CN117384292A - Preparation method and application of 68Ga labeled Trop2 immunoPET imaging probe - Google Patents

Abstract

The invention relates to the technical fields of molecular imaging, nuclear medicine and single domain antibodies for tumor diagnosis and treatment, in particular to a method for detecting tumor ⁶⁸ Preparation method and application of Ga-marked Trop2 immune PET imaging probe. The Trop2 specific single domain antibody has an amino acid sequence shown as SEQ ID No.1 or SEQ ID No. 3. Trop2 specificity prepared by the invention ⁶⁸ Ga-marked single-domain antibody probe for immune PET imaging, realizing Trop2 in-vivo noninvasive visualization, and further realizingNoninvasive diagnosis of Trop 2-expressing positive tumors is now presented. 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 68Ga labeled Trop2 immunoPET imaging probe

Technical field

The 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 preparation method and application of a ⁶⁸ Ga labeled Trop2 immunoPET imaging probe.

Background technique

In 1993, Belgian scientists Hamers et al. first reported the existence of an antibody with a naturally missing light chain in the peripheral blood of alpacas in the magazine Nature (Nature. 1993; 363 (6428): 446-8.). This kind of antibody has special characteristics. Antibodies with structural domains are heavy-chain antibodies (HCAbs). Through molecular biology methods, cloning the variable region of a heavy chain antibody can obtain an antigen-binding fragment with only the variable region of the heavy chain, which is a nanobody or 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 nanobody ( Registered trade name of Ablynx Corporation). Single domain antibodies are the smallest antibody units currently known that can bind target antigens. They have high affinity, small molecular weight, and low preparation costs (they can be expressed in E. coli or in eukaryotic expression systems such as yeast and Chinese hamster ovary cells. ), and has the advantages of easy clinical translation and popularization and application.

Single domain antibodies have been a popular targeting vector 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 nuclides 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 single-domain antibody probe targeting programmed death ligand 1 (PD-L1) has been successfully translated into clinical practice and used for non-invasive treatment of patients with non-small cell lung cancer. Diagnosis (J Nucl Med. 2019; 60(9):1213-1220.); Gallium-68 ( ⁶⁸ Ga; T _1/2 = 1.1h) labels single domain antibodies targeting human epidermal growth factor receptor (HER2) The probe has also been successfully translated into clinical practice for non-invasive diagnosis of breast cancer (J NuclMed. 2016; 57(1):27-33.). The above examples illustrate 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 monoclonal Evaluation of efficacy after antibody treatment.

Trophoblast cell surface antigen 2 (Trop2) is a cell membrane surface glycoprotein formed from a 36kDa nascent polypeptide that is translated through N-linked glycosylation. It regulates tumor proliferation, invasion, and migration through multiple signaling pathways and plays a role in stem cell biology. In a retrospective study including 197 paraffin-embedded pancreatic cancer primary tumor tissues, the expression of Trop2 antigen was analyzed. Immunohistochemistry results found that 55% of the antigens were overexpressed, which was associated with lymph node metastasis and poor tumor differentiation. Significantly related to poor prognosis (P<0.05). The expression of Trop2 is also related to the biological aggressiveness 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 differential expression of Trop2 in normal tissues and tumors makes it a potential tumor-specific marker that can avoid potential side effects. Currently, antibody-drug conjugate drugs targeting Trop2 have entered clinical trials. Therefore, there is an urgent need to develop a diagnostic tool targeting Trop2 to enable non-invasive visualization and monitoring of Trop2 expression in solid tumors. Based on research on companion diagnostic tools, new treatments targeting Trop2 can be further developed.

The applicant's preliminary series of basic and clinical studies have shown that by cleverly fusing the extraordinary target specificity of antibodies with the superior sensitivity and resolution of positron emission tomography (PET), immunoPET is superior to immunohistochemical staining (IHC). Or other traditional predictive markers 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 (Chem Rev .2020;120(8):3787-3851.). For example, the value of immunoPET imaging probes targeting human epidermal growth factor receptor 2 in breast cancer has been clinically proven. Based on the above evidence and our previous findings, we hypothesized that Trop2-targeted immunoPET imaging probes could non-invasively display intratumoral Trop2 expression 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 achieve long-term remission and even eradicate multiple cancer types.

