CN117624366A - 5T4 nanobody and application thereof - Google Patents

Abstract

The invention provides a 5T 4-targeted nanobody and application thereof. Specifically, the invention provides an anti-5T 4 nanobody and a long-acting anti-5T 4 nanobody formed by connecting the anti-5T 4 nanobody with an anti-serum albumin nanobody in series. Further, the antibodies of the invention can be used to prepare antibody-drug conjugates, construct chimeric antigen receptors, and engineered immune cells expressing chimeric antigen receptors. The antibodies, antibody-drug conjugates, and engineered immune cells of the invention are useful in the preparation of a medicament for the treatment and/or prevention of 5T 4-related diseases or disorders, such as solid tumors and hematological malignancies.

Description

5T4 nanobody and application thereof

Technical Field

The invention relates to the field of antibody medicines, in particular to a 5T4 nano antibody and application thereof.

Background

5T4 is an extracellular highly glycosylated type I single-transmembrane protein comprising 420 amino acids. It is also known as trophoblast glycoprotein, a cell surface antigen that internalizes rapidly. 5T4 has the dual effect of blocking both canonical Wnt/beta-catenin pathways and non-canonical Wnt signaling pathways. The Wnt signaling pathway is in a closed state in normally mature cells, but has increased activity in tumor cells, on the one hand 5T4 inhibits activation transduction of the Wnt/β -catenin canonical pathway through interaction with LRP 6; on the other hand it affects the Wnt pathway by activating a non-canonical Wnt signaling pathway. In normal adult tissues, 5T4 is expressed in only a few epithelial cells, such as basal lamina squamous epithelium, glandular and ductal epithelium, and retinal secondary neurons. However, 5T4 is highly expressed in a variety of cancer cells (e.g., uterine, colon, stomach, ovarian, oral, prostate, lung or kidney tissue). Thus, this target is considered to be an ideal therapeutic target.

Drug development targeting 5T4 has entered clinical research stages including therapeutic vaccines, diabodies, triabodies, ADCs, CAR-NK, etc. Among them, the 5T4/CD3 diabody developed by Genmab is being used for treating bladder cancer, esophageal cancer, non-small cell lung cancer, etc., and is being subjected to phase II clinical study. ADC drugs developed by Asana Bioscience LLC are being used to treat a variety of solid tumors in phase I clinical studies. Such drugs have been reported to have good therapeutic prospects.

The nanometer antibody is a kind of small molecular antibody from camelid and shark animals, has the size of only one tenth of that of conventional monoclonal antibody, and has good stability and drug property. Meanwhile, the nano antibody has simple structure, is easy to reform into multivalent or multifunctional antibody, and can be expressed and produced by various cell systems. Therefore, nanobodies are considered as an emerging force of a new generation of antibodies, and have a wide application prospect.

Based on the prior art, the 5T4 targeted nano antibody prepared by the method has good functional activity and drug property, and lays a good foundation for the development of different subsequent medicaments.

Disclosure of Invention

The invention aims to provide a 5T 4-targeted nanobody and a drug conjugate thereof, and application of the antibody and the drug conjugate thereof in disease diagnosis and treatment.

In a first aspect of the invention there is provided a VHH chain of an anti-5T 4 nanobody, the VHH chain comprising complementarity determining regions CDRs 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 (b)

(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 embodiment, the framework region FR comprises camelid FR and human FR.

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

(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 (b)

(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 group consisting of:

(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 (b)

(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 embodiment, the VHH chain of the anti-5T 4 nanobody has an amino acid sequence as set forth in any one of SEQ ID NOs 8, 15, 23, 28, 31 or 38.

In a second aspect of the invention there is provided an anti-5T 4 nanobody having a VHH chain according to the first aspect of the invention.

In another preferred example, the anti-5T 4 nanobody comprises a camelid nanobody, a chimeric nanobody, or a humanized nanobody.

In another preferred example, the anti-5T 4 nanobody comprises a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and/or a multivalent body (multivalent antibody).

In another preferred embodiment, the anti-5T 4 nanobody comprises one or more VHH chains having the amino acid sequence shown as SEQ ID NO. 8, 15, 23, 28, 31 or 38.

In a third aspect of the present invention, there is provided an anti-5T 4 nanobody Fc fusion protein having a structure from N-terminus to C-terminus as shown in formula Ia or Ib:

A-L-B(Ia)；

B-L-A(Ib)；

Wherein,

a is an anti-5T 4 nanobody according to the second aspect of the invention;

b is the Fc fragment of IgG; and

l is a non-or flexible linker.

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

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

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

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

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

In another preferred embodiment, the Fc fragment of IgG is selected from the group consisting of: an Fc fragment of IgG1, igG2, igG3, igG4, or a combination thereof.

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

In a fourth aspect of the invention there is provided a multispecific antibody comprising a VHH chain of an anti-5T 4 nanobody according to the first aspect of the invention, an anti-5T 4 nanobody according to the second aspect of the invention, and/or an anti-5T 4 nanobody Fc fusion protein according to the third aspect of the invention.

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

In a fifth aspect of the invention there is provided a long-acting antibody against a 5T4 nanobody comprising a VHH chain of an anti-5T 4 nanobody according to the first aspect of the invention or an anti-5T 4 nanobody according to the second aspect of the invention, and an anti-serum albumin nanobody moiety.

In another preferred embodiment, the structure of the long-acting antibody from the N-terminus to the C-terminus is represented by formula IIa or IIb:

Ab1-P-Ab2(IIa)；

Ab2-P-Ab1(IIb)；

wherein,

ab1 comprises a VHH chain of an anti-5T 4 nanobody according to the first aspect of the invention, or an anti-5T 4 nanobody according to the second aspect of the invention;

ab2 is an antisera albumin nanobody; and

p is a no or flexible linker.

In another preferred embodiment, the serum albumin comprises Human Serum Albumin (HSA).

In another preferred embodiment, the anti-serum albumin nanobody is selected from the group consisting of: the amino acid sequence of the antiserum albumin nanometer antibody is shown as SEQ ID NO. 55 or 56.

In another preferred embodiment, ab1 has the amino acid sequence shown as SEQ ID NO. 28 and Ab2 has the amino acid sequence shown as SEQ ID NO. 55.

In another preferred embodiment, ab1 has the amino acid sequence shown as SEQ ID NO. 28 and Ab2 has the amino acid sequence shown as SEQ ID NO. 56.

In another preferred embodiment, 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.

In another preferred embodiment, 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.

In another preferred embodiment, ab1 has the amino acid sequence shown as SEQ ID NO. 35 and Ab2 has the amino acid sequence shown as SEQ ID NO. 55.

In another preferred embodiment, 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.

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

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

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

In another preferred embodiment, the peptide linker is (GGGGS) 4, the amino acid sequence of which is shown in SEQ ID NO. 57.

In another preferred embodiment, the amino acid sequence of the long-acting antibody is as set forth in any one of SEQ ID NOs 43, 45, 47, 49, 51 or 53.

In a sixth aspect of the invention, there is provided a polynucleotide encoding a protein selected from the group consisting of: the VHH chain of an anti-5T 4 nanobody according to the first aspect of the invention, the anti-5T 4 nanobody according to the second aspect of the invention, the anti-5T 4 nanobody Fc fusion protein according to the third aspect of the invention, the multispecific antibody according to the fourth aspect of the invention, or the long-acting antibody of an anti-5T 4 nanobody according to the fifth aspect of the invention.

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

In another preferred embodiment, the polynucleotide encodes an anti-5T 4 nanobody according to the second aspect of the invention and has a nucleotide sequence as set forth in any one of SEQ ID NOs 9, 16, 24, 29, 32 or 36.

In another preferred embodiment, the polynucleotide encodes a long-acting antibody against the 5T4 nanobody of the fifth aspect of the invention and has the nucleotide sequence set forth in any one of SEQ ID NOs 44, 46, 48, 50, 52 or 54.

In a seventh aspect of the invention there is provided an expression vector comprising a polynucleotide according to the sixth aspect of the invention.

In another preferred embodiment, the expression vector is selected from the group consisting of: DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

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

In an eighth aspect of the invention there is provided a host cell comprising an expression vector according to the seventh aspect of the invention, or having integrated into its genome a polynucleotide according to the sixth aspect of the invention.

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

In another preferred embodiment, the host cell is selected from the group consisting of: coli, yeast cells, mammalian cells, phage, or combinations thereof.

In another preferred embodiment, the prokaryotic cell is selected from the group consisting of: coli, bacillus subtilis, lactobacillus, streptomyces, proteus mirabilis, or combinations thereof.

In another preferred embodiment, the eukaryotic cell is selected from the group consisting of: pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, trichoderma, or a combination thereof.

In another preferred embodiment, the eukaryotic cell is selected from the group consisting of: insect cells such as myxoplasma gondii, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.

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

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

In a ninth aspect of the present invention, there is provided a method for producing an anti-5T 4 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 invention under conditions suitable for protein expression, thereby obtaining a culture comprising said anti-5T 4 nanobody, or Fc fusion protein thereof, or long-acting antibody thereof;

(b) Isolating or recovering the anti-5T 4 nanobody, or Fc fusion protein thereof, or long-acting antibody thereof from the culture; and

(c) Optionally purifying and/or modifying to obtain the anti-5T 4 nanobody obtained in step (b), or an Fc fusion protein thereof, or a long-acting antibody thereof.

