Anti-Trop2 antibody, conjugate containing antibody and application thereof

Abstract

Anti-Trop2 antibodies, conjugates comprising the antibodies, and uses thereof are provided. Specifically, the invention provides an anti-Trop2 single-domain antibody, a long-acting anti-Trop2 antibody formed by connecting the anti-Trop2 single-domain antibody and an antiserum albumin single-domain antibody in series, and an antibody-drug conjugate constructed based on the long-acting anti-Trop2 antibody, and also provides a coding sequence for coding the antibody, a corresponding expression vector and a host cell capable of expressing the antibody. The anti-Trop2 antibodies and conjugates thereof can be used for the treatment or diagnosis of Trop2 related diseases.

Description

Anti-Trop2 antibodies, conjugates containing said antibodies and uses thereof Technical Field

[0001] The present invention relates to the field of biomedicine or biopharmaceutical technology, and more particularly to an anti-Trop2 antibody, a conjugate comprising the antibody, and applications thereof. Background Art

[0002] Trop2 belongs to the TACSTD family and is a cell surface glycoprotein encoded by the TACSTD2 gene. It is also known as tumor-associated calcium signal transducer 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1), gastrointestinal tumor-associated antigen (GA733-1), and surface marker 1 (M1S1). TROP-2 primarily promotes tumor cell growth, proliferation, and metastasis by regulating calcium signaling pathways, cell cycle protein expression, and reducing fibronectin adhesion. Trop2 also interacts with β-catenin in the Wnt signaling cascade, thereby affecting nuclear oncogene transcription and cell proliferation.

[0003] Studies have shown that Trop2 is highly expressed in tumors such as gastric cancer, cervical cancer, breast cancer, lung cancer, prostate cancer, colon cancer, and endometrial mucosal serous papillary carcinoma. It has been shown to correlate significantly with tumor metastasis and prognosis, and plays a crucial role in tumorigenesis. Trop2 is a transmembrane protein whose extracellular domain is overexpressed in a variety of tumors, making it a natural candidate for targeted therapy development. The tissue-localized expression of Trop2 reduces the toxicity of treatment, which is an advantage of Trop2-targeted therapies. A variety of therapeutic approaches targeting Trop2 are under development, including antibodies, antibody conjugates, and combination therapies. The efficacy of anti-Trop2 antibodies conjugated to other chemotherapeutic agents has been demonstrated in various preclinical studies. IMMU-132, an antibody-drug conjugate (ADC), is in Phase II / III clinical trials for the treatment of epithelial malignancies with Trop2 overexpression. Sacituzumab govitecan (IMMU-132), a novel antibody-drug conjugate targeting Trop2, utilizes the humanized antibody hRS7 as a targeting vector conjugated to irinotecan's active metabolite, SN38. It can be used to treat a variety of epithelial malignancies, such as breast cancer (triple-negative breast cancer), ovarian cancer, and small cell lung cancer. In addition, other humanized anti-Trop2 IgG-SN-38 conjugates, such as the anti-Trop2 hRS7-CL2A-SN-38 antibody-drug conjugate, have been demonstrated to exhibit significant and specific anti-cancer effects in xenograft models of various tumor cell lines (Calu-3, Capan-1, BxPC-3, and COLO-205).

[0004] To date, nanobodies (Nb), consisting of a single heavy-chain variable domain antibody (VHH)—a heavy-chain antibody (HCAb) naturally lacking light chains found in camels—are the smallest functional, stable, antigen-binding units currently available. Single-domain antibodies offer high stability, good water solubility, simple humanization, high targeting, and strong penetrability, demonstrating unimaginable capabilities in immunoassays, diagnosis, and therapy. They are becoming an emerging force in the next generation of antibody diagnostics and therapeutics.

[0005] Therefore, there is a need in the art to develop an anti-Trop2 single domain antibody, especially an anti-Trop2 single domain antibody with good Trop2 antigen binding.

[0006] Summary of the Invention

[0007] The object of the present invention is to provide an anti-Trop2 antibody, a conjugate comprising the antibody, and uses thereof.

[0008] Specifically, the present invention aims to provide a single-domain antibody that can specifically bind to Trop2 protein, a long-acting antibody composed of the single-domain antibody and an anti-serum albumin single-domain antibody, and a conjugate comprising the aforementioned antibody.

[0009] In a first aspect, the present invention provides a complementary determining region (CDR) of an anti-Trop2 single-domain antibody VHH chain, characterized in that the complementary determining region (CDR) of the VHH chain comprises CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 7, and CDR3 shown in SEQ ID NO: 8.

[0010] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by framework regions FR1, FR2, FR3 and FR4.

[0011] The second aspect of the present invention provides a VHH chain of an anti-Trop2 single-domain antibody, characterized in that the VHH chain comprises a framework region FR and the complementarity determining region CDR described in the first aspect of the present invention.

[0012] In another preferred embodiment, the framework region FR includes:

[0013] (a) FR1 shown in SEQ ID NO: 2, FR2 shown in SEQ ID NO: 3, FR3 shown in SEQ ID NO: 4, and FR4 shown in SEQ ID NO: 5; or

[0014] (b) FR1 shown in SEQ ID NO: 10, FR2 shown in SEQ ID NO: 11, FR3 shown in SEQ ID NO: 12, and FR4 shown in SEQ ID NO: 13.

[0015] In another preferred example, the VHH chain of the anti-Trop2 single-domain antibody is as shown in SEQ ID NO: 1 or 9.

[0016] The third aspect of the present invention provides an anti-Trop2 single-domain antibody, characterized in that the single-domain antibody has the VHH chain described in the second aspect of the present invention.

[0017] In another preferred embodiment, the anti-Trop2 single domain antibody includes a camel-derived single domain antibody, a chimeric single domain antibody or a humanized single domain antibody.

[0018] In another preferred embodiment, the anti-Trop2 single domain antibody includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and / or a multivalent body (multivalent antibody).

[0019] In another preferred example, the anti-Trop2 single-domain antibody comprises one or more VHH chains having an amino acid sequence as shown in SEQ ID NO: 1 or 9.

[0020] In another preferred example, the VHH chain sequence of the anti-Trop2 single-domain antibody is as shown in SEQ ID NO: 1 and / or 9.

[0021] In another preferred example, the anti-Trop2 single-domain antibody comprises two VHH chains having the amino acid sequence shown in SEQ ID NO: 1 or 9.

[0022] In another preferred example, the anti-Trop2 single-domain antibody comprises four VHH chains having the amino acid sequence shown in SEQ ID NO: 1 or 9.

[0023] In another preferred embodiment, the two VHH chains having the amino acid sequence shown in SEQ ID NO: 1 or 9 are connected via a connecting peptide.

[0024] In another preferred embodiment, the four VHH chains having the amino acid sequence shown in SEQ ID NO: 1 or 9 are connected via a connecting peptide.

[0025] In another preferred embodiment, the connecting peptide is selected from the following sequences: (G a S b ) x —(G m S n ) y , where a, b, m, n, x, y = 0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (preferably, a = 4 and b = 1, m = 3 and n = 1).

[0026] A fourth aspect of the present invention provides a VHH chain of a humanized anti-Trop2 single-domain antibody, characterized in that the VHH chain is as shown in SEQ ID NO:14.

[0027] The fifth aspect of the present invention provides an anti-Trop2 single-domain antibody, characterized in that the antibody has the VHH chain described in the fourth aspect of the present invention.

[0028] In a sixth aspect, the present invention provides a VHH chain of an anti-serum albumin single-domain antibody, characterized in that the complementarity determining region CDR of the VHH chain includes CDR1 shown in SEQ ID NO: 24, CDR2 shown in any one of SEQ ID NO: 25, 29, 30 or 31, and CDR3 shown in SEQ ID NO: 26.

[0029] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by framework regions FR1, FR2, FR3 and FR4.

[0030] In another preferred example, the complementarity determining region (CDR) of the VHH chain includes CDR1 shown in SEQ ID NO: 24, CDR2 shown in SEQ ID NO: 25, and CDR3 shown in SEQ ID NO: 26.

