CN117327183A

CN117327183A - Preparation method and application of nuclide-labeled Trop2 specific single-domain antibody probe

Info

Publication number: CN117327183A
Application number: CN202311251659.XA
Authority: CN
Inventors: 魏伟军; 黄未; 刘建军
Original assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2023-09-26
Filing date: 2023-09-26
Publication date: 2024-01-02

Abstract

The invention relates to the technical fields of molecular images, nuclear medicine and single-domain antibodies for tumor diagnosis and treatment, in particular to a preparation method and application of a nuclide marker Trop2 specific single-domain antibody probe. The Trop2 specific single domain antibody has a CDR1 with an amino acid sequence shown as SEQ ID No.1, a CDR2 with an amino acid sequence shown as SEQ ID No.2 and a CDR3 with an amino acid sequence shown as SEQ ID No. 3. Trop2 specificity prepared by the invention ¹⁸ F-labeled single-domain antibody probe is used for immune PET imaging, so that Trop2 in-vivo noninvasive visualization is realized, and Trop2 expression positive is further realizedNoninvasive diagnosis of sexual neoplasms. The probe has the advantages of simple preparation process, low cost, high specificity, high stability, short imaging period, low radiation dose, easy clinical transformation and the like.

Description

Preparation method and application of nuclide-labeled Trop2 specific single-domain antibody probe

Technical Field

The invention relates to the technical fields of molecular images, nuclear medicine and single-domain antibodies for tumor diagnosis and treatment, in particular to a preparation method and application of a nuclide marker Trop2 specific single-domain antibody probe.

Background

In 1993, belgium scientists Hamers et al reported for the first time in Nature journal that an antibody with a naturally deleted light chain was present in alpaca peripheral blood (Nature.1993; 363 (6428): 446-8.), an antibody with a specific domain, namely Heavy chain antibodies (HCAbs). By molecular biology means, the antigen binding fragment of only the heavy chain variable region can be obtained by cloning the variable region of the heavy chain antibody, namely the single domain antibody (VHH, variable Domain of Heavy Chain of Heavy Chain Antibody). VHH crystals are 2.5nm wide and 4nm long and have a molecular weight of only 15kDa and are therefore also known as single domain antibodies [ ]Ablynx corporation registers trade names). The single domain antibody is the currently known minimum antibody unit capable of combining the target antigen, and has the advantages of high affinity, small molecular weight, low preparation cost (not only can be expressed by using escherichia coli, but also can be expressed by using eukaryotic expression systems such as yeast, chinese hamster ovary cells and the like), and easy clinical transformation and popularization and application.

The single domain antibody is a popular targeting vector for constructing molecular image probes in recent years (theranostics.2014; 4 (4): 386-98.;) J Nucl Med.2022Oct;63 (10):1705-1709.). Currently, a variety of short half-life nuclides have been used to label single domain antibodies, producing single domain antibody molecular imaging probes. Technetium-99 m% ^99m Tc；T _1/2 =6.02 h) single domain antibody probes labeled targeting programmed death ligand 1 (PD-L1) have been successfully transformed into the clinic for non-invasive diagnosis of non-small cell lung cancer patients (J nucleic med.2019;60 1213-1220.); gallium-68% ⁶⁸ Ga；T _1/2 =1.1h) single domain antibody probes labeled to target human epidermal growth factor receptor (HER 2) have also been successfully transformed into clinic for non-invasive diagnosis of breast cancer (J nucleic med.2016;57 (1):27-33.). The above examples illustrate that the radionuclide-labeled single-domain antibody probe has great clinical transformation application prospects, and can be used for early noninvasive diagnosis of human malignant tumors, visualization of key pathogenic targets, screening of patients treated by monoclonal antibodies (mAbs) and evaluation of curative effects after monoclonal antibody treatment.

Trophoblast cell surface antigen 2 (Trop 2) is a cell membrane surface glycoprotein formed from a 36kDa nascent polypeptide modified by N-linked glycosylation translation. It regulates tumor proliferation, invasion and migration through a variety of signaling pathways and plays a role in stem cell biology. In a retrospective study of 197 paraffin-embedded pancreatic cancer primary tumor tissues, trop2 antigen expression was analyzed, and immunohistochemical results found that 55% of the antigen was overexpressed, which was clearly associated with lymph node metastasis, poor tumor differentiation and poor prognosis (P < 0.05). Trop2 expression is also associated with biological invasiveness and poor prognosis of malignant tumors such as gastric cancer, female reproductive system tumors, prostate cancer, colorectal cancer and the like, indicating that the malignant tumors are promoted to develop and develop. The difference of Trop2 expression in normal tissues and tumors makes the Trop2 a tumor-specific marker with great potential, and potential side effects can be avoided. At present, antibody drug conjugate drugs taking Trop2 as a target point enter clinical tests. Therefore, there is an urgent need to develop a diagnostic tool that targets Trop2 to enable noninvasive visualization and monitoring of Trop2 expression in solid tumors. On the basis of research accompanying diagnostic tools, novel therapeutic approaches to Trop2 can be further developed.

Early series of basic and clinical studies by the applicant showed that by subtly fusing the extraordinary targeting specificity of antibodies with the superior sensitivity and resolution of Positron Emission Tomography (PET), immunopet can better show the distribution and abundance of targets of interest in vivo, particularly heterogeneous expression, and better predict response to targeted or immunotherapy (Chem rev.2020;120 (8): 3787-3851.) compared to Immunohistochemical staining (IHC) or other conventional predictive markers. For example, the value of immune PET imaging probes targeting HER 2 in breast cancer has been clinically demonstrated. Based on the evidence and our previous findings, we hypothesize that Trop 2-targeting immune PET imaging probes can non-invasively display Trop2 expression in tumors and provide a better method for diagnosis and monitoring of Trop 2-positive solid tumors. Furthermore, there has been evidence that Radioimmunotherapy (RIT) and pretargeted radioimmunotherapy (pr it) may help tumor patients alleviate the condition for a long period of time, even eradicating multiple cancer types.

