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

Abstract

The present invention relates to the field of molecular imaging, nuclear medicine and single-domain antibody technology for tumor diagnosis and treatment, and in particular to a method for preparing a radionuclide-labeled Trop2-specific single-domain antibody probe and its application. The Trop2-specific single-domain antibody has a CDR1 having an amino acid sequence shown in SEQ ID No.1, a CDR2 having an amino acid sequence shown in SEQ ID No.2, and a CDR3 having an amino acid sequence shown in SEQ ID No.3. The Trop2-specific single-domain antibody prepared by the present invention 18 The F-labeled single-domain antibody probe is used for immuno-PET imaging, which realizes the non-invasive visualization of Trop2 in vivo and further realizes the non-invasive diagnosis of Trop2-positive tumors. The probe has the advantages of simple preparation process, low cost, high specificity, high stability, short imaging cycle, low radiation dose, and easy clinical transformation.

Description

Technical Field

[0001] The present invention relates to the technical fields of molecular imaging, nuclear medicine and single-domain antibodies for tumor diagnosis and treatment, and in particular to a method for preparing a radionuclide-labeled Trop2-specific single-domain antibody probe and its application. Background Art

[0002] In 1993, Belgian scientists Hamers et al. reported for the first time in Nature magazine that there is a naturally missing light chain antibody in the peripheral blood of alpacas (Nature. 1993; 363 (6428): 446-8.). This antibody with a special structural domain is a heavy chain antibody (Heavy-chain antibodies, HCAbs). Through molecular biological methods, cloning the variable region of the heavy chain antibody can obtain an antigen-binding fragment with only the heavy chain variable region, which is a single domain antibody (VHH, Variable Domain of Heavy Chain of Heavy Chain Antibody). The VHH crystal is 2.5nm wide, 4nm long, and has a molecular weight of only 15kDa, so it is also called a single domain antibody ( A registered trade name of Ablynx.) Single-domain antibodies are the smallest antibody units known to bind to target antigens. They have the advantages of high affinity, small molecular weight, low preparation cost (can be expressed in E. coli, yeast, Chinese hamster ovary cells and other eukaryotic expression systems), and easy clinical transformation and promotion and application.

[0003] Single-domain antibodies are popular targeting vectors for constructing molecular imaging probes in recent years (Theranostics. 2014; 4(4):386-98.; J Nucl Med. 2022 Oct; 63(10):1705-1709.). Currently, a variety of short-half-life radionuclides have been used to label single-domain antibodies and prepare single-domain antibody molecular imaging probes. Technetium-99m ( 99m Tc; T 1 / 2 =6.02h) labeled with a single-domain antibody probe targeting programmed death ligand 1 (PD-L1) has been successfully translated into clinical practice for non-invasive diagnosis of patients with non-small cell lung cancer (J Nucl Med. 2019; 60(9): 1213-1220.); Gallium-68 ( 68 Ga; T 1 / 2=1.1h) labeled single-domain antibody probe targeting human epidermal growth factor receptor (HER2) has also been successfully transformed into clinical practice for non-invasive diagnosis of breast cancer (J Nucl Med. 2016; 57(1): 27-33.). The above examples show that radionuclide labeled single-domain antibody probes have great clinical application prospects and can be used for early non-invasive diagnosis of human malignant tumors, visualization of key pathogenic targets, screening of patients for monoclonal antibody (mAb) treatment, and evaluation of the efficacy of monoclonal antibody treatment.

[0004] Trophoblast cell surface antigen 2 (Trop2) is a cell membrane surface glycoprotein, which is formed by a 36kDa nascent polypeptide through N-linked glycosylation translation modification. It regulates tumor proliferation, invasion and migration through multiple signal transduction pathways and plays a role in stem cell biology. In a retrospective study of 197 paraffin-embedded pancreatic cancer primary tumor tissues, the expression of Trop2 antigen was analyzed. Immunohistochemistry results showed that 55% of the antigen was overexpressed, which was significantly associated with lymph node metastasis, poor tumor differentiation and poor prognosis (P<0.05). The expression of Trop2 is also associated with the biological invasiveness and poor prognosis of malignant tumors such as gastric cancer, female reproductive system tumors, prostate cancer and colorectal cancer, indicating that it promotes the occurrence and development of tumors. The difference in expression of Trop2 in normal tissues and tumors makes it a highly potential tumor-specific marker and can avoid potential side effects. At present, antibody-drug conjugate drugs targeting Trop2 have entered clinical trials. Therefore, there is an urgent need to develop a diagnostic tool targeting Trop2 to achieve non-invasive visualization and monitoring of Trop2 expression in solid tumors. Based on the research of companion diagnostic tools, new therapeutic methods targeting Trop2 can also be further developed.