[0031] In another preferred embodiment, the sequence of the VHH chain is shown in SEQ ID NO: 15.

[0032] In another preferred embodiment, the sequence of the VHH chain is shown in any one of SEQ ID NOs: 34-36.

[0033] In another preferred embodiment, the serum albumin includes human serum albumin.

[0034] The seventh aspect of the present invention provides an anti-serum albumin single-domain antibody, characterized in that the antibody has the VHH chain described in the sixth aspect of the present invention.

[0035] In another preferred embodiment, the VHH chain of the antibody is shown in SEQ ID NO: 15.

[0036] In another preferred embodiment, the sequence of the VHH chain of the antibody is shown in any one of SEQ ID NOs: 34-36.

[0037] In an eighth aspect, the present invention provides an anti-Trop2 single-domain antibody Fc fusion protein, characterized in that the structure of the fusion protein from N-terminus to C-terminus is as shown in Formula Ia or Ib: ALB (Ia); BLA (Ib);

[0038] in,

[0039] A is the anti-Trop2 single domain antibody according to the third or fifth aspect of the present invention;

[0040] B is the Fc fragment of IgG; and

[0041] L is no or flexible joint.

[0042] In another preferred embodiment, the flexible linker is a peptide linker.

[0043] In another preferred embodiment, the peptide linker has 1-50 amino acids, preferably 1-20 amino acids.

[0044] In another preferred embodiment, the IgG Fc fragment includes the human IgG Fc fragment.

[0045] In another preferred embodiment, the peptide linker has a structure of (GGGGS)n, wherein n is a positive integer of 1-5.

[0046] In another preferred embodiment, the IgG Fc fragment includes the human IgG Fc fragment.

[0047] In another preferred embodiment, the IgG Fc fragment is selected from the following group: IgG1, IgG2, IgG3, IgG4 Fc fragment, or a combination thereof.

[0048] In another preferred embodiment, the Fc fragment of IgG is IgG4.

[0049] The ninth aspect of the present invention provides a multispecific antibody, characterized in that the multispecific antibody comprises the CDR region of the anti-Trop2 single-domain antibody VHH chain according to the first aspect of the present invention, the VHH chain of the anti-Trop2 single-domain antibody according to the second or fourth aspect of the present invention, the anti-Trop2 single-domain antibody according to the third or fifth aspect of the present invention, and / or the fusion protein according to the eighth aspect of the present invention.

[0050] In another preferred embodiment, the multispecific antibody includes a bispecific antibody, a trispecific antibody, and the like.

[0051] The tenth aspect of the present invention provides a bivalent antibody, characterized in that the bivalent antibody comprises the CDR region of the VHH chain of the anti-Trop2 single-domain antibody according to the first aspect of the present invention, the VHH chain of the anti-Trop2 single-domain antibody according to the second or fourth aspect of the present invention, the anti-Trop2 single-domain antibody according to the third or fifth aspect of the present invention, or the fusion protein according to the eighth aspect of the present invention.

[0052] As used herein, "diabody" and "bispecific antibody" are used interchangeably.

[0053] In another preferred embodiment, the structure of the bivalent antibody from N-terminus to C-terminus is as shown in Formula IIa or IIb: Ab1-P-Ab2 (IIa); Ab2-P-Ab1 (IIb);

[0054] in,

[0055] Ab1 comprises the CDR region of the VHH chain of the anti-Trop2 single-domain antibody according to the first aspect of the present invention, or the VHH chain of the anti-Trop2 single-domain antibody according to the second or fourth aspect of the present invention, or the anti-Trop2 single-domain antibody according to the third or fifth aspect of the present invention;

[0056] Ab2 is another single domain antibody; and

[0057] P is no or flexible joint.

[0058] In another preferred embodiment, the Ab2 comprises an anti-serum albumin single domain antibody.

[0059] In another preferred embodiment, the Ab2 comprises: an anti-serum albumin single-domain antibody comprising the VHH chain according to the sixth aspect of the present invention, or the anti-serum albumin single-domain antibody according to the seventh aspect of the present invention.

[0060] In another preferred embodiment, the serum albumin includes human serum albumin.

[0061] In another preferred embodiment, the Ab1 has the amino acid sequence shown in SEQ ID NO:9, and Ab2 has the amino acid sequence shown in SEQ ID NO:15.

[0062] In another preferred embodiment, the flexible linker is a peptide linker.

[0063] In another preferred embodiment, the peptide linker has 1-50 amino acids, preferably 1-20 amino acids.

[0064] In another preferred embodiment, the peptide linker has a structure of (GGGGS)n, wherein n is a positive integer of 1-5.

[0065] In another preferred embodiment, the peptide linker is (GGGGS)4.

[0066] In another preferred embodiment, the amino acid sequence of the bivalent antibody is shown in SEQ ID NO: 16.

[0067] The eleventh aspect of the present invention provides a polynucleotide, characterized in that the polynucleotide encodes a protein selected from the following group: the CDR region of the anti-Trop2 single-domain antibody VHH chain of the first aspect of the present invention, the VHH chain of the anti-Trop2 single-domain antibody of the second or fourth aspect of the present invention, the anti-Trop2 single-domain antibody of the third or fifth aspect of the present invention, the fusion protein of the eighth aspect of the present invention, the multispecific antibody of the ninth aspect of the present invention, or the bivalent antibody of the tenth aspect of the present invention.

[0068] In another preferred embodiment, the polynucleotide comprises DNA or RNA.

[0069] In another preferred embodiment, the polynucleotide encodes the bivalent antibody according to the ninth aspect of the present invention and has a nucleotide sequence as shown in SEQ ID NO: 22.

[0070] The twelfth aspect of the present invention provides an expression vector, characterized in that the expression vector contains the polynucleotide described in the eleventh aspect of the present invention.

[0071] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or a combination thereof.

[0072] Preferably, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0073] The thirteenth aspect of the present invention provides a host cell, characterized in that the host cell contains the expression vector described in the twelfth aspect of the present invention, or the polynucleotide described in the eleventh aspect of the present invention is integrated into its genome.

[0074] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0075] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or a combination thereof.

[0076] In another preferred embodiment, the prokaryotic cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or a combination thereof.

[0077] In another preferred embodiment, the eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, or a combination thereof.

[0078] In another preferred embodiment, the eukaryotic cells are selected from the following groups: insect cells such as fall armyworm, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or a combination thereof.

[0079] In another preferred embodiment, the host cell is preferably a mammalian cell, more preferably a HEK293 cell, a CHO cell, a BHK cell, a NSO cell or a COS cell.

[0080] In another preferred embodiment, the host cell is Pichia pastoris.

[0081] A fourteenth aspect of the present invention provides a method for producing an anti-Trop2 single domain antibody or an Fc fusion protein thereof, comprising the steps of:

[0082] (a) culturing the host cell of the thirteenth aspect of the present invention under conditions suitable for producing the single domain antibody or Fc fusion protein thereof, thereby obtaining a culture containing the anti-Trop2 single domain antibody or Fc fusion protein thereof;

[0083] (b) isolating or recovering the anti-Trop2 single domain antibody or Fc fusion protein thereof from the culture; and

[0084] (c) Optionally, purifying and / or modifying the anti-Trop2 single domain antibody or Fc fusion protein thereof obtained in step (b).

[0085] The fifteenth aspect of the present invention provides an immunoconjugate, which comprises:

[0086] (a) the VHH chain of the anti-Trop2 single domain antibody according to the second or fourth aspect of the present invention, the anti-Trop2 single domain antibody according to the third or fifth aspect of the present invention, the fusion protein according to the eighth aspect of the present invention, the multispecific antibody according to the ninth aspect of the present invention, or the bivalent antibody according to the tenth aspect of the present invention; and

[0087] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

[0088] In another preferred embodiment, the radioactive nuclides include:

[0089] (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or

[0090] (ii) therapeutic isotopes selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.

[0091] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0092] In another preferred embodiment, the drug is a cytotoxic drug.

[0093] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or a combination thereof.

[0094] In another preferred embodiment, particularly useful examples of cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or a combination thereof.

