5t4 nanobody and use thereof

Abstract

Provided are a nanobody targeting 5T4 and a use thereof. Specifically, provided are an anti-5T4 nanobody, and a long-acting anti-5T4 nanobody formed by linking the anti-5T4 nanobody with an anti-serum-albumin nanobody in tandem. Furthermore, the antibody can be used for preparing an antibody-drug conjugate and constructing a chimeric antigen receptor and an engineered immune cell expressing the chimeric antigen receptor. The antibody, the antibody-drug conjugate, and the engineered immune cell can be used for preparing a drug for treating and / or preventing diseases or disorders associated with 5T4, such as solid tumors and malignant hemopathies.

Description

5T4 nanobody and its application Technical Field

[0001] The present invention relates to the field of antibody drugs, and in particular, to a 5T4 nanobody and applications thereof. Background Art

[0002] 5T4 is a highly glycosylated, extracellular, type I, single-pass transmembrane protein consisting of 420 amino acids. Also known as trophoblast glycoprotein, 5T4 is a rapidly internalized cell surface antigen. 5T4 has dual effects, blocking both the canonical Wnt / β-catenin pathway and the non-canonical Wnt signaling pathway. The Wnt signaling pathway is inactive in normal mature cells, but its activity is enhanced in tumor cells. 5T4 inhibits the activation of the canonical Wnt / β-catenin pathway by interacting with LRP6; it also influences the Wnt pathway by activating the non-canonical Wnt signaling pathway. In normal adult tissues, 5T4 is expressed only in a few epithelial cell types, such as the basal stratified squamous epithelium, glandular and ductal epithelium, and retinal secondary neurons. However, 5T4 is highly expressed in a variety of cancer cells, including uterine, colon, gastric, ovarian, oral, prostate, lung, and renal cancers. Therefore, this target is considered an ideal therapeutic target.

[0003] Drug development targeting 5T4 has entered the clinical research stage, including therapeutic vaccines, bispecific antibodies, trispecific antibodies, ADCs, and CAR-NK cells. Among them, Genmab's 5T4 / CD3 bispecific antibody is currently in Phase II clinical trials for the treatment of bladder cancer, esophageal cancer, and non-small cell lung cancer. An ADC developed by Asana Bioscience LLC is currently in Phase I clinical trials for the treatment of various solid tumors. These drugs have been reported to show promising therapeutic potential.

[0004] Nanobodies are a class of small-molecule antibodies derived from camelids and sharks. They are only one-tenth the size of conventional monoclonal antibodies and possess excellent stability and druggability. Furthermore, their simple structure allows for easy modification into multivalent or multifunctional antibodies and can be expressed and produced using a variety of cell systems. Therefore, nanobodies are considered an emerging force in the next generation of antibodies with broad application prospects.

[0005] Based on the existing technology, the present invention prepared a nano-antibody targeting 5T4, which has good functional activity and drugability, laying a good foundation for the subsequent development of different types of drugs.

[0006] Summary of the Invention

[0007] The object of the present invention is to provide a nanobody targeting 5T4 and a drug conjugate thereof, as well as the use of the antibody and the drug conjugate thereof in disease diagnosis and treatment.

[0008] In a first aspect of the present invention, a VHH chain of an anti-5T4 nanobody is provided, wherein the VHH chain comprises a complementarity determining region (CDR) selected from the group consisting of:

[0009] (1) CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3;

[0010] (2) CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, and CDR3 shown in SEQ ID NO: 12; or

[0011] (3) CDR1 shown in SEQ ID NO: 17, CDR2 shown in SEQ ID NO: 18, and CDR3 shown in SEQ ID NO: 19.

[0012] In another preferred embodiment, the VHH chain further comprises a framework region FR.

[0013] In another preferred embodiment, the framework region FR includes camel-derived FR and human-derived FR.

[0014] In another preferred embodiment, the framework region FR is selected from the following group:

[0015] (1) FR1 shown in SEQ ID NO: 4, FR2 shown in SEQ ID NO: 5, FR3 shown in SEQ ID NO: 6, and FR4 shown in SEQ ID NO: 7;

[0016] (2) FR1 shown in SEQ ID NO: 4, FR2 shown in SEQ ID NO: 13, FR3 shown in SEQ ID NO: 14, and FR4 shown in SEQ ID NO: 7; or

[0017] (3) FR1 shown in SEQ ID NO: 20, FR2 shown in SEQ ID NO: 21, FR3 shown in SEQ ID NO: 22, and FR4 shown in SEQ ID NO: 7.

[0018] In another preferred embodiment, the framework region FR is selected from the following group:

[0019] (1) FR1 shown in SEQ ID NO: 25, FR2 shown in SEQ ID NO: 26, FR3 shown in SEQ ID NO: 6, and FR4 shown in SEQ ID NO: 27;

[0020] (2) FR1 shown in SEQ ID NO: 25, FR2 shown in SEQ ID NO: 30, FR3 shown in SEQ ID NO: 14, and FR4 shown in SEQ ID NO: 27; or

[0021] (3) FR1 shown in SEQ ID NO: 33, FR2 shown in SEQ ID NO: 34, FR3 shown in SEQ ID NO: 22, and FR4 shown in SEQ ID NO: 27.

[0022] In another preferred example, the VHH chain of the anti-5T4 Nanobody has an amino acid sequence as shown in any one of SEQ ID NO: 8, 15, 23, 28, 31 or 38.

[0023] The second aspect of the present invention provides an anti-5T4 nanobody, which has the VHH chain as described in the first aspect of the present invention.

[0024] In another preferred embodiment, the anti-5T4 nanobody includes a camel-derived nanobody, a chimeric nanobody or a humanized nanobody.

[0025] In another preferred embodiment, the anti-5T4 nanobody includes monomers, bivalents (bivalent antibodies), tetravalents (tetravalent antibodies), and / or multivalents (multivalent antibodies).

[0026] In another preferred example, the anti-5T4 nanobody comprises one or more VHH chains having an amino acid sequence as shown in SEQ ID NO: 8, 15, 23, 28, 31 or 38.