Currently, there are no reports on Trop2-specific single-domain antibody molecular imaging probes or radionuclide-labeled integrated diagnosis and treatment probes in clinical practice and literature reports. There are molecular imaging probes based on Trop2 monoclonal antibodies (Eur J Nucl MedMol Imaging. 2022Feb; 49(3):861-870.) and radionuclide-labeled integrated diagnosis and treatment probes (Eur J Nucl MedMol Imaging. 2022Dec; 50( 1):168-183.). But the use of radiolabeled monoclonal antibodies has been severely hampered by high cost, the necessity to use long-half-life radionuclides, a tedious imaging process within a week, and associated 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. In the small antibody format, Nanobodies or single domain antibodies from the family Camelidae are the smallest antigen-binding moieties with a molecular weight of approximately 15 kDa. Small size, high affinity, and ease of engineering make single-domain antibodies an excellent alternative for molecular imaging (J NuclMed. 2022 Oct; 63(10):1705-1709.). In recent years, we have focused on the development and clinical translation of single-domain antibody-derived tracers to leverage their superior molecular imaging properties. At present, there are no Trop2-specific single domain antibody molecular imaging probes at home and abroad.

Contents of the invention

To fill this gap in the field, here we describe the construction of Trop2-targeted theranostic 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 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 translation and application. Specifically, the present invention provides a preparation method and application of a ⁶⁸ Ga-labeled Trop2 immunoPET imaging probe.

Trop2-specific single domain antibody

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

It has the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 3.

In the present invention, for the sake of simplicity, the Trop2-specific single domain antibodies having the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 3 are respectively called T4 and T5.

As used herein, the term "single-domain antibody (sdAb)" is also known as "nanobody" or VHH (Variable Domain of Heavy Chain of Heavy Chain Antibody), which are used interchangeably. Nanobodies have the meaning generally understood by those skilled in the art, which refers to antibody fragments composed of a single monomeric variable antibody domain (such as a single heavy chain variable region), usually derived from heavy chain antibodies (such as camelids). antibody or shark antibody). Typically, Nanobodies are composed of 4 framework regions and 3 complementarity determining regions, with the structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Nanobodies can be truncated at the N- or C-terminus so that they contain only part of FR1 and/or FR4, or lack one or both of those backbone regions, as long as they substantially retain antigen binding and specificity.

Specifically, the CDR1 sequence of the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 3 is "GLPYERYC", the CDR2 sequence is "ILSDGTT", and the CDR3 sequence is "AAEAFRPFTPSDGDCTTVLGIDY".

In some embodiments, the invention also encompasses antigen-binding fragments of Trop2-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 the same antigen to which the Nanobody binds, and/or competes with the Nanobody for specificity for the antigen Binding, which is also known as the "antigen binding moiety". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes. It can be obtained by recombinant DNA technology Or an antigen-binding fragment of an antibody of the invention is generated by enzymatic or chemical cleavage of a Nanobody of the invention. In some embodiments, the "antigen-binding fragment" of the Nanobody can be at the N-terminal or C-terminus compared to the full-length Nanobody. The ends are truncated so that they contain only part of FRl and/or FR4, or lack one or both of those backbone regions, as long as they substantially retain antigen binding and specificity.

Antigen-binding fragments of a Nanobody can be obtained from a given Nanobody (eg, a Nanobody provided by the invention) using conventional techniques known to those skilled in the art (eg, recombinant DNA technology or enzymatic or chemical fragmentation methods) and Antigen-binding fragments of Nanobodies are screened for specificity in the same manner as for intact Nanobodies.

In this document, when the term "Nanobody" is mentioned, it includes not only intact Nanobodies, but also antigen-binding fragments of Nanobodies, unless the context clearly indicates otherwise.

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. Nanobodies contain 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, for example according to 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), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883) or 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. 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).

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

As used herein, the term "Trop2 specific" refers to specific binding to Trop2.

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

The specific binding properties between two molecules can be determined using methods known in the art. One approach involves measuring the rate at which antigen binding site/antigen complexes form and dissociate. Both the "association rate constant" ( _ka or _kon ) and the "dissociation rate constant" (k _dis or k _off ) can be calculated from the concentration and the actual rates of association and dissociation (see MalmqvistM, Nature, 1993,361:186-187). The ratio k _dis /k _on is equal to the dissociation constant K _D (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). The K _D , _kon and k _dis values may be measured by any valid method. In certain embodiments, dissociation constants can be measured in Biacore using surface plasmon resonance (SPR). Alternatively, bioluminescence interferometry or Kinexa can be used to measure dissociation constants.

In some embodiments, the invention also provides variants of Trop2-specific single domain antibodies as described herein that have 70-100% sequence identity with the amino acid sequence set forth in SEQ ID No. 1 or 3 , such as at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity properties, and basically retains the biological function of the Nanobody from which it is derived (such as the biological activity of specifically binding Trop2).