At present, no report of Trop2 specific single-domain antibody molecule imaging probes or nuclide labeled diagnosis and treatment integrated probes exists in clinical practice and literature reports. There are reports of molecular imaging probes (Eur J Nucl Med Mol imaging.2022Feb;49 (3): 861-870.) based on Trop2 monoclonal antibodies and nuclide labeled diagnostic integrated probes (Eur J Nucl Med Mol imaging.2022Dec;50 (1): 168-183.). However, the use of radiolabeled monoclonal antibodies is severely hampered by the high cost, necessity of using long half-life radionuclides, cumbersome imaging procedures within a week, and associated radiation exposure. In order to improve the clinical application of antibody diagnostics, the molecular imaging field is actively exploring pretargeting imaging strategies or using smaller molecular weight antibody derivatives to achieve the current day molecular imaging (same-day imaging). In the small antibody format, single domain antibodies from the family camelidae are the smallest antigen binding portion with a molecular weight of about 15 kDa. Small size, high affinity and ease of engineering single domain antibodies as excellent alternatives to molecular imaging (J nucleic Med.2022Oct;63 (10): 1705-1709.). In recent years, we have focused on the development and clinical transformation of single domain antibody-derived tracers to exert their superior molecular imaging properties. There is no Trop2 specific single domain antibody molecular imaging probe at home and abroad.

Disclosure of Invention

To fill the gap in this field, we describe here the construction of Trop 2-targeted diagnostic pairs derived from single domain antibodies and characterize their diagnostic and therapeutic value in cell-derived xenograft (CDX) models. Therefore, those skilled in the art are working to develop a single domain antibody immune PET imaging probe which has low preparation cost, small molecular weight, short in vivo circulation time, short imaging period, low radiation dose and easy clinical transformation application. Specifically, the invention provides a preparation method and application of a nuclide-labeled Trop2 specific single-domain antibody probe.

Trop 2-specific single domain antibodies

In one aspect, the invention provides a Trop 2-specific single domain antibody comprising:

CDR1 having the amino acid sequence shown in SEQ ID No.1, CDR2 having the amino acid sequence shown in SEQ ID No.2, and CDR3 having the amino acid sequence shown in SEQ ID No. 3.

More specifically, the Trop 2-specific single domain antibody of the present invention has the amino acid sequence shown in SEQ ID No.4, 6, 8 or 10.

In the present invention, trop 2-specific single domain antibodies having the amino acid sequences shown in SEQ ID nos. 4, 6, 8 or 10 are referred to as T4, T5, RT4 or RT5, respectively, for the sake of simplicity.

As used herein, the term "single-domain antibody (sdAb)" is also referred to as "nanobody" or VHH (Variable Domain of Heavy Chain of Heavy Chain Antibody), which are used interchangeably. Nanobodies have the meaning commonly understood by those skilled in the art, which refers to antibody fragments consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), typically derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, nanobodies consist of 4 framework regions and 3 complementarity determining regions, having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Nanobodies may be truncated at the N-or C-terminus such that they comprise only a portion of FR1 and/or FR4, or lack one or both of those framework regions, so long as they substantially retain antigen binding and specificity.

In some embodiments, the invention also encompasses antigen binding fragments of Trop 2-specific single domain antibodies as described herein.

As used herein, the term "antigen-binding fragment" refers to a polypeptide comprising a fragment of a nanobody that retains the ability to specifically bind to the same antigen to which the nanobody binds, and/or competes with the nanobody for specific binding to an antigen, also referred to as an "antigen-binding portion. Generally, see Fundamental Immunology, ch.7 (Paul, W., ed., 2 nd edition, raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes, antigen binding fragments of the present antibodies may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the present nanobodies.

Antigen-binding fragments of nanobodies can be obtained from a given nanobody (e.g., a nanobody provided by the invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and specifically screened in the same manner as for whole nanobodies.

In this context, unless the context clearly indicates otherwise, when referring to the term "nanobody" it includes not only whole nanobodies but also antigen-binding fragments of nanobodies.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are responsible for antigen binding. Three CDRs are contained in the nanobody, designated CDR1, CDR2 and CDR3. The precise boundaries of these CDRs may be defined according to various numbering systems known in the art, e.g., as in the Kabat numbering system (Kabat et al, sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md, 1991), the Chothia numbering system (Chothia & Lesk (1987) J.mol. Biol.196:901-917; chothia et al (1989) Nature 342:878-883) or the IMGT numbering system (Lefranc et al, dev. Comparat. Immunol.27:55-77,2003). For a given nanobody, one skilled in the art will readily identify the CDRs defined by each numbering system. Also, the correspondence between the different numbering systems is well known to the person skilled in the art (see e.g. Lefranc et al, dev. Comparat. Immunol.27:55-77,2003).

As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

As used herein, the term "Trop 2-specific" refers to specifically binding Trop2.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction can be determined by the equilibrium dissociation constant (K _D ) And (3) representing. In the present invention, the term "K _D "refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and antigen.