[0005] The applicant's previous series of basic and clinical studies have shown that by cleverly combining the extraordinary targeting specificity of antibodies and the superior sensitivity and resolution of positron emission tomography (PET), immuno-PET can better display the distribution and abundance of targets of interest in the body, especially heterogeneous expression, and better predict the response to targeted therapy or immunotherapy compared to immunohistochemistry (IHC) or other traditional predictive markers (Chem Rev. 2020; 120 (8): 3787-3851.). For example, the value of immuno-PET imaging probes targeting human epidermal growth factor receptor 2 in breast cancer has been clinically verified. Based on the above evidence and our previous findings, we hypothesize that immuno-PET imaging probes targeting Trop2 can non-invasively display Trop2 expression in tumors and provide a better method for the diagnosis and monitoring of Trop2-positive solid tumors. In addition, there is evidence that radioimmunotherapy (RIT) and pre-targeted radioimmunotherapy (pRIT) may help cancer patients to relieve their condition for a long time and even eradicate various types of cancer.

[0006] At present, there are no reports of Trop2-specific single-domain antibody molecular imaging probes or radionuclide-labeled therapeutic probes in clinical practice and literature. There are reports of molecular imaging probes based on Trop2 monoclonal antibodies (Eur J Nucl Med Mol Imaging. 2022 Feb; 49(3): 861-870.) and radionuclide-labeled therapeutic probes (Eur J Nucl Med Mol Imaging. 2022 Dec; 50(1): 168-183.). However, the application of radiolabeled monoclonal antibodies is severely hindered by high costs, the necessity of using long half-life radionuclides, cumbersome imaging processes within a week, and related radiation exposure. In order to improve the clinical application of antibody diagnosis, the field of molecular imaging is actively exploring pre-targeted imaging strategies or using antibody derivatives with smaller molecular weights to achieve same-day molecular imaging. Among small antibody forms, single-domain antibodies from camelids are the smallest antigen-binding moieties with a molecular weight of approximately 15 kDa. Small size, high affinity and easy engineering design make single-domain antibodies an excellent alternative for molecular imaging (J Nucl Med. 2022 Oct; 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. At present, there are no Trop2-specific single-domain antibody molecular imaging probes at home and abroad. Summary of the invention

[0007] To fill the gap in this field, we describe here the construction of a Trop2-targeted diagnostic and therapeutic pair derived from a single-domain antibody, and characterize its diagnostic and therapeutic value in a cell-derived xenograft (CDX) model. Therefore, those skilled in the art are committed to developing a single-domain antibody immunoPET imaging probe with low preparation cost, small molecular weight, short in vivo circulation time, short imaging cycle, low radiation dose, and easy clinical transformation application. Specifically, the present invention provides a method for preparing a radionuclide-labeled Trop2-specific single-domain antibody probe and its application.

[0008] Trop2-specific single domain antibody

[0009] In one aspect, the present invention provides a Trop2-specific single domain antibody comprising:

[0010] CDR1 has the amino acid sequence shown in SEQ ID No.1, CDR2 has the amino acid sequence shown in SEQ ID No.2, and CDR3 has the amino acid sequence shown in SEQ ID No.3.

[0011] More specifically, the Trop2-specific single domain antibody of the present invention has the amino acid sequence shown in SEQ ID No. 4, 6, 8 or 10.

[0012] In the present invention, for the sake of simplicity, the Trop2-specific single domain antibody having the amino acid sequence shown in SEQ ID No. 4, 6, 8 or 10 is referred to as T4, T5, RT4 or RT5, respectively.

[0013] 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. Nanobody has the meaning generally understood by those skilled in the art, and refers to an antibody fragment composed of a single monomer variable antibody domain (e.g., a single heavy chain variable region), usually 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, with a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N-terminus or C-terminus so that they contain only part of FR1 and / or FR4, or lack one or two of those framework regions, as long as they substantially maintain antigen binding and specificity.

[0014] In some embodiments, the present invention also encompasses antigen-binding fragments of the Trop2-specific single domain antibodies as described herein.

[0015] 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 the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of the antibodies of the invention can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of the Nanobodies of the invention. In some embodiments, the "antigen-binding fragment" of the Nanobody may be truncated at the N-terminus or C-terminus compared to the full-length Nanobody so that it comprises only part of FR1 and / or FR4, or lacks one or two of those framework regions, as long as it substantially retains antigen binding and specificity.

[0016] Antigen-binding fragments of Nanobodies can be obtained from a given Nanobody (e.g., the Nanobodies provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and antigen-binding fragments of Nanobodies can be screened for specificity in the same manner as for intact Nanobodies.

[0017] In this document, unless the context clearly indicates otherwise, when referring to the term "Nanobody", it includes not only complete Nanobodies but also antigen-binding fragments of Nanobodies.

[0018] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. In nanobodies, there are three CDRs, named CDR1, CDR2 and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as 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, a person skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).

[0019] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0020] As used herein, the term "Trop2-specific" refers to specific binding to Trop2.

