construct is linked to the linker via a thiol group on a cysteine ​​residue.

[0050] In some embodiments, the polypeptide construct is linked to the linker via a thiol group on a cysteine ​​residue on the VHH.

[0051] In some embodiments, the polypeptide construct is linked to the linker via a sulfhydryl group on a cysteine ​​residue on a reduced disulfide bond in the hinge region.

[0052] In some embodiments, the polypeptide construct is linked to the linker via a thiol group on a cysteine ​​residue in the Fc domain.

[0053] In some embodiments, the polypeptide construct is linked to the linker via the sulfhydryl group on the cysteine ​​residues at positions 239 and / or 290 of the Fc domain.

[0054] In some embodiments, the cytotoxic drug is selected from a tubulin inhibitor and a DNA damaging drug.

[0055] In some embodiments, the tubulin inhibitor is selected from auristatins (e.g., MMAE, MMAF), maytansines (e.g., maytansine, maytansinol, DM1, DM4), taxanes (e.g., Taxol, Docetaxel, Cabazitaxel), vinca alkaloids (e.g., vinblastine, vincristine), eribulin, and colchicine.

[0056] In some embodiments, the DNA damaging agent is selected from DNA alkylating agents (calicheamicin γ11, N-acetyl-γ11 calicheamicin, anthramycin, PBD, dukamycin), DNA topoisomerase inhibitors (e.g., camptothecin compounds (specifically, camptothecin, SN-38, Dxd, irinotecan, belotecan, topotecan, PNU-159682), doxorubicin, daunorubicin, etoposide, mitoxantrone) and amanitin.

[0057] In some embodiments, the cytotoxic drug is MMAE.

[0058] In some embodiments, the linker is a cleavable or non-cleavable linker.

[0059] In some embodiments, the cleavable linker is selected from a protease-sensitive, pH-sensitive, and glutathione-sensitive linker.

[0060] In some embodiments, the linker is selected from MC (6-maleimidocaproyl), MCC (maleimidomethylcyclohexane-1-carboxylate), MP (maleimidopropionyl), Val-Cit (valine-citrulline), Val-Ala (valine-alanine), Ala-Phe (alanine-phenylalanine), PAB (p-aminobenzyloxycarbonyl), SPP (5-(succinimidyl)-4-(pyridine-2- 6-(2,5-dioxopyrrolidin-1-yl)-5-methyl-4-(pyridin-2-ylthio)hexanoate, SMCC (N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) or SIAB (N-succinimidyl (4-iodo-acetyl) aminobenzoate) and any combination thereof.

[0061] In some embodiments, the linker is MC-Val-Cit-PAB.

[0062] In some embodiments, each peptide chain of the polypeptide construct is connected to 0, 1, 2, 3, 4 or 5 of the following structures through a cysteine ​​residue in a disulfide bond after reduction of a VHH, a hinge region or a cysteine ​​residue in an Fc domain:

[0063]

[0064] In some embodiments, the antibody-drug conjugate is selected from:

[0065]

[0066] Wherein, x=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0067] Ab is a polypeptide construct as described in any of the above.

[0068] In some embodiments, the antibody-drug conjugate is:

[0069]

[0070] Wherein, x=1, 2, 3, 4, 5 or 6;

[0071] Ab comprises or consists of the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the antibody-drug conjugate is:

[0072]

[0073] Wherein, x=1, 2, 3, 4, 5, 6, 7 or 8;

[0074] Ab comprises or consists of the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the antibody-drug conjugate is:

[0075]

[0076] Wherein, x=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0077] Ab comprises or consists of the amino acid sequence shown in SEQ ID NO:8.

[0078] 5. Composition

[0079] In another aspect, the present application provides a composition comprising or consisting of one or more antibody-drug conjugates as described in any of the foregoing.

[0080] In some embodiments, the composition has a DAR value of 1-10, 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, 2-4. 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. .

[0081] In another aspect, the present application provides a composition comprising or consisting of one or more of the following antibody-drug conjugates:

[0082]

[0083] Wherein, x=1, 2, 3, 4, 5, 6, 7 or 8;

[0084] Ab comprises or consists of the amino acid sequence shown in SEQ ID NO:7.

[0085] In some embodiments, the composition has a DAR value of 1-8, 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, 1.5-2, 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 7.5-8.

[0086] In some embodiments, the composition has a DAR value of 1.5-3.0, such as 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0.

[0087] In another aspect, the present application provides a composition comprising or consisting of one or more of the following antibody-drug conjugates:

[0088]

[0089] Wherein, x=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0090] Ab comprises or consists of the amino acid sequence shown in SEQ ID NO:8.

[0091] In some embodiments, the composition has a DAR value of 1-10, 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, 4-6.5, 4-7, 4-7.5, 4-8, 4-8.5, 4-9, 4-9.5, 4-10, 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, 5.5-6, 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, 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 or 9.5-10;.

[0092] In some embodiments, the composition has a DAR value of 3.5-4.5, e.g., 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, or 4.5.

[0093] 6. Pharmaceutical Compositions

[0094] In another aspect, the present application provides a pharmaceutical composition, which includes the antibody-drug conjugate or composition described in any of the above items, and an optional carrier or excipient.

[0095] The excipients may be those 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, flavoring agents, sweeteners, coloring agents.

[0096] In some embodiments, suitable buffering agents include calcium bicarbonate, calcium carbonate, sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium gluconate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide and other calcium salts, etc. or combinations thereof.

[0097] 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, p-aminobenzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol and N-acetyl cysteine, etc.

[0098] 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 methyl cellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose; polyvinyl pyrrolidone (povidone); polyethylene glycol (PEG); waxes; calcium carbonate; calcium phosphate; alcohols such as sorbitol, xylitol, mannitol and water, or combinations thereof.

[0099] In some embodiments, suitable lubricants include metal stearates (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 oil, paraffin, hydrogenated vegetable oil, leucine, polyethylene glycol (PEG), metal dodecyl sulfate (e.g., sodium dodecyl sulfate, magnesium dodecyl sulfate), sodium chloride, sodium benzoate, sodium acetate, and talc, or combinations thereof.

[0100] In some embodiments, disintegrants can be non-effervescent disintegrants. Suitable non-effervescent disintegrants include starches, such as corn starch, potato starch, pregelatinized and modified starches, sweeteners, clays, such as bentonite, microcrystalline cellulose, alginate, sodium starch glycolate, gums, such as agar, guar fat, locust bean fat, karaya gum, pectin (pecitin) and tragacanth. In some embodiments, disintegrants can be effervescent disintegrants. Suitable effervescent disintegrants include sodium bicarbonate combined with citric acid and sodium bicarbonate combined with tartaric acid.

[0101] In some embodiments, suitable flavoring agents may be selected from cinnamon oil; wintergreen oil; peppermint oil; clover oil; hay oil; anise oil; eucalyptus oil; vanilla; citrus oils, such as lemon oil, orange oil, grape and grapefruit oil; and fruit flavors, including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple and apricot flavors.

[0102] 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; the dihydrochalcone compound, glycyrrhizin; Stevia Rebaudiana (stevioside); chlorinated derivatives of sucrose, such as sucralose; and sugar alcohols, such as sorbitol, mannitol, xylitol (sylitol), and the like.

[0103] In some embodiments, suitable colorants include food, drug, and cosmetic colors (FD&C), drug and cosmetic colors (D&C), and external drug and cosmetic colors (Ext. D&C).