More specifically, the variant differs from a Trop2-specific single domain antibody as described herein by only one or more (e.g., up to 20, up to 15, up to 10, up to 5, or up to 1 Conservative substitution of amino acids) Conservative substitution of 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 (for example, a position in each of two DNA molecules is occupied by adenine, or two A certain position in each polypeptide is occupied by lysine), then the molecules are identical at that position. "Percent identity" between two sequences is a function of the number of matching positions common to the two sequences divided by the number of positions compared × 100. For example, if 6 out of 10 positions of two sequences match, then the two sequences are 60% identical. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (matching at 3 positions out of a total of 6 positions). Typically, comparisons are made when two sequences are aligned to yield maximum identity. Such alignment can be accomplished 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.). It is also possible to use the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), using the PAM120 weight residue table (weight residue table), 12 A gap length penalty of 4 and a gap penalty of 4 are used to determine the percent identity between two amino acid sequences. Alternatively, the Needleman and Wunsch (JMoIBiol.48:444-453 (1970)) algorithm can be used using the Blossum 62 matrix or the PAM250 matrix, which has been integrated into the GAP program of the GCG software package (available at www.gcg.com) 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 to determine the percent identity between two amino acid sequences.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter 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 those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., one that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties, including 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 those 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), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acids, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids. Therefore, 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 is incorporated herein by reference).

polynucleotide

In another aspect, the present invention also provides polynucleotides encoding the above-mentioned Nanobodies or antigen-binding fragments thereof.

More specifically, the polynucleotide has the nucleotide sequence shown in SEQ ID No. 2 or SEQ ID No. 4.

More specifically, a polynucleotide encoding a Trop2-specific single domain antibody as described herein has the nucleotide sequence shown in SEQ ID No. 2 or SEQ ID No. 4.

The polynucleotides of the invention may be in DNA form or RNA form. Forms of DNA include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.

The term "polynucleotide encoding a polypeptide/protein/antibody" may include polynucleotides encoding the polypeptide/protein/antibody, or may also include polynucleotides that additionally include coding and/or non-coding sequences.

The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80%, most preferably at least 90% identity between the two sequences, and the polypeptides they encode /protein/antibody has essentially the same function and activity. The invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides of the invention. 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) adding There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only if the identity between the two sequences is at least 90%, more It is preferred that hybridization occurs only when 95% or more are present.

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

carrier

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

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages 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, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyoma vacuolating viruses (such as SV40). A vector can contain a variety of expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain an origin of replication site.

host cell

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, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. 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 can include single cells or populations of cells.

Introduction of the vector into the host cell can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method, using steps well known in the art. Another method is to use MgCl ₂ . If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

Nanobodies of the present invention can be used alone, or can be combined or coupled with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties, or any combination of these substances.

Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or those capable of producing a detectable product Enzymes. Preferred detectable labels are radionuclides.

Therapeutic agents that can be combined or conjugated with the antibodies of the present invention include but are not limited to: 1. Radionuclides; 2. Biological toxins; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses Particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. Chemotherapy agents ( For example, cisplatin) or any form of nanoparticles, etc.

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. ^{, 18} F or ⁶⁸ Ga labeling is preferred for non-invasive tumor diagnosis, and ²²⁵ Ac, ²¹¹ At, ²¹² Pt or ¹³¹ I labeling is preferred for tumor radioimmunotherapy.

Binding or conjugation of a detectable label or therapeutic agent to an antibody can be performed by conventional methods well known to those skilled in the art. For example, a detectable label can be directly or indirectly bound to the Nanobody, for example via a cleavable or non-cleavable linker peptide, or incorporated into the Nanobody. The detectable label may be bound to the Nanobody, inter alia, by substitution (eg by replacing H with I at the level of a tyrosine residue), by complexing or by chelation. For example, therapeutic agents can be conjugated to Nanobodies via cleavable linkers such as peptidyl, disulfide or hydrazone linkers.

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

Trop2-specific molecular imaging probe

In another aspect, the invention provides a human Trop2-specific ⁶⁸ Ga-labeled monovalent single domain antibody probe comprising a radionuclide-labeled Trop2-specific single domain antibody antibody as described herein.

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

As used herein, a bifunctional chelator is a type of chelator that has both a metal chelating end and a protein anchoring end. The bifunctional chelating agent can be selected from NOTA, MAA-NOTA, p-SCN-Bn-NOTA, p-SCN-Bn-desferrioxamine (DFO), p-SCN-NODA, MAA-GA-NODA, MAA- DOTA, DOTA-NHS, iEDTA or p-SCN-Bn-DTPA.

Preferably, the bifunctional chelating agent is selected from p-SCN-Bn-NOTA.