[0021] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D In the present invention, the term "K D ” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0022] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate at which the antigen binding site / antigen complex forms and dissociates. The "binding rate constant" (ka or k on ) and the “dissociation rate constant” (k dis or k off ) Both can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). dis / k on The ratio 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 In some embodiments, the dissociation constant can be measured using surface plasmon resonance (SPR) in Biacore. In addition, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0023] In some embodiments, the present invention also provides a variant of a Trop2-specific single domain antibody as described herein, which has 70-100% sequence identity with the amino acid sequence shown in SEQ ID No. 4, 9, 14 or 19, such as at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, and substantially retains the biological function of the Nanobody from which it is derived (e.g., the biological activity of specifically binding to Trop2).

[0024] More specifically, the variants differ from the Trop2-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) amino acid residues.

[0025] As used herein, the term "identity" is used to refer to the matching 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 the two DNA molecules is occupied by adenine, or a position in each of the 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 matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0026] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). 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 a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions 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).

[0027] Polynucleotide

[0028] In another aspect, the present invention also provides a polynucleotide encoding the above-mentioned Nanobody or its antigen-binding fragment.

[0029] More specifically, the polynucleotide has a nucleotide sequence shown in SEQ ID No. 5, 7, 9 or 11.

[0030] More specifically, the polynucleotide encoding the Trop2-specific single domain antibody as described herein has a nucleotide sequence shown in SEQ ID No. 5, 7, 9 or 11.

[0031] The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0032] The term "polynucleotide encoding a polypeptide / protein / antibody" may include a polynucleotide encoding the polypeptide / protein / antibody, or may also include additional coding and / or non-coding sequences.

[0033] The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% identity between the two sequences, and the polypeptides / proteins / antibodies encoded by them have substantially the same functions and activities. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, more preferably 95%.

[0034] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

[0035] Carrier

[0036] In another aspect, the present invention also provides a vector comprising a polynucleotide encoding the above-mentioned Trop2-specific single domain antibody or an antigen-binding fragment thereof.

[0037] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can 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 initiation site.

[0038] Host cells

[0039] In another aspect, the present invention also provides a host cell comprising the vector as described herein.

[0040] As used herein, the term "host cell" refers to cells that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or other human cells. Host cells may include a single cell or a cell population.

[0041] The vector can be introduced into the host cell using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0042] The Nanobodies of the invention can be used alone or in combination or coupled to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety or a combination of any of these.

[0043] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing a detectable product. Preferred detectable labels are radionuclides.

[0044] Therapeutic agents that can be combined or coupled to the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0045] 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., preferably 18 F or 68 Ga labeling is used for non-invasive diagnosis of tumors. 225 Ac, 211 At 212 Pt or 131 I labeling is used for tumor radioimmunotherapy.

[0046] The binding or coupling of a detectable marker or therapeutic agent to an antibody can be carried out by conventional methods well known to those skilled in the art. For example, a detectable marker can be directly or indirectly bound to a nanobody, for example, by a cleavable or non-cleavable linker peptide, or incorporated into a nanobody. The detectable marker can be bound to a nanobody, in particular, by replacement (for example, by replacing H with I at the level of a tyrosine residue), by complexing or by chelation. For example, a therapeutic agent can be conjugated to a nanobody via a cleavable linker (for example, a peptidyl, a disulfide or a hydrazone linker).

[0047] In a preferred embodiment, as described in more detail below, the Nanobodies of the invention are conjugated to a radionuclide for use as Trop2-specific molecular imaging probes.

[0048] Trop2-specific molecular imaging probe

[0049] In another aspect, the present invention provides a human Trop2-specific 18 The F-labeled monovalent single domain antibody probe comprises a Trop2-specific single domain antibody as described herein that is labeled with a radionuclide.

[0050] More specifically, the Trop2-specific single domain antibodies as described herein are radionuclide-labeled via a bifunctional chelator.

[0051] As used herein, a bifunctional chelator is a type of chelator having both a metal chelating end and a protein anchoring end. The bifunctional chelator can be selected from at least one of (±)-H3RESCA-TFP and (±)H3RESCA-Mal.

[0052] As used herein, the (±)-H3RESCA-TFP is a tetrafluorophenyl ester derivative of a constrained complexing agent (RESCA) that can be used to couple the chelator to biomolecules via amine coupling (e.g., the N-terminus and / or the ε-amino group of lysine);

[0053] The (±)H3RESCA-Mal is (±)H3RESCA-maleimide, a tetrafluorophenyl ester derivative of a constrained complexing agent (RESCA), which can be used to couple the chelator to biomolecules via amine coupling (e.g., N-terminus and / or ε-amino group of lysine).

[0054] More specifically, the Trop2-specific single domain antibodies as described herein are radionuclide-labeled via (±)-H3RESCA-TFP or (±)H3RESCA-Mal.

[0055] More particularly, the radionuclide is selected from F-18.

[0056] 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.