The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation. "binding Rate constant" (k) _a Or k _on ) And "dissociation rate constant" (k) _dis Or k _off ) Both can be calculated from the concentration and the actual rate of association and dissociation (see Malmqvist M, nature,1993, 361:186-187). k (k) _dis /k _on Is equal to the dissociation constant K _D (see Davies et al, annual Rev Biochem,1990; 59:439-473). K can be measured by any effective method _D 、k _on And k _dis Values. In certain embodiments, the dissociation constant may be measured in Biacore using Surface Plasmon Resonance (SPR). In addition to this, bioluminescence interferometry or Kinexa can be used to measure the dissociation constant.

In some embodiments, the invention also provides variants of a Trop 2-specific single domain antibody as described herein that have 70-100% sequence identity, such as at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, to the amino acid sequence set forth in SEQ ID nos. 4, 9, 14 or 19, and substantially retain the biological function (e.g., the biological activity of specifically binding Trop 2) of the nanobody from which it is derived.

More specifically, the variants differ from Trop 2-specific single domain antibodies as described herein only in conservative substitutions of one or more (e.g., conservative substitutions of up to 20, up to 15, up to 10, up to 5, or up to 1) amino acid residues.

As used herein, the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matched positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions in total are matched). Typically, the comparison is made when two sequences are aligned to produce maximum identity. Such alignment may be conveniently performed using, for example, a computer program such as the Align program (DNAstar, inc.) Needleman et al (1970) j.mol.biol.48: 443-453. The percent identity between two amino acid sequences can also be determined using the algorithms of E.Meyers and W.Miller (Comput. ApplBiosci.,4:11-17 (1988)) which have been integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), the gap length penalty of 12 and the gap penalty of 4. Furthermore, percent identity between two amino acid sequences may be determined using the Needleman and Wunsch (jmoibiol. 48:444-453 (1970)) algorithm that has been incorporated into the GAP program of the GCG software package (available on www.gcg.com), using the Blossum 62 matrix or PAM250 matrix, and GAP weights (GAP weights) of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the desired properties of a protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions may be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions that replace an amino acid residue with an amino acid residue having a similar side chain, such as substitutions with residues that are physically or functionally similar (e.g., of similar size, shape, charge, chemical nature, including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitutions of amino acids are well known in the art (see, e.g., brummell et al, biochem.32:1180-1187 (1993); kobayashi et al Protein Eng.12 (10): 879-884 (1999); and Burks et al Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

Polynucleotide

In another aspect, the invention also provides polynucleotides encoding the nanobodies described above or antigen-binding fragments thereof.

More specifically, the polynucleotide has the nucleotide sequence shown in SEQ ID No.5, 7, 9 or 11.

More specifically, the polynucleotide encoding a Trop 2-specific single domain antibody as described herein has the nucleotide sequence set forth in SEQ ID No.5, 7, 9 or 11.

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.

The term "polynucleotide encoding a polypeptide/protein/antibody" may include polynucleotides encoding such polypeptide/protein/antibody, as well as polynucleotides further comprising 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%, most preferably at least 90% identity between the two sequences, and which encode polypeptides/proteins/antibodies having substantially the same function and activity. The 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.

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.

Carrier body

In another aspect, the invention also provides a vector comprising a polynucleotide encoding a Trop 2-specific single domain antibody or antigen binding fragment thereof as described above.

As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide may be inserted. When a vector enables expression of a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction or transfection such that the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes, such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal viruses that may be used as vectors include, but are not limited to, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus, papilloma vacuolation virus (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin.

Host cells

In another aspect, the invention also provides a host cell comprising a vector as described herein.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as e.g. escherichia coli or bacillus subtilis, a fungal cell such as e.g. yeast cells or aspergillus, an insect cell such as e.g. S2 drosophila cells or Sf9, or an animal cell such as e.g. fibroblasts, CHO cells, COS cells, NSO cells, heLa cells, BHK cells, HEK 293 cells or other human cells. Host cells may include single cells or cell populations.

The vector may be introduced into the host cell by 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, it canThe following DNA transfection method is selected: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The nanobodies 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. The preferred detectable label is a radionuclide.

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)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticle, and the like.

The radionuclides include but are not limited to, ¹⁸ F、 ⁶⁸ Ga、 ¹⁷⁷ Lu、 ⁶⁴ Cu、 ⁸⁹ Zr、 ²²⁵ Ac、 ²¹¹ At、 ²¹² Pt、 ¹²⁴ I、 ¹³¹ I、 ¹²⁵ I、 ²²³ Ra、 ⁴⁴ sc, etc., is preferably ¹⁸ F or F ⁶⁸ Ga-labeling for non-invasive diagnosis of tumors, preferably ²²⁵ Ac、 ²¹¹ At、 ²¹² Pt or ¹³¹ The I label is used for tumor radioimmunotherapy.

Binding or coupling of the detectable label or therapeutic agent to the antibody can be performed by conventional methods well known to those skilled in the art. For example, the detectable label may be directly or indirectly bound to the nanobody, e.g., via a cleavable or non-cleavable linker peptide, or incorporated into the nanobody. The detectable label may be bound to the nanobody, in particular by substitution (e.g. by substituting H with I at the tyrosine residue level), by complexation or by chelation. For example, the therapeutic agent may be conjugated to the nanobody via a cleavable linker (e.g., a peptidyl, disulfide, or hydrazone linker).

In a preferred embodiment, the nanobody of the invention is conjugated with a radionuclide for use as a Trop 2-specific molecular imaging probe, as described in more detail below.

Trop2 specific molecular imaging probe

In another aspect, the invention provides a human Trop 2-specific ¹⁸ F-labeled monovalent single domain antibody probes comprising radionuclide-labeled Trop 2-specific single domain antibody antibodies as described herein.