In a tenth aspect of the present invention, there is provided an antibody-drug conjugate comprising:

(a) An antibody moiety selected from the group consisting of: the VHH chain of an anti-5T 4 nanobody according to the first aspect of the invention, the anti-5T 4 nanobody according to the second aspect of the invention, the anti-5T 4 nanobody Fc fusion protein according to the third aspect of the invention, the multispecific antibody according to the fourth aspect of the invention, or the long-acting antibody of an anti-5T 4 nanobody according to the fifth aspect of the invention; and

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

In another preferred embodiment, the radionuclide comprises: isotopes for diagnosis and/or isotopes for therapy.

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 group consisting of: an anti-tubulin drug, a DNA minor groove binding agent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folic acid antagonist, an antimetabolite, a chemosensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.

In another preferred example, 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 (auristatins), camptothecins (camptothecins), duocarmycin/doubly cancerous, etoposides (etoposides), maytansinoids (maytansines) and maytansinoids (maytansinoids) (e.g., DM1 and DM 4), taxanes (taxanes), benzodiazepines (benzodiazepines) or benzodiazepine-containing drugs (benzodiazepine containing drugs) (e.g., pyrrolo [1,4] benzodiazepines (PBDs), indoline benzodiazepines (indoxazepines) and oxazolidinbenzodiazepines (oxybenzodiazepines), vinca alkaloids (vinca), or combinations thereof.

In another preferred embodiment, the toxin is selected from the group consisting of: auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), aureomycin, mestaneol, ricin a-chain, combretastatin, docamicin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide (tenoposide), vincristine, vinblastine, colchicine, dihydroxyanthrax, diketo, actinomycin, diphtheria toxin, pseudomonas Exotoxin (PE) A, PE, abrin a chain, a-chain of jezosin, α -octacocin, gelonin, mitogellin, restrictocin (retproctrocin), phenol, enomycin, curcin, crotonin, calicheamicin, saporin (Sapaonaria officinalis), glucocorticoids, or combinations thereof.

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

In another preferred embodiment, the coupling moiety is selected from the group consisting of: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or enzymes capable of producing a detectable product.

In another preferred embodiment, the antibody-drug conjugate has the structure shown in formula III:

Ab-(J-U)m (III)

in the method, in the process of the invention,

ab is an anti-5T 4 antibody selected from the group consisting of: the VHH chain of an anti-5T 4 nanobody according to the first aspect of the invention, the anti-5T 4 nanobody according to the second aspect of the invention, the anti-5T 4 nanobody Fc fusion protein according to the third aspect of the invention, the multispecific antibody according to the fourth aspect of the invention, or the long-acting antibody of an anti-5T 4 nanobody according to the fifth aspect of the invention;

u is a drug;

j is a bond or linker;

m is a positive integer;

"-" is a bond or a linker.

In another preferred embodiment, the agent is attached to the terminal amino group or side chain amino group of the heavy chain constant region or heavy chain variable domain of the anti-5T 4 antibody.

In another preferred embodiment, the drug is linked to the C-terminal amino group of the anti-5T 4 antibody.

In another preferred embodiment, the drug is site-directed and/or randomly linked to the anti-5T 4 antibody (i.e., in formula III, the U site is and/or randomly linked to Ab).

In another preferred embodiment, the U site is attached to Ab.

In another preferred embodiment, J is a linker comprising, CC; (G) _n C, wherein n is an integer of 0 to 4; (A) _n C, wherein n is an integer of 1 to 4; (G) _n CG, wherein n is an integer from 0 to 4; (G) _n SC, wherein n is an integer of 0 to 4; (GGGGS) _n C, wherein n is an integer of 0 to 4; c (GGGGS) _n Wherein n is an integer from 0 to 4; (GGGS) _n C, wherein n is an integer of 0 to 4; c (GGGS) _n Wherein n is an integer from 0 to 4; (GGS) _n C, wherein n is an integer of 0 to 4, C (GGS) _n Wherein 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-eea, val-Cit-PABC, polyethylene glycol PEG, or combinations thereof.

In another preferred embodiment, the linker comprises a derivative compound of MPA-AEEA, MPA-AEEA-Val-Cit-PABC, or polyethylene glycol PEG, including, but not limited to, substitution, modification, or deletion of one or more groups on their respective basis.

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

In another preferred embodiment, m is a positive integer from 1 to 10.

In another preferred embodiment, the drug comprises a cytotoxic drug, an immunomodulator, an enzyme and a hormonal suppressor.

In another preferred example, the drug comprises monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), eribulin (Eribulin), irinotecan (exatecan), maytansine (maytansine), SN-38, and the like.

In an eleventh aspect of the invention there is provided a Chimeric Antigen Receptor (CAR) whose antigen binding domain comprises a VHH chain of an anti-5T 4 nanobody according to the first aspect of the invention, an anti-5T 4 nanobody according to the second aspect of the invention, a multispecific antibody according to the fourth aspect of the invention, or a long-acting antibody of an anti-5T 4 nanobody according to the fifth aspect of the invention.

In a twelfth aspect of the invention there is provided an engineered immune cell expressing a chimeric antigen receptor according to the eleventh aspect of the invention on the cell surface.

In another preferred embodiment, the immune cells comprise T cells, NK cells.

In another preferred embodiment, the immune cells are derived from a human or non-human mammal.

In a thirteenth aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) The VHH chain of an anti-5T 4 nanobody according to the first aspect of the invention, the anti-5T 4 nanobody according to the second aspect of the invention, the anti-5T 4 nanobody Fc fusion protein according to the third aspect of the invention, the multispecific antibody according to the fourth aspect of the invention, the long-acting antibody of an anti-5T 4 nanobody according to the fifth aspect of the invention, the antibody-drug conjugate according to the tenth aspect of the invention, and/or the engineered immune cell according to 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 for the preparation of a medicament for the prevention and/or treatment of a disease or disorder associated with 5T4.

In another preferred embodiment, the 5T 4-related disease or disorder is a solid tumor or hematological malignancy.

In another preferred embodiment, the solid tumor and hematological malignancy are highly expressed as 5T4.

In another preferred embodiment, the solid tumor includes, but is not limited to: lung cancer, stomach cancer, ovarian cancer, kidney cancer, colon cancer, uterine cancer, prostate cancer, breast cancer, pancreatic cancer, ductal adenocarcinoma of the pancreas, oral cancer, cholangiocarcinoma, 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 cancer.

In another preferred embodiment, the hematological malignancy includes, but is not limited to: acute lymphoblastic leukemia, acute myelogenous leukemia, chronic eosinophilic leukemia, chronic lymphocytic leukemia, hodgkin's lymphoma, myelodysplastic syndrome, non-hodgkin's lymphoma, mixed phenotype acute leukemia, myelofibrosis, primary thrombocythemia, and plasma cell leukemia.

In a fourteenth aspect of the invention there is provided the use of a VHH chain of an anti-5T 4 nanobody according to the first aspect of the invention, an anti-5T 4 nanobody according to the second aspect of the invention, an anti-5T 4 nanobody Fc fusion protein according to the third aspect of the invention, a multispecific antibody according to the fourth aspect of the invention, a long-acting antibody of an anti-5T 4 nanobody according to the fifth aspect of the invention, an antibody-drug conjugate according to the tenth aspect of the invention, or an engineered immune cell according to the twelfth aspect of the invention, for the preparation of:

(a) A medicament for the prevention and/or treatment of a disease or condition associated with 5T 4;

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

In another preferred embodiment, the 5T 4-related disease or disorder is a solid tumor or hematological malignancy.

In another preferred embodiment, the solid tumor and hematological malignancy are highly expressed as 5T4.

In another preferred embodiment, the solid tumor includes, but is not limited to: lung cancer, stomach cancer, ovarian cancer, kidney cancer, colon cancer, uterine cancer, prostate cancer, breast cancer, pancreatic cancer, ductal adenocarcinoma of the pancreas, oral cancer, cholangiocarcinoma, 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 cancer.

In another preferred embodiment, the hematological malignancy includes, but is not limited to: acute lymphoblastic leukemia, acute myelogenous leukemia, chronic eosinophilic leukemia, chronic lymphocytic leukemia, hodgkin's lymphoma, myelodysplastic syndrome, non-hodgkin's lymphoma, mixed phenotype acute leukemia, myelofibrosis, primary thrombocythemia, and plasma cell leukemia.

In another preferred embodiment, the assay comprises a flow assay, a cellular immunofluorescence assay, an ELISA assay.

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, there is provided a method of detecting a 5T4 protein in a sample, the method comprising the steps of:

(1) Contacting a VHH chain of an anti-5T 4 nanobody according to the first aspect of the invention, an anti-5T 4 nanobody according to the second aspect of the invention, an anti-5T 4 nanobody Fc fusion protein according to the third aspect of the invention, or an antibody-drug conjugate according to the tenth aspect of the invention;

(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a 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 a sixteenth aspect of the invention there is provided a method of treating a disease or condition associated with 5T4, the method comprising administering to a subject in need thereof a VHH chain according to the first aspect of the invention, an anti-5T 4 nanobody according to the second aspect of the invention, an anti-5T 4 nanobody Fc fusion protein according to the third aspect of the invention, a multispecific antibody according to the fourth aspect of the invention, a long-acting antibody against a 5T4 nanobody according to the fifth aspect of the invention, an antibody-drug conjugate according to the tenth aspect of the invention, or an engineered immune cell according to the twelfth aspect of the invention, or a pharmaceutical composition according to the thirteenth aspect of the invention.