[0057] The radionuclide fluorine-18 ( 18 F; T 1 / 2 = 110min) has nearly ideal nuclear decay characteristics and a half-life of 68Ga is longer and is more suitable for radiolabeling of antibody-derived fragments such as single-domain antibodies. Traditional radiofluorination strategies have harsh and cumbersome conditions, such as reaction in organic solvents, high temperature, and time-consuming synthesis steps (Nat Protoc. 2018 Oct; 13 (10): 2330-2347.). The radiolabeling method involved in this patent has the following advantages: room temperature reaction, short labeling process (30 min), and high product radiochemical purity (> 99%).

[0058] More specifically, the human Trop2 specific 18 F-labeled monovalent single-domain antibody probe is [ 18 F]F-H3RESCA-RT4, a Trop2-specific single domain antibody having an amino acid sequence as shown in SEQ ID No. 8, is labeled with F-18 via (±)-H3RESCA-TFP. It has lower kidney enrichment, lower liver enrichment, lower bone enrichment and lower muscle tissue enrichment, and thus has better diagnostic efficacy.

[0059] In another aspect, the present invention also provides a method for inhibiting the specificity of human Trop2. 18 The invention discloses a method for labeling a monovalent single-domain antibody probe with F, comprising modifying a Trop2-specific single-domain antibody with a bifunctional chelator to obtain a radionuclide-labeled precursor; and labeling the radionuclide-labeled precursor with a radionuclide to obtain a human Trop2-specific probe. 18 F labels the monovalent single domain antibody probe.

[0060] More specifically, when the bifunctional chelator is (±)H3RESCA-Mal, before preparing the radionuclide labeled precursor, a cysteine-containing peptide segment (GGGGS)nC needs to be expressed at the end of the Trop2-specific single domain antibody, where n=1-10; specifically, n=1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0061] Composition

[0062] In another aspect, the present invention provides a composition comprising a Trop2-specific single domain antibody, polynucleotide, vector, host cell or molecular imaging probe as described herein. The composition can be used to detect the expression level of Trop2, diagnose Trop2-related tumors, predict the treatment effect of Trop2-related tumors or treat Trop2-related tumors.

[0063] In some embodiments, the composition may be a pharmaceutical composition.

[0064] In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier and / or excipient.

[0065] In some embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent.

[0066] In some embodiments, the additional pharmaceutically active agent is an anti-inflammatory drug or an immunosuppressant.

[0067] In some embodiments, in the pharmaceutical composition, the Trop2-specific single domain antibody, polynucleotide, vector, host cell or molecular imaging probe as described herein and the additional pharmaceutically active agent can be provided as separate components or as mixed components. Therefore, the Trop2-specific single domain antibody, polynucleotide, vector, host cell or molecular imaging probe as described herein and the additional pharmaceutically active agent can be administered simultaneously, separately or sequentially.

[0068] 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 water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0069] The pharmaceutical composition of the present invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of a Trop2-specific single domain antibody, polynucleotide, vector, host cell or molecular imaging probe as described herein. A "prophylactically effective amount" refers to an amount sufficient to prevent, prevent or delay the occurrence of a disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount may vary depending on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and gender, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0070] Reagent test kit

[0071] The present invention also provides a kit comprising the Trop2-specific single domain antibody, polynucleotide, vector, host cell or molecular imaging probe as described herein.

[0072] The kit can be used to detect the expression level of Trop2, diagnose Trop2-related tumors, predict the treatment effect of Trop2-related tumors, or treat Trop2-related tumors.

[0073] The kit may also include a container, instructions for use, and other reagents and buffers required for practical applications, such as a lysis medium for dissolving the sample, various buffers, detection labels, detection substrates, etc.

[0074] Diagnostic and therapeutic applications

[0075] The Trop2-specific single-domain antibody of the present invention has extremely high affinity for Trop2, and thus can be used to detect the expression level of Trop2, diagnose Trop2-related tumors, predict the therapeutic effect of Trop2-related tumors, or treat Trop2-related tumors.

[0076] In particular, the Trop2-specific molecular imaging probe prepared from the Trop2-specific single domain antibody of the present invention has the characteristics of significantly improved affinity, significantly reduced nonspecific uptake of normal tissues, and significantly improved image quality, and can be used for non-invasive, accurate, and efficient detection of human Trop2 expression, and is therefore particularly suitable for diagnosing Trop2-related tumors and predicting the therapeutic effect of Trop2-related tumors. After selecting a suitable radionuclide for coupling, it can also be used for accurate treatment of Trop2-related tumors.

[0077] Therefore, in another aspect, the present invention also relates to the use of the Trop2-specific single domain antibody, polynucleotide, vector, host cell or molecular imaging probe as described herein in the preparation of a kit or drug for detecting the expression level of Trop2, diagnosing Trop2-related tumors, predicting the treatment effect of Trop2-related tumors or treating Trop2-related tumors.

[0078] As used herein, Trop2-related tumors may include various tumors or cancers well known in the art. For example, Trop2-related tumors may include breast cancer, lung cancer, gastric cancer, pancreatic cancer, colon cancer, prostate cancer, cervical cancer, etc.

