Multivalent trop2 diagnostic probes and methods of making and using the same

Abstract

The application provides a multivalent Trop2 diagnostic probe and a preparation method and application thereof, the probe comprising a Trop2 specific multivalent nanobody, the Trop2 specific multivalent nanobody being composed of a T4 monovalent nanobody, a linker and a tail tag; the amino acid sequences of the T4 monovalent nanobody, the linker and the tail tag are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3. The multivalent Trop2 diagnostic probe prepared by the application reduces non-target organ uptake and improves the signal-to-noise ratio during targeted imaging or treatment through a multivalent strategy; the probe has high affinity and high specificity to Trop2 in vivo, so that the probe can accurately identify a lesion. The probe can be used for non-invasive diagnosis of Trop2 expression positive tumors, and has the advantages of simple preparation process, low cost, high specificity, high stability and easy clinical transformation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular imaging probes, and particularly relates to a multivalent Trop2 diagnostic probe and a preparation method and application thereof. BACKGROUND

[0002] Trophoblast cell surface antigen 2 (Trop2) is a cell membrane surface glycoprotein formed by N-linked glycosylation translation modification of a 36 kDa nascent polypeptide. It regulates the proliferation, invasion and migration of tumors through various signal transduction pathways and plays a role in stem cell biology. In a retrospective study including 197 paraffin-embedded primary tumor tissues of pancreatic cancer, the expression of Trop2 antigen was analyzed, and the immunohistochemical results showed that 55% of the antigens were overexpressed, which was significantly related to lymph node metastasis, poor tumor differentiation and poor prognosis (P<0.05). The expression of Trop2 is also related to 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 the expression of Trop2 in normal tissues and tumors makes it a very potential tumor-specific marker and can avoid potential side effects. Currently, there are antibody drug conjugate drugs targeting Trop2 that have entered clinical trials. Therefore, there is an urgent need to develop diagnostic tools targeting Trop2 to achieve non-invasive visualization and monitoring of Trop2 expression in solid tumors. On the basis of studying companion diagnostic tools, new treatment methods targeting Trop2 can also be further developed.

[0003] The applicant team has previously submitted three related invention patents for monovalent nanobody immunopet imaging probes targeting Trop2 antigen (application patent names: 68Preparation method and application of Ga-labeled Trop2 immunoPET imaging probe; Publication number: CN117384292A; Application number: CN202311314186.3; Status: Under examination; Patent application name: Preparation method and application of Trop2 specific diagnostic and therapeutic molecular imaging probe; Publication number: CN116333142A; Application number: CN202310393659.7; Status: Under examination; Patent application name: Preparation method and application of radionuclide-labeled Trop2 specific single-domain antibody probe; Publication number: CN117327183A; Application number: CN202311251659.X; Status: Granted. While the monovalent nanobody immunoscanning targeting Trop2 antigen described in the aforementioned patents demonstrates excellent tumor targeting capabilities in various solid tumors such as thyroid cancer, breast cancer, lung cancer, and bladder cancer, the physiologically high expression of Trop2 in glands such as salivary glands, thyroid gland, and pancreas leads to a decreased image target-to-sample ratio, increased unnecessary radiation dose to patients, and even affects the detection rate of tumor lesions. Therefore, there is an urgent need to develop a novel Trop2 nanobody probe that reduces uptake by non-target organs, thereby reducing uptake by normal tissues and organs, especially glands, and thus reducing radiation damage to normal tissues and organs such as glands (e.g., oral mucosal inflammation, thyroiditis, pancreatitis, etc.), and potentially increasing the detection rate of lesions in one step. Based on previous basic research and related literature review, multivalent nanobodies may be a feasible strategy to reduce glandular uptake without affecting their affinity for the antigen, thus enabling the construction of Trop2-specific therapeutic probes. Summary of the Invention

[0004] This invention provides a multivalent Trop2 diagnostic method, its preparation method, and its application. The purpose of this invention is to construct a novel multivalent Trop2-specific nanobody molecular imaging probe, improve the image quality of nanobody immunoPET imaging, improve pharmacokinetics, thereby reducing the radiation dose to non-target organs and achieving better stratification of patients receiving Trop2-targeted therapy, monitoring of the efficacy of Trop2-targeted therapy, and radionuclide therapy targeting Trop2.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] In a first aspect, the present invention provides a Trop2-specific multivalent nanobody, wherein the Trop2-specific multivalent nanobody is composed of a T4 monovalent nanobody, a linker and a tail tag;

[0007] The amino acid sequence of the T4 monovalent nanobody is shown in SEQ ID NO.1; the number of T4 monovalent nanobodies is n, and n is equal to 2 or 3;

[0008] The amino acid sequence of the connector is shown as SEQ ID NO. 2; the number of connectors is n-1.

[0009] The amino acid sequence of the tail tag is shown as SEQ ID NO. 3.