More specifically, trop 2-specific single domain antibodies as described herein are labeled with radionuclides via bifunctional chelators.

As used herein, a bifunctional chelating agent is a class of chelating agents having both a metal chelating end and a protein anchoring end. The bifunctional chelating agent may be at least one selected from (+ -) -H3RESCA-TFP, (+ -) H3 RESCA-Mal.

As used herein, the (+ -) -H3RESCA-TFP is a tetrafluorophenyl ester derivative of a constrained complexing agent (RESCA) that can be used to couple a chelator to a biomolecule by amine coupling (e.g., N-terminal and/or epsilon-amino group of lysine);

the (+ -) H3RESCA-Mal is (+ -) H3 RESCA-maleimide, a tetrafluorophenyl ester derivative of a constrained complexing agent (RESCA), useful for coupling chelators to biomolecules via amine coupling (e.g., N-terminal and/or epsilon-amino groups of lysine).

More specifically, trop 2-specific single domain antibodies as described herein are labeled with a radionuclide via (±) -H3RESCA-TFP or (±) H3 RESCA-Mal.

More specifically, the radionuclide is selected from F-18.

More specifically, as described herein [ ¹⁸ F]F-H3RESCA-T4、[ ¹⁸ F]F-H3RESCA-T5、[ ¹⁸ F]F-H3RESCA-RT4 or [ ¹⁸ F]F-H3RESCA-RT5。

The radionuclide fluorine-18 adopted by the invention ¹⁸ F；T _1/2 =110 min) has near ideal nuclear decay characteristicsAnd half-life ratio ⁶⁸ Ga is longer and is more compatible with radiolabeling of antibody derived fragments, e.g. single domain antibodies. Traditional radiofluorination strategies are harsh and cumbersome, such as the disadvantages of reaction in organic solvents, high temperatures, time consuming synthetic steps, etc. (Nat Protoc.2018Oct;13 (10): 2330-2347.). The radiolabelling method according to the present patent has the following advantages: the reaction is carried out at room temperature, the labeling process is short (30 min), and the radioactive chemical purity of the product is high (> 99%).

More specifically, the human Trop2 specificity ¹⁸ F-labeled monovalent Single-Domain antibody Probe [ ¹⁸ F]F-H3RESCA-RT4 was labeled with F-18 via (+ -) -H3RESCA-TFP according to the Trop 2-specific single domain antibody having the amino acid sequence shown in SEQ ID No. 8. It has lower kidney enrichment, lower liver enrichment, lower bone enrichment and lower muscle tissue, and therefore has better diagnostic efficacy.

In another aspect, the invention also provides for human Trop2 specificity ¹⁸ F, a method for labeling a monovalent single-domain antibody probe comprises the steps of modifying a Trop2 specific single-domain antibody through a bifunctional chelating agent to obtain a radionuclide labeling precursor; and labeling the radionuclide-labeled precursor with a radionuclide to obtain human Trop2 specificity ¹⁸ F labeling monovalent single domain antibody probes.

More specifically, when the bifunctional chelating agent is (±) H3RESCA-Mal, a cysteine-containing peptide fragment (GGGGS) nC, n=1-10, is expressed at the end of Trop 2-specific single domain antibody before the preparation of the radionuclide-labeled precursor; in particular n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

Composition and method for producing the same

In another aspect, the invention provides a composition comprising a Trop 2-specific single domain antibody, polynucleotide, vector, host cell or molecular imaging probe as described herein. The composition can be used for detecting the expression level of Trop2, diagnosing Trop 2-related tumors, predicting the treatment effect of Trop 2-related tumors or treating Trop 2-related tumors.

In some embodiments, the composition may be a pharmaceutical composition.

In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and/or excipient.

In some embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent.

In some embodiments, the additional pharmaceutically active agent is an anti-inflammatory drug or an immunosuppressant.

In some embodiments, the Trop 2-specific single domain antibodies, polynucleotides, vectors, host cells, or molecular imaging probes as described herein and the additional pharmaceutically active agent in the pharmaceutical composition may be provided as separate components or as a mixed component. Thus, a Trop 2-specific single domain antibody, polynucleotide, vector, host cell or molecular imaging probe as described herein and the additional pharmaceutically active agent may be administered simultaneously, separately or sequentially.

In some embodiments, the pharmaceutically acceptable carrier and/or excipient may comprise a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w/v) NaCl), dextrose solutions (e.g., 5% dextrose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffered solutions (e.g., phosphate buffered solutions), ringer's solution, and any combination thereof.

The pharmaceutical compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of a Trop 2-specific single domain antibody, polynucleotide, vector, host cell, or molecular imaging probe as described herein. "prophylactically effective amount" means an amount sufficient to prevent, arrest or delay the onset of a disease. By "therapeutically effective amount" is meant an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount may vary depending on the factors: the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like.

Kit for detecting a substance in a sample

The invention also provides a kit comprising a Trop 2-specific single domain antibody, polynucleotide, vector, host cell or molecular imaging probe as described herein.

The kit can be used for detecting the expression level of Trop2, diagnosing Trop 2-related tumors, predicting the treatment effect of Trop 2-related tumors or treating Trop 2-related tumors.

The kit may further comprise further containers, instructions for use, and other reagents and buffers required for the actual application, such as lysis media for lysing the sample, various buffers, detection labels, detection substrates, etc.

Diagnostic and therapeutic applications

The Trop2 specific single domain antibody has extremely high affinity to Trop2, so that the Trop2 specific single domain antibody can be used for detecting the expression level of Trop2, diagnosing Trop 2-related tumors, predicting the treatment effect of Trop 2-related tumors or treating Trop 2-related tumors.