[0079] Beneficial effects of the present invention

[0080] 1) The Trop2-specific molecular probe prepared by the present invention is used for immunoPET imaging, which realizes the non-invasive visualization of human Trop2 molecule expression and further realizes the non-invasive diagnosis of Trop2-positive tumors (such as pancreatic cancer).

[0081] 2) The probe prepared by the present invention has the advantages of simple preparation process, low cost, high specificity, high stability, short imaging cycle, low radiation dose, and easy clinical transformation. With clinical transformation application, it is expected to realize non-invasive visualization of Trop2 heterogeneous expression, screen patients with high Trop2 expression, and further carry out Trop2-specific radioimmunotherapy, thereby realizing the integration of target-specific diagnosis and treatment of Trop2-positive tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 The results of SDS-PAGE and HPLC determination of the expression of single-domain antibodies T4 and T5 and SPR detection of the binding affinity of single-domain antibodies to targets are shown; Figure 1 A is the SDS-PAGE result of T4; Figure 1 B is the HPLC result of T4; Figure 1 C is the binding affinity result of T4 and the target; Figure 1 D is the SDS-PAGE result of T5; Figure 1 E is the HPLC result of T5; Figure 1 F is the binding affinity result of T5 and target;

[0083] Figure 2 Shows [ 18 F] Results of radiochemical purity determination of F-H3RESCA-T4 before and after purification;

[0084] Figure 3 Shows [ 18 F] PET / CT imaging, ROI analysis and in vitro biodistribution results of F-H3RESCA-T4 in T3M-4 tumor-bearing Balb / c mice 30 minutes after injection; Figure 3 A is the PET / CT imaging results of the unblocked group; Figure 3 B is the ROI analysis result of the unblocked group; Figure 3 C is the in vitro biodistribution result of the unblocked group; Figure 3 D is the PET / CT imaging result of the T4 closure group; Figure 3 E is the ROI analysis result of T4 closure group; Figure 3 F is the in vitro biodistribution results of the T4 blocking group;

[0085] Figure 4 Shows [ 18 F] Immunohistochemical staining of F-H3RESCA-T4 unblocked group and blocked group; Figure 4 A is the unclosed group; Figure 4 B is the closed group;

[0086] Figure 5The results of SDS-PAGE and HPLC determination of the expression of single-domain antibodies RT4 and RT5 and SPR detection of the binding affinity of single-domain antibodies to targets are shown; Figure 5 A is the SDS-PAGE result of RT4; Figure 5 B is the HPLC result of RT4; Figure 5 C is the binding affinity result of RT4 and the target; Figure 5 D is the SDS-PAGE result of RT5; Figure 5 E is the HPLC result of RT5; Figure 5 F is the binding affinity result of RT5 and target;

[0087] Figure 6 Shows [ 18 F] Results of radiochemical purity determination of F-H3RESCA-RT4 after purification;

[0088] Figure 7 Shows [ 18 F] PET / CT imaging and ROI analysis results of F-H3RESCA-RT4 in T3M-4 tumor-bearing Balb / c mice 45 minutes and 2.5 hours after injection; Figure 7 A is the PET / CT imaging results of the unblocked group; Figure 7 B is the ROI analysis result of the unblocked group; Figure 7 C is the PET / CT imaging results of the T4 closure group; Figure 7 D is the ROI analysis result of T4 closure group;

[0089] Figure 8 Shows [ 18 F] In vitro biodistribution results of F-H3RESCA-RT4 unblocked group and RT4 blocked group in T3M-4 tumor-bearing Balb / c mice;

[0090] Fig. 9 Shows [ 18 F]F-H3RESCA-T4 and [ 18 F] ROI delineation results and tumor / organ ratio comparison results of F-H3RESCA-RT4;

[0091] Fig.10 The chemical structure of the chelator (±)-H3RESCA-TFP is shown. DETAILED DESCRIPTION

[0092] For ease of understanding of the present invention, the present invention will be described in more detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the present invention is not limited to the ad hoc methods, schemes, cell lines, constructs and reagents described herein, and can be changed equally. Unless otherwise defined, all technical and scientific terms used in this specification are identical with the meanings generally understood by those skilled in the art belonging to the technical field of the present invention. The terms used in this specification in the specification of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention.

[0093] At present, there are no reports of Trop2-specific single-domain antibody molecular imaging probes and radionuclide-labeled integrated diagnostic and therapeutic probes in clinical practice and literature reports. There are reports of molecular imaging probes based on Trop2 monoclonal antibodies (Eur J Nucl Med Mol Imaging. 2022 Feb; 49 (3): 861-870.) and radionuclide-labeled integrated diagnostic and therapeutic probes (Eur J Nucl Med Mol Imaging. 2022 Dec; 50 (1): 168-183.). However, the clinical transformation application of monoclonal antibody immune PET imaging probes is severely limited by reasons such as high preparation cost, large molecular weight, long in vivo circulation time, long imaging cycle, high radiation dose, and large toxic side effects. In comparison, single-domain antibody immune PET imaging probes have the advantages of low preparation cost, small molecular weight, short in vivo circulation time, short imaging cycle, low radiation dose, and easy clinical transformation application.