[0104] In some embodiments, suitable chelating agents include ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA); disodium, trisodium, tetrasodium, dipotassium, tripotassium, dilithium, and diammonium salts of EDTA; barium, calcium, cobalt, copper, dysprosium, europium, iron, indium, lanthanum, magnesium, manganese, nickel, samarium, strontium, or zinc chelates of EDTA; trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid monohydrate; N,N-bis(2-hydroxyethyl)glycine; 1,3-diamino-2-hydroxypropane-N,N,N′,N′-tetraacetic acid; 1,3-diaminopropane-N,N,N′,N′-tetraacetic acid; ethylenediamine-N,N′-diacetic acid; ethylenediamine-N,N′-dipropionic acid dihydrochloride; ethylenediamine-N,N′-bis(methylenephosphonic acid) hemihydrate; N-(2-hydroxyethyl)ethylenediamine-N , N′, N′-triacetic acid; ethylenediamine-N, N, N′, N′-tetra(methylenephosphonic acid); O, O′-bis(2-aminoethyl)ethylenediol-N, N, N′, N′-tetraacetic acid; N, N-bis(2-hydroxybenzyl)ethylenediamine-N, N-diacetic acid; 1,6-hexanediamine-N, N, N′, N′-tetraacetic acid; N-(2-hydroxyethyl)iminodiacetic acid; iminodiacetic acid; 1,2- Diaminopropane-N,N,N′,N′-tetraacetic acid; nitrilotriacetic acid; nitrilotripropionic acid; trisodium salt of nitrilotris(methylenephosphoric acid); 7,19,30-trioxa-1,4,10,13,16,22,27,33-octaazabicyclo[11,11,11]pentatriacontane hexahydrobromide; or triethylenetetramine-N,N,N′,N″,N″′,N″′-hexaacetic acid, etc.

[0105] In some embodiments, suitable diluents include water, glycerol, methanol, ethanol, and other biocompatible diluents.

[0106] In some embodiments, suitable surfactants include polysorbates, sodium lauryl sulfate, sodium stearyl fumarate, polyoxyethylene alkyl ethers, sorbitan fatty acid esters, polyethylene glycol (PEG), polyoxyethylene castor oil derivatives, sugar esters of fatty acids, glycerides of fatty acids, or combinations thereof.

[0107] Pharmaceutical compositions described herein can be formulated into a variety of formulations and applied in a variety of different ways, such as oral, rectal or parenteral administration. The term "parenteral" used herein can include intra-arterial, intracardiac, intraventricular, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravenous, intravitreal, epidural, subcutaneous, inhaled, transdermal, mucosal, sublingual, buccal and local (including epidermis, dermis, enema, eye drops, ear drops, intranasal, vaginal) administration. In some exemplary embodiments, the route of administration can be by injection, such as intramuscular, intravenous, subcutaneous or intraperitoneal injection. Preparations for oral administration can include capsules, tablets, caplets, pills, lozenges, lozenges, powders and granules, etc.

[0108] 7. Medical applications

[0109] In another aspect, the present application provides the use of the Nanobodies or antigen-binding fragments thereof, polypeptide constructs, antibody-drug conjugates, compositions or pharmaceutical compositions described herein in the preparation of drugs for treating cancer.

[0110] In another aspect, the present application provides a Nanobody or antigen-binding fragment thereof, a polypeptide construct, an antibody-drug conjugate, a composition or a pharmaceutical composition described herein for use in treating cancer.

[0111] In another aspect, the present application provides a method for treating cancer, comprising administering to a subject in need thereof an effective amount of the Nanobody or its antigen-binding fragment, polypeptide construct, antibody-drug conjugate, composition or pharmaceutical composition described herein.

[0112] In some embodiments, the cancer is Trop2 positive.

[0113] In some embodiments, the cancer is selected from gastric cancer, pancreatic cancer, breast cancer (eg, triple-negative breast cancer), lung cancer, colon cancer, oral squamous cell carcinoma, ovarian cancer (eg, epithelial ovarian cancer), and bladder cancer.

[0114] Definition of terms

[0115] In this document, unless otherwise specified, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, cell culture, biochemistry, cell biology and other operating procedures used in this document are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0116] As used herein, the singular forms "a", "an" and "an" are intended to include the plural forms as well, unless the context clearly indicates otherwise. In addition, the terms "includes", "comprising", "having", "containing" or their variations are open-ended and not exclusive or exhaustive.

[0117] 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, referred to as a heavy chain variable region (VH) and a light chain variable region (VL), respectively. VH and VL are together responsible for binding to the antigen recognized by the antibody. There are five major heavy chain classes (or isotypes) of mammalian immunoglobulins, which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW, and IgNAR. IgY is a primary antibody produced by birds and reptiles, similar in function to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are present in amphibians.

[0118] The variable region of an antibody comprises a framework region (FR) and a hypervariable region (HVR), which is called a "complementarity determining region (CDR)". The CDR is mainly responsible for binding to the epitope of an antigen. VH and VL consist of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The distribution of amino acids in each region or domain can follow the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.

[0119] As used herein, the term "nanobody", "single-domain antibody", "VHH antibody" or "camelid antibody" refers to an antibody that has no additional antibody domains and has a single domain (variable region) that can specifically bind to an antigen or antigenic epitope. Nanobodies include, for example, VH Domain antibody, V NAR Antibodies, Camelidae VHH antibodies and V L Domain antibody. V NAR The antibodies are produced by cartilaginous fishes, such as the nurse shark, Wobegon shark, spiny dogfish, and bamboo shark. Camelid VHH antibodies are produced by a variety of species, including camels, llamas, alpacas, dromedaries, and guanacos, and they produce heavy chain antibodies that naturally lack light chains.

[0120] As used herein, the term "Fc region" or "Fc domain" refers to the portion of the heavy chain constant region that includes CH2 and CH3. In some embodiments, the Fc region includes a hinge, CH2, and CH3. In some embodiments, when the Fc region includes a hinge, the hinge mediates dimerization between two Fc-containing polypeptides. The Fc region can be any antibody heavy chain constant region isotype discussed herein. In some embodiments, the Fc region is IgG1, IgG2, IgG3, or IgG4.

[0121] 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.

[0122] As used herein, the term "conservative variant" refers to a protein containing 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 to Trop2 may include at most 1, at most 2, at most 5, at most 10, or at most 15 conservative substitutions and a polypeptide that specifically binds to Trop2. 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 of each other:

[0123] 1) Alanine (A), serine (S), threonine (T);

[0124] 2) Aspartic acid (D), Glutamic acid (E);

[0125] 3) Asparagine (N), glutamine (Q);

[0126] 4) Arginine (R), Lysine (K);

[0127] 5) isoleucine (I), leucine (L), methionine (M), valine (V); and

[0128] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0129] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

[0130] As used herein, the term "identity" refers to the matching 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 the two DNA molecules is occupied by adenine, or a position in each of the 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 matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can 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 incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0131] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules (i.e., a binding molecule and a target molecule), such as the reaction between an antibody and its antigen. The binding affinity between two molecules can be expressed as K. D Value description. K D The value refers to the dissociation constant obtained by the ratio of kd (dissociation rate of a specific binding molecule-target molecule interaction; also known as koff) to ka (association rate of a specific binding molecule-target molecule interaction; also known as kon), or kd / ka expressed as a molar concentration (M). DThe smaller the value, the tighter the two molecules bind and the higher the affinity. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) refers to an antibody that binds to an antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller K D Bind to the antigen. D The value can be determined by methods well known in the art, for example using surface plasmon resonance (SPR) in a BIACORE instrument.