In another preferred embodiment, the subject comprises a mammal, such as a human.

In another preferred embodiment, the 5T 4-related disease or disorder is a solid tumor or hematological malignancy.

In another preferred embodiment, the solid tumor and hematological malignancy are highly expressed as 5T4.

In another preferred embodiment, the solid tumor includes, but is not limited to: lung cancer, stomach cancer, ovarian cancer, kidney cancer, colon cancer, uterine cancer, prostate cancer, breast cancer, pancreatic cancer, ductal adenocarcinoma of the pancreas, oral cancer, cholangiocarcinoma, 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 cancer.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the binding activity of candidate 5T4 nanobodies to 5T4 protein. ELISA results showed good binding activity of 10 antibodies.

FIG. 2 shows endocytosis of candidate 5T4 nanobodies on A431 cells. The flow cytometry detection result shows that 3 strains of 5T4 nano antibodies have good endocytosis activity.

Detailed Description

The inventor has studied extensively and intensively, and through a large number of screening, unexpectedly found a class of anti-5T 4 nanobody for the first time. Experimental results show that the anti-5T 4 nanobody disclosed by the invention can specifically recognize 5T4 protein and has good binding specificity and affinity. Meanwhile, the anti-5T 4 nano antibody has good endocytic activity. The long-acting anti-5T 4 nanobody obtained by connecting the anti-5T 4 nanobody and the anti-serum albumin nanobody in series can be further coupled with a drug to form an antibody-drug conjugate, so that tumor cells expressing 5T4 can be effectively killed in vitro and in vivo. On this basis, the present invention has been completed.

Terminology

For a better understanding of the present invention, the following terms are defined.

The term "about" includes values within the standard deviation of the values unless the context clearly indicates otherwise.

Throughout the specification and claims, the words "comprise," "have," "include," and the like are to be construed as having an inclusive, rather than an exclusive or exhaustive, meaning unless the context clearly requires otherwise; that is, the meaning of "including but not limited to". Unless otherwise indicated, "comprising" includes "consisting of … ….

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 invention may also be applied in veterinary practice to any mammal or other animal in need of such 5T 4-targeted anti-cancer treatment. This may include, for example, non-human primates, dogs, felines, pigs, horses, and any other animals treated for 5T4 against cancer.

As used herein, the terms "nanobody of the invention", "anti-5T 4 nanobody of the invention", "anti-5T 4 nanobody", "5T4 nanobody" have the same meaning, and are used interchangeably, all refer to nanobodies that specifically recognize and bind to 5T4 protein.

As used herein, the term "antibody" or "immunoglobulin" is an iso-tetralin protein of about 150000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H), having identical structural features. Each light chain is linked to the heavy chain by a 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 a plurality of constant regions. One end of each light chain is provided with a variable region (VL) and the other end is provided with a constant region; 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.

As used herein, the terms "nanobody" and "single domain antibody (singledomain antibody, sdAb)" have the same meaning and are used interchangeably to refer to the variable region of a cloned antibody heavy chain, constructing a single domain antibody consisting of only one heavy chain variable region (VHH), which is the smallest antigen-binding fragment with complete function. Typically, after an antibody is obtained which naturally lacks the light and heavy chain constant region 1 (CH 1), the variable region of the heavy chain of the antibody is cloned, and a single domain antibody consisting of only one heavy chain variable region is constructed.

As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.

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

As used herein, the term "variable region" is used interchangeably with "complementarity determining region (complementarity determining region, CDR)".

In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably to refer to a polypeptide that specifically binds to a 5T4 protein, such as a protein or polypeptide having a heavy chain variable region. They may or may not contain an initiating methionine.

The invention also provides other proteins or fusion expression products having the antibodies of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., antibody-drug conjugate and fusion expression product) having a heavy chain comprising a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of an antibody of the invention.

In general, the antigen binding properties of antibodies can be described by 3 specific regions located in the heavy chain variable region, called Complementarity Determining Regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, the β -sheets formed by the FR therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody.

The variable regions of the heavy chains of the antibodies of the invention are of particular interest because they are involved, at least in part, in binding to antigens. Thus, the invention includes those molecules having antibody heavy chain variable regions with CDRs, so long as the CDRs are 90% or more (preferably 95% or more, most preferably 98% or more) homologous to the CDRs identified herein.

The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.

The antibody of the present invention refers to a polypeptide having 5T4 binding activity and comprising the above CDR regions. The term also includes variants of polypeptides comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.

The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with the encoding DNA of an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.

The invention also provides other polypeptides, such as fusion proteins comprising a single domain antibody or fragment thereof. In addition to nearly full length polypeptides, the invention also includes fragments of the single domain antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids, 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 of the antibody of the invention.

In the present invention, a "conservative variant of an antibody of the present invention" refers to a polypeptide in which at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids of similar or similar nature, as compared to the amino acid sequence of the antibody of the present invention. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table A.

Table A

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

Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; a coding sequence for a mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature polypeptide, and non-coding sequences.

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

The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, 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 a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.

At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; animal cells of CHO, COS7, 293 cells, and the like.

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 prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl ₂ The process is carried out using procedures well known in the art. Another approach is to use MgCl ₂ . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, 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 coupling with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.

Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or enzymes capable of producing a detectable product.

Therapeutic agents that may be conjugated or coupled to an antibody of the invention include, but are not limited to: 1. a radionuclide; 2. biological toxicity; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. a viral particle; 6. a liposome; 7. nano magnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), and the like.

anti-5T 4 nanobodies

The invention provides a multi-strain anti-5T 4 nanobody. Through a large number of screening, the invention provides a plurality of specific anti-5T 4 nano antibodies with high binding affinity. Further, by humanization, a corresponding humanized anti-5T 4 nanobody is provided.

In one embodiment, the anti-5T 4 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-5T 4 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 the VHH chain has an amino acid sequence shown as SEQ ID NO. 8. In one embodiment, the anti-5T 4 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 the VHH chain thereof has an amino acid sequence as shown in SEQ ID NO. 28.

In one embodiment, the anti-5T 4 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-5T 4 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 the VHH chain has an amino acid sequence shown as SEQ ID NO. 15. In one embodiment, the anti-5T 4 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 the VHH chain thereof has an amino acid sequence as shown in SEQ ID NO. 31.

In one embodiment, the anti-5T 4 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-5T 4 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 the VHH chain thereof has an amino acid sequence as shown in SEQ ID NO. 23. In one embodiment, the anti-5T 4 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 the VHH chain thereof has an amino acid sequence as shown in SEQ ID NO. 38.

Long-acting antibodies

The invention also provides an anti-5T 4 long-acting nano antibody. As used herein, the terms "anti-5T 4 long-acting nanobody", "long-acting 5T4 nanobody", "long-acting anti-5T 4 nanobody", "long-acting antibody against 5T4 nanobody" are used interchangeably and refer to a long-acting antibody obtained by tandem connection of an anti-5T 4 nanobody of the invention with an anti-serum albumin nanobody.

In an embodiment of the invention, the structure of the long-acting antibody from the N-terminal to the C-terminal is shown as formula IIa or IIb:

Ab1-P-Ab2(IIa)；

Ab2-P-Ab1(IIb)；

wherein,

ab1 comprises a VHH chain of an anti-5T 4 nanobody according to the first aspect of the invention, or an anti-5T 4 nanobody according to the second aspect of the invention;

ab2 is an antisera albumin nanobody; and

p is a no or flexible linker.

In a preferred embodiment of the invention, the structure from the N end to the C end of the long-acting antibody is shown as a formula IIa, wherein Ab1 is the anti-5T 4 nanobody and Ab2 is an anti-serum albumin nanobody; preferably, ab1 has the amino acid sequence shown in any one of SEQ ID NO 28, 31 or 38, ab2 has the amino acid sequence shown in SEQ ID NO 55 or 56, P is a peptide linker, and has the structure of (GGGGS) n, wherein n is a positive integer from 1 to 5.

Antibody-drug conjugates (ADC)

The invention also provides antibody-drug conjugates (ADCs) based on the antibodies of the invention, including nanobodies and long-acting antibodies of the invention.

The term antibody-drug conjugate (ADC) refers to a monoclonal antibody or antibody fragment linked to a biologically active toxic drug by a linking unit. The antibodies or antibody fragments described in the present disclosure may be conjugated to an effector molecule by any means. For example, an antibody or antibody fragment may be chemically or recombinantly attached to a toxic drug. The chemical means of preparing the fusion or conjugate are known in the art. The method used to couple an antibody or antibody fragment and a drug must be capable of linking the antibody to a toxic drug without interfering with the ability of the antibody or antibody fragment to bind to the target molecule.