As used herein, the NOTA is 1,4,7-triazacyclononane-1,4,7-triacetic acid;

The MAA-NOTA is (2,2'-(7-(2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino) -2-Oxyethyl)-1,4,7-triazacyclononane-1,4-diyl)diacetic acid;

The p-SCN-Bn-NOTA is 2-S-(4-isothiocyanophenyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid;

The p-SCN-Bn-deferoxamine (DFO) is 1-(4-isothiocyanophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo- 27-(N-acetylhydroxylamino)-6,11,17,22-tetraazaheptabiose]thiourea;

The p-SCN-NODA is 1,4,7-triazacyclooctane-1,4-diacetic acid-7-p-isothiocyanatobenzyl;

The MAA-GA-NODA is 2,2'-(7-(1-carboxy-4-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) Ethyl)amino)-4-oxobutyl)-1,4,7-triazacyclononane-1,4-diyl)diacetic acid;

The MAA-DOTA is 2,2',2″-(10-(1-carboxy-4-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl) )ethyl)amino)-4-oxobutyl)-1,4,7,10-triazacyclododecane-1,4,7-triyl)triacetic acid];

The DOTA-NHS is 2,2',2"-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxyethyl)-1,4,7 ,10-triazacyclododecane-1,4,7-triyl)triacetic acid;

The iEDTA is 1-(4-isothiocyanobenzyl)ethylenediamine-N,N,N',N'-tetraacetic acid;

The p-SCN-Bn-DTPA is 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentacetic acid.

More specifically, Trop2-specific single domain antibodies as described herein are radionuclide labeled via p-SCN-Bn-NOTA.

More specifically, the radionuclide is selected from ⁶⁸ Ga.

More specifically, [ ⁶⁸ Ga]Ga-NOTA-T4, [ ⁶⁸ Ga]Ga-NOTA-T5 as described herein.

In another aspect, the present invention also provides a method for preparing a Trop2-specific ⁶⁸ Ga-labeled monovalent single domain antibody probe, including modifying a Trop2-specific single domain antibody with a bifunctional chelator to obtain a radionuclide-labeled precursor; and The radionuclide-labeled precursor is labeled with a radionuclide to obtain a human Trop2-specific ⁶⁸ Ga-labeled monovalent single-domain antibody probe.

combination

In another aspect, the 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 therapeutic effect of Trop2-related tumors, or treat Trop2-related tumors.

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

In some embodiments, the pharmaceutical composition may further include pharmaceutically acceptable carriers and/or excipients.

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

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

In some embodiments, 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 used as separate agents in the pharmaceutical composition. components or provided as mixed components. Thus, a Trop2-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 (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w/v) NaCl), dextrose solutions (eg 5% glucose), surfactant containing solutions (eg 0.01% polysorbate 20), pH buffer solutions (eg phosphate buffer solution), Ringer's solution and any combination thereof.

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" means an amount sufficient to prevent, arrest or delay the onset of disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent disease and its complications in a patient already suffering from the disease. The therapeutically effective amount may vary depending on factors such as 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 manner in which the drug is administered, and other concurrent treatments administered. wait.

Reagent test kit

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

The kit 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.

The kit may also include a container, instructions for use, and other reagents and buffers required for practical applications, such as lysis media for dissolving samples, various buffers, detection labels, detection substrates, etc.

Diagnostic and therapeutic applications

The Trop2-specific single domain antibody of the present invention has extremely high affinity for Trop2, and therefore 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.

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 non-specific uptake in normal tissue, and significantly improved image quality, and can be used for non-invasive, accurate and efficient applications. Detecting the expression of human Trop2, it is therefore particularly suitable for diagnosing Trop2-related tumors and predicting the treatment effect of Trop2-related tumors. After selecting the appropriate radionuclide for conjugation, it can also be used to precisely treat Trop2-related tumors.

Therefore, in another aspect, the present invention also relates to the preparation of Trop2-specific single domain antibodies, polynucleotides, vectors, host cells or molecular imaging probes as described herein for detecting the expression level of Trop2 and diagnosing Trop2-related diseases. Tumors, predicting the therapeutic effect of Trop2-related tumors, or use in kits or drugs for treating Trop2-related tumors.

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.

Beneficial effects of the invention

1) The Trop2-specific ⁶⁸ Ga-labeled monovalent single domain antibody probes [ ⁶⁸ Ga]Ga-NOTA-T4 and [ ⁶⁸ Ga]Ga-NOTA-T5 prepared by the present invention for immunoPET imaging realize non-invasive visualization of Trop2 in vivo, This further enables the non-invasive diagnosis of Trop2-expressing tumors, especially pancreatic cancer.

2) The Trop2-specific molecular imaging diagnosis and treatment integrated system described in the present invention, namely [ ⁶⁸ Ga]Ga-NOTA-T4 and [ ⁶⁸ Ga]Ga-NOTA-T5 immunoPET imaging probes, has the advantages of simple preparation process and low cost. , high specificity, high stability, short imaging cycle, low radiation dose, and easy clinical translation. With clinical translation and application, it is expected to realize non-invasive visualization of heterogeneous expression of Trop2, 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.