[0132] 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, and generally can include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0133] As used herein, the term "isolated" means that a substance (eg, a nucleic acid molecule or a polypeptide) is separated from its source or environment, ie, does not substantially contain any other components.

[0134] As used herein, the term "vector" is a medium for introducing exogenous nucleic acid into a host cell, and when the vector is transformed into an appropriate host cell, the exogenous nucleic acid is amplified or expressed. The vector usually remains free, but can be designed to integrate a gene or part thereof into a chromosome of the genome. In this article, 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), etc.

[0135] As used herein, the term "expression vector" refers to a vector capable of expressing DNA, which is operably connected to a regulatory sequence (such as a promoter, a ribosome binding site) that can affect DNA expression. The regulatory sequence can include a promoter and a terminator sequence, and optionally can include an origin of replication, a selective marker, an enhancer, a polyadenylation signal, etc. The expression vector can be a plasmid, a phage vector, a recombinant virus or other vector that, when introduced into a suitable host cell, causes the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and are included in reproducible expression vectors in eukaryotic cells and / or prokaryotic cells and expression vectors that remain free or are integrated into the host cell genome.

[0136] As used herein, the term "host cell" is a cell that is used to receive, maintain, replicate or amplify a vector. A host cell can also be used to express a nucleic acid or a polypeptide encoded by a vector. A host cell can be a eukaryotic cell or a prokaryotic cell.

[0137] As used herein, the term "contacting" refers to direct physical association; including both solid and liquid forms.

[0138] As used herein, the term "cytotoxic drug" refers to any drug or compound that is capable of killing cells. "Cytotoxicity" refers to the toxicity of a molecule to the intended target cells, rather than to cells in the rest of the organism. In contrast, the term "toxicity" refers to the toxicity of a molecule to cells other than the intended target cells.

[0139] As used herein, the terms "subject", "patient" or "individual" include mammals and non-mammals. Mammals can be any member of the class Mammalia, including but not limited to humans; non-human primates, such as chimpanzees, apes or other monkeys; farm animals, such as cattle, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs (or canines), cats; experimental animals, including rodents, such as rats, mice and guinea pigs; and the like. Non-mammals can include birds, fish, etc. In some embodiments, the subject can be a mammal. In some embodiments, the subject can be a human. In some cases, the human can be an adult. In some cases, the human can be a child. In some cases, the human can be 0-17 years old. In some cases, the human can be 18-130 years old. In some cases, the subject can be a male. In some cases, the subject can 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 can be a patient or an individual. In some cases, the subject, patient or individual can be used interchangeably.

[0140] As used herein, the terms "treat," "treat," "improve," or "alleviate" include alleviating or relieving the symptoms of a disease, inhibiting a disease, such as arresting the development of a disease, relieving a disease, causing regression of a disease, alleviating symptoms caused by a disease, or stopping symptoms of a disease. The terms "treat," "treat," "improve," or "alleviate" may further include obtaining a therapeutic benefit. A therapeutic benefit may refer to the eradication of the disease being treated. Additionally, a therapeutic benefit may also be achieved by eradicating one or more physiological symptoms associated with the disease being treated, resulting in an observable improvement in the subject, although in some embodiments, the subject may still be afflicted with the underlying disease.

[0141] As used herein, the term "effective amount", "therapeutically effective amount" refers to a sufficient amount of the drug administered, which will at least partially alleviate the symptoms of the disease being treated. The dosage regimen can be adjusted to provide the best required response. For example, a single bolus can be administered, or several divided doses can be administered over time, or the dosage can be reduced or increased in proportion to the treatment situation. It should be noted that the dosage value can vary with the type and severity of the disease to be alleviated, and can include single or multiple doses. It is further understood that for any particular individual, the specific dosage regimen should be adjusted over time according to individual needs and drug instructions or clinical physician professional judgment. In general, the effective dose is about 0.0001 to about 50 mg per kg body weight per day, for example, about 0.01 to about 10 mg / kg / day (single or divided administration). For a 70 kg person, this will add up 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 no 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 harmful side effects, provided that the larger dose is first divided into several smaller doses for administration throughout the day.

[0142] Advantageous Effects of the Invention

[0143] The present application provides nanobodies, polypeptide constructs and antibody-drug conjugates targeting Trop2, as well as compositions and uses thereof. The antibody-drug conjugates have excellent affinity for Trop2 protein, exhibit good targeting and inhibitory activity for Trop2-positive tumor cells, and have good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0144] Figure 1 The structures of C2-VC-MMAE and C3-VC-MMAE are shown.

[0145] Figure 2 The results of detecting the binding activity of C2-VC-MMAE and C3-VC-MMAE to the antigen Trop2 using the ELISA method are shown.

[0146] Figure 3 The results of the detection of the DAR values ​​of C2-VC-MMAE and C3-VC-MMAE using hydrophobic interaction chromatography (HIC) are shown.

[0147] Figure 4 The results of detecting the DAR values ​​of C2-VC-MMAE and C3-VC-MMAE using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS) are shown.

[0148] Figure 5The affinity curves obtained by the Biocare method for the parent antibody and antibody-drug conjugates C2-VC-MMAE and C3-VC-MMAE affinity determination are shown.

[0149] Figure 6 The MFI value change curve obtained by flow cytometry for detecting the binding activity of the ADC parent antibody of the present application and the positive control RS7 with Trop2 high-expressing tumor cells.

[0150] Figure 7 The results of flow cytometry detection of the endocytic activity of C1, C2 and C3 in BxPC3 cells are shown.

[0151] Figure 8 The results of testing the penetration ability of C1, C2 and C3 in the BxPC3 tumor sphere model using an ultra-high resolution microscope system are shown.

[0152] Fig. 9 The in vitro killing rate curve of C2-VC-MMAE and C3-VC-MMAE against BxPC3 cells (Trop2 positive) is shown.

[0153] Fig.10 The results of in vivo targeting detection of C1, C2, and C3 in mice are shown.

[0154] Fig.11 The inhibitory effect and body weight change results of a single administration of C2-VC-MMAE and C3-VC-MMAE on BxPC3 tumor-bearing mice are shown. DETAILED DESCRIPTION

[0155] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0156] Sequence Listing

[0157] SEQ ID NO: describe SEQ ID NO: describe 1 Nano-C 5 Fc domain 2 Nano-C CDR1 6 Peptide construct C1 3 Nano-C CDR2 7 Peptide construct C2 4 Nano-C CDR3 8 Peptide construct C3

[0158] Example 1: Site-directed modification and binding activity of Trop2 nanobody

[0159] 1.1 In the early stage of the laboratory, a nano antibody specific to Trop2 was screened from the alpaca antibody library based on phage display technology and screening technology. The amino acid sequence is shown in Table 1. It was named "Nano-C".