[0027] In a third aspect of the present invention, an anti-5T4 nanobody Fc fusion protein is provided, wherein the structure of the fusion protein from N-terminus to C-terminus is as shown in Formula Ia or Ib:

[0028] ALB(Ia);

[0029] BLA(Ib);

[0030] in,

[0031] A is the anti-5T4 nanobody as described in the second aspect of the present invention;

[0032] B is the Fc fragment of IgG; and

[0033] L is no or flexible joint.

[0034] In another preferred embodiment, the flexible linker is a peptide linker.

[0035] In another preferred embodiment, the peptide linker has 1-50 amino acids, preferably 1-20 amino acids.

[0036] In another preferred embodiment, the IgG Fc fragment includes the human IgG Fc fragment.

[0037] In another preferred embodiment, the peptide linker has a structure of (GGGGS)n, wherein n is a positive integer of 1-5.

[0038] In another preferred embodiment, the IgG Fc fragment includes the human IgG Fc fragment.

[0039] In another preferred embodiment, the IgG Fc fragment is selected from the following group: IgG1, IgG2, IgG3, IgG4 Fc fragment, or a combination thereof.

[0040] In another preferred embodiment, the Fc fragment of IgG is IgG4.

[0041] The fourth aspect of the present invention provides a multispecific antibody, which comprises the VHH chain of the anti-5T4 nanobody as described in the first aspect of the present invention, the anti-5T4 nanobody as described in the second aspect of the present invention, and / or the anti-5T4 nanobody Fc fusion protein as described in the third aspect of the present invention.

[0042] In another preferred embodiment, the multispecific antibody includes a bispecific antibody, a trispecific antibody, and the like.

[0043] In a fifth aspect of the present invention, a long-acting antibody of an anti-5T4 nanobody is provided, wherein the long-acting antibody comprises the VHH chain of the anti-5T4 nanobody as described in the first aspect of the present invention or the anti-5T4 nanobody as described in the second aspect of the present invention, and an anti-serum albumin nanobody portion.

[0044] In another preferred embodiment, the structure of the long-acting antibody from N-terminus to C-terminus is as shown in Formula IIa or IIb:

[0045] Ab1-P-Ab2(IIa);

[0046] Ab2-P-Ab1(IIb);

[0047] in,

[0048] Ab1 comprises the VHH chain of the anti-5T4 nanobody as described in the first aspect of the present invention, or the anti-5T4 nanobody as described in the second aspect of the present invention;

[0049] Ab2 is an anti-serum albumin nanobody; and

[0050] P is no or flexible joint.

[0051] In another preferred embodiment, the serum albumin includes human serum albumin (HSA).

[0052] In another preferred embodiment, the anti-serum albumin nanobody is selected from: an anti-serum albumin nanobody having an amino acid sequence as shown in SEQ ID NO: 55 or 56.

[0053] In another preferred example, the Ab1 has the amino acid sequence shown in SEQ ID NO: 28, and Ab2 has the amino acid sequence shown in SEQ ID NO: 55.

[0054] In another preferred example, the Ab1 has the amino acid sequence shown in SEQ ID NO: 28, and Ab2 has the amino acid sequence shown in SEQ ID NO: 56.

[0055] In another preferred example, the Ab1 has the amino acid sequence shown in SEQ ID NO: 31, and Ab2 has the amino acid sequence shown in SEQ ID NO: 55.

[0056] In another preferred embodiment, the Ab1 has the amino acid sequence shown in SEQ ID NO: 31, and Ab2 has the amino acid sequence shown in SEQ ID NO: 56.

[0057] In another preferred example, the Ab1 has the amino acid sequence shown in SEQ ID NO: 35, and Ab2 has the amino acid sequence shown in SEQ ID NO: 55.

[0058] In another preferred embodiment, the Ab1 has the amino acid sequence shown in SEQ ID NO: 35, and Ab2 has the amino acid sequence shown in SEQ ID NO: 56.

[0059] In another preferred embodiment, the flexible linker is a peptide linker.

[0060] In another preferred embodiment, the peptide linker has 1-50 amino acids, preferably 1-20 amino acids.

[0061] In another preferred embodiment, the peptide linker has a structure of (GGGGS)n, wherein n is a positive integer of 1-5.

[0062] In another preferred embodiment, the peptide linker is (GGGGS)4, and its amino acid sequence is shown in SEQ ID NO:57.

[0063] In another preferred example, the amino acid sequence of the long-acting antibody is shown in any one of SEQ ID NO: 43, 45, 47, 49, 51 or 53.

[0064] In the sixth aspect of the present invention, a polynucleotide is provided, which encodes a protein selected from the following group: the VHH chain of the anti-5T4 nanobody as described in the first aspect of the present invention, the anti-5T4 nanobody as described in the second aspect of the present invention, the anti-5T4 nanobody Fc fusion protein as described in the third aspect of the present invention, the multispecific antibody as described in the fourth aspect of the present invention, or the long-acting antibody of the anti-5T4 nanobody as described in the fifth aspect of the present invention.

[0065] In another preferred embodiment, the polynucleotide comprises DNA or RNA.

[0066] In another preferred example, the polynucleotide encodes the anti-5T4 nanobody described in the second aspect of the present invention and has a nucleotide sequence as shown in any one of SEQ ID NO: 9, 16, 24, 29, 32 or 36.

[0067] In another preferred example, the polynucleotide encodes the long-acting antibody of the anti-5T4 nanobody described in the fifth aspect of the present invention, and has a nucleotide sequence as shown in any one of SEQ ID NO: 44, 46, 48, 50, 52 or 54.

[0068] The seventh aspect of the present invention provides an expression vector, which contains the polynucleotide as described in the sixth aspect of the present invention.

[0069] 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.

[0070] In another preferred embodiment, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0071] The eighth aspect of the present invention provides a host cell, which contains the expression vector as described in the seventh aspect of the present invention, or the polynucleotide as described in the sixth aspect of the present invention is integrated into its genome.

[0072] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] In another preferred embodiment, the host cell is Pichia pastoris.

