WO2025082447A1 - 5t4 nanobody and use thereof - Google Patents

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

The present invention relates to the field of antibody drugs, and in particular, to a 5T4 nano antibody and applications thereof.

Background Art

5T4 is an extracellular highly glycosylated type I single-pass transmembrane protein containing 420 amino acids. It is also called trophoblast glycoprotein and is a rapidly internalized cell surface antigen. 5T4 has the dual effect of blocking the canonical Wnt/β-catenin pathway and the non-canonical Wnt signaling pathway. The Wnt signaling pathway is in a closed state in normal mature cells, but its activity is enhanced in tumor cells. On the one hand, 5T4 inhibits the activation and conduction of the Wnt/β-catenin canonical pathway by interacting with LRP6; on the other hand, it affects the Wnt pathway by activating the non-canonical Wnt signaling pathway. In normal adult tissues, 5T4 is only expressed in several epithelial cells, such as basal stratified squamous epithelium, glandular and ductal epithelium, and retinal secondary neurons. However, 5T4 is highly expressed in a variety of cancer cells (such as uterine cancer, colon cancer, gastric cancer, ovarian cancer, oral cancer, prostate cancer, lung cancer or renal cancer tissue). Therefore, this target is considered to be an ideal therapeutic target.

The development of drugs targeting 5T4 has entered the clinical research stage, including therapeutic vaccines, bispecific antibodies, trispecific antibodies, ADC, CAR-NK and other types. Among them, the 5T4/CD3 bispecific antibody developed by Genmab is currently undergoing Phase II clinical trials for the treatment of bladder cancer, esophageal cancer, non-small cell lung cancer, etc. The ADC drug developed by Asana Bioscience LLC is currently undergoing Phase I clinical trials for the treatment of various solid tumors. Such drugs have been reported to have good therapeutic prospects.

Nanobodies are a type of small molecule antibodies derived from camelids and sharks. They are only one-tenth the size of conventional monoclonal antibodies and have good stability and drugability. At the same time, nanobodies have a simple structure and are easy to transform into multivalent or multifunctional antibodies. They can also be expressed and produced using various cell systems. Therefore, nanobodies are considered to be an emerging force in the new generation of antibodies with broad application prospects.

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

Summary of the invention

The object of the present invention is to provide a nano antibody 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.

In a first aspect of the present invention, a VHH chain of an anti-5T4 nanobody is provided, wherein the VHH chain comprises a complementary determining region CDR selected from the following group:

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

(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.

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

In another preferred example, the framework region FR includes camel-derived FR and human-derived FR.

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

(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;

(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

(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.

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

(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;

(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

(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.

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.

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

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

In another preferred embodiment, the anti-5T4 nanobody includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and/or a multivalent body (multivalent antibody).

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.

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 the N-terminus to the C-terminus is as shown in Formula Ia or Ib:

A-L-B(Ia);

B-L-A(Ib);

in,

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

B is the Fc fragment of IgG; and

L is no or flexible joint.

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

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

In another preferred embodiment, the IgG Fc fragment includes the Fc fragment of human IgG.

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

In another preferred embodiment, the IgG Fc fragment includes the Fc fragment of human IgG.

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

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

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.

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

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

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:

Ab1-P-Ab2(IIa);

Ab2-P-Ab1(IIb);

in,

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;

Ab2 is an anti-serum albumin nanobody; and

P is no or flexible joint.

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

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

In another preferred embodiment, the Ab1 has an amino acid sequence as shown in SEQ ID NO: 28, and Ab2 has The amino acid sequence shown in SEQ ID NO:55.

In another preferred example, Ab1 has an amino acid sequence as shown in SEQ ID NO:28, and Ab2 has an amino acid sequence as shown in SEQ ID NO:56.

In another preferred example, Ab1 has an amino acid sequence as shown in SEQ ID NO:31, and Ab2 has an amino acid sequence as shown in SEQ ID NO:55.