[0160] Table 1. Amino acid sequence of Nanobody Nano-C

[0161]

[0162] The method described by KR Abhinandan et al. (Abhinandan KR, Martin AC. Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains. Mol Immunol. 2008 Aug; 45(14): 3832-9. doi: 10.1016 / j.molimm.2008.05.022.) and IMGT (THE INTERNATIONAL IMMUNOGENETICS INFORMATION ) double strategy was used to determine the CDR sequence of Nano-C. The amino acid sequence of the variable region CDR of Nano-C is shown in Table 2.

[0163] Table 2. Amino acid sequences of the variable region CDRs of Nano-C

[0164] name CDR sequences SEQ ID NO Nano-C CDR1 EYNID 2 Nano-C CDR2 AVSRSGVVTKYVDSVKG 3 Nano-C CDR3 DREWTTPATGRGIDY 4

[0165] 1.2 The screened nano-antibody Nano-C VHH was expressed in series with the traditional antibody Fc segment CH2, CH3 (SEQ ID NO: 5) to extend the half-life. The expression vector used PTT5-H and was named "C1". The serine at position 239 was mutated to cysteine ​​and named "C2". Based on C2, the lysine at position 290 was mutated to cysteine ​​and named "C3". The amino acid sequences of C1, C2, and C3 (with Fc tags) are shown in Table 3.

[0166] Table 3. Amino acid sequences of C1, C2, and C3 (with Fc tag)

[0167]

[0168]

[0169] Example 2: Transient expression and affinity chromatography purification of C1, C2, and C3

[0170] 2.1 Obtaining plasmids C1, C2, and C3 plasmids were synthesized by Universal Biosystems (Anhui) Co., Ltd. The plasmids were introduced into DH-5α E. coli competent cells (Shenzhen Kangti) by rapid transfection, and cultured overnight at 37°C in an ampicillin + LB culture plate after heat shock for 90s. Monoclonal colonies were picked and amplified in ampicillin + LB medium at 37°C, 220rpm, 15h, and then the plasmids were extracted using an endotoxin-free plasmid extraction kit.

[0171] 2.2 Antibody expression: Single plasmid was used to transiently transfect Expi-293F cells to express C1, C2, and C3. The density was 4×10 6 / mL, 95% viability, 200mL Expi-293F cells. 0.5mg of each antibody single plasmid was filtered through 0.22μm and added to 5mL CD05 medium. At the same time, 2mgPEI was added to 5mL CD05 medium, immediately vortexed for 8s, and allowed to stand for 2min. Then 7ml PEI 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 cell fluid with a pipette, and lightly mixed while adding. Cultured in a constant temperature shaker at 5% CO2 and 37℃, taken out after 4h and supplemented with 200mL freestyle culture medium, and then cultured in a constant temperature shaker for expression for 7-8 days.

[0172] 2.3 Antibody Purification The cell expression supernatants of C1, C2, and C3 were collected and centrifuged at 10,000 rpm for 25 min. The cell supernatants were filtered through 0.22 μm for later use.

[0173] AKTA purification instrument operation: the software sets the flow rate to 8mL / min and the maximum pressure to 0.3MPa; first, flush the instrument pipeline with B solution (100mM citric acid monohydrate), reduce the flow rate to 2mL / min, install the proteinA medium-pressure chromatography column, and use 95% A solution (200mM disodium hydrogen phosphate dodecahydrate) at a flow rate of 8ml / min to balance the proteinA medium until the baseline level is stable. This process takes about 15min; load the sample at a flow rate of 8mL / min, and the UV value can be seen to rise and remain at a certain level. This peak is the penetration peak; after the loading is completed, it is balanced with 95% A solution again, and the peak value drops to the baseline level and stabilizes; elution is performed with 70% B solution. In this process, it can be seen that the peak value first climbs and then drops to the baseline. The process of elution peak formation is the process of eluting the target protein, and the eluate of this process is collected; 100% B solution is used to flush the pipeline with impurities, and then the pipeline and protein A column are filled with 20% ethanol, the column is removed, and stored at 4℃. The eluted protein was dialyzed in 20mM PBS through a dialysis bag at 4°C for 24h. The antibody concentration was detected by an ELISA reader or BCA. If the antibody concentration was less than 0.5mg / mL, it was concentrated by a 10Kd concentrator (Millipore) at 3000rpm for 10min. The antibody was aliquoted and stored at -20°C for later use.

[0174] Example 3: Preparation of antibody-drug conjugates C2-VC-MMAE and C3-VC-MMAE

[0175] The antimitotic agent monomethyl oleostatin E (MMAE, a tubulin inhibitor) was used as a toxic payload and linked to the site-directed mutagenesis site of the antibody via a lysosomal-cleavable MC-Val-Cit-PAB (maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarboxyl) linker to obtain an antibody-drug conjugate (e.g. Figure 1 The experimental details are as follows:

[0176] (1) Add 0.5 M EDTA to the antibody reaction system to make its working concentration reach 5 mM;

[0177] (2) Reduction: 10 eq of Tris (2-carboxyethyl) phosphine hydrochloride (TCEP; BEYOTIME) was added to the reaction system and incubated at 37°C for 2 h. The disulfide bonds on the engineered antibody and the cysteine-activated thiol groups at the mutation site were exposed, and the buffer was replaced by an ultrafiltration concentration tube (10Kd; Millipore);

[0178] (3) Oxidation: Add excess dehydroascorbic acid (DHAA) (50 eq) and incubate at room temperature for 3 h to reoxidize the reduced disulfide bonds and expose only the engineered cysteine ​​sites. Use ultrafiltration concentration tubes to perform buffer exchange to remove the oxidant DHAA.

[0179] (4) Coupling: MC-vc-PAB-MMAE (MCE, CAS No.: 646502-53-6) was dissolved in DMSO, the storage concentration was 10mM, the added DMSO accounted for 10% of the total reaction system, vortexed and mixed, incubated at 4°C, 3eq vc-MMAE was added to the reaction system of C2 engineered antibody, incubated at 4°C for 3h; 6eq MC-vc-PAB-MMAE was added to the reaction system of C3 engineered antibody, incubated at 4°C for 4h; ultrafiltration concentrator was used to remove excess small molecules, cysteine, DMSO and other impurities. Two antibody-conjugated drugs C2-VC-MMAE and C3-VC-MMAE were obtained.

[0180] Example 4: ELISA method to detect the binding activity of C1, C2, C3 and antigen Trop2

[0181] Antigen Trop2 (purchased from Sino Biological) was diluted to 1 μg / mL with PBS and spread on a 96-well plate, 100 μL per well, incubated at 4°C overnight, washed 5 times with PBST, blocked overnight with 2% BSA, removed the blocking solution, washed 5 times with PBST, and dried for 24 hours. C1, C2, C3, C2-VC-MMAE, C3-VC-MMAE and negative control antibody 125s were diluted to a series of different concentrations and added to the 96-well plate coated with Trop2 antigen, 100 μL per well, incubated at 37°C for 1 hour, and washed 5 times with PBST. Then Goat Anti-Human IgG (HRP) secondary antibody (1:2000 dilution), incubated at 37℃ for 30min, washed 5 times with PBST, added substrate color development solution A / B solution 1:1 mixture, 100μgL / well, color development for about 10min, showing a blue gradient change, then added 50μL / well stop solution, the color turned to yellow, and the absorbance value was measured at 450nm / 630nm wavelength. The results showed that C1, C2, and C3 bound to the antigen Trop2 in a concentration-dependent manner, and the coupling drug did not affect the binding activity of the antibody, EC 50 All of them were at the ng / mL level (as shown in Table 4), and the negative control 125s did not show a specific binding trend (as shown in Table 4). Figure 2 shown).