[0079] The ninth aspect of the present invention provides a method for producing an anti-5T4 nanobody, or an Fc fusion protein thereof, or a long-acting antibody thereof, comprising the steps of:

[0080] (a) culturing the host cell according to the eighth aspect of the present invention under conditions suitable for protein expression, thereby obtaining a culture containing the anti-5T4 nanobody, or its Fc fusion protein, or its long-acting antibody;

[0081] (b) isolating or recovering the anti-5T4 nanobody, or its Fc fusion protein, or its long-acting antibody from the culture; and

[0082] (c) Optionally, purifying and / or modifying the anti-5T4 nanobody, or its Fc fusion protein, or its long-acting antibody obtained in step (b).

[0083] The tenth aspect of the present invention provides an antibody-drug conjugate, wherein the antibody-drug conjugate comprises:

[0084] (a) an antibody portion selected from the group consisting of: a VHH chain of the anti-5T4 nanobody as described in the first aspect of the invention, an anti-5T4 nanobody as described in the second aspect of the invention, an anti-5T4 nanobody Fc fusion protein as described in the third aspect of the invention, a multispecific antibody as described in the fourth aspect of the invention, or a long-acting antibody of the anti-5T4 nanobody as described in the fifth aspect of the invention; and

[0085] (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.

[0086] In another preferred embodiment, the radioactive nuclide includes: diagnostic isotope and / or therapeutic isotope.

[0087] In another preferred embodiment, the drug is a cytotoxic drug.

[0088] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] In another preferred embodiment, the coupling moiety is a detectable label.

[0093] In another preferred embodiment, the coupling portion is selected from the following group: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products.

[0094] In another preferred embodiment, the structure of the antibody-drug conjugate is as shown in Formula III:

[0095] Ab-(JU)m (III)

[0096] Where,

[0097] Ab is an anti-5T4 antibody, the antibody being selected from the group consisting of the VHH chain of the anti-5T4 nanobody as described in the first aspect of the present invention, the anti-5T4 nanobody as described in the second aspect of the present invention, the anti-5T4 nanobody Fc fusion protein as described in the third aspect of the present invention, the multispecific antibody as described in the fourth aspect of the present invention, or the long-acting antibody of the anti-5T4 nanobody as described in the fifth aspect of the present invention;

[0098] U is the drug;

[0099] J is a chemical bond or linker;

[0100] m is a positive integer;

[0101] “-” represents a chemical bond or a linker.

[0102] 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-5T4 antibody.

[0103] In another preferred embodiment, the drug is linked to the C-terminal amino group of the anti-5T4 antibody.

[0104] In another preferred embodiment, the drug is site-specifically and / or randomly linked to the anti-5T4 antibody (ie, in Formula III, the U is site-specifically and / or randomly linked to Ab).

[0105] In another preferred embodiment, the U is site-specifically linked to Ab.

[0106] 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.

[0107] 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.

[0108] In another preferred embodiment, the linker is a combination of GSC and Val-Cit-PABC.

[0109] In another preferred embodiment, m is a positive integer of 1-10.

[0110] In another preferred embodiment, the drugs include cytotoxic drugs, immunomodulators, enzyme and hormone inhibitors.

[0111] In another preferred embodiment, the drugs include monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), Eribulin, exatecan, maytansine, SN-38, and the like.

[0112] In the eleventh aspect of the present invention, a chimeric antigen receptor (CAR) is provided, the antigen binding domain of the chimeric antigen receptor comprising the VHH chain of the anti-5T4 nanobody described in the first aspect of the present invention, the anti-5T4 nanobody as described in the second aspect of the present invention, the multispecific antibody as described in the fourth aspect of the present invention, or the long-acting antibody of the anti-5T4 nanobody as described in the fifth aspect of the present invention.

[0113] The twelfth aspect of the present invention provides an engineered immune cell, the cell surface expressing the chimeric antigen receptor as described in the eleventh aspect of the present invention.

[0114] In another preferred embodiment, the immune cells include T cells and NK cells.

[0115] In another preferred embodiment, the immune cells are from humans or non-human mammals.

[0116] The thirteenth aspect of the present invention provides a pharmaceutical composition, comprising:

[0117] (i) the VHH chain of the anti-5T4 nanobody as described in the first aspect of the invention, the anti-5T4 nanobody as described in the second aspect of the invention, the anti-5T4 nanobody Fc fusion protein as described in the third aspect of the invention, the multispecific antibody as described in the fourth aspect of the invention, the long-acting antibody of the anti-5T4 nanobody as described in the fifth aspect of the invention, the antibody-drug conjugate as described in the tenth aspect of the invention, and / or the engineered immune cell as described in claim 12; and

[0118] (ii) a pharmaceutically acceptable carrier.

[0119] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0120] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug for preventing and / or treating a disease or condition associated with 5T4.

[0121] In another preferred embodiment, the diseases or conditions associated with 5T4 are solid tumors and hematological malignancies.

[0122] In another preferred embodiment, the solid tumors and hematological malignancies highly express 5T4.

[0123] In another preferred embodiment, the solid tumors include but are not limited to: lung cancer, gastric cancer, ovarian cancer, kidney cancer, colon cancer, uterine cancer, prostate cancer, breast cancer, pancreatic cancer, pancreatic duct adenocarcinoma, oral cancer, bile duct cancer, bladder cancer, bone and soft tissue cancer, brain tumor, esophageal cancer, liver cancer, mesothelioma, malignant melanoma, osteosarcoma, thyroid cancer, rhabdomyosarcoma, skin cancer, gastric adenocarcinoma, glioblastoma, gynecological tumors, head and neck squamous cell carcinoma, soft tissue sarcoma, and urothelial carcinoma.

[0124] In another preferred embodiment, the malignant blood diseases include but are not limited to: acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic eosinophilic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, myelodysplastic syndrome, non-Hodgkin's lymphoma, mixed phenotype acute leukemia, myelofibrosis, essential thrombocythemia, and plasma cell leukemia.

[0125] In the fourteenth aspect of the present invention, there is provided a use of the VHH chain of the anti-5T4 nanobody according to the first aspect of the present invention, the anti-5T4 nanobody according to the second aspect of the present invention, the anti-5T4 nanobody Fc fusion protein according to the third aspect of the present invention, the multispecific antibody according to the fourth aspect of the present invention, the long-acting antibody of the anti-5T4 nanobody according to the fifth aspect of the present invention, the antibody-drug conjugate according to the tenth aspect of the present invention, or the engineered immune cell according to the twelfth aspect of the present invention, for preparing:

[0126] (a) medicaments for preventing and / or treating diseases or conditions associated with 5T4;

[0127] (b) Detection reagents, detection plates or detection kits for detecting 5T4 molecules.