In another preferred example, Ab1 has the amino acid sequence shown as SEQ ID NO:31, and Ab2 has the amino acid sequence shown as SEQ ID NO:56.

In another preferred example, Ab1 has an amino acid sequence as shown in SEQ ID NO:35, and Ab2 has an amino acid sequence as shown in SEQ ID NO:55.

In another preferred example, Ab1 has an amino acid sequence as shown in SEQ ID NO:35, and Ab2 has an amino acid sequence as shown in SEQ ID NO:56.

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

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

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

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

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.

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.

In another preferred embodiment, the polynucleotide includes DNA or RNA.

In another preferred embodiment, 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.

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.

The seventh aspect of the present invention provides an expression vector, wherein the expression vector contains the polynucleotide as described in the sixth aspect of the present invention.

In another preferred embodiment, the expression vector is selected from the following group: DNA, RNA, viral vector, plasmid, transposon, other gene transfer system, or a combination thereof.

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

The eighth aspect of the present invention provides a host cell, wherein the host cell 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 the genome.

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

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.

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.

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.

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.

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.

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

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:

(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;

(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).

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

(a) an antibody portion selected from the group consisting of: a VHH chain of an 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 an anti-5T4 nanobody as described in the fifth aspect of the invention; 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.

In another preferred embodiment, the radioactive nuclide includes: diagnostic isotopes and/or therapeutic isotopes.

In another preferred embodiment, the drug is a cytotoxic drug.

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

In another preferred embodiment, the cytotoxic drug is selected from the following group: 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.

In another preferred embodiment, examples of particularly useful 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.

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

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

In another preferred embodiment, the coupling part 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.

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

Ab-(J-U)m      (III)

In the formula,

Ab is an anti-5T4 antibody, and the antibody is 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;

U is the drug;

J is a chemical bond or linker;

m is a positive integer;

“-” is a chemical bond or linker.

In another preferred embodiment, the drug is linked to the heavy chain constant region or heavy chain variable structure of the anti-5T4 antibody The terminal amino group or side chain amino group of the domain.

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

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).

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

In another preferred embodiment, the J is a linker, which includes CC; (G) _n C, wherein n is an integer of 0-4; (A) _n C, wherein n is an integer of 1-4; (G) _n CG, wherein n is an integer of 0-4; (G) _n SC, wherein n is an integer of 0-4; (GGGGS) _n C, wherein n is an integer of 0-4; C(GGGGS) _n , wherein n is an integer of 0-4; (GGGS) _n C, wherein n is an integer of 0-4; C(GGGS) _n , wherein n is an integer of 0-4; (GGS) _n C, wherein n is an integer of 0-4, C(GGS) _n , wherein n is an integer of 0-4; GSCC; CDV; VDC; LPTEG; GGGGCGGGG; (G) _n CA, wherein n is an integer of 0-4; (A) _n CA, wherein n is an integer of 1-4; (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.

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.

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

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

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

In another preferred embodiment, the drug includes monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), Eribulin, exatecan, maytansine, SN-38, etc.

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 described in the second aspect of the present invention, the multispecific antibody described in the fourth aspect of the present invention, or the long-acting antibody of the anti-5T4 nanobody described in the fifth aspect of the present invention.

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.

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

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

The thirteenth aspect of the present invention provides a pharmaceutical composition, which contains:

(i) 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, 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, and/or the engineered immune cell as described in claim 12; and

(ii) a pharmaceutically acceptable carrier.

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

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.

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

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

In another preferred embodiment, the solid tumor includes but is 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.

In another preferred embodiment, the malignant blood disease includes but is 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.

In the fourteenth aspect of the present invention, there is provided a VHH chain of the anti-5T4 nanobody as described in the first aspect of the present invention, an anti-5T4 nanobody as described in the second aspect of the present invention, an anti-5T4 nanobody Fc fusion protein as described in the third aspect of the present invention, a multispecific antibody as described in the fourth aspect of the present invention, a long-acting antibody of the anti-5T4 nanobody as described in the fifth aspect of the present invention, an antibody-drug conjugate as described in the tenth aspect of the present invention, or a use of the engineered immune cell as described in the twelfth aspect of the present invention, 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.