[0182] Table 4. EC50 values ​​of C1, C2, C3 and ADC (ELISA)

[0183] 125s C1 C2 C3 C2-VC-MMAE C3-VC-MMAE <![CDATA[EC 50 (μg / mL)]]> ～ 0.1023 0.07497 0.05109 0.07456 0.1085 <![CDATA[LogEC 50 (μg / mL)]]> ～ -0.9902 -1.125 -1.292 -1.127 -0.9644

[0184] Example 5: Identification of Drug Antibody Coupling Ratio (DAR) of C2-VC-MMAE and C3-VC-MMAE

[0185] 5.1 UV spectrophotometer to identify the average DAR value of antibody-drug conjugates

[0186] (1) C2, C3, C2-VC-MMAE and C3-VC-MMAE were diluted to 0.6 mg / mL, and vc-MMAE was diluted to 80 μM;

[0187] (2) Determine the absorbance of VC-PABC-MMAE, C2, C3 antibodies, C2-VC-MMAE and C3-VC-MMAE at 280 nm and 248 nm respectively, measure three times and take the average value;

[0188] (3) According to the Beer-Lambert law formula: A = Ig(1 / T) = Kbc

[0189] A is absorbance, T is transmittance, which is the ratio of transmitted light intensity to incident light intensity, c is the concentration of the absorbing substance, and b is the absorption thickness. K is the molar extinction coefficient of the absorbing substance. Calculate the molar extinction coefficient of the absorbing substance at 280nm and 248nm;

[0190] (4) The concentrations of the antibody and small molecule drug (C) were calculated according to the following formula, and finally the average DAR values ​​of C2-VC-MMAE and C3-VC-MMAE were obtained.

[0191] The average DAR of C2-VC-MMAE is 2.6;

[0192] The average DAR of C3-VC-MMAE is 4.1;

[0193]

[0194]

[0195] The above two equations can be used to obtain the concentrations of antibodies and drugs respectively.

[0196]

[0197]

[0198] The DAR value calculation results of ADC are as follows:

[0199] DAR=c drug / c mAb .

[0200] 5.2 Average DAR values ​​of antibody-drug conjugates identified by hydrophobic interaction chromatography (HIC)

[0201] (1) A silica-based HPLC column (4.6 × 100 mm, 3.5 μm, Agilent) was used for HIC-UPLC (waters) analysis to determine the drug-antibody ratio (DAR);

[0202] (2) Antibodies and their conjugates were eluted by a 40-min linear gradient from buffer A (1.5 M ammonium sulfate, 50 mM sodium phosphate) to buffer B (80% sodium phosphate, 20% isopropanol), pH = 6.0 for group C2 and pH = 7.5 for group C3, 0.5 mL / min, 25°C. Figure 3 As shown: when pH = 6.0, DAR = 1.96 for C2-VC-MMAE, and when pH = 7.5, DAR = 3.96 for C3-VC-MMAE, which are close to the theoretical value and UV spectrophotometry.

[0203] 5.3 Average DAR value of antibody-drug conjugates identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS)

[0204] The sample was added with 0.5 μL PNGase F and incubated at 37 °C for 1 h. After desugaring, 1 μL 1 M DTT was added and reduced at room temperature for more than 30 min. The sample was analyzed by autoflex maX MALDI-TOF MS (Bruker). The SA sinapinic acid concentration was 10 mg / mL and mixed with the sample in a 1:1 ratio. 0.8 μg of sample was injected each time. The detection method was LP 30-210 K, the solvent was water and acetonitrile in a 1:1 ratio, the laser intensity was 100%, the ion source was 19.5 kV and 16.65 kV, and the detection window was 20 kDa to 100 kDa, as shown in Figure 2 . Figure 4 As shown: when pH = 6.0, DAR = 2.14 for C2-VC-MMAE, and when pH = 7.5, DAR = 3.78 for C3-VC-MMAE, which are close to the theoretical value, UV spectrophotometry and HIC analysis values.

[0205] Example 6: Identification of affinity and binding activity of C2-VC-MMAE and C3-VC-MMAE

[0206] 6.1 Biocare method for affinity determination of parental antibodies and ADCs

[0207] The Trop2 antigen was coupled to the CM5 chip, and different concentrations of antibodies (C2, C3, C2-VC-MMAE, C3-VC-MMAE) were injected into the system and flowed through the chip to bind to the antigen. 3M MgCl2 was used on the chip surface for dissociation. TM The affinity constant value was calculated by 8K (GE Healthcare Life Sciences) system. The affinity curve is shown in Figure 5 As shown in the table 5, the affinity values ​​are shown in Table 5. The KD values ​​are all at the nM level. The affinity of the antibody is not affected after coupling with small molecule drugs.

[0208] Table 5. KD(M) values ​​of C2, C3 and ADC

[0209] C2 C2-VC-MMAE C3 C3-VC-MMAE KD(M) 3.30e-9 8.21e-9 2.15e-9 5.77e-9

[0210] 6.2 Flow cytometry detection of the binding activity of ADC parent antibody and positive control RS7 to Trop2-overexpressing tumor cells

[0211] Sacituzumab govitecan is a first-in-class antibody-drug conjugate that targets the Trop2 receptor. It has been approved in more than 35 countries and regions under the trade name Trodelvy for the treatment of unresectable, locally advanced or metastatic triple-negative breast cancer (TNBC) in adult patients who have received at least two previous systemic therapies, at least one of which was for metastatic disease. We selected its antibody part, Sacituzumab, also known as RS7, as a positive antibody control. The parent antibody and the positive control RS7 were diluted in multiples, with 20 μg / mL in the first well, 4-fold dilutions, and a total of 15 to 20 gradients. The antibody was mixed with 5×10 5 After incubation at 4°C for 1 h, the cells were washed twice with 2% FBS / PBS and fluorescent secondary antibodies (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, DyLight TM 650; Invitrogen), incubated at 4°C for 30 min, washed twice and resuspended in 200 μL PBS, detected by analytical flow cytometry, and the MFI value was plotted as a fluorescence curve. The results showed that in BxPC3 tumor cells with high expression of Trop2, C1, C2, and C3 all had strong binding activity with the positive control RS7. The EC values ​​of C1, C2, and C3 were 50 The value is at the ng / mL level, and the EC of RS7 50 The value is at the μg / mL level, such as Figure 6 , as shown in Table 6.