[0128] In another preferred embodiment, the diseases or conditions associated with 5T4 are solid tumors and hematological malignancies.

[0129] In another preferred embodiment, the solid tumors and hematological malignancies highly express 5T4.

[0130] In another preferred embodiment, the solid tumors include but are not limited to: lung cancer, gastric cancer, ovarian cancer, kidney cancer, colon cancer, uterine cancer, prostate cancer, breast cancer, pancreatic cancer, pancreatic duct adenocarcinoma, oral cancer, bile duct cancer, bladder cancer, bone and soft tissue cancer, brain tumor, esophageal cancer, liver cancer, mesothelioma, malignant melanoma, osteosarcoma, thyroid cancer, rhabdomyosarcoma, skin cancer, gastric adenocarcinoma, glioblastoma, gynecological tumors, head and neck squamous cell carcinoma, soft tissue sarcoma, and urothelial carcinoma.

[0131] In another preferred embodiment, the malignant blood diseases include but are not limited to: acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic eosinophilic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, myelodysplastic syndrome, non-Hodgkin's lymphoma, mixed phenotype acute leukemia, myelofibrosis, essential thrombocythemia, and plasma cell leukemia.

[0132] In another preferred embodiment, the detection includes flow cytometry, cell immunofluorescence detection, and ELISA detection.

[0133] In another preferred embodiment, the use is diagnostic and / or non-diagnostic, and / or therapeutic and / or non-therapeutic.

[0134] A fifteenth aspect of the present invention provides a method for detecting 5T4 protein in a sample, the method comprising the steps of:

[0135] (1) contacting the VHH chain of the anti-5T4 nanobody as described in the first aspect of the present invention, the anti-5T4 nanobody as described in the second aspect of the present invention, the anti-5T4 nanobody Fc fusion protein as described in the third aspect of the present invention, or the antibody-drug conjugate as described in the tenth aspect of the present invention;

[0136] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of 5T4 protein in the sample.

[0137] In another preferred embodiment, the aspect is an in vitro method.

[0138] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

[0139] In the sixteenth aspect of the present invention, a method for treating a disease or condition associated with 5T4 is provided, the method comprising administering to a subject in need thereof the VHH chain as described in the first aspect of the present invention, the anti-5T4 nanobody as described in the second aspect of the present invention, the anti-5T4 nanobody Fc fusion protein as described in the third aspect of the present invention, the multispecific antibody as described in the fourth aspect of the present invention, the long-acting antibody of the anti-5T4 nanobody as described in the fifth aspect of the present invention, the antibody-drug conjugate as described in the tenth aspect of the present invention, or the engineered immune cell as described in the twelfth aspect of the present invention, or the pharmaceutical composition as described in the thirteenth aspect of the present invention.

[0140] In another preferred embodiment, the subject includes mammals, such as humans.

[0141] In another preferred embodiment, the diseases or conditions associated with 5T4 are solid tumors and hematological malignancies.

[0142] In another preferred embodiment, the solid tumors and hematological malignancies highly express 5T4.

[0143] In another preferred embodiment, the solid tumors include but are not limited to: lung cancer, gastric cancer, ovarian cancer, kidney cancer, colon cancer, uterine cancer, prostate cancer, breast cancer, pancreatic cancer, pancreatic duct adenocarcinoma, oral cancer, bile duct cancer, bladder cancer, bone and soft tissue cancer, brain tumor, esophageal cancer, liver cancer, mesothelioma, malignant melanoma, osteosarcoma, thyroid cancer, rhabdomyosarcoma, skin cancer, gastric adenocarcinoma, glioblastoma, gynecological tumors, head and neck squamous cell carcinoma, soft tissue sarcoma, and urothelial carcinoma.

[0144] 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

[0145] Figure 1 shows the binding activity of candidate 5T4 nanobodies to 5T4 protein. ELISA results showed that all 10 antibodies had good binding activity.

[0146] Figure 2 shows the endocytic activity of candidate 5T4 nanobodies on A431 cells. Flow cytometry results showed that the three 5T4 nanobodies had good endocytic activity. DETAILED DESCRIPTION

[0147] After extensive and intensive research and extensive screening, the inventors unexpectedly discovered a class of anti-5T4 nanobodies for the first time. Experimental results demonstrate that the anti-5T4 nanobodies of the present invention can specifically recognize the 5T4 protein, exhibiting excellent binding specificity and affinity. Furthermore, the anti-5T4 nanobodies of the present invention exhibit excellent antibody endocytosis activity. The long-acting anti-5T4 nanobodies obtained by combining the anti-5T4 nanobodies of the present invention with anti-serum albumin nanobodies can be further coupled with drugs to form antibody-drug conjugates, effectively killing 5T4-expressing tumor cells in vitro and in vivo. Based on this, the present invention was completed.

[0148] the term

[0149] In order to better understand the present invention, the following terms are defined.

[0150] Unless the context clearly dictates otherwise, the term "about" includes values ​​that are within a standard deviation of the stated value.

[0151] 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."

[0152] 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 5T4-targeted anti-cancer treatment. This may include, for example, non-human primates, canines, felines, porcines, horses, and any other animal for which 5T4-targeted anti-cancer treatment is desired.

[0153] As used herein, the terms "Nanoantibodies of the present invention", "Nanoantibodies of the present invention", "anti-5T4 Nanoantibodies of the present invention", "5T4 Nanoantibodies of the present invention", "anti-5T4 Nanoantibodies", and "5T4 Nanoantibodies" have the same meaning and are used interchangeably, all referring to Nanoantibodies that specifically recognize and bind to the 5T4 protein.

[0154] 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.

[0155] As used herein, the terms "nanobody" and "single domain antibody (sdAb)" have the same meaning and are used interchangeably. They refer to the construction of a single-domain antibody (sdAb) consisting solely of a single heavy chain variable region (VHH) 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 consisting solely of a single heavy chain variable region.