Particularly, the Trop2 specific molecular image probe prepared by the Trop2 specific single-domain antibody has the characteristics of obviously improved affinity, obviously reduced non-specific uptake of normal tissue uptake and obviously improved image quality, and can be used for noninvasively, accurately and efficiently detecting the expression of human Trop2, so that the Trop2 specific molecular image probe is particularly suitable for diagnosing Trop 2-related tumors and predicting the treatment effect of Trop 2-related tumors. After proper radionuclides are selected for coupling, the method can also be used for accurately treating Trop 2-related tumors.

Thus, in another aspect, the invention also relates to the use of a Trop 2-specific single domain antibody, polynucleotide, vector, host cell or molecular imaging probe as described herein for the preparation of a kit or medicament for detecting the expression level of Trop2, diagnosing a Trop 2-associated tumor, predicting the therapeutic effect of a Trop 2-associated tumor, or treating a Trop 2-associated tumor.

As used herein, trop 2-related tumors may include various tumors or cancers well known in the art. For example, trop 2-related tumors may include breast cancer, lung cancer, stomach cancer, pancreatic cancer, colon cancer, prostate cancer, cervical cancer, and the like.

The beneficial effects of the invention are that

1) The Trop2 specific molecular probe prepared by the invention is used for immune PET imaging, so that noninvasive visualization of human Trop2 molecular expression is realized, and noninvasive diagnosis of Trop2 expression positive tumors (such as pancreatic cancer) is further realized.

2) The probe prepared by the invention has the advantages of simple preparation process, low cost, high specificity, high stability, short imaging period, low radiation dose, easy clinical transformation and the like. The clinical transformation application is hopeful to realize noninvasive visualization of Trop2 heterogeneous expression, screen patients with high Trop2 expression, and further develop Trop2 specific radioimmunotherapy, thereby realizing target specific diagnosis and treatment integration of Trop2 expression positive tumors.

Drawings

FIG. 1 shows SDS-PAGE and HPLC results of measuring the expression of single domain antibodies T4 and T5 and SPR detecting the binding affinity of the single domain antibodies to the target; wherein FIG. 1A shows the SDS-PAGE result of T4; FIG. 1B shows the HPLC results of T4; FIG. 1C shows the binding affinity results of T4 to a target; FIG. 1D shows the SDS-PAGE result of T5; FIG. 1E shows the HPLC results of T5; FIG. 1F shows the binding affinity results of T5 to a target;

FIG. 2 shows [ ¹⁸ F]Radiochemical purity measurements before and after purification of F-H3 RESCA-T4;

FIG. 3 shows [ [ ¹⁸ F]PET/CT imaging, ROI analysis and in-vitro biodistribution results of F-H3RESCA-T4 after 30 minutes of injection of T3M-4 tumor-bearing Balb/c mice; wherein FIG. 3A is a PET/CT imaging result of an unsealed group; FIG. 3B is the result of the ROI analysis of the unsealed group; FIG. 3C is an in vitro biodistribution result for the unsealed group; FIG. 3D shows PET/CT imaging results for the T4 closed group; FIG. 3E is the result of the ROI analysis of the T4 closed group; FIG. 3F shows the in vitro biodistribution results of the T4-blocked group;

FIG. 4 shows [ [ ¹⁸ F]Immunohistochemical staining of F-H3RESCA-T4 non-blocked and blocked groupsColor; wherein, fig. 4A is an unsealed group; FIG. 4B is a closed group;

FIG. 5 shows SDS-PAGE and HPLC results of determining the expression of single domain antibodies RT4 and RT5 and SPR detection of binding affinity of single domain antibodies to targets; wherein FIG. 5A shows the SDS-PAGE result of RT 4; FIG. 5B is the HPLC results of RT 4; FIG. 5C shows the binding affinity results of RT4 to the target; FIG. 5D shows SDS-PAGE results of RT 5; FIG. 5E is the HPLC results of RT 5; FIG. 5F shows the binding affinity results of RT5 to the target;

FIG. 6 shows [ [ ¹⁸ F]Radiochemical purity measurement results after purification of F-H3RESCA-RT 4;

FIG. 7 shows [ [ ¹⁸ F]PET/CT imaging and ROI analysis results after injection of F-H3RESCA-RT4 into T3M-4 tumor-bearing Balb/c mice for 45 minutes and 2.5 hours; wherein FIG. 7A is PET/CT imaging results for an unsealed group; FIG. 7B is the result of the ROI analysis of the unsealed group; FIG. 7C shows PET/CT imaging results for the T4 closed group; FIG. 7D is the result of the ROI analysis of the T4 closed group;

FIG. 8 shows [ sic ] ¹⁸ F]In vitro biodistribution results of F-H3RESCA-RT4 non-blocked group and RT4 blocked group in T3M-4 tumor-bearing Balb/c mice;

FIG. 9 shows [ sic ] ¹⁸ F]F-H3RESCA-T4 and [ ¹⁸ F]ROI delineation results of F-H3RESCA-RT4 and tumor/organ ratio comparison results;

FIG. 10 shows the chemical structure of chelator (+ -) -H3 RESCA-TFP.