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

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

In another preferred embodiment, the solid tumor includes but is 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.

In another preferred embodiment, the malignant blood disease includes but is 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, bone marrow Fibrosis, essential thrombocythemia, plasma cell leukemia.

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

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

In a fifteenth aspect of the present invention, a method for detecting 5T4 protein in a sample is provided, the method comprising the steps of:

(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;

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

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

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

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.

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

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

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

In another preferred embodiment, the solid tumor includes but is 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.

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 specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the binding activity of candidate 5T4 nanobodies to 5T4 protein. ELISA results show that 10 antibodies The binding activity is good.

Figure 2 shows the endocytic activity of candidate 5T4 nanobodies on A431 cells. The flow cytometry results showed that the endocytic activity of the three 5T4 nanobodies was good.

DETAILED DESCRIPTION

The inventors have conducted extensive and in-depth research and, after a large number of screenings, unexpectedly discovered a class of anti-5T4 nano antibodies for the first time. Experimental results show that the anti-5T4 nano antibodies of the present invention can specifically recognize 5T4 protein and have good binding specificity and affinity. At the same time, the anti-5T4 nano antibodies of the present invention have good antibody endocytosis activity. The long-acting anti-5T4 nano antibodies obtained by using the anti-5T4 nano antibodies of the present invention in series with anti-serum albumin nano antibodies can be further coupled with drugs to form antibody-drug conjugates, which effectively kill tumor cells expressing 5T4 in vivo and in vitro. On this basis, the present invention has been completed.

the term

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

Unless the context clearly dictates otherwise, the term "about" includes values within a standard deviation of the stated value.

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 specified, "comprising" includes "consisting of".

"Subjects" or "patients" according to the present invention are animals, including human patients in need of anti-cancer treatment. In certain aspects, the present invention can also be applied to any mammal or other animal in veterinary practice that needs such 5T4-targeted anti-cancer treatment. This may include, for example, non-human primates, canines, felines, pigs, horses, and any other animals for which 5T4-targeted anti-cancer treatment is desired.

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 can be used interchangeably, all referring to Nanoantibodies that specifically recognize and bind to 5T4 protein.

As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 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 between the heavy chains of different immunoglobulin isotypes varies. 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 to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

As used herein, the terms "nanobody" and "single domain antibody (sdAb)" have the same meaning and are used interchangeably, referring to cloning the variable region of the antibody heavy chain to construct a single domain antibody consisting of only one heavy chain variable region (VHH), which is the smallest antigen-binding fragment with complete function. After obtaining an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.

As used herein, the term "variable" means that certain parts of the variable region in an antibody 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 complementary determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. 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 roughly 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 together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pp. 647-669 (1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cellular toxicity of the antibody.

As used herein, the term "heavy chain variable region" is used interchangeably with "VH".

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

In the present invention, the terms "antibodies of the present invention", "proteins of the present invention", or "polypeptides of the present invention" are used interchangeably, and all refer to polypeptides that specifically bind to 5T4 protein, such as proteins or polypeptides having a heavy chain variable region. They may or may not contain an initial methionine.

The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., antibody-drug conjugate and fusion expression product) having 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.

Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the complementarity determining region (CDR), which is divided into four framework regions (FR). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

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 binding to antigen. Therefore, the present invention includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

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.

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. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted. A polypeptide having a substitution group in an amino acid residue, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs 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 (such as a leader sequence or a secretory sequence or a sequence or a proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.

The antibody of the present invention refers to a polypeptide having 5T4 binding activity and including the above-mentioned CDR region. The term also includes variant forms of polypeptides having the same function as the antibody of the present invention and including the above-mentioned CDR region. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and/or substitutions, 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, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

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.