The drug may be any cytotoxic, cytostatic or immunosuppressive drug, such as. In embodiments, the linker connects the antibody and the drug, and the drug has a functional group that can bond to the linker. For example, the drug may have an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, or a ketone group that may be bonded to the linker. In the case of a drug directly attached to a linker, the drug has reactive groups prior to attachment to the antibody. Useful classes of drugs 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, and the like.

Cytotoxic drugs, i.e., substances that inhibit or prevent the function of cells and/or cause cell death or destruction. Cytotoxic drugs can in principle kill tumor cells at sufficiently high concentrations, but due to lack of specificity, they can also cause apoptosis in normal cells, leading to serious side effects. Cytotoxic drugsIncluding toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (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 agents, antibiotics, and nucleolytic enzymes.

The antibody of the invention and the cytotoxic drug may be conjugated via a coupling agent. Examples of the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent using a carboxyl group, a peptide chain, and a coupling agent using a disulfide bond. The nonselective coupling agent refers to a compound such as glutaraldehyde or the like that forms a covalent bond between the effector molecule and the antibody. The coupling agent using carboxyl can be any one or more of cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and acyl hydrazone coupling agent (the coupling site is acyl hydrazone).

Certain residues on antibodies (e.g., cys or Lys, etc.) may be used in conjunction with a variety of functional groups, including imaging agents (e.g., chromophores and fluorophores), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. The antibody may be conjugated to a functional agent to form an antibody-functional agent conjugate. Functional agents (e.g., drugs, detection reagents, stabilizers) are coupled (covalently linked) to the antibody. The functional agent may be directly attached to the antibody, or indirectly attached through a linker.

Antibodies can be conjugated to drugs to form Antibody Drug Conjugates (ADCs). Typically, an ADC comprises a linker (or linker) between the drug and the antibody. The term "linker unit" or "connecting fragment" or "connecting unit" refers to a chemical structural fragment or bond that is linked at one end to an antibody or antigen binding fragment thereof and at the other end to a drug, or can be linked to other linkers before being linked to a drug. The linker may be degradable or non-degradable. Degradable linkers typically degrade readily in the intracellular environment, e.g., the linker degrades at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidase. The peptidyl linker may include, for example, a dipeptide, such as valine-citrulline, phenylalanine-lysine or valine-alanine; or tripeptides such as glycine-phenylalanine-glycine; or tetrapeptides such as 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 bonds). The non-degradable linker typically releases the drug under conditions where the antibody is hydrolyzed by the protease.

Prior to attachment to the antibody, the linker has reactive groups capable of reacting with certain amino acid residues, the attachment being accomplished through the reactive groups. Thiol-specific reactive groups are preferred and include: such as maleimides, halogenated amides (e.g., iodine, bromine, or chlorine); halogenated esters (e.g., iodine, bromine, or chlorinated); halomethyl ketone (e.g., iodine, bromine, or chlorine), benzyl halide (e.g., iodine, bromine, or chlorine); vinyl sulfone, pyridyl disulfide; mercury derivatives such as 3, 6-di- (mercuromethyl) dioxane, while the counterion 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, the drug-linker compound may be used to form the ADC in a single step. In other embodiments, the bifunctional linker compounds may be used to form ADCs in two or more step processes. 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 is reacted with a drug, thereby forming an ADC.

Typically, the functional groups on the linker are selected to facilitate specific reaction with the appropriate reactive groups on the drug moiety. As a non-limiting example, an azide-based moiety may be used to specifically react with a reactive alkynyl group on a drug moiety. The drug is covalently bound to the linker by 1, 3-dipolar cycloaddition between the azide and the alkyne 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 described in bioconjugate techniques, second edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that for selective reaction of a drug moiety with a linker, when a complementary pair of reactive functional groups is selected, each member of the complementary pair can be used for both the linker and the drug.

The invention also provides a method of making an ADC, which may further comprise: the antibody is conjugated to a drug-linker compound (or drug-linker compound (LD), such as LD-1 through LD-17 as shown in the present invention, under conditions sufficient to form an antibody conjugate (ADC).

In certain embodiments, the methods of the invention comprise: the antibody is bound to the linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the method of the present invention further comprises: the antibody linker conjugate is conjugated to the drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody through the linker.

Drug loading, also known as Drug-to-Antibody Ratio (DAR), is the average number of drugs conjugated per Antibody in an ADC. It may be in the range of, for example, about 1 to about 10 drugs per antibody conjugate, and in certain embodiments, in the range of about 1 to about 8 drugs per antibody conjugate, preferably in the range of 2-8,2-7,2-6,2-5,2-4, 3-5,5-6,5-7,5-8, and 6-8. Illustratively, the drug loading may be an average of 1,2,3,4,5,6,7,8,9, 10. The ADC formulas of the present disclosure include a collection of antibody drug conjugates within the aforementioned ranges. In embodiments of the present disclosure, the drug loading may be expressed as n, which is a decimal or integer. The drug loading can be determined by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assays and HPLC.

In one embodiment of the invention, the cytotoxic drug is conjugated to the antibody through a linking unit.

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

(1) Controlling the molar ratio of the drug linker fragment to the monoclonal antibody,

(2) The reaction time and the temperature are controlled,

(3) Different reagents are selected.

Chimeric Antigen Receptor (CAR)

The Chimeric Antigen Receptor (CAR) of the invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. Extracellular domains include target-specific binding elements (also referred to as antigen binding domains). The intracellular domain includes a costimulatory signaling region and a zeta chain moiety. A costimulatory signaling region refers to a portion of an intracellular domain that comprises a costimulatory molecule. Costimulatory molecules are cell surface molecules that are required for the efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands.

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

In a preferred embodiment of the invention, the extracellular domain of the CAR provided by the invention comprises an antigen binding domain that targets 5T 4. The CARs of the invention, when expressed in T cells, are capable of antigen recognition based on antigen binding specificity. When it binds to its cognate antigen, affects tumor cells, causes tumor cells to not grow, to be caused to die or to be otherwise affected, and causes the patient's tumor burden to shrink or eliminate. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and zeta chain. For example, the antigen binding domain may be fused to the intracellular domain of the combination of the 4-1BB signaling domain, and the CD3ζ signaling domain.

As used herein, "antigen binding domain" refers to Fab fragments, fab 'fragments, F (ab') ₂ Fragments, single Fv (scFv) fragments, or single domain antibody fragments. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding. The antigen binding domain is typically a scFv (single-chain variable fragment). The size of scFv is typically 1/6 of that of an intact antibody. The single chain antibody is preferably an amino acid sequence encoded by a single nucleotide chain. In the present invention, the antigen binding domain comprises an antibody that specifically recognizes 5T4, including a 5T4 nanobody, a 5T4 long-acting nanobody, or a multispecific antibody that targets 5T4 according to the present invention.

For hinge and transmembrane regions (transmembrane domains), the CAR may be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain is used that naturally associates with one of the domains in the CAR. In some examples, the transmembrane domain may be selected, or modified by amino acid substitutions, to avoid binding such domain to the transmembrane domain of the same or a different surface membrane protein, thereby minimizing interactions with other members of the receptor complex.

Engineered immune cells

The engineered immune cells of the 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 therapies: (1) the course of action of CAR-T cells is not restricted by MHC; (2) In view of the fact that many tumor cells express the same tumor antigen, CAR gene construction for a certain tumor antigen can be widely utilized once completed; (3) The CAR can utilize not only tumor protein antigens but also glycolipid non-protein antigens, so that the target range of the tumor antigens is enlarged; (4) The use of autologous patient cells reduces the risk of rejection; (5) The CAR-T cells have an immunological memory function and can survive in vivo for a long time.

Natural Killer (NK) cells are a major class of immune effector cells that protect the body from viral infection and tumor cell invasion by non-antigen specific pathways. New functions may be obtained by engineered (genetically modified) NK cells, including the ability to specifically recognize tumor antigens and enhanced anti-tumor cytotoxicity.

CAR-NK cells also have advantages over autologous CAR-T cells, such as: (1) The perforin and the granzyme are released to directly kill tumor cells, and the perforin and granzyme have no killing effect on normal cells of the organism; (2) They release very small amounts of cytokines and thus reduce the risk of cytokine storms; (3) Is easy to expand and develop into a ready-made product in vitro. In addition, similar to CAR-T cell therapy.

Pharmaceutical composition

The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof, or a fusion protein thereof, or an antibody-drug conjugate thereof, as described above, or an engineered immune cell expressing a chimeric antigen receptor whose antigen binding domain comprises said antibody or active fragment thereof, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may 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 for binding to 5T42 protein molecules, and thus can be used for treating 5T 42-related diseases, such as solid tumors, hematological malignancies, and the like. In addition, other therapeutic agents may also be used simultaneously.

The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the antibodies (or conjugates thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

In one embodiment of the invention, where a pharmaceutical composition is used, a safe and effective amount of an antibody or antibody-drug conjugate or engineered immune cell of the invention is administered to a mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is from about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

Kit for detecting a substance in a sample

The invention also provides a kit comprising an antibody (or fragment thereof) or detection reagent of the invention, which in a preferred embodiment of the invention further comprises a container, instructions for use, buffers, etc.