Detailed Description

In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to specific embodiments that are illustrated in the appended drawings. It is noted that the invention is not limited to the particular methods, protocols, cell lines, constructs, and reagents described herein, and may vary as well. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

At present, no report of Trop2 specific single-domain antibody molecule imaging probes and nuclide labeled diagnosis and treatment integrated probes exists in clinical practice and literature reports. There are reports of molecular imaging probes (Eur J Nucl Med Mol imaging.2022Feb;49 (3): 861-870.) based on Trop2 monoclonal antibodies and nuclide labeled diagnostic integrated probes (Eur J Nucl Med Mol imaging.2022Dec;50 (1): 168-183.). However, the clinical transformation application of the monoclonal antibody immune PET imaging probe is severely limited by the reasons of high preparation cost, large molecular weight, long in vivo circulation time, long imaging period, high radiation dose, large toxic and side effects and the like. In comparison, the single domain antibody immune PET imaging probe has the advantages of low preparation cost, small molecular weight, short in vivo circulation time, short imaging period, low radiation dosage, easy clinical transformation and application and the like.

The Trop2 specificity diagnosis and treatment integrated single-domain antibody molecular image probe is constructed, the expression of Trop2 in tumors is displayed noninvasively, and a better method is provided for diagnosing and monitoring Trop2 positive solid tumors. On the basis, a novel therapeutic method targeting Trop2 is to be further developed.

Example 1: preparation of Trop 2-specific single domain antibodies

The Trop2 specific single domain antibody is obtained by immunizing alpaca with human Trop2 protein extracellular domain (TR 2-H5223S; acrobiosystems). The novel Trop 2-specific monovalent single domain antibodies T4, T5, RT4 and RT5 were expressed recombinantly according to the methods previously published by the inventors (see patent No. ZL202011131233.7 entitled "molecular imaging probe for diagnosis of multiple myeloma" which is incorporated herein by reference in its entirety). The amino acid sequence of the single domain antibody T4 is shown as SEQ ID NO.4 (wherein the CDR1 sequence is GLPYERYC (SEQ ID NO. 1), the CDR2 sequence is ILSDGTT (SEQ ID NO. 2), the CDR3 sequence is AAEAFRPFTPSDGDCTTVLGIDY (SEQ ID NO. 3)), and the gene sequence is shown as SEQ ID NO. 5; the amino acid sequence of the single-domain antibody T5 is shown as SEQ ID NO.6, and the gene sequence is shown as SEQ ID NO. 7; the amino acid sequence of the single domain antibody RT4 is shown as SEQ ID NO.8, and the gene sequence is shown as SEQ ID NO. 9; the amino acid sequence of the single domain antibody RT5 is shown as SEQ ID NO.10, and the gene sequence is shown as SEQ ID NO. 11.

SDS-PAGE is used for determining the expression of the single domain antibodies T4 and T5, and the specific steps are as follows: firstly, preparing a 1.5mm thick gel, namely 15well gel according to a SDS-PAGE gel kit method, preheating a metal bath to 100 ℃, and heating a protein sample containing loading buffer (5X) for 5min; after SDS-PAGE gel is assembled, adding 500ml of 1x SDS-PAGE buffer, slowly spotting a protein sample into the upper sample hole, carrying out constant-voltage electric bath for about 30min at 80V, adjusting the voltage to 120V after a bromophenol blue indicator passes through a concentrated gel, carrying out electrophoresis to the bottom of the gel, taking the gel out, heating and dyeing in a coomassie blue dye solution for 50min, taking out, decoloring the decolored solution until the background is clean, and carrying out photographing when the strip is clear. The expression of the single domain antibodies T4, T5 is shown in FIGS. 1A and 1D. The expression of the single domain antibodies RT4, RT5 is shown in FIGS. 5A and 5D.

The results of HPLC assay for single domain antibodies T4, T5 are shown in fig. 1B and 1E. The binding affinity results of SPR detection single domain antibodies T4, T5 and targets are shown in FIG. 1C and FIG. 1F, K _D The values were 965.9pM and 699.6pM, respectively.

The results of HPLC determination of the single domain antibodies RT4, RT5 are shown in FIGS. 5B and 5E. The binding affinity results of SPR detection single domain antibodies RT4, RT5 and target are shown in FIG. 5C and FIG. 5F, K _D The values were 915.7pM and 846.7pM, respectively.

Example 2: preparation of Trop2 specific single-domain antibody probe

1) (+ -.) -H3RESCA-TFP modification T4, T5, RT4 and RT5 intermediates H3RESCA-T4, H3RESCA-T5, H3RESCA-RT4 and H3RESCA-RT5 were prepared. The method comprises the following specific steps: with 4X 5mL of 0.05M NaHCO ₃ The solution pre-equilibrates the PD-10 column. 1mg of the single domain antibody (T4, T5, RT4, RT 5) solution was applied to PD-10 column, 0.05M NaHCO was added ₃ The solution was replenished to a volume of 2.5mL. Using 0.05M NaHCO ₃ The solution was eluted and 2.5mL of eluate was collected. 12-fold antibody molar amounts of (+ -) -H3RESCA-TFP (CAS number:1919794-40-3; having the chemical formula shown in FIG. 10) were weighed and dissolved in 40. Mu.L of DMSO. And (3) respectively adding (+/-) -H3RESCA-TFP solution into the single-domain antibody solution, and then fully homogenizing. The reaction was allowed to proceed for 2 hours at room temperature on a shaker. With 4X 5mL of 0.1MCH ₃ COONH ₄ The solution pre-equilibrates the PD-10 column. The reaction solution was applied to a PD-10 column using 0.1M CH ₃ COONH ₄ Eluting the solution and collecting3mL of eluate was pooled. The centrifugal filter was rinsed with DD water. And concentrated using an Amicon ultracentrifuge filter, and the concentrations of the products (H3 RESCA-T4, H3RESCA-T5, H3RESCA-RT4 and H3RESCA-RT 5) were determined using NanoDrop.