[0212] Table 6. EC values ​​of parental antibody and marketed antibody RS750 Value(Stream)

[0213] RS7 125s C1 C2 C3 <![CDATA[EC 50 (ng / mL)]]> 2393 ～ 22.29 96.9 792.5 <![CDATA[LogEC 50 (ng / mL)]]> 3.379 ～ 1.348 1.986 2.899

[0214] Example 7: Identification of internalization properties of C2 and C3

[0215] 7.1 Flow cytometry detection of C2 and C3 endocytic activity in BxPC3

[0216] BxPC3 (human pancreatic cancer cells) (2×10 5 / well) and antibody (10μg / mL) were resuspended in pre-cooled PBS, and three parallel samples were made, incubated at 4℃ for 1h, and washed by centrifugation at 1200rpm for 3min. Two parallel samples were taken and resuspended in 2% FBS1640, incubated at 37℃ for 30min and 3h respectively, and washed by centrifugation at 1200rpm for 3min. Secondary antibody (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, DyLight TM 650; Invitrogen) was diluted 1:400, incubated at 4°C for 30 min, and analyzed by analytical flow cytometry (LSRFortessaX-20; BD). Figure 7 As shown: In the BxPC3 cell line, after the target antibody was incubated with the cells for 30 minutes, the internalization efficiency of the C series antibody and the positive antibody control RS7 was only within 10%; after incubation for 3 hours, the internalization efficiency of RS7 was 51.5%, and the internalization efficiency of the C series antibody was between 40% and 45%. In the BxPC3 cell line, after the target antibody was incubated with the cells for 30 minutes, the internalization efficiency of the positive control RS7 was 29.51%, and the internalization efficiency of the C series antibody was about 20%; after incubation for 3 hours, the internalization efficiency of the C series antibody and RS7 was close, between 50% and 60%.

[0217] 7.2 Ultra-high resolution microscopy system to detect the penetration ability of C2 and C3 in the BxPC3 tumor sphere model

[0218] (1) Preparation of tumor spheres. Prepare 10 mg / mL agarose, heat in a microwave oven to melt, add 50-80 μL / well to a 90-well cell culture plate (Bioland), and dry to form a film. Culture BxPC3 cells to the logarithmic phase, digest and wash, and plate 5000 cells / well with 100 μL of culture medium on the solidified agarose. Change the medium every two days, culture for a total of 5 days, and observe the sphere formation.

[0219] (2) Carefully select tumor spheres with good spheroid formation and place them in a black 96-well cell culture plate (PhenoPlate 96-well, black; PerkinElmer). Add the target antibody at 10 μg / mL or 100 μL / well. Incubate at 37°C for 3 h. Wash twice with PBS. Add the secondary antibody (Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, DyLight TM 650; Invitrogen) diluted 1:400, incubated at 37°C for 30 min, washed twice with PBS, added with DAPI (diluted 1:2000), incubated at room temperature for 10 min, washed twice with PBS, and captured two-dimensional and three-dimensional spheroid images using an ultra-high-resolution microscope system (TCS SP8 X; Leica), and image processing was performed using the Leica matching software, such as Figure 8 As shown: In the BxPC3 tumor sphere model, the permeability of C1, C2, and C3 was stronger than that of the positive control RS7, and the negative control 125s had no specific permeability to Trop2 high-expressing cells.

[0220] Example 8: In vitro killing effects of C2-VC-MMAE and C3-VC-MMAE

[0221] BxPC3 cells in the logarithmic growth phase were centrifuged, resuspended and counted, and 5000 cells / well were inoculated in a 96-well cell culture plate (Bioland) and cultured overnight at 37°C. Then, the ADC was diluted 2-fold (the diluent was 2% 1640 culture medium) at 500nM in the first well, with a total of 20 gradients. Three parallel curves were made for each ADC, 100μL / well. After incubation at 37°C for 72h, 10μL CCK8 reagent (MCE; cat.No:HY-K0301) was added to each well and cultured for 1-2h. The color reaction was observed, and the absorbance at 450nm was measured with an enzyme reader. During the experiment, a blank group (without cells) and a negative control group (without drug treatment) were set up, and the cell survival rate was calculated according to the following formula:

[0222] Cell killing rate = [(Ac-As) / (Ac-Ab)] × 100%

[0223] As: absorbance of experimental wells (containing cells, culture medium, CCK-8 solution and drug solution);

[0224] Ac: absorbance of control well (containing cells, culture medium, CCK-8 solution, but no drug);

[0225] Ab: absorbance of blank wells (containing culture medium and CCK-8 solution, but not cells or drugs).

[0226] IC50 values ​​were calculated using GraphPad Prism software. Fig. 9 As shown in Table 7, C2-VC-MMAE, C3-VC-MMAE and the positive controls RS7-VC-MMAE and MMAE have similar IC 50 The values ​​were all at the pM / L level, while the killing curve of the negative control 125s-VC-MMAE had significant differences.

[0227] Table 7. IC50 values ​​of parental antibodies and ADCs (CCK8)

[0228] <![CDATA[IC 50 (nM / L)]]> RS7-VC-MMAE 0.08390 125s-VC-MMAE 30.81 C2-VC-MMAE 0.1851 C3-VC-MMAE 0.1173 MMAE 0.1699

[0229] Example 9: In vivo targeting of C1, C2 and C3

[0230] The antibody was coupled to Cyanine5.5 NHS ester (Luminex life science solutions) at a ratio of 1:10, mixed overnight at 4° rotation, and then eluted on a desalting column (Zeba TM Spin Desalting Columns,7K MWCO,0.5mL; Thermo Scientific TM ) was centrifuged to remove free fluorescein. Nude mice (5-6 weeks old, female) were selected and BxPC3 was added at 2×10 6 The number of cells was subcutaneously inoculated until the tumor volume reached 80 mm 3 The mice were divided into groups according to tumor size and injected with 200 μg of antibody per mouse through the tail vein. Small animal fluorescent in vivo imaging (Caliper IVIS Lumina II) was performed at 5 hours, 1, 3, 5, and 7 days. Fig.10 As shown: C series antibodies were enriched in the tumor site of BxPC3 tumor-bearing mice on the first day. After that, due to the metabolism in the mouse body, the antibodies in the other non-specific organ sites were metabolized, while the antibodies in the tumor site were still significantly enriched on the 7th day. In contrast, the negative control 125s did not show enrichment, and was completely metabolized out of the body on the 3rd day, and no fluorescent signal was detected. On the 7th day, the mice were dissected, and the heart, liver, spleen, lung, kidney, and tumor were removed. The fluorescent signals of various organs and tumor sites were detected. There was no antibody retention in the main organs, and all were enriched in the tumor site. This shows that if this series of antibodies is prepared into ADC, the toxicity to normal organs can be avoided to the greatest extent, and the ADC can quickly target and enrich in the tumor, producing specific killing of the tumor.

[0231] Example 10: Single-injection tumor inhibition effect of C2-VC-MMAE and C3-VC-MMAE

[0232] Nude mice (5-6 weeks old, female) were selected and BxPC3 was transfected at 2×10 6 The number of cells was subcutaneously inoculated until the tumor volume reached 80 mm 3 Around 1:10 pm, the mice were divided into groups according to tumor size. The C2 series groups were C2-VC-MMAE 5μM, C2-VC-MMAE 3μM, C2-VC-MMAE 1.5μM, PBS, RS7-VC-MMAE 5μM, 125s-VC-MMAE 5μM. The C3 series groups were C3-VC-MMAE10μM, C2-VC-MMAE 5μM, C2-VC-MMAE 2.5μM, C3 5μM, and PBS. The mice were injected into the tail vein with a single needle, and the tumor volume and body weight were monitored every 3 to 4 days.

[0233] Tumor size was measured using calipers and volume was calculated using the following formula: V = (W 2 × L) / 2, where V = tumor volume, W = smaller vertical diameter, L = larger vertical diameter. When the tumor size reaches 1500mm 3 , euthanize the mice. Fig.11 As shown: C2-VC-MMAE can significantly inhibit 80-90 mm at a dose of 3 μM. 3 In the growth of BxPC3 tumors, at a dose of 5 μM, C2-VC-MMAE and RS7-VC-MMAE had comparable tumor inhibition effects, almost completely eliminating 80-90 mm 3 BxPC3 tumors, and no weight loss or other abnormalities occurred. However, C3-VC-MMAE at a dose of 2 μM can significantly inhibit 80-90 mm 3 The growth of BxPC3 tumors was completely eliminated at a dose of 2.5 μM, with a size of 80 to 90 mm 3 BxPC3 tumors, and there was no weight loss or other abnormalities, with good safety.