[0094] The construction of a Trop2-specific integrated diagnostic and therapeutic single-domain antibody molecular imaging probe can non-invasively display the expression of Trop2 in tumors and provide a better method for the diagnosis and monitoring of Trop2-positive solid tumors. On this basis, we plan to further develop new therapeutic methods targeting Trop2.

[0095] Example 1: Preparation of Trop2-specific single domain antibodies

[0096] The Trop2-specific single-domain antibody of the present invention is obtained by immunizing alpacas with the extracellular domain of human Trop2 protein (TR2-H5223S; Acrobiosystems). According to the method previously published by the inventor (reference invention titled "A molecular imaging probe for diagnosing multiple myeloma" patent number ZL202011131233.7, which is incorporated herein by reference in its entirety), novel Trop2-specific monovalent single-domain antibodies T4, T5, RT4 and RT5 are recombinantly expressed. The amino acid sequence of the single-domain antibody T4 is shown in SEQ ID NO.4 (wherein the CDR1 sequence is GLPYERYC (SEQ ID NO.1), the CDR2 sequence is ILSDGTT (SEQ ID NO.2), and the CDR3 sequence is AAEAFRPFTPSDGDCTTVLGIDY (SEQ ID NO.3)), and the gene sequence is shown in SEQ ID NO.5; the amino acid sequence of the single-domain antibody T5 is shown in SEQ ID NO.6, and the gene sequence is shown in SEQ ID NO.7; the amino acid sequence of the single-domain antibody RT4 is shown in SEQ ID NO.8, and the gene sequence is shown in SEQ ID NO.9; the amino acid sequence of the single-domain antibody RT5 is shown in SEQ ID NO.10, and the gene sequence is shown in SEQ ID NO.11.

[0097] The expression of single-domain antibodies T4 and T5 was determined by SDS-PAGE. The specific steps are as follows: First, prepare a 1.5mm thick, 15well gel according to the method of the SDS-PAGE gel kit, preheat the metal bath to 100°C, and heat the protein sample containing loading buffer (5X) for 5 minutes; after the SDS-PAGE gel is assembled, add 1x SDS-PAGE buffer 500ml, slowly spot the protein sample into the loading well, and bathe at 80V constant voltage for about 30 minutes. After the bromophenol blue indicator passes over the concentrated gel, adjust the voltage to 120V, electrophoresed to the bottom of the gel, remove the gel, heat and stain it in Coomassie blue dye for 50 minutes, then take it out, and take pictures when the decolorization solution is decolorized until the background is clean and the bands are clear. The expression of single-domain antibodies T4 and T5 is shown in Figure 2. Figure 1 A and Figure 1 D. The expression of single domain antibodies RT4 and RT5 is shown in Figure 5 A and Figure 5 As shown in D.

[0098] The results of HPLC determination of single domain antibodies T4 and T5 are as follows Figure 1 B and Figure 1 E. The results of SPR detection of the binding affinity between single domain antibodies T4 and T5 and the target are shown in Figure 1 C and Figure 1 F, K DThe values ​​were 965.9 pM and 699.6 pM respectively.

[0099] The results of HPLC determination of single domain antibody RT4 and RT5 are as follows Figure 5 B and Figure 5 E. The results of SPR detection of the binding affinity between single domain antibodies RT4 and RT5 and the target are shown in Figure 5 C and Figure 5 F, K D The values ​​are 915.7 pM and 846.7 pM respectively.

[0100] Example 2: Preparation of Trop2-specific single domain antibody probe

[0101] 1) (±)-H3RESCA-TFP modified T4, T5, RT4 and RT5 to prepare intermediates H3RESCA-T4, H3RESCA-T5, H3RESCA-RT4 and H3RESCA-RT5. The specific steps are as follows: Pre-equilibrate the PD-10 column with 4×5mL 0.05M NaHCO3 solution. Add 1mg of single-domain antibody (T4, T5, RT4, RT5) solution to the PD-10 column, and add 0.05M NaHCO3 solution to make up the volume to 2.5mL. Elute with 0.05M NaHCO3 solution and collect 2.5mL of eluate. Weigh 12 times the molar amount of antibody (±)-H3RESCA-TFP (CAS number: 1919794-40-3; which has the following characteristics: Fig.10 Chemical structure shown) was dissolved in 40 μL DMSO. After adding (±)-H3RESCA-TFP solution to the above single-domain antibody solution, mix thoroughly. Place on a shaker at room temperature for 2 hours. Pre-equilibrate the PD-10 column with 4×5mL 0.1MCH3COONH4 solution. Add the reaction solution to the PD-10 column, elute with 0.1M CH3COONH4 solution, and collect 3mL of eluate. Rinse the centrifugal filter with DD water. Concentrate using an Amicon ultracentrifugal filter, and use NanoDrop to measure the concentration of the product (H3RESCA-T4, H3RESCA-T5, H3RESCA-RT4 and H3RESCA-RT5).