Description of the drawings

Figure 1 shows 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 the target; Figure 1A is the SDS-PAGE result of T4; Figure 1B is the result of T4 HPLC results; Figure 1C shows the binding affinity results of T4 and human Trop2 recombinant protein; Figure 1D shows the SDS-PAGE results of T5; Figure 1E shows the HPLC results of T5; Figure 1F shows the binding affinity results of T5 and human Trop2 recombinant protein;

Figure 2 shows the fluorescence intensity results of pancreatic cancer cell line T3M-4;

Figure 3 shows the radiochemical purity measurement results of [ ⁶⁸ Ga]Ga-NOTA-T4 and [ ⁶⁸ Ga]Ga-NOTA-T5 before and after purification;

Figure 4 shows the in vitro biodistribution results of [ ⁶⁸ Ga]Ga-NOTA-T4 and [ ⁶⁸ Ga]Ga-NOTA-T5 in normal Balb/c mice;

Figure 5 shows the PET/CT imaging, ROI analysis and in vitro biodistribution results of [ ⁶⁸ Ga]Ga-NOTA-T4 45 minutes after injection into T3M-4 tumor-bearing Balb/c mice; Figure 5A shows the PET/CT imaging Results; Figure 5B shows the ROI analysis results; Figure 5C shows the in vitro biodistribution results;

Figure 6 shows the PET/CT imaging, ROI analysis and in vitro biodistribution results of [ ⁶⁸ Ga]Ga-NOTA-T5 45 minutes after injection into T3M-4 tumor-bearing Balb/c mice; Figure 6A shows the PET/CT imaging Results; Figure 6B shows the ROI analysis results; Figure 6C shows the in vitro biodistribution results;

Figure 7 shows immunohistochemical staining of [ ⁶⁸ Ga]Ga-NOTA-T4 and [ ⁶⁸ Ga]Ga-NOTA-T5;

Figure 8 shows the results of PET/CT imaging and ROI analysis 45 minutes and 2.5 hours after injection of [ ⁶⁸ Ga]Ga-NOTA-T4 in T3M-4 tumor-bearing Balb/c mice in different doses of blocking group;

Figure 9 shows the comparison of ROI delineation results between the unblocked group and the blocked group at different doses.

Detailed ways

In order to facilitate 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 this invention is not limited to the specific methods, protocols, cell lines, constructs, and reagents described herein, and may vary as such. Unless otherwise defined, all technical and scientific terms used in this specification have the same meanings commonly understood by those skilled in the technical field belonging to the present invention. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention.

Currently, antibody-drug conjugate drugs targeting Trop2 have entered clinical trials. ImmunoPET (ImmunoPET) imaging, which organically combines the high sensitivity of PET imaging and the high affinity of antibodies, is a new molecular imaging modality that can be used for non-invasive visualization of tumor targets and target-specific diagnosis of tumors (Chem Rev .2020;120(8):3787-3851.).

There are no reports on Trop2-specific single-domain antibody molecular imaging probes and radionuclide-labeled integrated diagnostic and therapeutic probes in clinical practice and literature reports. There are molecular imaging probes based on Trop2 monoclonal antibodies (Eur J Nucl Med MolImaging. 2022Feb; 49(3):861-870.) and radionuclide-labeled integrated diagnosis and treatment probes (Eur J Nucl Med MolImaging. 2022Dec; 50( 1):168-183.). However, the clinical translation and application of monoclonal antibody immunoPET imaging probes are severely limited by reasons such as expensive preparation cost, large molecular weight, long circulation time in the body, long imaging cycle, high radiation dose, and high toxic and side effects. In comparison, single-domain antibody immunoPET 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 translation and application.

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

Example 1: Preparation of Trop2-specific single domain antibodies

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 (refer to the patent number ZL202011131233.7 with the invention title "A molecular imaging probe for diagnosing multiple myeloma", which is incorporated herein by reference in its entirety), the novel Trop2 specificity was recombinantly expressed. Monovalent single domain antibodies T4 and T5. The amino acid sequence of the single domain antibody T4 is shown in SEQ ID NO.1 (wherein the CDR1 sequence is GLPYERYC, the CDR2 sequence is ILSDGTT, and the CDR3 sequence is AAEAFRPFTPSDGDCTTVLGIDY), and the gene sequence is shown in SEQ ID NO.2; single domain antibody T5 The amino acid sequence is shown in SEQ ID NO.3 (the CDR1 sequence is GLPYERYC, the CDR2 sequence is ILSDGTT, and the CDR3 sequence is AAEAFRPFTPSDGDCTTVLGIDY), and the gene sequence is shown in SEQ ID NO.4.

SDS-PAGE determines the expression of single domain antibodies T4 and T5. 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 loading buffer. (5X) protein sample for 5 minutes; after assembling the SDS-PAGE gel, add 500ml of 1x SDS-PAGE buffer, slowly spot the protein sample into the loading well, and keep in an 80V constant voltage electrobath for about 30 minutes until the bromophenol blue indicator crosses After concentrating the gel, adjust the voltage to 120V, electrophorese to the bottom of the gel, remove the gel, heat and stain it in Coomassie blue staining solution for 50 minutes, take it out, destain it with the destaining solution until the background is clean and the bands are clear, and then take pictures. The expression of single domain antibodies T4 and T5 is shown in Figure 1A and Figure 1D.