[0095] In another preferred embodiment, the toxin is selected from the group consisting of auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansin, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxybenzoate, daptomycin, acetaminophen, chlortetracycline ... anthracnose dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, a Sapaonaria officinalis inhibitor, a glucocorticoid, or a combination thereof.

[0096] In another preferred embodiment, the coupling moiety is a detectable label.

[0097] In another preferred embodiment, the coupling portion is selected from the following group: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)) or any form of nanoparticles.

[0098] In another preferred embodiment, the immunoconjugate contains: a multivalent (such as bivalent or tetravalent) VHH chain of the anti-Trop2 single domain antibody as described in the second or fourth aspect of the present invention, the anti-Trop2 single domain antibody as described in the third or fifth aspect of the present invention, or the fusion protein as described in the eighth aspect of the present invention.

[0099] In another preferred embodiment, the multivalency refers to the presence of multiple repeats of the VHH chain of the anti-Trop2 single domain antibody described in the second or fourth aspect of the present invention, the anti-Trop2 single domain antibody described in the third or fifth aspect of the present invention, or the fusion protein described in the eighth aspect of the present invention in the amino acid sequence of the immunoconjugate.

[0100] In a sixteenth aspect, the present invention provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the structure of the antibody-drug conjugate is shown in Formula III: Ab-(JU)n (III)

[0101] Where,

[0102] Ab is anti-Trop2 antibody;

[0103] U is the drug;

[0104] J is a chemical bond or linker;

[0105] n is a positive integer;

[0106] “-” represents a chemical bond or linker.

[0107] In another preferred embodiment, the Trop2 is human Trop2 or non-human mammal Trop2 (such as camel Trop2).

[0108] In another preferred embodiment, the Ab is an anti-Trop2 antibody or an antibody derived therefrom.

[0109] In another preferred embodiment, the derivative antibody is a modified anti-Trop2 antibody, including but not limited to linking the Trop2 antibody to an Fc fragment, human serum albumin, a polypeptide specifically binding to serum albumin, polyethylene glycol (PEG), to form a bivalent antibody and / or a multivalent antibody.

[0110] In another preferred embodiment, the antibodies include humanized antibodies, camel-derived antibodies, and chimeric antibodies.

[0111] In another preferred embodiment, the Ab comprises the VHH chain of the anti-Trop2 single domain antibody according to the second or fourth aspect of the present invention, the anti-Trop2 single domain antibody according to the third or fifth aspect of the present invention, the fusion protein according to the eighth aspect of the present invention, the multispecific antibody according to the ninth aspect of the present invention, or the bivalent antibody according to the tenth aspect of the present invention.

[0112] In another preferred embodiment, the sequence of the Ab comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence similarity to the amino acid sequence shown in any one of SEQ ID NO: 1, 9, or 14.

[0113] In another preferred example, the sequence of the Ab comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence similarity to the amino acid sequence shown in EQ ID NO: 16.

[0114] In another preferred embodiment, the sequence of the Ab is shown in EQ ID NO:16.

[0115] In another preferred embodiment, the drug is linked to the terminal amino group or side chain amino group of the heavy chain constant region or heavy chain variable domain of the anti-Trop2 antibody.

[0116] In another preferred embodiment, the drug is linked to the C-terminal amino group of the anti-Trop2 antibody.

[0117] In another preferred embodiment, the drug is site-specifically and / or randomly linked to the anti-Trop2 antibody (ie, in Formula III, the U is site-specifically and / or randomly linked to Ab).

[0118] In another preferred embodiment, the U is site-specifically linked to Ab.

[0119] In another preferred embodiment, the J is a linker, which includes CC; (G) n C, wherein n is an integer from 0 to 4; (A) n C, wherein n is an integer from 1 to 4; (G) n CG, wherein n is an integer from 0 to 4; (G) n SC, where n is an integer from 0 to 4; (GGGGS) n C, where n is an integer from 0 to 4; C(GGGGS) n , where n is an integer from 0 to 4; (GGGS) n C, where n is an integer from 0 to 4; C(GGGS) n , where n is an integer from 0 to 4; (GGS) n C, where n is an integer from 0 to 4, C(GGS) n , where n is an integer from 0 to 4; GSCC; CDV; VDC; LPTEG; GGGGCGGGG; (G) n CA, wherein n is an integer from 0 to 4; (A) n CA, wherein n is an integer from 1 to 4; (G) n CGA, wherein n is an integer from 0 to 4; GGGGCGGGGA; MPA-AEEA, Val-Cit-PABC, polyethylene glycol PEG, or a combination thereof.

[0120] In another preferred embodiment, the linker includes a derivative compound of MPA-AEEA, MPA-AEEA-Val-Cit-PABC, or polyethylene glycol PEG, including but not limited to replacement, modification or deletion of one or more groups based on each of them.

[0121] In another preferred embodiment, the linker is a combination of GGC and Val-Cit-PABC.

[0122] In another preferred embodiment, the degree of polymerization of the chemical bond is a positive integer greater than or equal to 1.

[0123] In another preferred embodiment, the drugs include cytotoxic drugs, immunomodulators, enzyme and hormone inhibitors.

[0124] In another preferred embodiment, the drugs include monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), Eribulin, exatecan, maytansine, SN-38, and the like.

[0125] A seventeenth aspect of the present invention provides a pharmaceutical composition comprising:

[0126] (i) the VHH chain of the anti-Trop2 single domain antibody of the second or fourth aspect of the present invention, the anti-Trop2 single domain antibody of the third or fifth aspect of the present invention, or the fusion protein of the eighth aspect of the present invention, the multispecific antibody of the ninth aspect of the present invention, the bivalent antibody of the tenth aspect of the present invention, or the immunoconjugate of the fifteenth aspect of the present invention, and / or the antibody-drug conjugate of the sixteenth aspect of the present invention; and

[0127] (ii) a pharmaceutically acceptable carrier.

[0128] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0129] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug for preventing and / or treating diseases or conditions associated with Trop2.

[0130] In another preferred embodiment, the Trop2-related disease or condition is cancer or tumor.

[0131] In another preferred embodiment, the cancer or tumor includes but is not limited to: gastric cancer, cervical cancer, breast cancer, lung cancer, prostate cancer, colon cancer, endometrial serous papillary carcinoma, ovarian cancer, glioma, medulloblastoma, urothelial carcinoma, head and neck cancer, kidney cancer, Kaposi's sarcoma, pancreatic cancer, etc.

[0132] In an eighteenth aspect, the present invention provides a use of the VHH chain of the anti-Trop2 single domain antibody according to the second or fourth aspect of the present invention, the anti-Trop2 single domain antibody according to the third or fifth aspect of the present invention, or the fusion protein according to the eighth aspect of the present invention, the multispecific antibody according to the ninth aspect of the present invention, the bivalent antibody according to the tenth aspect of the present invention, the immunoconjugate according to the fifteenth aspect of the present invention, or the antibody-drug conjugate according to the sixteenth aspect of the present invention, for preparing:

[0133] (a) a drug for preventing and / or treating a disease or condition associated with Trop2;

[0134] (b) A detection reagent, a detection plate or a detection kit for detecting human Trop2 molecules.

[0135] In another preferred embodiment, the Trop2-related disease or condition is cancer or tumor.

[0136] In another preferred embodiment, the cancer or tumor includes but is not limited to: gastric cancer, cervical cancer, breast cancer, lung cancer, prostate cancer, colon cancer, endometrial serous papillary carcinoma, ovarian cancer, glioma, medulloblastoma, urothelial carcinoma, head and neck cancer, kidney cancer, Kaposi's sarcoma, pancreatic cancer, etc.

[0137] In another preferred embodiment, the detection includes flow cytometry, cell immunofluorescence detection, and ELISA detection.

[0138] In another preferred embodiment, the use is diagnostic and / or non-diagnostic, and / or therapeutic and / or non-therapeutic.