The invention also provides a detection kit for detecting the 5T4 level, which comprises an antibody (the antibody disclosed by the invention) for recognizing the 5T4 protein, a lysis medium for dissolving a sample, and general reagents and buffers required for detection, such as various buffers, detection marks, detection substrates and the like. The detection kit may be an in vitro diagnostic device.

Detection method

The invention also relates to a method for detecting 5T4 protein. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving a sample in a medium; detecting the level of 5T4 protein in the solubilized sample.

In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a cell-containing sample present in a cell preservation solution.

Application of

As described above, the single domain antibody of the present invention has a wide range of biological and clinical applications, and its application relates to various fields such as diagnosis and treatment of 5T 4-related diseases, basic medical research, biological research, etc. One preferred application is for clinical diagnosis and targeted therapy of 5T4, such as treatment and diagnosis of solid tumors or hematological malignancies (including but not limited to lung cancer, stomach cancer, ovarian cancer, kidney cancer, colon cancer, uterine cancer, prostate cancer, breast cancer, pancreatic ductal adenocarcinoma, oral cancer, cholangiocarcinoma, 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, gynaecological tumor, head and neck squamous cell carcinoma, soft tissue sarcoma, urothelial cancer, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic eosinophilic leukemia, chronic lymphocytic leukemia, hodgkin's lymphoma, myelodysplastic syndrome, non-hodgkin's lymphoma, mixed phenotype acute leukemia, myelofibrosis, primary thrombocythemia, plasma cell leukemia, etc.).

Sequence information of the present invention:

Nb1-40(SEQ ID NO:8)

QVQLQESGGGSVQAGGSLRLSCAASGLTYCDSLMYWYRQGPGKEREFVSFIDRAGRTSYADSVQGRFTISQDNAKNTVYLQMNNLKPEDTAMYYCKITCYNSGYQQWGQGTQVTVSS

Nb1-40(SEQ ID NO:9)

CAGGTGCAGCTGCAGGAGTCTGGGGGAGGTTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCGTCTGGACTTACCTACTGTGACTCCCTCATGTACTGGTATCGACAGGGGCCAGGGAAGGAGCGCGAGTTCGTCTCATTTATTGATCGCGCTGGTAGGACTAGCTACGCAGACTCCGTGCAGGGCCGATTCACCATCTCCCAAGACAACGCCAAGAACACGGTGTATCTACAAATGAACAACCTGAAACCTGAGGACACGGCCATGTATTACTGTAAAATCACCTGCTACAATAGCGGCTATCAACAGTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

Nb2-26(SEQ ID NO:15)

QVQLQESGGGSVQAGGSLRLSCAASGLTYCSSLMSWYRQVPGKEREFVSFIDRLGRTSYDDSVKGRFTISQDNAKNTVYLQMNNLKPEDTATYYCKITCYDSGFQQWGQGTQVTVSS

Nb2-26(SEQ ID NO:16)

CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCGTCTGGACTTACCTACTGTAGCTCTCTCATGAGCTGGTATCGACAGGTTCCAGGGAAGGAGCGCGAGTTCGTCTCATTTATTGATCGGCTTGGTCGCACAAGCTACGACGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAACGCCAAGAACACGGTGTATCTACAAATGAACAACCTGAAACCTGAGGACACGGCCACGTATTACTGTAAAATCACGTGCTACGATAGCGGCTTTCAACAGTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

Nb10-59(SEQ ID NO:23)

QVQLQESGGGSVQAGGSLTLSCAASGYRGCKAIKTWYRQAPGKEREFVSFIDDDGRTSYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTATYRCKVDSTAGGTWCPYGKWGQGTQVTVSS

Nb10-59(SEQ ID NO:24)

CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGTCTCTGACACTCTCCTGTGCAGCCTCTGGATATCGTGGCTGTAAAGCCATCAAGACCTGGTACCGCCAGGCTCCAGGAAAGGAGCGCGAGTTCGTCTCATTTATTGATGATGATGGTAGAACAAGCTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCACGTATCGCTGTAAAGTAGACTCTACCGCGGGTGGTACCTGGTGCCCCTACGGGAAATGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

HuNb1-40(SEQ ID NO:28)

EVQLLESGGGLVQPGGSLRLSCAASGLTYCDSLMYWYRQGPGKGLEFVSFIDRAGRTSYADSVQGRFTISQDNAKNTVYLQMNNLKPEDTAMYYCKITCYNSGYQQWGQGTLVTVSS

HuNb1-40(SEQ ID NO:29)

GAGGTTCAATTGTTGGAGTCTGGTGGTGGTTTGGTTCAGCCAGGTGGTTCTTTGAGATTGTCTTGTGCTGCTTCTGGTTTGACTTACTGTGACTCTTTGATGTACTGGTACAGACAAGGTCCAGGAAAGGGTTTGGAGTTCGTTTCTTTCATTGATAGAGCTGGTAGAACTTCTTACGCTGATTCTGTTCAAGGTAGATTCACTATCTCTCAAGATAACGCTAAGAACACCGTTTACTTGCAAATGAACAACTTGAAGCCAGAAGATACTGCTATGTACTACTGTAAGATTACTTGTTACAACTCTGGTTACCAACAATGGGGTCAAGGTACTTTGGTTACTGTTTCTTCT

HuNb2-26(SEQ ID NO:31)

EVQLLESGGGLVQPGGSLRLSCAASGLTYCSSLMSWYRQVPGKGLEFVSFIDRLGRTSYDDSVKGRFTISQDNAKNTVYLQMNNLKPEDTATYYCKITCYDSGFQQWGQGTLVTVSS

HuNb2-26(SEQ ID NO:32)

GAAGTTCAATTGTTGGAATCCGGTGGTGGTTTGGTTCAACCAGGTGGTTCTTTGAGATTGTCTTGTGCTGCTTCTGGTTTGACTTACTGTTCTTCTTTGATGTCTTGGTACAGACAAGTTCCAGGAAAGGGTTTGGAATTTGTTTCTTTTATTGATAGATTGGGTAGAACTTCTTACGATGATTCTGTTAAGGGTAGATTCACTATTTCTCAAGATAACGCTAAGAACACTGTTTACTTGCAAATGAACAACTTGAAGCCAGAAGATACTGCTACTTACTACTGTAAGATTACTTGTTACGATTCTGGTTTTCAACAATGGGGTCAAGGTACTTTGGTTACTGTTTCTTCT

HuNb10-59(SEQ ID NO:35)

EVQLLESGGGLVQPGGSLTLSCAASGYRGCKAIKTWYRQAPGKGLEFVSFIDDDGRTSYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTATYRCKVDSTAGGTWCPYGKWGQGTLVTVSS

HuNb10-59(SEQ ID NO:36)

GAGGTTCAATTGTTGGAGTCTGGTGGTGGTTTGGTTCAACCAGGTGGTTCTTTGACTTTGTCTTGTGCTGCTTCTGGTTACAGAGGTTGTAAGGCTATTAAGACTTGGTACAGACAAGCTCCAGGTAAAGGTTTGGAGTTCGTTTCTTTCATTGATGATGATGGTAGAACTTCTTACGCTGATTCTGTTAAGGGTAGATTCACTATTTCTCAAGACAACGCTAAGAACACTGTTTACCTGCAAATGAACTCTTTGAAGCCTGAAGATACTGCTACTTACAGATGTAAGGTTGATTCTACCGCTGGTGGTACTTGGTGTCCATACGGAAAGTGGGGTCAAGGTACTTTGGTTACTGTTTCTTCT

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

HuNb1-40-GSC(SEQ ID NO:37)

EVQLLESGGGLVQPGGSLRLSCAASGLTYCDSLMYWYRQGPGKGLEFVSFIDRAGRTSYADSVQGRFTISQDNAKNTVYLQMNNLKPEDTAMYYCKITCYNSGYQQWGQGTLVTVSSGSC

HuNb1-40-GSC(SEQ ID NO:38)

GAGGTTCAATTGTTGGAGTCTGGTGGTGGTTTGGTTCAGCCAGGTGGTTCTTTGAGATTGTCTTGTGCTGCTTCTGGTTTGACTTACTGTGACTCTTTGATGTACTGGTACAGACAAGGTCCAGGAAAGGGTTTGGAGTTCGTTTCTTTCATTGATAGAGCTGGTAGAACTTCTTACGCTGATTCTGTTCAAGGTAGATTCACTATCTCTCAAGATAACGCTAAGAACACCGTTTACTTGCAAATGAACAACTTGAAGCCAGAAGATACTGCTATGTACTACTGTAAGATTACTTGTTACAACTCTGGTTACCAACAATGGGGTCAAGGTACTTTGGTTACTGTTTCTTCCGGTTCTTGT

HuNb2-26-GSC(SEQ ID NO:39)

EVQLLESGGGLVQPGGSLRLSCAASGLTYCSSLMSWYRQVPGKGLEFVSFIDRLGRTSYDDSVKGRFTISQDNAKNTVYLQMNNLKPEDTATYYCKITCYDSGFQQWGQGTLVTVSSGSC

HuNb2-26-GSC(SEQ ID NO:40)