The results of HPLC determination of single domain antibodies T4 and T5 are shown in Figure 1B and Figure 1E. The results of SPR detection of the binding affinity of single domain antibodies T4 and T5 to human Trop2 recombinant protein are shown in Figure 1C and Figure 1F. The affinity of single domain antibodies T4 and T5 to human Trop2 was measured, and the K _D values were 965.9pM and 699.6pM respectively.

Example 2: Preparation of Trop2-specific single domain antibody probes

1) p-SCN-Bn-NOTA modified T4 and T5 to prepare intermediate NOTA-T4 and NOTA-T5. The specific steps are as follows: Dissolve 1mg T4 and T5 in 1mL phosphate buffer saline (PBS), 0.1mL 0.1M sodium carbonate (Na ₂ CO ₃ , pH=11.4) buffer, adjust the pH of the single domain antibody solution to 9.0–10, The volume of the reaction system is 1.1 mL. p-SCN-Bn-NOTA (CAS Number: 170597-66-8) freshly dissolved in dimethyl sulfoxide (DMSO) at a molar ratio of p-SCN-Bn-NOTA:single domain antibody = 10:1 ; Macrocyclics) was added to the above single domain antibody solution. The reaction system was allowed to react at room temperature for 2 hours, then PBS was used as the mobile phase, and a pre-equilibrated PD-10 desalting column (GE Healthcare) was used to purify the NOTA-modified single domain antibody, and NOTA-T4 and NOTA-T5 were collected; The ultrafiltration tube (Merck Millipore) with a cutoff value of 10kDa was used to concentrate, and the NOTA-T4 and NOTA-T5 concentrations were measured using NanoDrop, and the aliquots were stored at -20°C for later use.

2) ⁶⁸ Ga labeled NOTA-T4 and NOTA-T5 to prepare [ ⁶⁸ Ga]Ga-NOTA-T4 and [ ⁶⁸ Ga]Ga-NOTA-T5. The specific steps are as follows: Use 4mL of 0.05M hydrochloric acid solution (HCl) to elute the germanium and gallium generator (Eckert&Ziegler RadiopharmaInc), collect the same volume of ⁶⁸ Ga eluent with an activity of about 370-555MBq; take the middle section of ⁶⁸ Ga with the highest activity to elute. 2mL of washing solution, add 0.1mL of 1M sodium acetate solution (NaoAc) to adjust the pH of the ⁶⁸ Ga eluent to 4.0–4.5; take 200 μg of NOTA-T4 and NOTA-T5 that have been used for coupling and add to the ⁶⁸ Ga eluent, and the volume of the reaction system <2.5mL; place the reaction system in a constant-temperature oscillator and react at room temperature for 5-10 minutes; after the labeling reaction, use PBS as the mobile phase, and again use the pre-equilibrated PD-10 desalting column to separate the free ⁶⁸ Ga and purify the final product; follow The unattenuated corrected radiochemical yield (RCY) obtained by the above steps is >50%.

3) [ ⁶⁸ Ga]Ga-NOTA-T4 and [ ⁶⁸ Ga]Ga-NOTA-T5 quality control. The specific steps are as follows: Take 10 μL of [ ⁶⁸ Ga]Ga-NOTA-T4 and [ ⁶⁸ Ga]Ga-NOTA-T5 and spot them on the silica gel plate, use 0.1M sodium citrate solution (pH=5) as the mobile phase, and use radioactive Thin layer chromatography (Radio-TLC, Eckert&Ziegler Radiopharma Inc) was used to determine the radiochemical purity (RCP) of the probe. As shown in Figure 3, the purified RCP of freshly prepared [ ⁶⁸ Ga]Ga-NOTA-T4 and [ ⁶⁸ Ga]Ga-NOTA-T5 was both greater than 99%.

Example 3: Imaging of [ ⁶⁸ Ga]Ga-NOTA-T4 and [ ⁶⁸ Ga]Ga-NOTA-T5 in normal 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). Each mouse was injected with 3.7-7.4MBq [ ⁶⁸ Ga]Ga-NOTA-T4 and [ ⁶⁸ Ga]Ga-NOTA-T5 (4 mice in each group) via the tail vein, and isoflurane (concentration 2%), and sacrifice the mice that entered deep anesthesia. The Gamma counter detects the radioactive activity of the collected blood and various organs (heart, lungs, liver, spleen, kidneys, pancreas, muscles, etc.), thereby displaying the metabolic distribution of the probe in the body. The in vitro biodistribution results of [ ⁶⁸ Ga]Ga-NOTA-T4 and [ ⁶⁸ Ga]Ga-NOTA-T5 in normal Balb/c mice are shown in Figure 4. The results showed that the probe was mainly excreted through the urinary system. [ ⁶⁸ Ga]Ga-NOTA-T4 and [ ⁶⁸ Ga]Ga-NOTA-T5 have the highest uptake in the kidneys, which is due to the need for tracer clearance in the kidneys.