[0139] The nineteenth aspect of the present invention provides a recombinant protein, wherein the recombinant protein has:

[0140] (i) the VHH chain of the second or fourth aspect of the present invention, the anti-Trop2 single domain antibody of the third or fifth aspect of the present invention, the fusion protein of the eighth aspect of the present invention, the multispecific antibody of the ninth aspect of the present invention, and / or the bivalent antibody of the tenth aspect of the present invention; and

[0141] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0142] In another preferred embodiment, the tag sequence includes an Fc tag, an HA tag and a 6His tag.

[0143] In another preferred embodiment, the recombinant protein specifically binds to Trop2 protein.

[0144] The twentieth aspect of the present invention provides a Trop2 protein detection reagent, the detection reagent comprising:

[0145] (i) the VHH chain of the second or fourth aspect of the invention, the single domain antibody of the third or fifth aspect of the invention, the fusion protein of the eighth aspect of the invention, the immunoconjugate of the fifteenth aspect of the invention, or the recombinant protein of the nineteenth aspect of the invention; and

[0146] (ii) a carrier that is acceptable for detection.

[0147] In another preferred embodiment, the conjugated portion of the immunoconjugate is a diagnostic isotope.

[0148] In another preferred embodiment, the assay-acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0149] In another preferred embodiment, the detection reagent is one or more reagents selected from the following group: isotope tracers, contrast agents, flow cytometry detection reagents, cell immunofluorescence detection reagents, nanomagnetic particles and imaging agents.

[0150] In another preferred embodiment, the detection reagent is used for in vivo detection or in vitro detection.

[0151] In another preferred embodiment, the dosage form of the detection reagent is liquid or powder (such as aqueous solution, injection, lyophilized powder, tablet, buccal preparation, inhaler).

[0152] The twenty-first aspect of the present invention provides a kit for detecting Trop2 protein, wherein the kit contains the detection reagent according to the twentieth aspect of the present invention and instructions.

[0153] In another preferred embodiment, the instructions state that the kit is used for non-invasively detecting Trop2 expression in a subject.

[0154] The twenty-second aspect of the present invention provides a method for treating a disease or condition associated with Trop2, the method comprising administering to a subject in need thereof the VHH chain of the second or fourth aspect of the present invention, the single-domain antibody of the third or fifth aspect of the present invention, the fusion protein of the eighth aspect of the present invention, the multispecific antibody of the ninth aspect of the present invention, the bivalent antibody of the tenth aspect of the present invention, the immunoconjugate of the fifteenth aspect of the present invention, and / or the antibody-drug conjugate of the sixteenth aspect of the present invention, or the pharmaceutical composition of the seventeenth aspect of the present invention.

[0155] In another preferred embodiment, the subject includes mammals, such as humans.

[0156] The twenty-third aspect of the present invention provides a method for detecting Trop2 protein in a sample, the method comprising the steps of:

[0157] (1) contacting a sample with the VHH chain of the second or fourth aspect of the present invention, the single domain antibody of the third or fifth aspect of the present invention, the fusion protein of the eighth aspect of the present invention, or the immunoconjugate of the fifteenth aspect of the present invention;

[0158] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of Trop2 protein in the sample.

[0159] In another preferred embodiment, the aspect is an in vitro method.

[0160] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

[0161] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0162] FIG1 is the result of FACS detection of the binding activity of Trop2 single domain antibody to A431 cells.

[0163] FIG2 is the result of FACS detection of the binding activity of humanized Trop2 single domain antibody to A431 cells.

[0164] FIG3 is the result of FACS detection of the binding activity of humanized Trop2 single domain antibody to BxPC3 cells.

[0165] FIG4 is the result of FACS detection of the endocytic activity of humanized Trop2 single domain antibody in BxPC3 cells.

[0166] FIG5 is the result of FACS detection of the binding activity of long-acting humanized Trop2 single-domain antibody-drug conjugates to A431 cells.

[0167] FIG6 is the result of FACS detection of the binding activity of long-acting humanized Trop2 single-domain antibody-drug conjugates to BxPC3 cells.

[0168] Figure 7 shows the results of CCK8 detection of the cytotoxicity of long-acting humanized Trop2 single-domain antibody-drug conjugates against A431 cells.

[0169] FIG8 is the result of CCK8 detection of the killing activity of long-acting humanized Trop2 single-domain antibody-drug conjugate against BxPC3 cells.

[0170] FIG9 shows the anti-tumor activity results of the long-acting humanized Trop2 single-domain antibody-drug conjugate in BxPC3 tumor-bearing mice. DETAILED DESCRIPTION

[0171] After extensive and in-depth research and extensive screening, the inventors unexpectedly discovered a class of anti-Trop2 single-domain antibodies for the first time. Experimental results show that the anti-Trop2 single-domain antibodies of the present invention can specifically recognize Trop2 and have good binding specificity and affinity. The long-acting anti-Trop2 antibodies obtained by combining the anti-Trop2 single-domain antibodies of the present invention with anti-serum albumin single-domain antibodies can be further coupled with drugs to form antibody-drug conjugates, which effectively kill Trop2-expressing tumor cells in vitro and in vivo. On this basis, the present invention was completed.

[0172] the term

[0173] In order to better understand the present invention, the following terms are defined.

[0174] Unless otherwise stated, all singular terms also include the plural, active and past tenses of the terms.

[0175] Unless the context clearly dictates otherwise, the term "about" includes values ​​that are within a standard deviation of the stated value.

[0176] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be understood to have an inclusive sense, rather than an exclusive or exhaustive sense; that is, the sense of "including, but not limited to." Unless otherwise stated, "comprising" includes "consisting of."

[0177] A "subject" or "patient" according to the present invention is an animal, including a human patient, in need of anti-cancer treatment. In certain aspects, the present invention can also be applied in veterinary practice to any mammal or other animal in need of such TROP2-targeted anti-cancer treatment. This may include, for example, non-human primates, canines, felines, porcines, horses, and any other animal for which TROP2-targeted anti-cancer treatment is desired.

[0178] As used herein, the terms “single domain antibody of the present invention,” “single domain antibody of the present invention,” “anti-Trop2 single domain antibody of the present invention,” “Trop2 single domain antibody of the present invention,” “anti-Trop2 single domain antibody,” and “Trop2 single domain antibody” have the same meaning and can be used interchangeably, all referring to single domain antibodies that specifically recognize and bind to Trop2 (including human Trop2).

[0179] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0180] As used herein, the terms "single domain antibody (sdAb)", "VHH", and "nanobody" have the same meaning and are used interchangeably. They refer to the construction of a single domain antibody (VHH) consisting solely of a single heavy chain variable region by cloning the variable region of an antibody heavy chain. This is the smallest fully functional antigen-binding fragment. Typically, antibodies naturally lacking the light chain and heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting solely of a single heavy chain variable region.

[0181] As used herein, the term "variable" refers to certain parts of the variable region in an antibody that are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the entire antibody variable region. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conservative parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-folded configuration, connected by three CDRs that form a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0182] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates formed by the combination of drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules with the antibodies of the present invention or fragments thereof.

[0183] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

[0184] As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0185] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.

[0186] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.

[0187] In the present invention, the terms "antibody of the present invention," "protein of the present invention," or "polypeptide of the present invention" are used interchangeably to refer to polypeptides that specifically bind to the Trop2 protein, such as proteins or polypeptides having a heavy chain variable region. These may or may not contain an initial methionine.

[0188] The present invention also provides other proteins or fusion expression products comprising the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) comprising a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.

[0189] Generally, an antibody's antigen-binding properties are described by three specific regions located in the heavy chain variable region, known as complementarity-determining regions (CDRs). This region is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a loop structure, spatially close to each other through the β-sheet formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antibody's antigen-binding site. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0190] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because they are at least partially involved in antigen binding. Thus, the present invention includes molecules having antibody heavy chain variable regions with CDRs that are 90% or more (preferably 95% or more, and most preferably 98% or more) homologous to the CDRs identified herein.