[0234] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A nanobody or antigen-binding fragment thereof that specifically binds to Trop2, 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: 2, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence as shown in SEQ ID NO: 2; (b) CDR2 having: a sequence as shown in SEQ ID NO: 3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence as shown in SEQ ID NO: 3; and (c) CDR3 having: a sequence as shown in SEQ ID NO: 4, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids) compared to the sequence as shown in SEQ ID NO: 4; Preferably, the substitution is a conservative substitution; Preferably, the Nanobody or antigen-binding fragment thereof comprises: CDR1 as shown in SEQ ID NO: 2, CDR2 as shown in SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4; Preferably, the CDR sequences are defined using the Kabat, IMGT, Chothia or Abm numbering systems.

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

3. A polypeptide construct that specifically binds to Trop2, comprising the Nanobody or antigen-binding fragment thereof according to claim 1 or 2, and comprising or not comprising an immunoglobulin Fc domain; Preferably, the immunoglobulin Fc domain is directly or via a peptide linker connected to the N-terminus or C-terminus of the Nanobody or its antigen-binding fragment; Preferably, the immunoglobulin Fc domain is directly or via a peptide linker connected to the C-terminus of the Nanobody or its antigen-binding fragment; Preferably, the immunoglobulin Fc domain A sequence comprising SEQ ID NO: 5, 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 more 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 the S239 and / or K290 position, and the mutation comprises, for example, 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 an amino acid sequence as shown in SEQ ID NO: 6, 7 or 8; Preferably, the polypeptide construct is a dimer; Preferably, the polypeptide construct comprises but is not limited to one or more of single domain antibodies, single chain antibodies, antibody Fab, full length antibody protein, antigen binding fragment, bispecific antibody, multispecific antibody, bi / multivalent single domain antibody, bi / multivalent single chain antibody and bi / multivalent antibody Fab.

4. An isolated nucleic acid molecule encoding the Nanobody or antigen-binding fragment thereof according to claim 1 or 2, or the polypeptide construct according to claim 3. An expression vector comprising the nucleic acid molecule according to claim 4 . A host cell comprising the nucleic acid molecule of claim 4 or the expression vector of claim 5.

7. An antibody-drug conjugate (ADC), comprising: A targeting moiety selected from the polypeptide construct of claim 3; Cytotoxic drug moiety; and A linker is used to connect the targeting part and the cytotoxic drug part.

8. The antibody-drug conjugate of claim 7, wherein the polypeptide construct is linked to the linker via a thiol group on a cysteine ​​residue; Preferably, the polypeptide construct is connected to the linker via the thiol group on the cysteine ​​residue in the VHH or Fc domain or the thiol group exposed by the cysteine ​​residue in the reduced disulfide bond in the hinge region; Preferably, the polypeptide construct is linked to the linker via the thiol group on the cysteine ​​residues in the VHH, hinge region disulfide bond after reduction or the cysteine ​​residues at positions 239 and / or 290 of the Fc domain.

9. The antibody-drug conjugate of claim 7 or 8, wherein the cytotoxic drug is selected from a microtubule inhibitor and a DNA damaging drug; Preferably, the microtubule inhibitor is selected from auristatins (e.g., MMAE, MMAF), maytansines (e.g., maytansine, maytansinol, DM1, DM4), taxanes (e.g., paclitaxel Taxol, docetaxel Docetaxel, cabazitaxel Carbazitaxel), vinca alkaloids (e.g., vinblastine, vincristine), eribulin and colchicine; Preferably, the DNA damaging agent is selected from DNA alkylating agents (calicheamicin γ11, N-acetyl-γ11 calicheamicin, anthramycin, PBD, dukamycin), DNA topoisomerase inhibitors (e.g., camptothecin compounds (specifically, camptothecin, SN-38, Dxd, irinotecan, belotecan, topotecan, PNU-159682), doxorubicin, daunorubicin, etoposide, mitoxantrone) and amanitin; Preferably, the cytotoxic drug is MMAE.

10. The antibody-drug conjugate according to any one of claims 7 to 9, wherein the linker is a cleavable or non-cleavable linker; Preferably, the cleavable linker is selected from protease-sensitive, pH-sensitive and glutathione-sensitive linkers; Preferably, the linker is selected from MC (6-maleimidocaproyl), MCC (maleimidomethylcyclohexane-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-ylthio) ) pentanoate), 6-(2,5-dioxopyrrolidin-1-yl)-4-(pyridin-2-ylthio)hexanoate, 6-(2,5-dioxopyrrolidin-1-yl)-5-methyl-4-(pyridin-2-ylthio)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.

11. The antibody-drug conjugate according to any one of claims 7 to 10, wherein each peptide chain of the polypeptide construct is connected to 0, 1, 2, 3, 4 or 5 of the following structures through a cysteine ​​residue in a disulfide bond after reduction of a VHH, a hinge region or a cysteine ​​residue in an Fc domain:

12. The antibody-drug conjugate according to any one of claims 7 to 11, which is selected from: in, x=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Ab is the polypeptide construct according to claim 3.

13. The antibody-drug conjugate according to any one of claims 7 to 12, which is: in, x = 1, 2, 3, 4, 5 or 6; Ab comprises or consists of the amino acid sequence shown in SEQ ID NO:

6.

14. The antibody-drug conjugate according to any one of claims 7 to 12, which is: in, x = 1, 2, 3, 4, 5, 6, 7 or 8; Ab comprises or consists of the amino acid sequence shown in SEQ ID NO:

7.

15. The antibody-drug conjugate according to any one of claims 7 to 12, which is: in, x=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Ab comprises or consists of the amino acid sequence shown in SEQ ID NO:

8.