The present invention also provides other polypeptides, such as fusion proteins comprising single domain antibodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of single domain antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids 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 amino acid substitution according to Table A.

Table A

The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

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

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

The present invention also relates to polynucleotides that hybridize with 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 can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of denaturing agents during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.

At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The 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 the protein sequence of the present invention by chemical synthesis.

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.

The host cell may be a prokaryotic cell, such as a bacterial cell, or a lower eukaryotic cell, such as a yeast cell; or It is a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.

Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl ₂ method, the steps used are well known in the art. Another method is to use MgCl _2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture 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 by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using 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, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

The antibodies of the invention may be used alone or in combination or conjugated to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

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.

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, etc.; 4. gold nanoparticles/nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

Anti-5T4 Nanobody

The present invention provides multiple strains of anti-5T4 nanobodies. After a large number of screenings, the present invention provides multiple strains of specific anti-5T4 nanobodies with high binding affinity. Further, after humanization transformation, corresponding humanized anti-5T4 nanobodies are provided.

In one embodiment, the anti-5T4 nanobody comprises CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3. In one embodiment, the anti-5T4 nanobody comprises 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; and its VHH chain has the amino acid sequence shown in SEQ ID NO: 8 In one embodiment, the anti-5T4 nanobody comprises 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; and its VHH chain has an amino acid sequence as shown in SEQ ID NO:28.

In one embodiment, the anti-5T4 nanobody comprises CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, and CDR3 shown in SEQ ID NO: 12. In one embodiment, the anti-5T4 nanobody comprises 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; and its VHH chain has the amino acid sequence shown in SEQ ID NO: 15. In one embodiment, the anti-5T4 nanobody comprises 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; and its VHH chain has the amino acid sequence shown in SEQ ID NO: 31.

In one embodiment, the anti-5T4 nanobody comprises CDR1 shown in SEQ ID NO:17, CDR2 shown in SEQ ID NO:18, and CDR3 shown in SEQ ID NO:19. In one embodiment, the anti-5T4 nanobody comprises 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; and its VHH chain has the amino acid sequence shown in SEQ ID NO:23. In one embodiment, the anti-5T4 nanobody comprises 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; and its VHH chain has the amino acid sequence shown in SEQ ID NO:38.

Long-acting antibodies

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 antibody of anti-5T4 nanobody" can be used interchangeably, all referring to the long-acting antibody obtained by connecting the anti-5T4 nanobody of the present invention in series with the anti-serum albumin nanobody.

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:

Ab1-P-Ab2(IIa);

Ab2-P-Ab1(IIb);

in,

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;

Ab2 is an anti-serum albumin nanobody; and

P is no or flexible joint.

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, and P is a peptide linker having a structure of (GGGGS)n, wherein n is a positive integer of 1-5.

Antibody-drug conjugates (ADCs)

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).

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 antibody or antibody fragment described in the present disclosure can be coupled to an effector molecule by any means. For example, the antibody or antibody fragment can be attached to the toxic drug by chemical or recombinant means. Chemical means for preparing fusions or conjugates are known in the art. The method for coupling antibodies or antibody fragments and drugs must be able to connect the antibody to the toxic drug without interfering with the ability of the antibody or antibody fragment to bind to the target molecule.

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 keto group that can form a bond with the connector. 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.

Cytotoxic drugs are substances that inhibit or prevent the function of cells and/or cause cell death or destruction. Cytotoxic drugs can kill tumor cells in principle at sufficiently high concentrations, but due to lack of specificity, while killing tumor cells, they can also cause apoptosis of normal cells, leading to serious side effects. Cytotoxic drugs include toxins, such as small molecule toxins or enzyme-active toxins from bacteria, fungi, plants or animals, radioactive isotopes (e.g., 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.

The antibody of the present invention and the cytotoxic drug can be coupled via a coupling agent. Examples of the coupling agent can be 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 be any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (the coupling site is an acylhydrazone).