[0191] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0192] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. A polypeptide fragment, derivative, or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence, or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0193] The antibodies of the present invention refer to polypeptides having Trop2 binding activity and comprising the above-mentioned CDR regions. The term also includes variant forms of polypeptides comprising the above-mentioned CDR regions that have the same function as the antibodies of the present invention. These variant forms include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids with similar or similar properties generally does not change the function of the protein. For another example, adding one or several amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0194] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0195] The present invention also provides other polypeptides, such as fusion proteins comprising single-domain antibodies or fragments thereof. In addition to substantially full-length polypeptides, the present invention also encompasses fragments of the single-domain antibodies of the invention. Typically, the fragment comprises at least about 50 contiguous amino acids of an antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

[0196] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties, compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A.

[0197] Table A

[0198] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies, fragments thereof, or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0199] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0200] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0201] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0202] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0203] Once the relevant sequence is obtained, recombinant methods can be used to obtain it in large quantities. This is typically accomplished by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) referred to in the present invention include biomolecules in isolated form.

[0204] Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into protein sequences of the present invention by chemical synthesis.

[0205] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0206] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 or Sf9; and animal cells such as CHO, COS7, and 293 cells.

[0207] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated using the CaCl2 method, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0208] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0209] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0210] The antibodies of the present invention may be used alone or in combination with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

[0211] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0212] Therapeutic agents that can be combined or coupled with the antibodies of the present invention include but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0213] Anti-Trop2 single domain antibody

[0214] In the present invention, an anti-Trop2 single domain antibody is provided, wherein the anti-Trop2 single domain antibody includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and / or a multivalent body (multivalent antibody).

[0215] In one aspect of the present invention, an anti-Trop2 single-domain antibody is provided, wherein the complementarity determining region (CDR) of the VHH chain of the antibody comprises CDR1 as shown in SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8. In another preferred embodiment, the anti-Trop2 single-domain antibody comprises a VHH chain as shown in SEQ ID NO: 1 or 9.

[0216] In another aspect of the present invention, an anti-Trop2 single-domain antibody is provided. The anti-Trop2 single-domain antibody is a humanized antibody comprising a VHH chain as shown in SEQ ID NO: 14.

[0217] In a preferred embodiment of the present invention, the anti-Trop2 single-domain antibody comprises one, two or more VHH chains having an amino acid sequence as shown in SEQ ID NO: 1 or 9, and / or SEQ ID NO: 14.

[0218] In another preferred example, the anti-Trop2 single-domain antibody comprises two VHH chains having the amino acid sequences shown in SEQ ID NO: 1, 9 and / or 14.

[0219] In another preferred example, the anti-Trop2 single-domain antibody comprises four VHH chains having the amino acid sequences shown in SEQ ID NO: 1, 9 and / or 14.

[0220] In a preferred embodiment of the present invention, two or four VHH chains are connected via a connecting peptide.

[0221] In a preferred embodiment of the present invention, the connecting peptide is selected from the following sequences: (G a S b ) x —(G m S n ) y , where a, b, m, n, x, y = 0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (preferably, a = 4 and b = 1, m = 3 and n = 1).

[0222] Labeled single domain antibodies

[0223] In a preferred embodiment of the present invention, the single domain antibody carries a detectable label. More preferably, the label is selected from the group consisting of an isotope, a colloidal gold label, a colored label, or a fluorescent label.

[0224] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, a single domain antibody against Trop2 is labeled with colloidal gold to obtain a colloidal gold-labeled single domain antibody.

[0225] Anti-serum albumin single domain antibody

[0226] The present invention also provides an anti-serum albumin single-domain antibody, the VHH chain of which comprises the following complementarity determining regions: CDR1 shown in SEQ ID NO: 24, CDR2 shown in any one of SEQ ID NO: 25, 29, 30 or 31, and CDR3 shown in SEQ ID NO: 26.

[0227] In another preferred embodiment, the VHH chain of the antibody comprises CDR1 shown in SEQ ID NO: 24, CDR2 shown in SEQ ID NO: 25, and CDR3 shown in SEQ ID NO: 26; preferably, the amino acid sequence of its VHH chain is shown in SEQ ID NO: 15.

[0228] In another preferred embodiment, the VHH chain of the antibody comprises CDR1 shown in SEQ ID NO: 24, CDR2 shown in SEQ ID NO: 29, and CDR3 shown in SEQ ID NO: 26; preferably, the amino acid sequence of its VHH chain is shown in SEQ ID NO: 34.

[0229] In another preferred embodiment, the VHH chain of the antibody comprises CDR1 shown in SEQ ID NO: 24, CDR2 shown in SEQ ID NO: 30, and CDR3 shown in SEQ ID NO: 26; preferably, the amino acid sequence of its VHH chain is shown in SEQ ID NO: 35.

[0230] In another preferred embodiment, the VHH chain of the antibody comprises CDR1 shown in SEQ ID NO: 24, CDR2 shown in SEQ ID NO: 31, and CDR3 shown in SEQ ID NO: 26; preferably, the amino acid sequence of its VHH chain is shown in SEQ ID NO: 36.

[0231] bivalent antibodies

[0232] The present invention also provides a bivalent antibody comprising the anti-Trop2 single-domain antibody of the present invention, wherein the structure of the bivalent antibody from N-terminus to C-terminus is as shown in Formula IIa or IIb: Ab1-P-Ab2 (IIa); Ab2-P-Ab1 (IIb);

[0233] in,

[0234] Ab1 is the anti-Trop2 single-domain antibody of the present invention;

[0235] Ab2 is another single domain antibody; and

[0236] P is no or flexible joint.

[0237] In one embodiment of the present invention, Ab2 is an anti-serum albumin single-domain antibody, which is connected in series with the anti-Trop2 single-domain antibody of the present invention to form the long-acting anti-Trop2 single-domain antibody of the present invention. In a preferred embodiment of the present invention, the structure of the bivalent antibody from N-terminus to C-terminus is as shown in Formula IIa, wherein Ab1 is the anti-Trop2 single-domain antibody of the third or fifth aspect of the present invention, and Ab2 is the anti-serum albumin single-domain antibody of the seventh aspect of the present invention; preferably, Ab1 has the amino acid sequence shown in SEQ ID NO: 9, and Ab2 has the amino acid sequence shown in SEQ ID NO: 15, P is a peptide linker having a structure of (GGGGS)n, wherein n is a positive integer of 1-5.

[0238] Antibody-drug conjugates (ADCs)

[0239] The present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.

[0240] The term antibody-drug conjugate (ADC) refers to a monoclonal antibody or antibody fragment connected to a biologically active toxic drug via a linker. The antibodies or antibody fragments described in the present disclosure can be coupled to effector molecules by any means. For example, the antibody or antibody fragment can be attached to the toxic drug by chemical or recombinant means. Chemical methods for preparing fusions or conjugates are known in the art. The method for coupling the antibody or antibody fragment and the drug must be able to connect the antibody and the toxic drug without interfering with the ability of the antibody or antibody fragment to bind to the target molecule.

[0241] The drug can be any cytotoxic, cell growth inhibiting or immunosuppressive drug, such as. In an embodiment, a linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug can have an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, or a ketone group that can form a bond with the linker. In the case where the drug is directly connected to the linker, the drug has a reactive group that reacts before being connected to the antibody. Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc.

[0242] Cytotoxic drugs are substances that inhibit or prevent cell function and / or cause cell death or destruction. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations. However, due to their lack of specificity, they can also cause apoptosis of normal cells while killing tumor cells, leading to serious side effects. Cytotoxic drugs include toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes (such as At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 and radioactive isotopes of Lu), chemotherapeutic drugs, antibiotics, and nucleolytic enzymes.

[0243] The antibody of the present invention and the cytotoxic drug can be coupled via a coupling agent. Examples of the coupling agent can include any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent utilizing a carboxyl group can include any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (where the coupling site is an acylhydrazone).