16. A composition comprising or consisting of one or more antibody-drug conjugates according to any one of claims 7 to 15; Preferably, the DAR value of the composition is 1-10, for example 1-1.5, 1-2, 1-2.5, 1-3, 1-3.5, 1-4, 1-4.5, 1-5, 1-5.5, 1-6, 1-6.5, 1-7, 1-7.5, 1-8, 1-8.5, 1-9, 1-9.5, 1-10, 1.5-2, 1.5-2.5, 1.5-3, 1.5-3.5, 1.5-4, 1.5-4.5, 1.5-5, 1.5-5.5, 1.5-6, 1.5-6.5, 1.5-7, 1.5-7.5, 1.5-8, 1.5-8.5, 1.5-9, 1.5-9.5, 1.5-10, 2-2.5, 2-3, 2-3.5, 2-4, 2-4.5, 2-5, 2-5.5, 2-6, 2-6.5, 2-7, 2-7.5, 2-8, 2-8.5, 2-9, 2-9.5, 2-10, 2.5-3, 2.5-3.5, 2.5-4, 2.5-4.5, 2.5-5, 2.5-5.5, 2.5-6, 2.5-6.5, 2.5 -7, 2.5-7.5, 2.5-8, 2.5-8.5, 2.5-9, 2.5-9.5, 2.5-10, 3-3.5, 3-4, 3-4.5, 3-5, 3-5.5, 3-6, 3-6.5, 3-7, 3-7.5, 3-8, 3-8.5, 3-9, 3-9.5, 3-10, 3.5-4, 3 .5-4.5, 3.5-5, 3.5-5.5, 3.5-6, 3.5-6.5, 3.5-7, 3.5-7.5, 3.5-8, 3.5-8.5, 3.5-9, 3.5-9.5, 3.5-10, 4-4.5, 4-5, 4-5.5, 4-6, 4.5-5, 4.5-5.5, 4.5-6, 4.5-6.5, 4.5-7, 4.5-7.5, 4.5-8, 4.5-8.5, 4.5-9, 4.5-9.5, 4.5-10, 5-5.5, 5-6, 5-6.5, 5-7, 5-7.5, 5-8, 5-8.5, 5-9, 5-9.5, 5-10, 5.5-6, 5.5-6.5, 5. 5-7, 5.5-7.5, 5.5-8, 5.5-8.5, 5.5-9, 5.5-9.5, 5.5-10, 6-6.5, 6-7, 6-7.5, 6-8, 6-8.5, 6-9, 6-9.5, 6-10, 6.5-7, 6.5-7.5, 6.5-8, 6.5-8.5, 6.5-9, 6. 5-9.5, 6.5-10, 7-7.5, 7-8, 7-8.5, 7-9, 7-9.5, 7-10, 7.5-8, 7.5-8.5, 7.5-9, 7.5-9.5, 7.5-10, 8-8.5, 8-9, 8-9.5, 8-10, 8.5-9, 8.5-9.5, 8.5-10, 9-9.5、9-10、9.5-10。.

17. A composition comprising or consisting of one or more antibody-drug conjugates according to claim 14; Preferably, the DAR value of the composition is 1-8, for example 1-1.5, 1-2, 1-2.5, 1-3, 1-3.5, 1-4, 1-4.5, 1-5, 1-5.5, 1-6, 1-6.5, 1-7, 1-7.5, 1-8, 1.5-2, 1.5-2.5, 1.5-3, 1.5-3.5, 1.5-4, 1.5-4.5, 1.5-5, 1.5-5.5, 1.5-6, 1.5-6. 5, 1.5-7, 1.5-7.5, 1.5-8, 2-2.5, 2-3, 2-3.5, 2-4, 2-4.5, 2-5, 2-5.5, 2-6, 2-6.5, 2-7, 2-7.5, 2-8, 2.5-3, 2.5-3.5, 2.5-4, 2.5-4.5, 2.5-5, 2.5-5.5, 2.5-6, 2.5-6.5, 2.5-7, 2.5-7.5, 2.5-8, 3 -3.5, 3-4, 3-4.5, 3-5, 3-5.5, 3-6, 3-6.5, 3-7, 3-7.5, 3-8, 3.5-4, 3.5-4.5, 3.5-5, 3.5-5.5, 3.5-6, 3.5-6.5, 3.5-7, 3.5-7.5, 3.5-8, 4-4.5, 4-5, 4-5.5, 4-6, 4-6.5, 4-7, 4-7.5, 4-8, 4.5-5, 4. 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, 7.5-8; Preferably, the composition has a DAR value of 1.5-3.0, such as 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 or 3.

0.

18. A composition comprising or consisting of one or more antibody-drug conjugates according to claim 15; Preferably, the DAR value of the composition is 1-10, for example 1-1.5, 1-2, 1-2.5, 1-3, 1-3.5, 1-4, 1-4.5, 1-5, 1-5.5, 1-6, 1-6.5, 1-7, 1-7.5, 1-8, 1-8.5, 1-9, 1-9.5, 1-10, 1.5-2, 1.5-2.5, 1.5-3, 1.5-3.5, 1.5-4, 1.5-4.5, 1.5-5, 1.5-5.5, 1.5-6, 1.5-6.5, 1.5-7, 1.5-7.5, 1.5-8, 1.5-8.5, 1.5-9, 1.5-9.5, 1.5-10, 2-2.5, 2-3, 2-3.5, 2 -4, 2-4.5, 2-5, 2-5.5, 2-6, 2-6.5, 2-7, 2-7.5, 2-8, 2-8.5, 2-9, 2-9.5, 2-10, 2.5-3, 2.5-3.5, 2.5-4, 2.5-4.5, 2.5-5, 2.5-5.5, 2.5-6, 2.5-6.5, 2.5-7 , 2.5-7.5, 2.5-8, 2.5-8.5, 2.5-9, 2.5-9.5, 2.5-10, 3-3.5, 3-4, 3-4.5, 3-5, 3-5.5, 3-6, 3-6.5, 3-7, 3-7.5, 3-8, 3-8.5, 3-9, 3-9.5, 3-10, 3.5-4, 3.5- 4.5, 3.5-5, 3.5-5.5, 3.5-6, 3.5-6.5, 3.5-7, 3.5-7.5, 3.5-8, 3.5-8.5, 3.5-9, 3.5-9.5, 3.5-10, 4-4.5, 4-5, 4-5.5, 4-6, 4-6.5, 4-7, 4-7.5, 4-8, 4-8. 5, 4-9, 4-9.5, 4-10, 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, 5.5-6, 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, 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 or 9.5-10;. Preferably, the composition has a DAR value of 3.5-4.5, 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.

19. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 7 to 15 or the composition according to any one of claims 16 to 18, and an optional carrier or excipient.

20. Use of the Nanobody or antigen-binding fragment thereof according to claim 1 or 2, the polypeptide construct according to claim 3, the antibody-drug conjugate according to any one of claims 7 to 15, the composition according to any one of claims 16 to 18, or the pharmaceutical composition according to claim 19 in the preparation of a drug for treating cancer; Preferably, the cancer is Trop2 positive; Preferably, the cancer is selected from gastric cancer, pancreatic cancer, breast cancer (eg, triple-negative breast cancer), lung cancer, colon cancer, oral squamous cell carcinoma, ovarian cancer (eg, epithelial ovarian cancer) and bladder cancer.

21. The Nanobody or antigen-binding fragment thereof of claim 1 or 2, the polypeptide construct of claim 3, the antibody-drug conjugate of any one of claims 7 to 15, the composition of any one of claims 16 to 18, or the pharmaceutical composition of claim 19, for use in treating cancer; Preferably, the cancer is Trop2 positive; Preferably, the cancer is selected from gastric cancer, pancreatic cancer, breast cancer (eg, triple-negative breast cancer), lung cancer, colon cancer, oral squamous cell carcinoma, ovarian cancer (eg, epithelial ovarian cancer) and bladder cancer.

22. A method for treating cancer, comprising administering to a subject in need thereof an effective amount of a Nanobody or antigen-binding fragment thereof according to claim 1 or 2, a polypeptide construct according to claim 3, an antibody-drug conjugate according to any one of claims 7 to 15, a composition according to any one of claims 16 to 18, or a pharmaceutical composition according to claim 19; Preferably, the cancer is Trop2 positive; Preferably, the cancer is selected from gastric cancer, pancreatic cancer, breast cancer (eg, triple-negative breast cancer), lung cancer, colon cancer, oral squamous cell carcinoma, ovarian cancer (eg, epithelial ovarian cancer) and bladder cancer.

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