[0156] 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 variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved 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-pleated configuration and are connected by three CDRs that form a connecting loop, and in some cases can form a partial β-pleated 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.

[0157] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

[0158] As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0159] 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 5T4 protein, such as proteins or polypeptides having a heavy chain variable region. These may or may not contain an initial methionine.

[0160] The present invention also provides other proteins or fusion products comprising the antibodies of the present invention. Specifically, the present invention encompasses any protein or protein conjugate and fusion product (i.e., antibody-drug conjugate and fusion 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 an antibody of the present invention, preferably at least 95% homologous.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] The antibodies of the present invention refer to polypeptides that have 5T4 binding activity and include the above-mentioned CDR regions. The term also includes variant forms of polypeptides that have the same function as the antibodies of the present invention and include the above-mentioned CDR regions. 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 more (usually within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids with similar or similar properties generally does not alter the function of the protein. For another example, addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] Table A

[0170] 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.

[0171] 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.

[0172] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0173] 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%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] Anti-5T4 nanobody

[0186] The present invention provides multiple anti-5T4 nanobodies. After extensive screening, the present invention provides multiple specific anti-5T4 nanobodies with high binding affinity. Furthermore, after humanization, corresponding humanized anti-5T4 nanobodies are provided.

[0187] In one embodiment, the anti-5T4 nanobody comprises the CDR1 shown in SEQ ID NO: 1, the CDR2 shown in SEQ ID NO: 2, and the CDR3 shown in SEQ ID NO: 3. In one embodiment, the anti-5T4 nanobody comprises the FR1 shown in SEQ ID NO: 4, the FR2 shown in SEQ ID NO: 5, the FR3 shown in SEQ ID NO: 6, and the FR4 shown in SEQ ID NO: 7; and its VHH chain has the amino acid sequence shown in SEQ ID NO: 8. In one embodiment, the anti-5T4 nanobody comprises the FR1 shown in SEQ ID NO: 25, the FR2 shown in SEQ ID NO: 26, the FR3 shown in SEQ ID NO: 6, and the FR4 shown in SEQ ID NO: 27; and its VHH chain has the amino acid sequence shown in SEQ ID NO: 28.

[0188] In one embodiment, the anti-5T4 nanobody comprises the CDR1 shown in SEQ ID NO: 10, the CDR2 shown in SEQ ID NO: 11, and the CDR3 shown in SEQ ID NO: 12. In one embodiment, the anti-5T4 nanobody comprises the FR1 shown in SEQ ID NO: 4, the FR2 shown in SEQ ID NO: 13, the FR3 shown in SEQ ID NO: 14, and the FR4 shown in SEQ ID NO: 7; and its VHH chain has the amino acid sequence shown in SEQ ID NO: 15. In one embodiment, the anti-5T4 nanobody comprises the FR1 shown in SEQ ID NO: 25, the FR2 shown in SEQ ID NO: 30, the FR3 shown in SEQ ID NO: 14, and the FR4 shown in SEQ ID NO: 27; and its VHH chain has the amino acid sequence shown in SEQ ID NO: 31.

[0189] In one embodiment, the anti-5T4 nanobody comprises the CDR1 shown in SEQ ID NO: 17, the CDR2 shown in SEQ ID NO: 18, and the CDR3 shown in SEQ ID NO: 19. In one embodiment, the anti-5T4 nanobody comprises the FR1 shown in SEQ ID NO: 20, the FR2 shown in SEQ ID NO: 21, the FR3 shown in SEQ ID NO: 22, and the FR4 shown in SEQ ID NO: 7; and its VHH chain has the amino acid sequence shown in SEQ ID NO: 23. In one embodiment, the anti-5T4 nanobody comprises the FR1 shown in SEQ ID NO: 33, the FR2 shown in SEQ ID NO: 34, the FR3 shown in SEQ ID NO: 22, and the FR4 shown in SEQ ID NO: 27; and its VHH chain has the amino acid sequence shown in SEQ ID NO: 38.

[0190] Long-acting antibodies

[0191] The present invention also provides a long-acting anti-5T4 nanobody. As used herein, the terms "long-acting anti-5T4 nanobody," "long-acting 5T4 nanobody," "long-acting anti-5T4 nanobody," and "long-acting anti-5T4 nanobody" are used interchangeably and refer to the long-acting antibody obtained by combining the anti-5T4 nanobody of the present invention with the anti-serum albumin nanobody in series.

[0192] In an embodiment of the present invention, the structure of the long-acting antibody from N-terminus to C-terminus is as shown in Formula IIa or IIb:

[0193] Ab1-P-Ab2(IIa);

[0194] Ab2-P-Ab1(IIb);

[0195] in,

[0196] Ab1 comprises the VHH chain of the anti-5T4 nanobody as described in the first aspect of the present invention, or the anti-5T4 nanobody as described in the second aspect of the present invention;

[0197] Ab2 is an anti-serum albumin nanobody; and

[0198] P is no or flexible joint.

[0199] In a preferred embodiment of the present invention, the structure of the long-acting antibody from N-terminus to C-terminus is as shown in Formula IIa, wherein Ab1 is the anti-5T4 nanobody of the present invention, and Ab2 is an anti-serum albumin nanobody; preferably, Ab1 has an amino acid sequence as shown in any one of SEQ ID NO: 28, 31 or 38, Ab2 has an amino acid sequence as shown in SEQ ID NO: 55 or 56, P is a peptide linker having a structure of (GGGGS)n, wherein n is a positive integer of 1-5.

[0200] Antibody-drug conjugates (ADCs)

[0201] The present invention also provides antibody-drug conjugates (ADCs) based on the antibodies of the present invention (including the Nanobodies and long-acting antibodies of the present invention).

[0202] 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.

[0203] 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.

[0204] 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 、Y90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 and radioactive isotopes of Lu), chemotherapeutic drugs, antibiotics, and nucleolytic enzymes.

[0205] 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).

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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).

[0212] In certain embodiments, the methods of the present invention comprise conjugating the antibody to the 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 the drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody via the linker.

[0213] 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.

[0214] In one embodiment of the present invention, the cytotoxic drug is coupled to the antibody via a linker unit.