[0244] Certain residues on antibodies (such as Cys or Lys, etc.) can be used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection reagents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0245] Antibodies can be coupled to drugs to form antibody-drug conjugates (ADCs). Typically, ADCs contain a linker (or linker) located between the drug and the antibody. The term "linker unit" or "linker fragment" or "linker unit" refers to a chemical structure fragment or bond that is connected to an antibody or its antigen-binding fragment at one end and to a drug at the other end, and can also be connected to other linkers before being connected to the drug. The linker can be a degradable or non-degradable linker. Degradable linkers are typically easily degraded in the intracellular environment, for example, the linker degrades at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including linkers containing peptide groups that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as linkers containing glucuronides that can be degraded by glucuronidases. Peptide linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine; or tripeptides such as glycine-phenylalanine-glycine; or tetrapeptides such as glycine-glycine-phenylalanine-glycine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0246] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved via the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethylketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, where the counter ion is acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. Linkers may include, for example, maleimides attached to the antibody via thiosuccinimide.

[0247] In the present invention, drug-linker compounds can be used to form ADCs in a single step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step process. For example, a cysteine ​​residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, the functional group on the linker reacts with the drug to form an ADC.

[0248] Typically, the functional group on the linker is selected to facilitate specific reaction with an appropriate reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that, when a complementary pair of reactive functional groups is selected for selective reaction between the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0249] The present invention also provides a method for preparing an ADC, which may further comprise: combining an antibody with a drug-linker compound (or a drug-linker compound (LD), such as LD-1 to LD-17 shown in the present invention) under conditions sufficient to form an antibody conjugate (ADC).

[0250] In certain embodiments, the methods of the present invention comprise conjugating an antibody to a linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the present invention further comprise conjugating the antibody-linker conjugate to a drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody via the linker.

[0251] Drug loading, also known as drug-to-antibody ratio (DAR), is the average number of drugs coupled to each antibody in the ADC. It can be, for example, in the range of about 1 to about 10 drugs coupled to each antibody, and in certain embodiments, in the range of about 1 to about 8 drugs coupled to each antibody, preferably in the range of 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 5-6, 5-7, 5-8 and 6-8. Exemplary, the drug loading can be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The ADC formula disclosed herein includes a collection of antibody drug conjugates within the aforementioned range. In embodiments disclosed herein, the drug loading can be expressed as n, which is a decimal or integer. Conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays and HPLC can be used to determine the drug loading.

[0252] In one embodiment of the present disclosure, the cytotoxic drug is coupled to the antibody via a linker unit.

[0253] The loading capacity of the ligand drug conjugate can be controlled by the following non-limiting methods, including:

[0254] (1) Control the molar ratio of drug linker fragment and monoclonal antibody,

[0255] (2) Control reaction time and temperature,

[0256] (3) Select different reaction reagents.

[0257] Pharmaceutical composition

[0258] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above-mentioned antibody or active fragment thereof, or fusion protein thereof, or immunoconjugate thereof, and a pharmaceutically acceptable carrier. Typically, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intraperitoneal, intravenous, or topical administration.

[0259] The pharmaceutical composition of the present invention can be used to directly bind to Trop2 protein molecules, and thus can be used to treat Trop2-related diseases, such as cancer, etc. In addition, other therapeutic agents can also be used simultaneously.

[0260] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0261] In one embodiment of the present invention, when using a pharmaceutical composition, a safe and effective amount of the immunoconjugate of the present invention is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight, preferably the dose is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0262] Reagent test kit

[0263] The present invention also provides a kit containing the antibody (or fragment thereof) or detection reagent of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc.

[0264] The present invention also provides a detection kit for detecting Trop2 levels, which includes an antibody that recognizes the Trop2 protein, a lysis medium for dissolving the sample, and common reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. The detection kit can be an in vitro diagnostic device.

[0265] Detection method

[0266] The present invention also relates to a method for detecting Trop2 protein. The method generally comprises the following steps: obtaining a cell and / or tissue sample; dissolving the sample in a medium; and detecting the level of Trop2 protein in the dissolved sample.

[0267] In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a sample containing cells in a cell storage medium.

[0268] application

[0269] As described above, the single-domain antibodies of the present invention have broad biological and clinical applications, encompassing a variety of fields, including the diagnosis and treatment of Trop2-related diseases, basic medical research, and biological research. A preferred application is in the clinical diagnosis and targeted therapy of Trop2, such as the treatment and diagnosis of Trop2-overexpressing cancers or tumors (including, but not limited to, gastric cancer, cervical cancer, breast cancer, lung cancer, prostate cancer, colon cancer, endometrial serous papillary carcinoma, ovarian cancer, mitochondria, medulloblastoma, urothelial carcinoma, head and neck cancer, renal cancer, Kaposi's sarcoma, and pancreatic cancer).

[0270] The main advantages of the present invention include:

[0271] (1) The Trop2 single-domain antibody of the present invention has good cell binding activity.

[0272] (2) The Trop2 single-domain antibody of the present invention has good endocytic activity.

[0273] (3) The anti-serum albumin single-domain antibody provided by the present invention has good binding activity and high expression yield, and has higher purity and better quality uniformity after one-step purification, which is conducive to drug development.

[0274] (4) The Trop2 single-domain antibody of the present invention can be used to construct a long-acting Trop2 single-domain antibody-drug conjugate. The long-acting Trop2 single-domain antibody-drug conjugate has good binding activity with both A431 cells and BxPC3 cells and is not affected by the presence of HSA protein. Moreover, the long-acting Trop2 single-domain antibody-drug conjugate has a significant killing effect on A431 cells and BxPC3 cells. It has significant BxPC3 tumor killing activity, and the tumor in the high-dose group completely regressed.

[0275] The following specific examples further illustrate the present invention. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions, such as those described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (3rd Edition) (2001) CSHL Press), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0276] Sequences of the present invention:

[0277] Amino acid sequence (wavy lines represent CDR regions, and underlined lines represent linkers or connectors)

[0278] Trop2 Nb1 (SEQ ID NO: 1)

[0279] Trop2 Nb1 FR1 (SEQ ID NO: 2)

[0280] Trop2 Nb1 FR2 (SEQ ID NO: 3)

[0281] Trop2 Nb1 FR3 (SEQ ID NO:4)

[0282] Trop2 Nb1 FR4 (SEQ ID NO: 5)

[0283] Trop2 Nb1 CDR1(SEQ ID NO:6)

[0284] Trop2 Nb1 CDR2(SEQ ID NO:7)

[0285] Trop2 Nb1 CDR3(SEQ ID NO:8)

[0286] Trop2 HuNb1(SEQ ID NO:9)

[0287] Trop2 HuNb1 FR1(SEQ ID NO:10)

[0288] Trop2 HuNb1 FR2(SEQ ID NO:11)

[0289] Trop2 HuNb1 FR3(SEQ ID NO:12)

[0290] Trop2 HuNb1 FR4(SEQ ID NO:13)

[0291] Trop2 HuNb2(SEQ ID NO:14)

[0292] Alb Nb1(SEQ ID NO:15)

[0293] Alb Nb1 CDR1(SEQ ID NO:24)

[0294] Alb Nb1 CDR2(SEQ ID NO:25)

[0295] Alb Nb1 CDR3(SEQ ID NO:26)

[0296] HuNb1‐Alb Nb1(SEQ ID NO:16)

[0297] HuNb1‐Alb Nb1‐GGC(SEQ ID NO:17)

[0298] base sequence

[0299] Trop2 HuNb1 (SEQ ID NO: 19)

[0300] Trop2 HuNb2 (SEQ ID NO: 20)

[0301] Alb Nb1 (SEQ ID NO: 21)

[0302] HuNb1-Alb Nb1 (SEQ ID NO: 22)

[0303] HuNb1-Alb Nb1-GGC (SEQ ID NO:23)

[0304] Example 1: Screening and identification of single-domain antibodies targeting Trop2

[0305] A Xinjiang Bactrian camel was immunized with highly purified hTrop2(ECD)-Fc protein. After seven immunizations, total RNA was isolated from the camel's peripheral blood. VHH genes were amplified by reverse transcription and PCR. These VHH genes were cloned into the phage vector pMECS and transformed into TG1 host cells to construct a phage-displayed single-domain antibody library. After three to four rounds of "bind-wash-elute" screening, Trop2-specific phages were obtained. Six hundred clones were randomly selected from these clones for PE-ELISA analysis. All positive clones were sequenced and purified. The candidate single-domain antibodies were then expressed and purified from E. coli strains.