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 agents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, ADCs contain a linker between the drug and the antibody. The term "linker unit" or "linker fragment" or "linker unit" refers to a chemical structure fragment that is connected to an antibody or its antigen-binding fragment at one end and to a drug at the other end. Or bond, it can also be connected to other linkers and then 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 is degraded at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. 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 (such as linkers that are hydrolyzed when the pH is less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (such as disulfide bond linkers). Non-degradable linkers typically release drugs under conditions where the antibody is hydrolyzed by proteases.

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 through the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimide compounds, halogenated amides (e.g., iodinated, brominated or chlorinated); halogenated esters (e.g., iodinated, brominated or chlorinated); halogenated methyl ketones (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, and the counter ion is acetate, chloride or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, maleimide attached to the antibody via thiosuccinimide.

In the present invention, drug-linker compounds can be used to form ADCs in one simple 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, a functional group on the linker reacts with a drug to form an ADC.

Typically, the functional groups on the linker are selected to react specifically with the appropriate reactive groups on the drug moiety. As a non-limiting example, an azide-based moiety can be used to react specifically with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker by a 1,3-dipolar cycloaddition between the azide and the alkynyl group. 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 attachment 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 of the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

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

In certain embodiments, the methods of the invention comprise: under conditions sufficient to form an antibody-linker conjugate, Conjugating the Antibody to the Linker Compound In these embodiments, the method of the invention further comprises conjugating the antibody-linker conjugate to the drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody via the linker.

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. Exemplarily, the drug loading can be the mean 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 certain range. In the embodiments disclosed herein, the drug loading can be expressed as n, which is a decimal or an integer. The drug loading can be determined by conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA test and HPLC.

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

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

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

(2) Control the reaction time and temperature,

(3) Select different reaction reagents.

Chimeric Antigen Receptor (CAR)

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 a costimulatory molecule. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a joint may be incorporated. As used herein, the term "joint" 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 joint may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

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 tumor cells not to grow, to be prompted to die or otherwise affected, and causes the patient's tumor load to be reduced or 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 an intracellular domain of a combination of a 4-1BB signaling domain and a CD3ζ signaling domain.

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 antibody contains the variable region of the antibody heavy chain and the variable region of the light chain, but no constant region, and is the smallest antibody fragment with all antigen binding sites. In general, Fv The antibody also comprises a polypeptide linker between the VH and VL domains, and is capable of forming a structure required for antigen binding. The antigen binding domain is usually a scFv (single-chain variable fragment). The size of a 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 a multispecific antibody targeting 5T4 described in the present invention.

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 binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

Engineered immune cells

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

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 targeting 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; and (5) CAR-T cells have immune memory function and can survive in the body for a long time.

Natural killer (NK) cells are a major type of immune effector cells that protect 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 have enhanced anti-tumor cytotoxic effects.

Compared with autologous CAR-T cells, CAR-NK cells also have the following advantages, such as: (1) they directly kill tumor cells by releasing perforin and granzyme, 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 very easy to expand in vitro and develop into "ready-made" products. Other than that, it is similar to CAR-T cell therapy.

Pharmaceutical composition

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.

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

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 (or its conjugate) of the present invention 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, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. 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 milligrams/kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

In one embodiment of the present invention, when using a pharmaceutical composition, a safe and effective amount of the antibody or antibody-drug conjugate of the present invention or an engineered immune cell is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases does not exceed about 50 milligrams/kg body weight, preferably the dose is about 10 micrograms/kg body weight to about 10 milligrams/kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

Reagent test kit

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.

The present invention also provides a detection kit for detecting 5T4 levels, which includes an antibody that recognizes 5T4 protein (the antibody described in the present invention), a lysis medium for dissolving the sample, and universal reagents and buffers required for detection, such as various buffers, detection markers, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

Detection Methods

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.