2) ¹⁸ F marks H3RESCA-T4, H3RESCA-T5, H3RESCA-RT4 and H3RESCA-RT5 are prepared by the following specific steps: the QMA column was activated with 5mL of sterile injectable water-5 mL of air-0.9% 5mL of physiological saline-5 mL of air in sequence. 500 mu L of the accelerator ¹⁸ Enrichment of F ions [ ¹⁸ O]The water was passed through a QMA column, and the filtrate was discarded, 500. Mu.l of 0.9% physiological saline was passed through the QMA column, and the filtrate was collected to measure the activity as 24mCi. mu.L of 2mM AlCl was added ₃ (in 0.1M CH ₃ COONH ₄ ) The solution was allowed to stand for 5 minutes. 160. Mu.L (7.6 mCi) ¹⁸⁺¹⁹ F-/Al3+ solution, 150. Mu.L (200. Mu.g) intermediate, 690. Mu.L 0.1M CH ₃ COONH ₄ The solutions were mixed and reacted at room temperature for 12 minutes. During this time, the PD-10 column was pre-equilibrated with 4X 5mL of 0.9% NaCl eluent (pH 5.9-6.1) containing 5mg/mL ascorbic acid. The reaction solution was added to PD-10, the eluate was used to replenish to 2.5mL, and then 0.5mL each time was added for elution, and the products were measured separately for a total of 5 tubes (i.e. [ i.e. ¹⁸ F]F-H3RESCA-T4、[ ¹⁸ F]F-H3RESCA-T5、[ ¹⁸ F]F-H3RESCA-RT4 [ ¹⁸ F]F-H3RESCA-RT 5).

3)[ ¹⁸ F]F-H3RESCA-T4、[ ¹⁸ F]Quality control of F-H3RESCA-RT 4. The method comprises the following specific steps: suction of small amounts using capillary glass tubes ¹⁸ F]F-H3RESCA-T4、[ ¹⁸ F]F-H3RESCA-RT4 was spotted on a silica gel plate using physiological saline as the mobile phase, and using a radioactive thin layer chromatograph (Radio-TLC, eckert)&Ziegler Radiopharma Inc) the radiochemical purity of the probe was determined (Radiochemical purity, RCP). Freshly prepared [ as shown in FIG. 2 ] ¹⁸ F]The RCP of F-H3RESCA-T4 is more than 99%. Freshly prepared [ as shown in FIG. 6 ] ¹⁸ F]The RCP of F-H3RESCA-RT4 is more than 99%.

Example 3: [ ¹⁸ F]F-H3RESCA-T4、[ ¹⁸ F]Imaging of F-H3RESCA-RT4 in T3M-4 tumor-bearing Balb/c mice

1) And (5) establishing a Trop2 expression positive tumor-bearing mouse model. Tool withThe method comprises the following steps: will be 2X 10 ⁶ T3M-4 cells were suspended in a mixture of PBS and matrigel (Corning) (1:1 ratio) and injected into the left shoulder of 4-5 week old Balb/c nude mice to model subcutaneous pancreatic cancer (i.e., T3M-4 tumor-bearing Balb/c mice).

2)[ ¹⁸ F]Imaging of F-H3RESCA-T4 in T3M-4 tumor-bearing Balb/c mice. The method comprises the following specific steps: the PET/CT imaging acquisitions of the animals involved in this study were all done using an IRIS small animal PET/CT scanner (Inviscan Imaging Systems). Mice were divided into T4-blocked and unblocked groups, T4-blocked groups were injected [ ¹⁸ F]F-H3RESCA-T4 was injected with unlabeled T4 (20 mg/kg) and the non-blocked group was not injected with T4. Each mouse was injected via the tail vein with 3.7-7.4MBq [ ¹⁸ F]F-H3RESCA-T4 (4 in each group), mice were anesthetized with isoflurane (concentration 2%) at 0.5 hours after injection, and mice entered into a deep anesthesia state were placed on a PET/CT scanning bed in a supine position, PET and CT images were continuously acquired, image reconstruction was completed with the IRIS system self-contained software, the heart and main tissue organs (liver, lung, kidney, bone, muscle) and other regions of interest (Region of interest, ROI) were delineated on the reconstructed PET images with an OsiriX Lite image processing workstation (Pixmeo SARL), the radioactive uptake values of the important tissue organs were calculated in units of% ID/g (percent of injected dose per gram), and the main tissue organ uptake values were plotted. As shown in fig. 3A and 3B, the probe is excreted mainly through the urinary system and is taken up higher at the tumor site. In addition, the in vitro biodistribution test results (shown in FIG. 3C) further revealed the distribution of the probes in the major tissue organs in vivo. And by comparing the ROI data and biodistribution data of tumor model imaging of the unblocked group (FIG. 3A, FIG. 3B, FIG. 3C) and the T4 blocked group (FIG. 3D, FIG. 3E, FIG. 3F), the binding specificity of T4 to the tumor cell surface target was shown [ ¹⁸ F]The F-H3RESCA-T4 probe can realize noninvasive visualization of Trop2 expression.

3) Immunohistochemical staining was performed using Trop 2-specific antibody (sc-376746,Santa Cruz Biotechnology), and staining results of the unblocked group and the T4 blocked group are shown in fig. 4A and 4B, confirming the expression of Trop2 in tumor tissues.