[0306] Example 2: Binding activity of candidate Trop2 antibodies to A431 cells

[0307] Flow cytometry was used to test the binding activity of the candidate single-domain antibodies against the Trop2-high-expressing cell line A431. Cultured A431 cells were trypsinized and neutralized with complete medium. The cells were washed once with PBS, harvested, counted, and plated into a 96V well plate at 2E5 cells / well. Diluted candidate single-domain antibodies and control antibodies BMK1, BMK2, and BMK3 (control antibody sequences are described in patent CN111518212B, where the BMK1 sequence is the amino acid sequence set forth in SEQ ID NO:78, the BMK2 sequence is the amino acid sequence set forth in SEQ ID NO:65, and the BMK3 sequence is the amino acid sequence set forth in SEQ ID NO:68) were added and incubated at 4°C for 40 minutes. After centrifugation, the cells were washed twice with PBS and then incubated with an APC anti-HA antibody at 4°C for 40 minutes. After centrifugation, the cells were washed twice with PBS, the supernatant removed, and the cells resuspended in 200 μL / well of PBS. The APC signal in each sample was measured by flow cytometry.

[0308] The results are shown in Figure 1 , showing that the candidate antibody Nb1 has a very significant advantage in A431 cell binding activity compared to the other three control antibodies.

[0309] Example 3: Humanization of Trop2 single-domain antibody

[0310] The candidate antibody Nb1 was humanized. The humanization method is described in Example 4 of patent CN2018101517526. The sequence of the modified antibody is shown in SEQ ID NO: 9 (labeled as HuNb1).

[0311] In addition, the amino acid sequence shown in SEQ ID NO: 47 in patent CN111518212B was rehumanized, and the modified antibody sequence was shown in SEQ ID NO: 14 (labeled as HuNb2). The binding activity of the humanized antibody was again detected by flow cytometry using the same detection method as in Example 2. As shown in Figure 2, the humanized Trop2 single domain antibody HuNb2 has better cell binding activity than the original humanized antibody (SEQ ID NO: 78 in CN111518212B, i.e., BMK1 in Figure 2).

[0312] Example 4: Binding activity of humanized Trop2 single domain antibody to BxPC3 cells

[0313] Flow cytometry was used to detect the binding activity of the prepared humanized Trop2 antibody to the Trop2-overexpressing cell line BxPC3. The detection method was the same as that described in Example 2. Cultured BxPC3 cells were incubated with the diluted antibody at 4°C for 40 minutes. After washing the cells with PBS, an APC anti-HA antibody was added and incubated at 4°C for 40 minutes. After centrifugation, the cells were washed and the supernatant removed. The cells were resuspended in PBS and the APC signal in each sample was measured by flow cytometry.

[0314] As shown in FIG3 , the humanized Trop2 single domain antibodies HuNb1 and HuNb2 had good BxPC3 cell binding activity.

[0315] Example 5: Identification of endocytic activity of humanized Trop2 single domain antibody

[0316] Cultured A431 and BXPC-3 cells were harvested, counted, and aliquoted into U-shaped plates. Diluted single-domain antibodies were added to the cells and incubated at 4°C for 30 minutes. After washing with PBS, the cells were added with an APC anti-HA antibody and incubated at 4°C for 30 minutes. After washing, medium containing 1% FBS was added to the non-endocytosis (0 hour) and maximum endocytosis (0 hour) experimental groups, the cells were resuspended, and incubated at 4°C. Simultaneously, medium containing 1% FBS was added to the endocytosis (0.25 hour, 0.5 hour, and 1 hour) experimental groups, and the cells were placed in a 37°C CO2 incubator. Samples were removed at 0.25 hour, 0.5 hour, and 1 hour, and refrigerated at 4°C. Finally, the supernatant was removed by centrifugation, and stripping buffer was added and incubated at 4°C. After washing, the cells were resuspended in PBS, and the APC signal in each sample was measured by flow cytometry.

[0317] The results are shown in FIG4 , showing that both humanized Trop2 single-domain antibodies HuNb1 and HuNb2 have good endocytic activity.

[0318] Example 6: Screening of anti-serum albumin single domain antibodies

[0319] The albumin single-domain antibody sequence HuNb3-11 and its optimized mutant sequence from patent CN2022113943304 were selected to extend the half-life of the Trop2 single-domain antibody. HuNb3-11 and its mutants (CDR2 single amino acid mutation) were expressed in Pichia pastoris. The vector construction method and protein expression method refer to Example 4 of patent CN202110164376.6.

[0320] The HuNb3-11 sequence is shown below, where the underlined parts are CDRs:

[0321] Table 1 Comparison of HuNb3-11 and mutant CDR sequences

[0322] Table 2 Expression yield and binding activity of anti-serum albumin single domain antibodies

[0323] The results showed that the HuNb3-11 mutant expressed in yeast had a higher expression yield, higher purity and better quality uniformity after one-step purification, and maintained its original binding activity (Table 2).

[0324] Example 7: Construction and expression of long-acting Trop2 antibodies and their conjugated drugs

[0325] The candidate Trop2 single-domain antibody HuNb1 and the albumin single-domain antibody (Alb Nb1, SEQ ID NO: 15) were constructed in series to form a bivalent body, giving it a long-lasting function. A linker sequence (such as GG) and a free cysteine ​​(C) were designed at its C-terminus for site-directed coupling of loaded drugs. The amino acid sequence of the constructed long-acting Trop2 single-domain antibody-drug conjugate is shown in SEQ ID NO: 17. The amino acid sequence was cloned into pPICZaA according to the base sequence optimized for Pichia pastoris codons (SEQ ID NO: 23), and then linearized and electroporated into X33 competent cells to construct a stable expression cell line. The long-acting Trop2 antibody HuNb1-Alb Nb1-GGC was then purified from the supernatant of the induced culture of the cell line.

[0326] Using the same method, the candidate Trop2 single-domain antibody HuNb1 was tandemly linked with the albumin single-domain antibodies Alb Nb2 (SEQ ID NO: 34), Alb Nb3 (SEQ ID NO: 35), and Alb Nb4 (SEQ ID NO: 36), respectively, to construct the long-acting Trop2 single-domain antibodies HuNb1-Alb Nb2-GGC, HuNb1-Alb Nb3-GGC, and HuNb1-Alb Nb4-GGC. The yeast expression yields of the four antibodies are shown in Table 3. HuNb1-Alb Nb1-GGC exhibited the highest expression yield.

[0327] Table 3 Yeast expression yield of long-acting Trop2 single domain antibodies

[0328] Based on the above data, the purified long-acting Trop2 antibody HuNb1-Alb Nb1-GGC was reduced with 10mM TCEP at 4°C for 16 hours. The excess TCEP was removed by filtration, and then a 2-fold molar amount of vc-PAB-MMAE (Monomethyl auristatin E) was added and incubated at room temperature for 1 hour. The reaction was terminated by the addition of an appropriate amount of acetylcysteine, and the solution was ultrafiltrated into PBS for activity analysis. The resulting long-acting Trop2 antibody-drug conjugate was designated HuNb1-Alb Nb1-MMAE.

[0329] Example 8: Identification of Cell Binding Activity of Long-Acting Trop2 Antibody-Drug Conjugates

[0330] The constructed long-acting Trop2 antibody-drug conjugate HuNb1-Alb Nb1-MMAE was incubated with A431 and BxPC3 cells, and the binding activity of the antibody-drug conjugate to the cells was detected by flow cytometry. The detection method is the same as that in Example 2. The cultured A431 cells and BxPC3 cells were incubated with the diluted antibody at 4°C for 40 minutes (human albumin was added during the incubation process). After washing the cells with PBS, goat anti-VHH antibody was added and incubated at 4°C for 40 minutes. After washing the cells, Donkey-anti-Goat antibody (Alexa Fluor488) was incubated at 4°C for 40 minutes. After centrifugation, the cells were washed and the supernatant was removed. After resuspending the cells with PBS, the Alexa Fluor488 signal of each sample was detected by flow cytometry.