GAAGTTCAATTGTTGGAATCCGGTGGTGGTTTGGTTCAACCAGGTGGTTCTTTGAGATTGTCTTGTGCTGCTTCTGGTTTGACTTACTGTTCTTCTTTGATGTCTTGGTACAGACAAGTTCCAGGAAAGGGTTTGGAATTTGTTTCTTTTATTGATAGATTGGGTAGAACTTCTTACGATGATTCTGTTAAGGGTAGATTCACTATTTCTCAAGATAACGCTAAGAACACTGTTTACTTGCAAATGAACAACTTGAAGCCAGAAGATACTGCTACTTACTACTGTAAGATTACTTGTTACGATTCTGGTTTTCAACAATGGGGTCAAGGTACTTTGGTTACTGTTTCTTCTGGTTCTTGT

HuNb10-59-GSC(SEQ ID NO:41)

EVQLLESGGGLVQPGGSLTLSCAASGYRGCKAIKTWYRQAPGKGLEFVSFIDDDGRTSYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTATYRCKVDSTAGGTWCPYGKWGQGTLVTVSSGSC

HuNb10-59-GSC(SEQ ID NO:42)

GAGGTTCAATTGTTGGAGTCTGGTGGTGGTTTGGTTCAACCAGGTGGTTCTTTGACTTTGTCTTGTGCTGCTTCTGGTTACAGAGGTTGTAAGGCTATTAAGACTTGGTACAGACAAGCTCCAGGTAAAGGTTTGGAGTTCGTTTCTTTCATTGATGATGATGGTAGAACTTCTTACGCTGATTCTGTTAAGGGTAGATTCACTATTTCTCAAGACAACGCTAAGAACACTGTTTACCTGCAAATGAACTCTTTGAAGCCTGAAGATACTGCTACTTACAGATGTAAGGTTGATTCTACCGCTGGTGGTACTTGGTGTCCATACGGAAAGTGGGGTCAAGGTACTTTGGTTACTGTTTCTTCTGGTTCTTGT

HuNb1-40-HSA Nb3-11-GSC(SEQ ID NO:43)

EVQLLESGGGLVQPGGSLRLSCAASGLTYCDSLMYWYRQGPGKGLEFVSFIDRAGRTSYADSVQGRFTISQDNAKNTVYLQMNNLKPEDTAMYYCKITCYNSGYQQWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLQESGGGLVEPGGSLRLSCVASGFTFQHYAMSWVRQAPGKGLEWVSSISLVGGGTSYADSVKGRFTMSRDNAKNTLYLQLNSLKSEDTAMYYCVRGWHPDVPRPLGQGTLVTVSSGSC

HuNb1-40-HSA Nb3-11-GSC(SEQ ID NO:44)

GAGGTGCAGCTCCTCGAATCCGGAGGAGGACTGGTGCAGCCCGGCGGATCTCTGAGGCTCTCCTGTGCTGCTTCCGGACTGACATACTGTGACTCCCTCATGTACTGGTATAGGCAGGGACCTGGGAAAGGCCTCGAATTTGTTAGTTTTATCGACAGGGCAGGCCGAACCTCCTACGCAGACAGTGTGCAGGGTCGGTTTACAATTTCACAGGACAATGCCAAGAATACTGTTTACCTGCAGATGAACAACTTGAAACCCGAGGATACTGCAATGTATTATTGCAAAATCACCTGCTACAATAGCGGATATCAGCAGTGGGGCCAGGGAACCCTGGTTACTGTCTCCTCTGGTGGCGGAGGGTCTGGTGGTGGAGGATCAGGGGGTGGAGGTTCAGGAGGCGGGGGAAGTGAAGTTCAGCTGCAGGAATCAGGAGGGGGGTTGGTCGAACCCGGGGGGAGTCTCCGGTTGAGTTGTGTTGCATCCGGGTTCACTTTCCAGCATTACGCTATGTCTTGGGTGCGGCAGGCTCCTGGAAAGGGCTTGGAATGGGTGTCTAGTATCTCTCTCGTGGGTGGCGGTACCTCATACGCTGACTCAGTGAAAGGTCGGTTCACTATGTCTCGCGACAATGCTAAGAATACTCTGTATCTCCAGCTGAATAGCCTGAAAAGCGAGGACACAGCCATGTACTACTGCGTCCGAGGATGGCACCCTGATGTGCCCCGACCTCTCGGGCAGGGGACCTTGGTCACAGTCTCATCCGGAAGTTGC

HuNb2-26-HSA Nb3-11-GSC(SEQ ID NO:45)

EVQLLESGGGLVQPGGSLRLSCAASGLTYCSSLMSWYRQVPGKGLEFVSFIDRLGRTSYDDSVKGRFTISQDNAKNTVYLQMNNLKPEDTATYYCKITCYDSGFQQWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLQESGGGLVEPGGSLRLSCVASGFTFQHYAMSWVRQAPGKGLEWVSSISLVGGGTSYADSVKGRFTMSRDNAKNTLYLQLNSLKSEDTAMYYCVRGWHPDVPRPLGQGTLVTVSSGSC

HuNb2-26-HSA Nb3-11-GSC(SEQ ID NO:46)

GAAGTCCAGCTCCTCGAAAGCGGCGGAGGCCTGGTGCAGCCCGGCGGCAGCCTCAGGCTGAGCTGCGCCGCCAGCGGCCTGACATATTGTAGCAGCCTGATGAGCTGGTATAGGCAGGTGCCCGGGAAAGGACTGGAATTTGTCTCCTTCATCGACCGTCTCGGCAGAACTTCTTACGACGACTCCGTCAAAGGAAGATTCACCATTTCCCAGGACAACGCTAAGAACACAGTGTACCTGCAGATGAATAACCTCAAACCAGAGGATACAGCTACTTACTACTGCAAAATCACTTGCTATGACAGTGGTTTCCAGCAGTGGGGACAGGGTACCCTCGTGACCGTTTCATCCGGTGGCGGAGGGTCTGGTGGTGGAGGATCAGGGGGTGGAGGTTCAGGAGGCGGGGGAAGTGAGGTCCAGTTGCAGGAGAGCGGGGGTGGACTGGTGGAGCCAGGGGGAAGTTTGAGACTCAGTTGTGTTGCCAGCGGTTTCACATTTCAGCATTACGCCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGACTGGAGTGGGTTAGCAGCATTTCTCTCGTCGGAGGGGGTACAAGCTATGCCGATTCAGTGAAGGGGCGTTTCACTATGTCCAGAGACAACGCAAAGAACACCTTGTACTTGCAGCTGAACTCCCTCAAAAGTGAGGACACAGCTATGTACTACTGCGTCCGGGGTTGGCACCCAGACGTTCCCAGGCCACTGGGGCAGGGAACATTGGTGACCGTGTCATCTGGCTCCTGT

HuNb10-59-HSA Nb3-11-GSC(SEQ ID NO:47)

EVQLLESGGGLVQPGGSLTLSCAASGYRGCKAIKTWYRQAPGKGLEFVSFIDDDGRTSYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTATYRCKVDSTAGGTWCPYGKWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLQESGGGLVEPGGSLRLSCVASGFTFQHYAMSWVRQAPGKGLEWVSSISLVGGGTSYADSVKGRFTMSRDNAKNTLYLQLNSLKSEDTAMYYCVRGWHPDVPRPLGQGTLVTVSSGSC

HuNb10-59-HSA Nb3-11-GSC(SEQ ID NO:48)

GAGGTCCAGCTCCTGGAAAGCGGCGGCGGCCTGGTCCAGCCTGGCGGAAGCCTGACACTGAGCTGCGCCGCTTCTGGCTACCGGGGATGTAAAGCTATCAAGACCTGGTACAGGCAGGCCCCTGGAAAAGGCCTGGAATTTGTGTCTTTCATTGACGATGACGGCCGCACCAGCTATGCAGACTCCGTGAAAGGTAGATTTACTATTTCTCAGGACAACGCTAAGAACACTGTGTATCTGCAGATGAATAGTCTCAAACCTGAGGACACTGCAACTTATAGATGTAAGGTGGACAGCACTGCCGGCGGAACTTGGTGTCCATACGGAAAATGGGGACAGGGTACCCTGGTCACAGTGTCATCAGGTGGCGGAGGGTCTGGTGGTGGAGGATCAGGGGGTGGAGGTTCAGGAGGCGGGGGAAGTGAGGTGCAGTTGCAGGAGTCCGGTGGTGGCTTGGTTGAGCCCGGAGGCAGTTTGCGTTTGTCCTGCGTCGCCTCTGGCTTCACCTTTCAGCATTATGCTATGAGTTGGGTGCGGCAGGCTCCAGGGAAAGGGTTGGAATGGGTTAGTTCAATTTCTCTCGTGGGTGGCGGGACTAGCTATGCCGATTCCGTGAAGGGCAGATTCACCATGTCACGCGATAACGCAAAGAATACCCTGTACCTCCAGCTCAATAGCCTGAAGAGCGAAGATACCGCTATGTACTACTGCGTCCGCGGCTGGCACCCTGACGTGCCCAGGCCATTGGGCCAGGGGACATTGGTGACCGTTAGCAGCGGTAGCTGT

HuNb1-40-HSA Nb1-60-GSC(SEQ ID NO:49)