[0215] The loading capacity of the ligand drug conjugate can be controlled by the following non-limiting methods, including:

[0216] (1) Control the molar ratio of drug linker fragment and monoclonal antibody,

[0217] (2) Control reaction time and temperature,

[0218] (3) Select different reaction reagents.

[0219] Chimeric Antigen Receptor (CAR)

[0220] The chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes a costimulatory signaling region and a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain including costimulatory molecules. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0221] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a linker can be incorporated. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The linker may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0222] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen binding domain targeting 5T4. When the CAR of the present invention is expressed in T cells, it is possible to perform antigen recognition based on antigen binding specificity. When it binds to its associated antigen, it affects tumor cells, causing them to not grow, be caused to die or be affected in other ways, and causing the patient's tumor load to shrink or be eliminated. The antigen binding domain is preferably fused with one or more intracellular domains from a costimulatory molecule and a ζ chain. For example, the antigen binding domain can be fused with the intracellular domain of a combination of a 4-1BB signaling domain and a CD3ζ signaling domain.

[0223] As used herein, "antigen-binding domain" refers to a Fab fragment, Fab' fragment, F(ab')2 fragment, single Fv (scFv) fragment, or single-domain antibody fragment with antigen-binding activity. Fv antibodies contain the variable region of the heavy chain and the variable region of the light chain, but no constant region, and are the smallest antibody fragments with all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains, and are capable of forming the structure required for antigen binding. The antigen-binding domain is usually scFv (single-chain variable fragment). The size of scFv is generally 1 / 6 of a complete antibody. A single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain. In the present invention, the antigen-binding domain comprises an antibody that specifically recognizes 5T4, including the 5T4 nanobody, 5T4 long-acting nanobody, or multispecific antibody targeting 5T4 described in the present invention.

[0224] For hinge region and transmembrane region (transmembrane domain), CAR can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used. In some examples, a transmembrane domain can be selected, or modified by amino acid replacement to avoid such a domain being bound to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0225] Engineered immune cells

[0226] The engineered immune cells of the present invention express the aforementioned chimeric antigen receptor, including CAR-T cells and CAR-NK cells.

[0227] CAR-T cells have the following advantages over other T-cell-based treatments: (1) The action of CAR-T cells is not restricted by MHC; (2) Since many tumor cells express the same tumor antigens, once the CAR gene construction for a certain tumor antigen is completed, it can be widely used; (3) CAR can utilize both tumor protein antigens and glycolipid non-protein antigens, expanding the target range of tumor antigens; (4) The use of the patient's own cells reduces the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.

[0228] Natural killer (NK) cells are a major type of immune effector cell that protects the body from viral infection and tumor cell invasion through non-antigen-specific pathways. Engineered (genetically modified) NK cells may acquire new functions, including the ability to specifically recognize tumor antigens and possess enhanced anti-tumor cytotoxicity.

[0229] Compared with autologous CAR-T cells, CAR-NK cells have the following advantages: (1) they directly kill tumor cells by releasing perforin and granzymes, but have no killing effect on normal cells in the body; (2) they release very small amounts of cytokines, thereby reducing the risk of cytokine storms; (3) they are extremely easy to expand in vitro and develop into "ready-made" products. Other than that, it is similar to CAR-T cell therapy.

[0230] Pharmaceutical composition

[0231] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment, or its fusion protein, or its antibody-drug conjugate, or an engineered immune cell expressing a chimeric antigen receptor whose antigen-binding domain contains the antibody or its active fragment, and a pharmaceutically acceptable carrier. Generally, 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.

[0232] The pharmaceutical composition of the present invention can be used to directly bind to 5T42 protein molecules, and thus can be used to treat 5T42-related diseases, such as solid tumors and hematological malignancies, etc. In addition, other therapeutic agents can also be used simultaneously.

[0233] 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.

[0234] In one embodiment of the present invention, when using a pharmaceutical composition, a safe and effective amount of an antibody or antibody-drug conjugate or engineered immune cell 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 will also take into account factors such as the route of administration and the patient's health condition, which are all within the skill of a skilled physician.

[0235] Reagent test kit

[0236] 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.

[0237] The present invention also provides a detection kit for detecting 5T4 levels, comprising an antibody that recognizes the 5T4 protein (the antibody described herein), a lysis medium for dissolving the sample, and general reagents and buffers required for detection, such as various buffers, a detection marker, and a detection substrate. The detection kit can be an in vitro diagnostic device.

[0238] Detection method

[0239] The present invention also relates to a method for detecting 5T4 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 5T4 protein in the dissolved sample.

[0240] 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.

[0241] application

[0242] As described above, the single-domain antibody of the present invention has broad biological and clinical application value, and its application involves multiple fields such as diagnosis and treatment of 5T4-related diseases, basic medical research, and biological research. A preferred application is for clinical diagnosis and targeted therapy of 5T4, such as the treatment and diagnosis of solid tumors or hematological malignancies with high expression of 5T4 (including but not limited to lung cancer, gastric cancer, ovarian cancer, kidney cancer, colon cancer, uterine cancer, prostate cancer, breast cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, oral cancer, bile duct cancer, bladder cancer, bone and soft tissue cancer, brain tumor, esophageal cancer, liver cancer, mesothelioma, malignant melanoma, osteosarcoma, thyroid cancer, rhabdomyosarcoma, skin cancer, gastric adenocarcinoma, glioblastoma, gynecological tumors, head and neck squamous cell carcinoma, soft tissue sarcoma, urothelial carcinoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic eosinophilic leukemia, chronic lymphocytic leukemia, Hodgkin's lymphoma, myelodysplastic syndrome, non-Hodgkin's lymphoma, mixed phenotype acute leukemia, myelofibrosis, essential thrombocythemia, plasma cell leukemia, etc.).

[0243] Sequence information of the present invention:

[0244] In the above sequence, the underlined parts are CDR1, CDR2 and CDR3 of the antibody; FR1, FR2, FR3 and FR4 are separated by CDRs.

[0245] Summary of 5T4 nanobody and its long-acting antibody sequence information:

[0246] The main advantages of the present invention include:

[0247] (1) The anti-5T4 nanobody of the present invention has excellent antigen-binding activity and good endocytosis activity.