Example 4: Imaging of [ ⁶⁸ Ga]Ga-NOTA-T4 and [ ⁶⁸ Ga]Ga-NOTA-T5 in T3M-4 tumor-bearing Balb/c mice

1) Establish a tumor-bearing mouse model with positive Trop2 expression. Specifically, it includes the following steps: Using anti-human Trop2 monoclonal antibody (10428-MM01-F, Sino Biological Inc.) as the primary antibody, the pancreatic cancer cell line T3M-4 (high expression of Trop2) was found through flow cytometry experiments, as shown in the figure As shown in 2; 2×10 ⁶ T3M-4 cells were suspended in PBS and Matrigel (Corning) at a ratio of 1:1, and injected into the left shoulder of 4–5 week old Balb/c nude mice to establish a subcutaneous model. Pancreatic cancer model.

2) Immuno-PET imaging of [ ⁶⁸ Ga]Ga-NOTA-T4 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 ImagingSystems). Each mouse was injected with 3.7-7.4 MBq [ ⁶⁸ Ga]Ga-NOTA-T4 (4 mice per group) through the tail vein, and 45 minutes after the injection, the mice were anesthetized with isoflurane mixed with oxygen (concentration: 2%). The mice in a state of deep anesthesia were placed on the PET/CT scanning bed in a supine position, and PET and CT images were collected continuously, and the image reconstruction was completed using the software that comes with the IRIS system. Use the OsiriX Lite image processing workstation (Pixmeo SARL) to outline the region of interest (ROI) such as the heart and major tissues and organs (liver, lung, kidney, muscle) on the reconstructed PET image, and use %ID/g The radioactive uptake value of important tissues and organs is calculated in units of (percent of injected dose per gram). Figure 5 shows the PET/CT imaging results 45 minutes after [ ⁶⁸ Ga]Ga-NOTA-T4 probe injection (Figure 5A), ROI quantification results (Figure 5B), and in vitro biodistribution data (Figure 5C). It can be seen that the Trop2-specific single domain antibody probe [ ⁶⁸ Ga]Ga-NOTA-T4 has higher uptake in tumor tissues that highly express Trop2, and is also higher in the main excretory (kidney) and metabolic (liver) tissues. Non-specific uptake. By delineating the ROI to analyze the distribution of [ ⁶⁸ Ga]Ga-NOTA-T4 in the body, the tumor part showed a higher uptake of 4.60±1.42% ID/g. In addition, the in vitro biodistribution experiment results further revealed that the probe has a higher uptake in the body. Distribution of major tissues and organs.

3) Immuno-PET imaging of [ ⁶⁸ Ga]Ga-NOTA-T5 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 ImagingSystems). Each mouse was injected with 3.7-7.4MBq[ ⁶⁸ Ga]Ga-NOTA-T5 through the tail vein (4 mice per group), and 45 minutes after the injection, the mice were anesthetized with isoflurane mixed with oxygen (concentration: 2%). The mice in a state of deep anesthesia were placed on the PET/CT scanning bed in a supine position, and PET and CT images were collected continuously, and the image reconstruction was completed using the software that comes with the IRIS system. Use the OsiriX Lite image processing workstation (Pixmeo SARL) to outline the region of interest (ROI) such as the heart and major tissues and organs (liver, lung, kidney, muscle) on the reconstructed PET image, and use %ID/g The radioactive uptake value of important tissues and organs is calculated in units of (percent of injected dose per gram). Figure 6 shows the PET/CT imaging results 45 minutes after [ ⁶⁸ Ga]Ga-NOTA-T5 probe injection (Figure 6A), ROI quantification results (Figure 6B), and in vitro biodistribution data (Figure 6C). It can be seen that the Trop2-specific single domain antibody probe [ ⁶⁸ Ga]Ga-NOTA-T5 has higher uptake in tumor tissues that highly express Trop2, and is also higher in the main excretory (kidney) and metabolic (liver) tissues. Non-specific uptake. By delineating the ROI to analyze the distribution of [ ⁶⁸ Ga]Ga-NOTA-T5 in the body, the tumor part showed a higher uptake, which was 2.10±0.59% ID/g. In addition, the in vitro biodistribution experimental results further revealed that the probe has a higher uptake in the body. Distribution of major tissues and organs.