EVQLLESGGGLVQPGGSLRLSCAASGLTYCDSLMYWYRQGPGKGLEFVSFIDRAGRTSYADSVQGRFTISQDNAKNTVYLQMNNLKPEDTAMYYCKITCYNSGYQQWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLQESGGGLVQPGGSLRLSCAASGFRFSSYWMYWVRQAPGKGLEWVSAINSSGGYTRYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCATDSGDGKRYWSGEYFYRSRGQGTLVTVSSGSC

HuNb1-40-HSA Nb1-60-GSC(SEQ ID NO:50)

GAGGTGCAGCTGCTGGAGAGCGGAGGCGGCCTGGTCCAGCCTGGAGGCAGCCTCCGGCTGTCCTGTGCCGCTTCTGGACTCACATACTGTGACAGCCTCATGTACTGGTATCGGCAGGGACCCGGTAAAGGACTGGAATTTGTCAGTTTTATCGACCGAGCAGGGCGCACATCTTACGCAGATAGTGTGCAGGGTCGCTTTACCATCAGTCAGGATAATGCAAAGAACACTGTGTATCTCCAGATGAACAATCTGAAGCCTGAAGATACCGCTATGTATTACTGCAAAATTACTTGTTATAACTCAGGATACCAGCAGTGGGGGCAGGGAACTCTGGTCACTGTTTCTAGCGGTGGCGGAGGGTCTGGTGGTGGAGGATCAGGGGGTGGAGGTTCAGGAGGCGGGGGAAGTGAGGTCCAGCTGCAGGAGTCCGGCGGAGGATTGGTTCAGCCTGGTGGGTCCCTGCGTTTGAGCTGCGCAGCCTCAGGCTTTAGATTCTCATCATACTGGATGTATTGGGTGCGCCAGGCACCCGGTAAGGGCCTCGAATGGGTTAGTGCTATTAATTCAAGTGGCGGTTATACCCGGTATGCTGATTCAGTCAAGGGGCGGTTTACTATTTCTCGCGACAACGCTAAGAATACTTTGTACTTGCAGATGAATAGCCTGCGGGCAGAGGATACTGCAGTCTACTACTGTGCCACTGATAGTGGGGATGGTAAGCGTTATTGGTCTGGCGAATATTTTTACCGGTCCAGAGGTCAGGGCACCCTCGTGACAGTGTCAAGTGGCAGTTGC

HuNb2-26-HSA Nb1-60-GSC(SEQ ID NO:51)

EVQLLESGGGLVQPGGSLRLSCAASGLTYCSSLMSWYRQVPGKGLEFVSFIDRLGRTSYDDSVKGRFTISQDNAKNTVYLQMNNLKPEDTATYYCKITCYDSGFQQWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLQESGGGLVQPGGSLRLSCAASGFRFSSYWMYWVRQAPGKGLEWVSAINSSGGYTRYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCATDSGDGKRYWSGEYFYRSRGQGTLVTVSSGSC

HuNb2-26-HSA Nb1-60-GSC(SEQ ID NO:52)

GAGGTCCAGCTCCTGGAGAGCGGCGGAGGCCTGGTCCAGCCCGGCGGATCTCTGAGGCTGAGCTGCGCTGCCTCCGGCCTGACCTATTGTTCCAGCCTGATGTCCTGGTACCGGCAGGTCCCAGGCAAAGGTTTGGAGTTCGTTTCCTTTATCGATCGGCTGGGTCGAACATCCTACGATGATAGTGTTAAGGGCAGGTTTACTATTTCCCAGGATAACGCTAAGAATACCGTGTACTTGCAGATGAATAATCTCAAACCAGAGGACACAGCTACTTACTACTGCAAAATCACATGTTATGATAGCGGTTTCCAGCAGTGGGGTCAGGGCACACTCGTTACCGTTTCAAGCGGTGGCGGAGGGTCTGGTGGTGGAGGATCAGGGGGTGGAGGTTCAGGAGGCGGGGGAAGTGAGGTTCAGCTGCAGGAATCTGGGGGAGGGCTGGTGCAGCCTGGGGGGAGTCTGCGCCTGTCTTGTGCTGCTTCCGGCTTCCGTTTCTCATCCTACTGGATGTATTGGGTGAGACAGGCACCTGGAAAGGGGTTGGAATGGGTTAGTGCAATCAATTCCTCAGGGGGGTACACTAGGTACGCTGATTCTGTCAAAGGTCGGTTTACAATTAGCAGAGACAATGCTAAGAACACTCTCTATCTCCAGATGAACAGTCTGAGAGCCGAGGACACAGCCGTGTATTACTGTGCCACTGACTCCGGAGACGGAAAACGATATTGGAGCGGAGAATACTTTTACCGCTCCAGGGGACAGGGTACACTCGTGACAGTGTCTAGCGGCTCTTGT

HuNb10-59-HSA Nb1-60-GSC(SEQ ID NO:53)

EVQLLESGGGLVQPGGSLTLSCAASGYRGCKAIKTWYRQAPGKGLEFVSFIDDDGRTSYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTATYRCKVDSTAGGTWCPYGKWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLQESGGGLVQPGGSLRLSCAASGFRFSSYWMYWVRQAPGKGLEWVSAINSSGGYTRYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCATDSGDGKRYWSGEYFYRSRGQGTLVTVSSGSC

HuNb10-59-HSA Nb1-60-GSC(SEQ ID NO:54)

GAAGTGCAGCTCCTGGAGAGCGGAGGAGGCCTGGTCCAGCCCGGCGGCTCCCTCACCCTGTCCTGCGCCGCTTCTGGCTATCGGGGCTGCAAAGCCATTAAAACATGGTACAGGCAGGCCCCCGGAAAAGGGCTCGAATTTGTTTCCTTTATTGACGACGATGGTCGTACCTCCTATGCCGACAGTGTCAAGGGTCGTTTTACAATCAGTCAGGACAATGCAAAGAATACAGTCTACCTCCAGATGAATAGCCTGAAGCCAGAAGATACAGCTACATACCGCTGCAAGGTTGACAGTACAGCCGGGGGTACATGGTGTCCTTACGGTAAATGGGGGCAGGGAACCTTGGTGACCGTCAGCTCCGGTGGCGGAGGGTCTGGTGGTGGAGGATCAGGGGGTGGAGGTTCAGGAGGCGGGGGAAGTGAGGTTCAGCTGCAGGAAAGCGGTGGGGGCCTCGTTCAGCCCGGAGGATCTTTGAGACTGAGTTGCGCAGCCAGTGGGTTTAGATTCTCTTCATATTGGATGTACTGGGTGCGGCAGGCACCCGGAAAGGGGTTGGAATGGGTGTCAGCAATTAACTCAAGCGGAGGTTATACAAGATACGCAGATAGTGTGAAGGGACGGTTCACCATCTCCAGGGACAATGCCAAGAACACCCTGTATCTGCAGATGAACAGTTTGCGTGCCGAGGACACTGCTGTTTATTATTGCGCAACTGACTCTGGGGACGGTAAGCGCTATTGGTCCGGCGAGTATTTTTACCGCTCACGGGGCCAGGGAACACTCGTGACTGTGTCTAGTGGAAGTTGC

HSA Nb1-60(SEQ ID NO:55)

EVQLQESGGGLVQPGGSLRLSCAASGFRFSSYWMYWVRQAPGKGLEWVSAINSSGGYTRYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCATDSGDGKRYWSGEYFYRSRGQGTLVTVSS

HSA Nb3-11(SEQ ID NO:56)

EVQLQESGGGLVEPGGSLRLSCVASGFTFQHYAMSWVRQAPGKGLEWVSSISLVGGGTSYADSVKGRFTMSRDNAKNTLYLQLNSLKSEDTAMYYCVRGWHPDVPRPLGQGTLVTVSS

Connecting peptide (SEQ ID NO: 57):

GGGGSGGGGSGGGGSGGGGS

5T4 extracellular portion protein (SEQ ID NO: 58)

SSPTSSASSFSSSAPFLASAVSAQPPLPDQCPALCECSEAARTVKCVNRNLTEVPTDLPAYVRNLFLTGNQLAVLPAGAFARRPPLAELAALNLSGSRLDEVRAGAFEHLPSLRQLDLSHNPLADLSPFAFSGSNASVSAPSPLVELILNHIVPPEDERQNRSFEGMVVAALLAGRALQGLRRLELASNHFLYLPRDVLAQLPSLRHLDLSNNSLVSLTYVSFRNLTHLESLHLEDNALKVLHNGTLAELQGLPHIRVFLDNNPWVCDCHMADMVTWLKETEVVQGKDRLTCAYPEKMRNRVLLELNSADLDCDPILPPSLQTS

Summarizing sequence information of 5T4 nanobodies and long-acting antibodies:

the main advantages of the invention include:

(1) The anti-5T 4 nanobody has excellent antigen binding activity and good endocytic activity.

(2) The anti-5T 4 nano antibody has high expression yield in host cells, high purity after purification and good quality uniformity, and is beneficial to drug-forming.

(3) The anti-5T 4 nano antibody is modified to introduce free cysteine, is used for coupling other substances at fixed points, comprises linker-connected small molecule drugs, isotopes and the like, and is used for developing image products and nuclear drugs.

(4) The anti-5T 4 nanobody can be used for constructing an antibody-drug conjugate, and can be connected with an antiserum albumin nanobody in series to form a long-acting antibody, so that the long-acting antibody-drug conjugate can be constructed.