[0102] 2) 18 The specific steps for the preparation of F-labeled H3RESCA-T4, H3RESCA-T5, H3RESCA-RT4 and H3RESCA-RT5 are as follows: 5 mL of sterile water for injection-5 mL of air-0.9% 5 mL of saline-5 mL of air are used to activate the QMA column. 500 μL of the column containing 18 F ion enrichment 18O] water passed through the QMA column, discarded the filtrate, took 500μl 0.9% saline to pass through the QMA column, collected the filtrate, and measured the activity to be 24mCi. Add 8μL 2mM AlCl3 (in 0.1M CH3COONH4) solution and let it stand for 5 minutes. Add 160μL (7.6mCi) 18+19 F– / Al3+ solution, 150μL (200μg) intermediate, 690μL 0.1M CH3COONH4 solution were mixed and reacted at room temperature for 12 minutes. During this period, 4×5mL of 0.9% NaCl eluent (pH 5.9-6.1) containing 5mg / mL ascorbic acid was used to pre-equilibrate the PD-10 column. The reaction solution was added to PD-10, and the eluent was added to 2.5mL, and then 0.5mL was added each time for elution, for a total of 5 tubes, and the product (i.e. [ 18 F]F-H3RESCA-T4, [ 18 F]F-H3RESCA-T5, [ 18 F]F-H3RESCA-RT4 and [ 18 F]F-H3RESCA-RT5) activity.

[0103] 3)[ 18 F]F-H3RESCA-T4, [ 18 F]F-H3RESCA-RT4 quality control. The specific steps are as follows: use a capillary glass tube to draw a small amount of [ 18 F]F-H3RESCA-T4, [ 18 F]F-H3RESCA-RT4 was spotted on a silica gel plate, and physiological saline was used as the mobile phase. Radiochemical purity (RCP) of the probe was determined by radio-thin layer chromatography (Radio-TLC, Eckert & Ziegler Radiopharma Inc). Figure 2 As shown, freshly prepared [ 18 F] The RCP of F-H3RESCA-T4 is greater than 99%. Figure 6 As shown, freshly prepared [ 18 F] The RCP of F-H3RESCA-RT4 is greater than 99%.

[0104] Example 3: 18 F]F-H3RESCA-T4, [ 18 F]F-H3RESCA-RT4 imaging in T3M-4 tumor-bearing Balb / c mice

[0105] 1) Establish a Trop2-positive tumor-bearing mouse model. The specific steps include: 2×10 6T3M-4 cells were suspended in a mixture of PBS and Matrigel (Corning) (the ratio of the two was 1:1) and injected into the left shoulder of 4-5 week-old Balb / c nude mice to establish a subcutaneous pancreatic cancer model (i.e., T3M-4 tumor-bearing Balb / c mice).

[0106] 2)[ 18 F]F-H3RESCA-T4 imaging in T3M-4 tumor-bearing Balb / c mice. The specific steps are as follows: The small animal PET / CT imaging acquisition involved in this study was completed using the IRIS small animal PET / CT scanner (Inviscan Imaging Systems). The mice were divided into a T4-blocked group and an unblocked group. The T4-blocked group was injected with [ 18 F]F-H3RESCA-T4 was injected with unlabeled T4 (20 mg / kg) at the same time, while the unblocked group was not injected with T4. Each mouse was injected with 3.7-7.4 MBq [ 18 F]F-H3RESCA-T4 (4 mice in each group), 0.5 hours after injection, mice were anesthetized with isoflurane (concentration of 2%), and the mice in deep anesthesia were placed in supine position on the PET / CT scanning bed, PET and CT images were collected, and image reconstruction was completed using the IRIS system's own software. OsiriX Lite image processing workstation (Pixmeo SARL) was used to outline the heart and major tissues and organs (liver, lungs, kidneys, bones, muscles) and other regions of interest (ROI) on the reconstructed PET images, and the radioactive uptake values ​​of important tissues and organs were calculated in units of %ID / g (percent of injected dose per gram), and the uptake values ​​of major tissues and organs were plotted. Figure 3 A and Figure 3 As shown in B, the probe is mainly excreted through the urinary system and has a high uptake in the tumor site. In addition, the results of in vitro biodistribution experiments ( Figure 3 C) further revealed the distribution of the probe in the main tissues and organs in the body. And by comparing the unblocked group ( Figure 3 A. Figure 3 B. Figure 3 C) and T4 closed group ( Figure 3 D. Figure 3 E. Figure 3 F) ROI data and biodistribution data of tumor model imaging, indicating the binding specificity of T4 to tumor cell surface targets. 18 F]F-H3RESCA-T4 probe can non-invasively visualize Trop2 expression.