4) Afterwards, immunohistochemical staining was performed using Trop2-specific antibody (sc-376746, Santa Cruz Biotechnology), which confirmed the expression of Trop2 inside the tumor in the subcutaneous tumor model (Figure 7).

5) Immuno-PET imaging results of [ ⁶⁸ Ga]Ga-NOTA-T4 co-injected with different doses of unlabeled T4 blocking group. The imaging steps are basically the same as the aforementioned step 2), the only difference is: 1) The T4 blocking group was injected with unlabeled T4 at the same time as [ ⁶⁸ Ga]Ga-NOTA-T4; 2) In addition to the 45-minute imaging, Add 2.5 hours delayed imaging time point. Figure 8 shows the PET/CT imaging results at different time points (45 minutes and 2.5 hours) and different dose blocking groups (co-injection of unlabeled T4 50μg, 200μg, 400μg). It can be seen that at the 45 minutes and 2.5 hour time points, Tumor uptake did not decrease significantly and remained stable. The probe was tightly bound to the target and had strong stability. The tumor uptake in the high-dose blocking group was significantly lower than that in the low-dose blocking group, and the tumor uptake in the medium-dose blocking group was between the two. Figure 9 shows the comparison of ROI delineation results. Compared with the group treated with the method of step 2 (step 2), that is, [ ⁶⁸ Ga]Ga-NOTA-T4 shown in Figure 9), all closed groups ([ ⁶⁸ Ga]Ga-NOTA shown in Figure 9 -T4 (50 μg), [ ⁶⁸ Ga]Ga-NOTA-T4 (200 μg), [ ⁶⁸ Ga]Ga-NOTA-T4 (400 μg)) tumor uptake was significantly reduced, and renal uptake decreased with increasing blocking dose. At the same time, the target binding specificity of [ ⁶⁸ Ga]Ga-NOTA-T4 was also confirmed.

It should be noted that the preferred embodiments of the present invention are given in the description 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 to the content of the present invention, and are provided for the purpose of making the disclosure of the present invention more thorough and comprehensive. Furthermore, the above technical features can be continuously combined with each other to form various embodiments not listed above, which are all deemed to be within the scope of the description of the present invention; further, for those of ordinary skill in the art, they can be improved or transformed according to the above description. , and all these improvements and transformations should fall within the protection scope of the appended claims of the present invention.

Claims (10)

1. A Trop2-specific single domain antibody, characterized by comprising: It has the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 3. 2. The Trop2-specific single domain antibody according to claim 1, characterized in that the Trop2-specific single domain antibody has at least 70-100 amino acid sequences with the amino acid sequence described in SEQ ID No. 1 or SEQ ID No. 3. % homology to the sequence. 3. A polynucleotide, characterized in that it encodes the Trop2-specific single domain antibody according to claim 1 or 2; Preferably, the polynucleotide has the nucleotide sequence shown in SEQ ID No. 2 or 4. 4. A vector, characterized by comprising the polynucleotide according to claim 3. 5. A host cell, characterized by comprising the vector according to claim 4. 6. A human Trop2-specific ⁶⁸ Ga-labeled monovalent single-domain antibody probe, characterized by comprising a radionuclide-labeled Trop2-specific single-domain antibody according to claim 1 or 2. 7. The human Trop2-specific ⁶⁸ Ga-labeled monovalent single domain antibody probe according to claim 6, characterized in that the Trop2-specific single domain antibody according to claims 1 or 2 is radioactively nucleated via a bifunctional chelator. prime mark. 8. The human Trop2-specific ⁶⁸ Ga labeled monovalent single domain antibody probe according to claim 7, characterized in that the bifunctional chelating agent is selected from NOTA, MAA-NOTA, p-SCN-Bn-NOTA, p - at least one of SCN-Bn-deferoxamine, p-SCN-NODA, MAA-GA-NODA, MAA-DOTA, DOTA-NHS, iEDTA or p-SCN-Bn-DTPA, The radionuclide is selected from ⁶⁸ Ga. 9. A kit or composition for visualizing the expression of Trop2, diagnosing Trop2-related tumors, predicting the progression and prognosis of Trop2-related tumors, predicting the therapeutic effect of Trop2-related tumors, or treating Trop2-related tumors, characterized by comprising The Trop2-specific single domain antibody according to claim 1 or 2, the polynucleotide according to claim 3, the vector according to claim 4, the host cell according to claim 5, the host cell according to claim 6 The probe described in any one of -8. 10. The Trop2-specific single domain antibody according to claim 1 or 2, the polynucleotide according to claim 3, the vector according to claim 4, the host cell according to claim 5 or the host cell according to claim 5. The probe described in any one of claims 6 to 8 is used for visualizing the expression of Trop2, diagnosing Trop2-related tumors, predicting the progression and prognosis of Trop2-related tumors, predicting the therapeutic effect of Trop2-related tumors, or treating Trop2-related tumors. Use in kits or compositions.