[0248] (2) The anti-5T4 nanobody of the present invention has high expression yield in host cells, high purity after purification, good quality uniformity, and is conducive to drug development.

[0249] (3) The anti-5T4 nanobody of the present invention is modified to introduce free cysteine ​​for site-specific coupling of other substances, including linker-connected small molecule drugs and isotopes, etc., for the development of imaging products and nuclear medicines.

[0250] (4) The anti-5T4 nanobody of the present invention can be used to construct antibody-drug conjugates, and can be connected in series with anti-serum albumin nanobody to form long-acting antibodies, which can then be used to construct long-acting antibody-drug conjugates.

[0251] 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.

[0252] Example 1: Screening of 5T4 Nanobodies

[0253] Two Xinjiang Bactrian camels were immunized with a highly purified 5T4 extracellular segment protein (SEQ ID NO: 58). After seven immunizations, peripheral blood was collected from the camels and PBMCs were isolated. RNA was then extracted and reverse transcribed into cDNA. VHH gene fragments were then obtained by nested PCR and cloned into a phage vector to construct a phage display library. The library capacity of both libraries reached 5×10 9 CFU, with a fragment insertion rate exceeding 90%. Subsequently, phage display technology was used to select antigen-specific nanobodies, and after four rounds of panning, a more than 10-fold enrichment of specific phages was achieved. Randomly selected clones were identified by PE-ELISA, and positive clones were sequenced to ultimately obtain antibodies from 11 different families.

[0254] Example 2: Binding activity determination of 5T4 nanobody

[0255] The antibodies of the above 11 families were expressed in E. coli cells and purified using nickel columns, and then the binding activity of the candidate antibodies was identified by ELISA.

[0256] The human 5T4 extracellular segment protein was coated on a 96-well ELISA plate at 37°C for 2 hours; after washing with PBST, BSA blocking solution was added and sealed at 37°C for 2 hours; after washing with PBST, a gradient dilution of the 5T4 nanoantibody to be tested was added and incubated at 37°C for 1 hour; after washing with PBST, diluted biotinylated anti-his antibody was added and incubated, followed by diluted SA-HRP; after washing with PBST, a color development solution and a stop solution were added, and the absorbance was detected by a microplate reader.

[0257] The results are shown in Figure 1 , and the 10 candidate nanobodies can effectively bind to the 5T4 protein.

[0258] Example 3: Determination of cell binding activity of 5T4 nanobody

[0259] Flow cytometry was used to detect the binding activity of three 5T4 nanobodies (Nb1-40, Nb2-26 and Nb10-59) with better ELISA binding activity to different 5T4 high-expressing cell lines A431, BxPC3 and A549.

[0260] Cultured A431, BxPC3, and A54 cells were incubated with diluted test antibodies at 4°C for 40 minutes. After washing with PBS, the cells were incubated with an APC anti-HA antibody 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. The results, shown in Table 1, showed that all three candidate nanobodies exhibited good binding activity against various tumor cell types.

[0261] Table 1 Cell binding activity of 5T4 nanobody

[0262] Example 4: Determination of endocytic activity of 5T4 nanobody

[0263] Cultured A431 cells were collected, counted, and aliquoted into U-shaped plates. Diluted 5T4 nanoantibodies were added to the cells and incubated at 4°C for 30 minutes. After washing the cells with PBS, an APC anti-HA antibody was added and incubated at 4°C for 30 minutes. After washing the cells, medium containing 1% FBS was added to the non-endocytosis (0 h) and maximum endocytosis (0 h) experimental groups, the cells were resuspended, and incubated at 4°C. Simultaneously, medium containing 1% FBS was added to the endocytosis (0.5 h, 1 h, and 2 h) experimental groups, and the cells were placed in a 37°C CO2 incubator. Samples were removed at 0.5 h, 1 h, and 2 h, and placed in a 4°C refrigerator. Finally, the supernatant was removed by centrifugation, and stripping buffer was added and incubated at 4°C. After washing the cells, the cells were resuspended in PBS, and the APC signal in each sample was measured by flow cytometry.

[0264] The results are shown in Figure 2 , and all three 5T4 nanobodies (Nb1-40, Nb2-26, and Nb10-59) have good endocytic activity.

[0265] Example 5: Design and expression of humanized 5T4 nanobody

[0266] The three 5T4 nanobodies (Nb1-40, Nb2-26, and Nb10-59) were humanized, with the CDR regions remaining unchanged and only the framework regions of the candidate antibodies being humanized. The sequences of the modified antibodies are shown in Table 2 below.

[0267] The humanized nanobody was then expressed in Pichia pastoris, and its expression and fermentation yield were evaluated.

[0268] Specifically, the amino acid sequence of the humanized nanobody was cloned into the pPICZaA vector according to the base sequence optimized for Pichia pastoris codons. The linearized plasmid was then electroporated into X-33 competent cells and cultured for 3 days on YPD culture plates containing bleomycin resistance. A single clone was picked and induced for 3 days. The expression of the antibody was detected by SDS-PAGE gel and its yield in the 7L fermentor was evaluated. The results showed that all three humanized 5T4 nanobodies had good expression yields in yeast cells.

[0269] Table 2 Expression levels of humanized nanobodies in Pichia pastoris

[0270] The binding activity of the humanized antibodies was assessed. The binding activity of the candidate antibodies to the 5T4 extracellular segment protein was tested using ELISA. Briefly, the 5T4 protein was coated onto an ELISA plate overnight. After blocking with blocking solution, a gradient dilution of the humanized antibody was added and incubated for 1 hour. After washing, a goat anti-VHH antibody was added and incubated for 1 hour. After washing again, an HRP-labeled mouse anti-goat antibody was added and incubated for 1 hour. Finally, a stop solution was added and the OD450 absorbance was read on a microplate reader. The results are shown in Table 3 below. The results show that the humanized nanobodies all maintained good antigen binding activity.

[0271] Table 3 Binding activity of humanized antibodies

[0272] Example 6: Design and expression of long-acting 5T4 nanobody

[0273] Long-acting antibodies with different structures were designed and expressed in Pichia pastoris, and their expression and fermentation yields were evaluated.