[0010] Preferably, the amino acid sequence of the Trop2-specific multivalent nanobody is shown as SEQ ID NO. 4 or 6.

[0011] Preferably, the nucleotide sequence encoding the Trop2-specific multivalent nanobody is shown as SEQ ID NO. 5 or 7.

[0012] In a second aspect, the present application provides a Trop2-specific multivalent nanobody for use in the preparation of a multivalent Trop2 diagnosis and treatment synergistic probe.

[0013] In a third aspect, the present application provides a multivalent Trop2 diagnosis and treatment synergistic probe, which comprises a tumor targeting group, a radionuclide and a bifunctional chelating agent; the tumor targeting group is the aforementioned Trop2-specific multivalent nanobody.

[0014] Preferably, the tumor targeted by the tumor targeting gene is a Trop2-expressing tumor.

[0015] More preferably, the Trop2-expressing tumor is a high Trop2-expressing malignant tumor; the high Trop2-expressing malignant tumor includes at least one of urothelial carcinoma, renal clear cell carcinoma, nasopharyngeal carcinoma, lymphoma, multiple myeloma, breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, prostate cancer, cervical cancer, head and neck cancer, lung cancer, oral cancer, throat cancer, esophageal cancer, bladder cancer, uterine cancer, ovarian cancer, brain glioma, glioblastoma, thyroid cancer, liver cancer, kidney cancer, skin cancer, melanoma, and penile cancer.

[0016] Preferably, the radionuclide is selected from Tc-99m, Ga-68, F-18, I-123, I-125, I-131, I-124, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, Y-86, Mn-52, Sc-44, Y-90, Ac-225, At-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra-223, Ru-106, Na-24, Sr-89, Tb-149, Tb-161, Th-227, Xe-133, Yb-169 or Yb-177. More preferably, the radionuclide is Ga-68 or F-18.

[0017] Preferably, the bifunctional chelator is selected from (±) -H3RESCA-TFP, (±) H3RESCA-Mal, NOTA, MAA-NOTA, p -SCN-Bn-NOTA, p -SCN-Bn-DFO, p -SCN-NODA, MAA-GA-NODA, MAA-DOTA, DOTA-NHS, p -SCN-Bn-DOTA, iEDTA, p -SCN-Bn-DTPA, Iso -SGMIB or Boc2-SGMTB. More preferably, the bifunctional chelator is selected from (±) -H3RESCA-TFP or p -SCN-Bn-NOTA.

[0018] In a sixth aspect, the present application provides a preparation method of a multivalent Trop2 diagnosis and treatment synergistic probe, the preparation method comprising the following steps:

[0019] (1) modifying a tumor targeting group with a bifunctional chelator to obtain a conjugated tumor targeting group;

[0020] (2) labeling the conjugated tumor targeting group with a radionuclide to obtain the probe.

[0021] The present application will be described in more detail below.

[0022] The present application provides a Trop2 specific multivalent nanobody, which is BT4 or TT4.

[0023] The amino acid sequence of the BT4 is obtained by connecting the amino acid sequences of two T4 monovalent nanobodies (shown in SEQ ID No. 1) through the amino acid sequence of one linker (shown in SEQ ID No. 2), and then connecting the nucleotide sequence of a C-terminal tag (shown in SEQ ID No. 3) at the C-terminus; specifically, the amino acid sequence of the BT4 is shown in SEQ ID No. 4, and the nucleotide sequence is shown in SEQ ID No. 5.

[0024] The amino acid sequence of the TT4 is obtained by connecting the amino acid sequences of three T4 monovalent nanobodies (shown in SEQ ID No. 1) through the amino acid sequences of two linkers (shown in SEQ ID No. 2), and then connecting the nucleotide sequence of a C-terminal tag (shown in SEQ ID No. 3) at the C-terminus; specifically, the amino acid sequence of the TT4 is shown in SEQ ID No. 6, and the nucleotide sequence is shown in SEQ ID No. 7.

[0025] In some embodiments, the linker in the BT4 or TT4 is used to connect two T4 monovalent nanobodies, and the amino acid sequence of the linker comprises the amino acid sequence of GGGS or GGGGS, and the number of repetitions of GGGS or GGGGS is not fixed and can be any number. For example, the linker with the amino acid sequence shown in SEQ ID No. 2 is specifically connected by two GGGGS and one GGGS.

[0026] In some embodiments, the C-terminal tag in the BT4 or TT4 can be used for site-specific coupling, and the amino acid sequence of the C-terminal tag comprises the amino acid sequence of GGGS or GGGGS, and the number of repetitions of GGGS or GGGGS is not fixed and can be any number. For example, the C-terminal tag with the amino acid sequence shown in SEQ ID No. 3 specifically comprises the amino acid sequence connected by two GGGGS and one GGGS.