[0107] 3) Immunohistochemical staining was performed using Trop2-specific antibody (sc-376746, Santa Cruz Biotechnology). The staining results of the unblocked group and the T4-blocked group are shown in Figure 4 A. Figure 4 As shown in B, the expression of Trop2 in tumor tissue was confirmed.

[0108] 5)[ 18 F]F-H3RESCA-RT4 imaging in T3M-4 tumor-bearing Balb / c mice. The specific steps are as follows: The small animal PET / CT imaging acquisition involved in this study was completed using the IRIS small animal PET / CT scanner (Inviscan Imaging Systems). The mice were divided into an RT4-blocked group and an unblocked group. The RT4-blocked group was injected with [ 18 F]F-H3RESCA-RT4 was injected with unlabeled RT4 (20 mg / kg) at the same time, while the unblocked group was not injected with T4. Each mouse was injected with 3.7-7.4 MBq [ 18 F]F-H3RESCA-RT4 (4 mice in each group), mice were anesthetized with isoflurane (concentration of 2%) 45 minutes and 2.5 hours after injection, and the mice in deep anesthesia were placed in supine position on the PET / CT scanning bed, and PET and CT images were collected. Image reconstruction was completed using the IRIS system's own software, and the OsiriX Lite image processing workstation (Pixmeo SARL) was used to outline the heart and major tissues and organs (liver, lungs, kidneys, bones, muscles) and other regions of interest (ROI) on the reconstructed PET images. The radioactive uptake values ​​of important tissues and organs were calculated in units of %ID / g (percent of injected dose pergram), and the uptake values ​​of major tissues and organs were plotted over time. The PET / CT scan results and ROI results are shown in Figure 2. Figure 7 As shown in the figure, the results showed that in the unblocked group, the 2.5h renal uptake value was lower than the 45min renal uptake value, and the probe was excreted through the kidneys. In addition, the tumor uptake value was high and stable at both time points, with no downward trend in uptake. Figure 8 As shown, the results showed that the tumor uptake in the RT4-blocked group was significantly lower than that in the non-blocked group, which was the same as the ROI delineation result.

[0109] 6)[ 18 F]F-H3RESCA-T4 and [ 18 F]F-H3RESCA-RT4 ROI delineation results and tumor / organ ratio comparison, such as Fig. 9 As shown. 18F]F-H3RESCA-RT4 tumor uptake was lower than [ 18 F]F-H3RESCA-T4, but there was no statistical difference. 18 The tumor / liver, tumor / kidney, tumor / bone, and tumor / muscle ratios of F]F-H3RESCA-RT4 were superior to those of [ 18 F]F-H3RESCA-T4. The above data show that 18 F-labeled anti-Trop2 single-domain antibody probe is a potential in vivo non-invasive diagnostic and therapeutic tool targeting Trop2.

[0110] It should be noted that the [ 18 F]F-H3RESCA-T5 and [ 18 F]F-H3RESCA-RT5 also has the same function as [ 18 F]F-H3RESCA-T4 and [ 18 F]F-H3RESCA-RT4 showed similar PET imaging results, which is also a potential in vivo non-invasive diagnostic and therapeutic tool targeting Trop2.

[0111] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not used as additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all regarded as the scope of the present invention; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. A human Trop2-specific 18 F-labeled monovalent single-domain antibody probe, characterized in that A Trop2-specific single-domain antibody labeled with a radionuclide, wherein the Trop2-specific single-domain antibody comprises CDR1, CDR2 and CDR3, wherein the amino acid sequence of the CDR1 is shown in SEQ ID No.1, the amino acid sequence of the CDR2 is shown in SEQ ID No.2, and the amino acid sequence of the CDR3 is shown in SEQ ID No.3, and the Trop2-specific single-domain antibody is labeled with a radionuclide via a bifunctional chelator; the bifunctional chelator is (±)-H3RESCA-TFP, and the radionuclide is selected from F-18; The amino acid sequence of the Trop2-specific single domain antibody is shown in SEQ ID No.4 or SEQ ID No.

8.

2. The human Trop2-specific 18 F-labeled monovalent single-domain antibody probe, characterized in that The Trop2-specific single domain antibody having the amino acid sequence shown in SEQ ID No. 8 is labeled with F-18 via (±)-H3RESCA-TFP.

3. A kit or composition for visualizing Trop2 expression, diagnosing pancreatic cancer, predicting the progression and prognosis of pancreatic cancer, predicting the therapeutic effect of pancreatic cancer or treating pancreatic cancer, characterized in that: Comprising the human Trop2 specific 18 F labels the monovalent single domain antibody probe.

4. The human Trop2-specific 18 Use of a F-labeled monovalent single-domain antibody probe in preparing a kit or composition for visualizing Trop2 expression, diagnosing pancreatic cancer, predicting the progression and prognosis of pancreatic cancer, predicting the therapeutic effect of pancreatic cancer, or treating pancreatic cancer.

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