The following specific examples further illustrate the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address specific conditions in the examples below, is generally followed by conventional conditions, for example those described in the laboratory Manual (Molecular Cloning-ALaboratory Manual) (third edition) (2001) CSHL Press, or by the manufacturer's recommendations (Sambrook and Russell et al, molecular cloning). Percentages and parts are by weight unless otherwise indicated.

Example 1: screening of 5T4 nanobodies

Immunizing 2 Bactrian camels with high-purity 5T4 extracellular domain protein (SEQ ID NO: 58) for 7 timesCamel peripheral blood was taken after epidemic and PBMCs were isolated therefrom, from which RNA was subsequently extracted and reverse transcribed into cDNA. And obtaining VHH gene fragments through nested PCR and cloning the VHH gene fragments to phage vectors to construct phage display libraries. The library capacity of both libraries was identified to be 5X 10 ⁹ CFU, fragment insertion rate is above 90%. Then, the antigen-specific nanobody is panned by using phage display technology, and more than 10 times of specific phage enrichment is obtained after 4 rounds of panning. Clones were randomly selected for PE-ELISA identification, and positive clones were sequenced to finally obtain 11 different families of antibodies.

Example 2: determination of binding Activity of 5T4 nanobody

The antibodies of the above 11 families were expressed with e.coli cells and purified with nickel columns, followed by identification of the binding activity of the candidate antibodies using ELISA.

Coating a 96-well ELISA plate coated with human 5T4 extracellular protein at 37 ℃ for 2 hours; adding BSA blocking solution after PBST washing, and sealing for 2 hours at 37 ℃; adding a gradient diluted 5T4 nano antibody to be detected after PBST washing, and incubating for 1 hour at 37 ℃; adding diluted biotinylated anti-his antibody for incubation after PBST washing, and then adding diluted SA-HRP; and (3) adding a color development solution and a stop solution after PBST washing, and detecting the light absorption value by using an enzyme-labeled instrument.

The results are shown in figure 1, where 10 candidate nanobodies were able to bind efficiently to 5T4 protein.

Example 3: determination of cell binding Activity of 5T4 nanobodies

The binding activity of 3 5T4 nanobodies (Nb 1-40, nb2-26 and Nb 10-59) with better ELISA binding activity and different 5T4 high-expression cell strains A431, bxPC3 and A549 is detected by using flow cytometry.

The cultured A431, bxPC3 and A54 cells were incubated with the diluted antibodies to be tested at 4℃for 40min, respectively. After washing cells with PBS, adding APC anti-HA anti-antibody, incubating for 40min at 4 ℃, centrifuging, washing cells to remove supernatant, and detecting APC signals of each sample by a flow cytometer after cell resuspension with PBS. The results are shown in table 1, and the 3 candidate nanobodies have good binding activity with different types of tumor cells.

TABLE 1 cell binding Activity of 5T4 nanobodies

Example 4: endocytic activity assay for 5T4 nanobodies

Collecting cultured A431 cells, counting, and then sub-packaging the cells to a U-shaped plate; adding the diluted 5T4 nano antibody into cells, and incubating for 30 minutes at 4 ℃; after washing cells by PBS, adding APC anti-HA anti-ibody and incubating for 30min at 4 ℃; washing cells, adding a culture medium containing 1% FBS to a non-endocytosis (0 h) experimental group and an endocytosis maximum (0 h) experimental group, and placing the cells at 4 ℃ after re-suspending; simultaneously adding 1% FBS-containing culture medium into endocytosis (0.5 h, 1h and 2 h) experimental group, placing into 37 deg.C CO ₂ An incubator; taking out corresponding samples at 0.5h, 1h and 2h respectively, and placing the samples in a refrigerator at 4 ℃; finally, the supernatant is removed by unified centrifugation, and a Stripping buffer is added for incubation at 4 ℃. After washing the cells, PBS was added to resuspend the cells and the flow cytometer was used to detect the APC signal for each sample.

As shown in FIG. 2, all three 5T4 nanobodies (Nb 1-40, nb2-26 and Nb 10-59) had good endocytosis.

Example 5: design and expression of humanized 5T4 nanobodies

The three 5T4 nanobodies (Nb 1-40, nb2-26 and Nb 10-59) are subjected to humanized design, the CDR regions are kept unchanged, and only the framework regions of the candidate antibodies are subjected to humanized modification. The sequences of the engineered antibodies are shown in table 2 below.

The humanized nanobody was then expressed using pichia pastoris, and its expression and fermentation yield were assessed.

Specifically, the amino acid sequence of the humanized nanobody is cloned on a pPICZaA vector according to the base sequence optimized by pichia pastoris codons. The linearized plasmid was then electrotransformed into X-33 competent cells and cultured in YPD plates containing bleomycin resistance for 3 days. The monoclonal was picked up and induced for 3 days, and the expression of the antibodies was detected by SDS-PAGE gel and evaluated for 7L fermenter yield. As a result, the 3 humanized 5T4 nanobodies have good expression yield in yeast cells.

TABLE 2 expression level of humanized nanobodies in Pichia pastoris

And (3) carrying out binding activity identification on the humanized antibody. The binding activity of the candidate antibodies to 5T4 extracellular domain protein was detected by ELISA. Briefly, the 5T4 protein is coated on an ELISA plate overnight, and after the blocking solution is blocked, the humanized antibody with gradient dilution is added for incubation for 1 hour; adding sheep anti-VHH antibody after washing, and incubating for 1 hour; after washing again, HRP-labeled murine anti-sheep antibody was added and incubated for 1 hour, and finally stop solution was added and OD450 absorbance was read in a microplate reader, with the results shown in table 3 below. The results show that the humanized nano antibodies all maintain good antigen binding activity.

TABLE 3 binding Activity of humanized antibodies

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

The long-acting antibodies with different structures are designed and expressed by pichia pastoris, and the expression and fermentation yield of the long-acting antibodies are evaluated.

Specifically, long-acting antibody amino acid sequences with different structures are cloned on a pPICZaA vector according to the base sequence optimized by pichia pastoris codons. The linearized plasmid was then electrotransformed into X-33 competent cells and cultured in YPD plates containing bleomycin resistance for 3 days. The monoclonal was picked up and induced for 3 days, and the expression of the antibody was detected by SDS-PAGE and evaluated for 7L fermenter yield.

TABLE 4 expression level of long-acting nanobodies in Pichia pastoris

The result shows that the constructed long-acting 5T4 nano antibody has good expression yield in yeast cells.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims (10)

1. A VHH chain of an anti-5T 4 nanobody, characterized in that said VHH chain comprises complementarity determining regions CDRs 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 (b)

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

2. An anti-5T 4 nanobody having a VHH chain according to claim 1.

3. An anti-5T 4 nanobody Fc fusion protein, which is characterized in that the structure from the N end to the C end of the fusion protein is shown as formula Ia or Ib:

A-L-B(Ia)；

B-L-A(Ib)；

Wherein,

a is the anti-5T 4 nanobody of claim 2;

b is the Fc fragment of IgG; and

l is a non-or flexible linker.

4. A multispecific antibody comprising a VHH chain of an anti-5T 4 nanobody according to claim 1, an anti-5T 4 nanobody according to claim 2, and/or an anti-5T 4 nanobody Fc fusion protein according to claim 3.

5. A long-acting antibody against a 5T4 nanobody, comprising a VHH chain of an anti-5T 4 nanobody according to claim 1 or an anti-5T 4 nanobody according to claim 2, and an anti-serum albumin nanobody moiety.

6. A polynucleotide encoding a protein selected from the group consisting of: the VHH chain of an anti-5T 4 nanobody of claim 1, the anti-5T 4 nanobody of claim 2, the anti-5T 4 nanobody Fc fusion protein of the present claim 3, the multispecific antibody of claim 4, or the long-acting antibody of the anti-5T 4 nanobody of claim 5.

7. An antibody-drug conjugate, the antibody-drug conjugate comprising:

(a) An antibody moiety selected from the group consisting of: the VHH chain of an anti-5T 4 nanobody of claim 1, the anti-5T 4 nanobody of claim 2, the anti-5T 4 nanobody Fc fusion protein of the present claim 3, the multispecific antibody of claim 4, or the long-acting antibody of the anti-5T 4 nanobody of claim 5; and

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

8. A Chimeric Antigen Receptor (CAR) whose antigen binding domain comprises a VHH chain of the anti-5T 4 nanobody of claim 1, the anti-5T 4 nanobody of claim 2, the multispecific antibody of claim 4, or the long-acting antibody of the anti-5T 4 nanobody of claim 5.

9. An engineered immune cell that expresses the chimeric antigen receptor of claim 8 on the cell surface.

10. A pharmaceutical composition comprising:

(i) The VHH chain of anti-5T 4 nanobody of claim 1, the anti-5T 4 nanobody of claim 2, the anti-5T 4 nanobody Fc fusion protein of the present claim 3, the multispecific antibody of claim 4, the long-acting antibody of anti-5T 4 nanobody of claim 5, the antibody-drug conjugate of claim 7, and/or the engineered immune cell of claim 9; and

(ii) A pharmaceutically acceptable carrier.