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 solution.

application

As described above, the single domain antibody of the present invention has a wide range of biological application value and clinical application value, and its application involves multiple fields such as diagnosis and treatment of diseases related to 5T4, basic medical research, and biological research. A preferred application is for clinical diagnosis and targeted therapy of 5T4, such as solid tumors or malignant blood diseases 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, Treatment and diagnosis of gastric adenocarcinoma, glioblastoma, gynecological tumors, head and neck squamous cell carcinoma, soft tissue sarcoma, urothelial carcinoma, 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, plasma cell leukemia, etc.

Sequence information of the present invention:

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

Summary of 5T4 nanoantibody and its long-acting antibody sequence information:

The main advantages of the present invention include:

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

(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.

(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.

(4) The anti-5T4 nanobody of the present invention can be used to construct antibody-drug conjugates, and can be tandem with anti-serum albumin nanobody to form a long-acting antibody, which can then be used to construct a long-acting antibody-drug conjugate.

The following specific examples further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions, such as the conditions described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (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.

Example 1: Screening of 5T4 Nanobodies

Two Xinjiang Bactrian camels were immunized with highly purified 5T4 extracellular segment protein (SEQ ID NO: 58). After 7 immunizations, peripheral blood was taken from the camels and PBMC was isolated from them. RNA was then extracted and reverse transcribed into cDNA. VHH gene fragments were obtained by nested PCR and cloned into phage vectors to construct phage display libraries. After identification, the library capacity of both libraries reached 5×10 ⁹ CFU, and the fragment insertion rate was above 90%. Subsequently, phage display technology was used to select antigen-specific nanoantibodies, and after 4 rounds of selection, more than 10 times of specific phage enrichment was obtained. Clones were randomly selected for PE-ELISA identification, and the positive clones were sequenced to finally obtain antibodies from 11 different families.

Example 2: Binding activity determination of 5T4 nanobody

The antibodies of the above 11 families were expressed by E. coli cells and purified by nickel column, and then the binding activity of the candidate antibodies was identified by ELISA.

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, gradient dilutions of the 5T4 nanoantibody to be tested were added and incubated at 37°C for 1 hour; after washing with PBST, diluted biotinylated anti-his antibody was added and incubated, and then diluted SA-HRP was added; after washing with PBST, color development solution and stop solution were added, and the absorbance value was detected by an ELISA instrument.

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

Example 3: Determination of cell binding activity of 5T4 nanobody

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

The cultured A431, BxPC3, and A54 cells were incubated with the diluted test antibodies at 4°C for 40 min. After washing the cells with PBS, APC anti-HA antibody was added and incubated at 4°C for 40 min. After centrifugation, the cells were washed and the supernatant was removed. After resuspending the cells with PBS, the APC signal of each sample was detected by flow cytometry. The results are shown in Table 1. The three candidate nanoantibodies have good binding activity with different types of tumor cells.

Table 1 Cell binding activity of 5T4 nanobody

Example 4: Determination of endocytic activity of 5T4 nanobody

After collecting the cultured A431 cells and counting them, the cells were divided into U-shaped plates; the diluted 5T4 nanoantibody was added to the cells and incubated at 4°C for 30 minutes; the cells were washed with PBS and the APC anti-HA antibody was added and incubated at 4°C for 30 minutes; after washing the cells, the culture medium containing 1% FBS was added to the non-endocytosis (0h) experimental group and the maximum endocytosis (0h) experimental group, and the cells were resuspended and placed at 4°C; at the same time, the culture medium containing 1% FBS was added to the endocytosis (0.5h, 1h and 2h) experimental group, and placed in a 37°C _CO2 incubator; the corresponding samples were taken out at 0.5h, 1h and 2h and placed in a 4°C refrigerator; finally, the supernatant was removed by centrifugation, and the Stripping buffer was added and incubated at 4°C. After washing the cells, PBS was added to resuspend the cells, and the APC signal of each sample was detected by flow cytometry.