[0274] Specifically, the amino acid sequences of long-acting antibodies of varying structures were cloned into the pPICZaA vector using a Pichia pastoris codon-optimized base sequence. The linearized plasmids were then electroporated into X-33 competent cells and cultured for three days on YPD plates containing bleomycin resistance. Single clones were selected and induced for three days. Antibody expression was assessed by SDS-PAGE, and yields were estimated in a 7-L fermentor.

[0275] Table 4 Expression levels of long-acting nanobodies in Pichia pastoris

[0276] The results showed that the constructed long-acting 5T4 nanoantibodies had good expression yields in yeast cells.

[0277] 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-5T4 nanobody, characterized in that The VHH chain comprises a complementarity determining region CDR selected from the group consisting of: (1) CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3; (2) CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, and CDR3 shown in SEQ ID NO: 12; or (3) CDR1 shown in SEQ ID NO: 17, CDR2 shown in SEQ ID NO: 18, and CDR3 shown in SEQ ID NO:

19.

2. The VHH chain of the anti-5T4 nanobody according to claim 1, characterized in that The VHH chain of the anti-5T4 nanobody has an amino acid sequence as shown in any one of SEQ ID NO: 8, 15, 23, 28, 31 or 38.

3. An anti-5T4 nanobody, characterized in that The Nanobody has the VHH chain as claimed in claim 1.

4. An anti-5T4 nanobody 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-5T4 nanobody as claimed in claim 3; B is the Fc fragment of IgG; and L is no or flexible joint.

5. A multispecific antibody, characterized in that: The multispecific antibody comprises the VHH chain of the anti-5T4 nanobody according to claim 1, the anti-5T4 nanobody according to claim 3, and / or the anti-5T4 nanobody Fc fusion protein according to claim 4.

6. A long-acting antibody against 5T4 nanobody, characterized in that: The long-acting antibody comprises the VHH chain of the anti-5T4 nanobody according to claim 1 or the anti-5T4 nanobody according to claim 3, and an anti-serum albumin nanobody portion.

7. The long-acting antibody against 5T4 nanobody according to claim 6, characterized in that, The structure of the long-acting 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-5T4 nanobody as described in claim 1, or the anti-5T4 nanobody as described in claim 3; Ab2 is an anti-serum albumin nanobody; and P is no or flexible joint.

8. The long-acting antibody against 5T4 nanobody according to claim 7, characterized in that, The anti-serum albumin nanobody is selected from: an anti-serum albumin nanobody with an amino acid sequence as shown in SEQ ID NO: 55 or 56.

9. The long-acting antibody against 5T4 nanobody according to claim 7, characterized in that, The flexible linker is a peptide linker, and the peptide linker has a structure of (GGGGS)n, wherein n is a positive integer of 1-5.

10. The long-acting antibody against 5T4 nanobody according to claim 6, characterized in that, The amino acid sequence of the long-acting antibody is shown in any one of SEQ ID NO: 43, 45, 47, 49, 51 or 53.

11. A polynucleotide, characterized in that The polynucleotide encodes a protein selected from the following group: the VHH chain of the anti-5T4 nanobody as described in claim 1, the anti-5T4 nanobody as described in claim 3, the anti-5T4 nanobody Fc fusion protein as described in claim 4, the multispecific antibody as described in claim 5, or the long-acting antibody of the anti-5T4 nanobody as described in claim 6.

12. An expression vector comprising the polynucleotide according to claim 11.

13. A host cell, comprising the expression vector according to claim 12, or the polynucleotide according to claim 11 integrated into its genome.

14. A method for producing an anti-5T4 nanobody, or an Fc fusion protein thereof, or a long-acting antibody thereof, comprising the steps of: (a) culturing the host cell as claimed in claim 13 under conditions suitable for protein expression, thereby obtaining a culture containing the anti-5T4 nanobody, or its Fc fusion protein, or its long-acting antibody; (b) separating or recovering the anti-5T4 nanobody, or its Fc fusion protein, or its long-acting antibody from the culture; and (c) Optionally, purifying and / or modifying the anti-5T4 nanobody, or its Fc fusion protein, or its long-acting antibody obtained in step (b).

15. An antibody-drug conjugate, comprising: (a) an antibody portion selected from the group consisting of: a VHH chain of an anti-5T4 nanobody as described in claim 1, an anti-5T4 nanobody as described in claim 3, an anti-5T4 nanobody Fc fusion protein as described in claim 4, a multispecific antibody as described in claim 5, or a long-acting antibody of an anti-5T4 nanobody as described in claim 6; 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.

16. A chimeric antigen receptor (CAR), the antigen binding domain of which comprises the VHH chain of the anti-5T4 nanobody according to claim 1, the anti-5T4 nanobody according to claim 3, the multispecific antibody according to claim 5, or the long-acting antibody of the anti-5T4 nanobody according to claim 6.

17. An engineered immune cell, the cell surface expressing the chimeric antigen receptor according to claim 16.

18. A pharmaceutical composition comprising: (i) the VHH chain of the anti-5T4 nanobody according to claim 1, the anti-5T4 nanobody according to claim 3, the anti-5T4 nanobody Fc fusion protein according to claim 4, the multispecific antibody according to claim 5, the long-acting antibody of the anti-5T4 nanobody according to claim 6, the antibody-drug conjugate according to claim 15, and / or the engineered immune cell according to claim 17; and (ii) a pharmaceutically acceptable carrier.

19. Use of the VHH chain of the anti-5T4 nanobody according to claim 1, the anti-5T4 nanobody according to claim 3, the anti-5T4 nanobody Fc fusion protein according to claim 4, the multispecific antibody according to claim 5, the long-acting antibody of the anti-5T4 nanobody according to claim 6, the antibody-drug conjugate according to claim 15, or the engineered immune cell according to claim 17, for preparing: (a) a drug for preventing and / or treating a disease or condition associated with 5T4; (b) A detection reagent, a detection plate or a detection kit for detecting 5T4 molecules.

20. A method for detecting 5T4 protein in a sample, the method comprising the steps of: (1) contacting the VHH chain of the anti-5T4 nanobody according to claim 1, the anti-5T4 nanobody according to claim 3, the anti-5T4 nanobody Fc fusion protein according to claim 4, or the antibody-drug conjugate according to claim 15; (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of 5T4 protein in the sample.

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