[0331] The results are shown in Figures 5 and 6 , and the long-acting Trop2 antibody-drug conjugate HuNb1-Alb Nb1-MMAE has good binding activity to both A431 cells and BxPC3 cells, and is not affected by the presence of HSA protein.

[0332] Example 9: Identification of the Cell Killing Activity of Long-Acting Trop2 Antibody-Drug Conjugates

[0333] The long-acting Trop2 single-domain antibody conjugate HuNb1-Alb Nb1-MMAE was serially diluted in cell culture medium and mixed with human serum albumin for 20 minutes. The cells were then incubated with A431 cells and BxPC3 cells, respectively. After 96 hours of incubation at 37°C in a 5% CO2 incubator, the cells were added with the Cell Counting Kit-8 and incubated in a cell culture incubator for 2.5 hours. The data (OD450) was then read using a microplate reader and the IC was calculated. 50 .

[0334] The results are shown in Figures 7 and 8 , showing that the long-acting Trop2 single-domain antibody-drug conjugate HuNb1-Alb Nb1-MMAE has a significant killing effect on both A431 cells and BxPC3 cells.

[0335] Example 10: Identification of the anti-tumor activity of long-acting Trop2 antibody-drug conjugates

[0336] NCG mice were inoculated with BxPC3 pancreatic cancer cells to form tumors. 3 Around 6:00 p.m., mice were divided into groups, with 6 mice in each group. Three dose groups were set up, with intraperitoneal injections of 0.2 mg / kg, 1 mg / kg, and 5 mg / kg of HuNb1-Alb Nb1-MMAE three times a week. The negative control group was intraperitoneally injected with PBS. The positive control group was intraperitoneally injected with 150 mg / kg of Gemcitabine three times a week.

[0337] The results are shown in Figure 9. The long-acting Trop2 antibody-drug conjugate HuNb1-Alb Nb1-MMAE has significant BxPC3 tumor killing activity. The tumor in the high-dose group completely regressed after 20 days of treatment, which is significantly better than the therapeutic effect of Gemcitabine.

[0338] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A VHH chain of an anti-Trop2 single domain antibody, characterized in that The VHH chain includes a framework region FR and a complementarity determining region CDR, and the complementarity determining region CDR of the VHH chain includes CDR1 shown in SEQ ID NO: 6, CDR2 shown in SEQ ID NO: 7, and CDR3 shown in SEQ ID NO:

8.

2. The VHH chain according to claim 1, wherein The amino acid sequence of the VHH chain of the anti-Trop2 single domain antibody is shown in SEQ ID NO: 1 or 9.

3. An anti-Trop2 single domain antibody, characterized in that The single-domain antibody comprises the VHH chain according to claim 1 .

4. A VHH chain of a humanized anti-Trop2 single domain antibody, characterized in that: The amino acid sequence of the VHH chain is shown in SEQ ID NO:

14.

5. An anti-Trop2 single domain antibody, characterized in that The antibody has the VHH chain according to claim 4.

6. A VHH chain of an anti-serum albumin single-domain antibody, characterized in that The complementarity determining region (CDR) of the VHH chain includes CDR1 shown in SEQ ID NO: 24, CDR2 shown in any one of SEQ ID NO: 25, 29, 30 or 31, and CDR3 shown in SEQ ID NO:

26.

7. An anti-serum albumin single domain antibody, characterized in that The antibody has the VHH chain according to claim 6.

8. An anti-Trop2 single domain antibody Fc fusion protein, characterized in that: The structure of the fusion protein from N-terminus to C-terminus is as shown in Formula Ia or Ib: ALB (Ia); BLA (Ib); in, A is the anti-Trop2 single domain antibody according to claim 3 or claim 5; B is the Fc fragment of IgG; and L is no or flexible joint.

9. A multispecific antibody, characterized in that The multispecific antibody comprises the VHH chain of the anti-Trop2 single domain antibody according to claim 1 or 4, the anti-Trop2 single domain antibody according to claim 3 or 5, and / or the fusion protein according to claim 8.

10. A bivalent antibody, characterized in that: The bivalent antibody comprises the VHH chain of the anti-Trop2 single-domain antibody according to claim 1 or 4, the anti-Trop2 single-domain antibody according to claim 3 or 5, or the fusion protein according to claim 8.

11. The bivalent antibody according to claim 10, wherein The structure of the bivalent antibody from N-terminus to C-terminus is as shown in Formula IIa or IIb: Ab1-P-Ab2 (IIa); Ab2-P-Ab1 (IIb); in, Ab1 comprises the VHH chain of the anti-Trop2 single domain antibody according to claim 1 or 4, or the anti-Trop2 single domain antibody according to claim 3 or 5; Ab2 is another single domain antibody, preferably, the Ab2 comprises an anti-serum albumin single domain antibody; more preferably, the Ab2 comprises: an anti-serum albumin single domain antibody comprising the VHH chain of claim 6, or the anti-serum albumin single domain antibody of claim 7; and P is no or flexible joint.

12. The bivalent antibody according to claim 11, wherein The Ab1 has the amino acid sequence shown in SEQ ID NO:9, and Ab2 has the amino acid sequence shown in SEQ ID NO:

15.

13. A polynucleotide, characterized in that The polynucleotide encodes a protein selected from the group consisting of the VHH chain of the anti-Trop2 single domain antibody according to claim 1 or 4, the anti-Trop2 single domain antibody according to claim 3 or 5, the fusion protein according to claim 8, the multispecific antibody according to claim 9, or the bivalent antibody according to claim 10.

14. An expression vector, characterized in that The expression vector contains the polynucleotide according to claim 13.

15. A host cell, characterized in that The host cell contains the expression vector according to claim 14, or the polynucleotide according to claim 13 is integrated into its genome.

16. An immunoconjugate comprising: (a) the VHH chain of the anti-Trop2 single domain antibody according to claim 1 or 4, the anti-Trop2 single domain antibody according to claim 3 or 5, the fusion protein according to claim 8, the multispecific antibody according to claim 9, or the bivalent antibody according to claim 10; and (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

17. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the structure of the antibody-drug conjugate is as shown in Formula III: Ab-(JU)n (III) Where, Ab is an anti-Trop2 antibody, comprising the VHH chain of the anti-Trop2 single-domain antibody according to claim 1 or 4, the anti-Trop2 single-domain antibody according to claim 3 or 5, the fusion protein according to claim 8, the multispecific antibody according to claim 9, or the bivalent antibody according to claim 10; U is the drug; J is a chemical bond or linker; n is a positive integer; "-" represents a chemical bond or linker.

18. A pharmaceutical composition comprising: (i) the VHH chain of the anti-Trop2 single domain antibody according to claim 1 or 4, the anti-Trop2 single domain antibody according to claim 3 or 5, the fusion protein according to claim 8, the multispecific antibody according to claim 9, the bivalent antibody according to claim 10, the immunoconjugate according to claim 16, and / or the antibody-drug conjugate according to claim 17; and (ii) a pharmaceutically acceptable carrier.

19. Use of the VHH chain of the anti-Trop2 single domain antibody according to claim 1 or 4, the anti-Trop2 single domain antibody according to claim 3 or 5, the fusion protein according to claim 8, the multispecific antibody according to claim 9, the bivalent antibody according to claim 10, the immunoconjugate according to claim 16, or the antibody-drug conjugate according to claim 17 for preparing: (a) a drug for preventing and / or treating a disease or condition associated with Trop2; (b) A detection reagent, a detection plate or a detection kit for detecting human Trop2 molecules.

20. A method for treating a disease or condition associated with Trop2, the method comprising administering to a subject in need thereof the VHH chain of the anti-Trop2 single-domain antibody of claim 1 or 4, the anti-Trop2 single-domain antibody of claim 3 or 5, the fusion protein of claim 8, the multispecific antibody of claim 9, the bivalent antibody of claim 10, the immunoconjugate of claim 16, the antibody-drug conjugate of claim 17, or the pharmaceutical composition of claim 18.

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