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

Example 5: Design and expression of humanized 5T4 nanobody

The above three 5T4 nanobodies (Nb1-40, Nb2-26 and Nb10-59) were humanized, keeping the CDR region unchanged and only humanizing the framework region of the candidate antibodies. The sequences of the modified antibodies are shown in Table 2 below.

The humanized nanobodies were then expressed using Pichia pastoris, and their expression and fermentation yields were evaluated.

Specifically, the amino acid sequence of the humanized nanobody was cloned into the pPICZaA vector according to the base sequence optimized by the Pichia pastoris codon. The linearized plasmid was then electrotransformed into X-33 competent cells and cultured for 3 days in a YPD culture plate containing bleomycin resistance. A single clone was selected and induced for 3 days, and the expression of the antibody was detected by SDS-PAGE gel image and its 7L fermentation tank yield was evaluated. As can be seen from the results, all three humanized 5T4 nanobodies had good expression yields in yeast cells.

Table 2 Expression levels of humanized nanobodies in Pichia pastoris

The above humanized antibodies were identified for binding activity. ELISA was used to detect the binding activity of candidate antibodies to the extracellular segment protein of 5T4. Briefly, the 5T4 protein was coated on the ELISA plate overnight, and after blocking with the blocking solution, gradient dilutions of humanized antibodies were added and incubated for 1 hour; after washing, sheep anti-VHH antibodies were added and incubated for 1 hour; after washing again, HRP-labeled mouse anti-sheep antibodies were added and incubated for 1 hour, and finally the stop solution was added and the OD450 absorbance was read in the ELISA instrument. The results are shown in Table 3 below. The results show that the humanized nanoantibodies all maintain good antigen binding activity.

Table 3 Binding activity of humanized antibodies

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

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

Specifically, the amino acid sequences of long-acting antibodies with different structures were cloned into the pPICZaA vector according to the base sequence optimized by Pichia pastoris codons. The linearized plasmid was then electroporated into X-33 competent cells and cultured for 3 days in a YPD culture plate containing bleomycin resistance. A single clone was selected and induced for 3 days, and the expression of the antibody was detected by SDS-PAGE and its 7L fermentation tank yield was evaluated.

Table 4 Expression of long-acting nanobodies in Pichia pastoris

The results show that the constructed long-acting 5T4 nanoantibodies have good expression yields in yeast cells.

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims (20)

A VHH chain of an anti-5T4 nanobody, characterized in that the VHH chain comprises a complementary determining region CDR selected from the following group: (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. The VHH chain of the anti-5T4 nanobody as described in claim 1 is 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. An anti-5T4 nanobody, characterized in that the nanobody has the VHH chain as claimed in claim 1. 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. 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. A long-acting antibody against 5T4 nanoantibody, characterized in that the long-acting antibody comprises the VHH chain of the anti-5T4 nanoantibody according to claim 1 or the anti-5T4 nanoantibody according to claim 3, and an anti-serum albumin nanoantibody part. 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. The long-acting antibody against 5T4 nanoantibody as described in claim 7 is characterized in that the anti-serum albumin nanoantibody is selected from: an anti-serum albumin nanoantibody with an amino acid sequence as shown in SEQ ID NO: 55 or 56. 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. The long-acting antibody against 5T4 nanobody as described in claim 6 is 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. 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. An expression vector comprising the polynucleotide according to claim 11. A host cell, wherein the host cell contains the expression vector according to claim 12, or the polynucleotide according to claim 11 is integrated into its genome. 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). 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. 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. An engineered immune cell, the cell surface expressing the chimeric antigen receptor according to claim 16. 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. Use of the anti-5T4 nanobody VHH chain of claim 1, the anti-5T4 nanobody of claim 3, the anti-5T4 nanobody Fc fusion protein of claim 4, the multispecific antibody of claim 5, the long-acting antibody of the anti-5T4 nanobody of claim 6, the antibody-drug conjugate of claim 15, or the engineered immune cell of 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. 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.