5)[ ¹⁸ F]Imaging of F-H3RESCA-RT4 in T3M-4 tumor-bearing Balb/c mice. The method comprises the following specific steps: the PET/CT imaging acquisitions of the animals involved in this study were all done using an IRIS small animal PET/CT scanner (Inviscan Imaging Systems). Mice were divided into RT 4-blocked and unblocked groups, with RT 4-blocked on injection [ ¹⁸ F]F-H3RESCA-RT4 was injected simultaneously with unlabeled RT4 (20 mg/kg), while the non-blocked group was not injected with T4. Each mouse was injected via the tail vein with 3.7-7.4MBq [ ¹⁸ F]F-H3RESCA-RT4 (4 in each group), mice were anesthetized with isoflurane (concentration 2%) at 45min and 2.5 hours after injection, and the mice entering the deep anesthesia state were placed on a PET/CT scanning bed in a supine position, PET and CT images were continuously acquired, image reconstruction was completed with the IRIS system self-contained software, the image of the heart and major tissue organs (liver, lung, kidney, bone, muscle) were delineated on the reconstructed PET images with an OsiriX Lite image processing workstation (Pixmeo SARL) and other regions of interest (Region of interest, ROI), the radioactive uptake values of the major tissue organs were calculated in units of% ID/g (percent of injected dose per gram), and the major tissue organ uptake values were plotted over time. The PET/CT scan results and the ROI results are shown in FIG. 7, and the results show that in the unblocked group, the 2.5h renal uptake value is lower than 45min renal uptake and the probe is excreted through the kidney. In addition, tumor uptake values were higher and remained stable at both time points, with no tendency to decrease uptake. The in vitro biodistribution results are shown in fig. 8, and the results show that the tumor uptake of the RT4 blocked group is significantly lower than that of the unblocked group, and the same result as the ROI delineation result.

6)[ ¹⁸ F]F-H3RESCA-T4 and [ ¹⁸ F]The result of the ROI delineation of F-H3RESCA-RT4 is compared with the tumor/organ ratio, as shown in FIG. 9. [ ¹⁸ F]Tumor uptake of F-H3RESCA-RT4 was lower than [ ¹⁸ F]F-H3RESCA-T4, but no statistical differences. And [ t ] ¹⁸ F]The ratio of F-H3RESCA-RT4 tumor/liver, tumor/kidney, tumor/bone and tumor/muscle is better than that of [ ¹⁸ F]F-H3RESCA-T4. The above data indicate that ¹⁸ The F-labeled anti-Trop 2 single-domain antibody probe is an in-vivo noninvasive diagnosis and treatment tool with certain potential for targeting Trop 2.

The invention is to be used for preparing [ ¹⁸ F]F-H3RESCA-T5 and [ ¹⁸ F]F-H3RESCA-RT5 also has an effect on Balb/c tumor-bearing mice ¹⁸ F]F-H3RESCA-T4 and [ ¹⁸ F]F-H3RESCA-RT4 similar PET imaging results are also in-vivo noninvasive diagnosis and treatment tools with certain potential for targeting Trop 2.

It should be noted that the description of the present invention and the accompanying drawings illustrate preferred embodiments of the present invention, but the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, which are not to be construed as additional limitations of the invention, but are provided for a more thorough understanding of the present invention. The above-described features are further combined with each other to form various embodiments not listed above, and are considered to be the scope of the present invention described in the specification; further, modifications and variations of the present invention may be apparent to those skilled in the art in light of the foregoing teachings, and all such modifications and variations are intended to be included within the scope of this invention as defined in the appended claims.

Claims

1. A Trop 2-specific single domain antibody comprising:

2. The Trop 2-specific single domain antibody of claim 1, wherein said Trop 2-specific single domain antibody has the amino acid sequence set forth in SEQ ID No.4, 6, 8 or 10.

3. A polynucleotide encoding a Trop 2-specific single domain antibody according to claim 1 or 2;

preferably, the polynucleotide has the nucleotide sequence shown in SEQ ID No.5, 7, 9 or 11.

4. A vector comprising the polynucleotide of claim 3.

5. A host cell comprising the vector of claim 4.

6. Human Trop2 specificity ¹⁸ F-labeled monovalent single domain antibody probe, characterized in that it comprises a radionuclide-labeled Trop 2-specific single domain antibody according to claim 1 or 2.

7. A human Trop 2-specific according to claim 6 ¹⁸ F-labeled monovalent single-domain antibody probe, characterized in that Trop 2-specific single-domain antibodies according to claim 1 or 2 are labeled with radionuclides via bifunctional chelators.

Preferably, the bifunctional chelating agent is selected from at least one of (+ -) -H3RESCA-TFP, (+ -) H3RESCA-Mal,

preferably, the radionuclide is selected from F-18.

8. A human Trop 2-specific according to claim 7 ¹⁸ F-labeled monovalent single domain antibody probe, characterized in that Trop 2-specific single domain antibody having the amino acid sequence shown in SEQ ID No.8 according to claim 2 is labeled with F-18 via (+ -) -H3 RESCA-TFP.

9. A kit or composition for visualizing Trop2 expression, diagnosing Trop 2-associated tumors, predicting the progression and prognosis of Trop 2-associated tumors, predicting the therapeutic effect of Trop 2-associated tumors or treating Trop 2-associated tumors, characterized by comprising a Trop 2-specific single domain antibody according to claim 1 or 2, a polynucleotide according to claim 3, a vector according to claim 4, a host cell according to claim 5, a probe according to any one of claims 6-8.

10. Use of a Trop 2-specific single domain antibody according to claim 1 or 2, a polynucleotide according to claim 3, a vector according to claim 4, a host cell according to claim 5 or a probe according to any one of claims 6-8 for the preparation of a kit or composition for visualizing Trop2 expression, diagnosing Trop 2-related tumors, predicting the progression and prognosis of Trop 2-related tumors, predicting the therapeutic effect of Trop 2-related tumors or treating Trop 2-related tumors.