[0027] The present application also provides a variant of the Trop2-specific multivalent nanobody as described herein, which has 80-99% or more sequence identity to the amino acid sequence of the Trop2-specific nanobody, and substantially retains the biological function (e.g., the biological activity of specifically binding to the target) of the nanobody from which it is derived.

[0028] More specifically, the variant differs from the Trop2-specific multivalent nanobody as described herein only in that 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 acid residues) conservative substitutions of amino acid residues.

[0029] As used herein, the (±) -H3RESCA-TFP is 2,3,5,6-tetrafluorophenyl 3-[4-[(2S,5R,8R,11S)-2,5,8-tris(carboxymethyl)-11-(4-(di(2-hydroxyethyl)amino)benzyl)azocan-1-yl]phenyl]propanoate;

[0030] The (±) H3RESCA-Mal is (±)-2,2',2''-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-trisyl)triacetic acid;

[0031] The NOTA is 1,4,7-triazacyclononane-1,4,7-triacetic acid;

[0032] The MAA-NOTA is (2,2'-(7-(2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-2-oxoethyl)-1,4,7-triazacyclononane-1,4-diyl)diacetic acid;

[0033] The p -SCN-Bn-NOTA is 2-S-(4-isothiocyanatophenyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid;

[0034] The p -SCN-Bn-DFO is 1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetyloxylamino)-6,11,17,22-tetraazahexacosane]thiourea;

[0035] The p -SCN-NODA is 1,4,7-triazacyclononane-1,4-diethyl acid-7-p-isothiocyanatobenzyl;

[0036] The MAA-GA-NODA is 2,2'-(7-(1-carboxy-4-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-oxobutyl)-1,4,7-triazacyclononane-1,4-diyl)diacetic acid;

[0037] The MAA-DOTA is 2,2',2''-(10-(1-carboxy-4-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-oxobutyl)-1,4,7,10-triazacyclododecane-1,4,7-trisyl)triacetic acid];

[0038] The DOTA-NHS is 2,2',2”-(10-(2-((2,5-dioxopyrrolidine-1-yl)oxy)-2-oxoethyl)-1,4,7,10-triazacyclododecane-1,4,7-triyl)triacetic acid;

[0039] The p -SCN-Bn-DOTA is 1-(4-isothiocyanophenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid;

[0040] The iEDTA is 1-(4-isothiocyanobenzyl)vinyldiamine-N,N,N',N'-tetraacetic acid;

[0041] The p -SCN-Bn-DTPA is 2-(4-isothiocyanobenzyl)-diethylenetriaminepentaacetic acid;

[0042] The Iso -SGMIB is N-[4-(iodophenylcarbamoylmethyl)-succinimide] p-guanidinyl methyl benzoate;

[0043] The Boc2-SGMTB is N-succinimide-4-(N,N-di-tert-butoxycarbonylguanidinylmethyl)-3-(tert-butoxycarbonylaminomethyl)benzoate.

[0044] In one specific implementation, a human Trop2-specific [method / initiative] is provided. 68 Ga-labeled multivalent nanobody probes [ 68 Ga]Ga-NOTA-BT4.

[0045] In another specific implementation, a human Trop2-specific [method / approach] is provided. 68 Ga-labeled multivalent nanobody probes [ 68 Ga]Ga-NOTA-TT4.

[0046] In another specific implementation, a human Trop2-specific [method / approach] is provided. 18 F-labeled multivalent nanobody probes [ 18 F]AIF-RESCA-BT4.

[0047] In another specific implementation, a human Trop2-specific [method / approach] is provided. 18 F-labeled multivalent nanobody probes [ 18 F]AIF-RESCA-TT4.

[0048] Compared with the prior art, the beneficial effects that at least one technical solution adopted in the embodiments of this specification can achieve include at least:

[0049] (1) The four kinds of nanobody probes prepared by the application[ 18 F]AlF-RESCA-BT4, 18 F]AlF-RESCA-TT4, 68 Ga]Ga-NOTA-BT4 and 68 Ga]Ga-NOTA-TT4 reduce the non-target organ uptake through the multivalent strategy, and improve the signal-to-noise ratio in targeted imaging or treatment. The comparison of the results of immunopet imaging shows that the nanobody probe[ 18 F]AlF-RESCA-TT4 prepared by the application is mainly excreted through the urinary system, so there is high uptake of the imaging agent in the urinary organs such as kidneys and bladder, and different degrees of uptake of the imaging agent are also observed in the parotid gland, submaxillary gland, thyroid gland and pancreas. Compared with the immunopet imaging results of the[ 18 F]AlF-RESCA-T4 probe prepared by the monovalent nanobody T4, the uptake of the imaging agent in the parotid gland and submaxillary gland is obviously reduced, and the uptake in the parotid gland is reduced by about 8-10 times. The above results show that the[ 18 F]AlF-RESCA-BT4 probe has good safety and potential effect of reducing the uptake of non-target organs. In the modification process of the multivalent nanobody, the binding site of the nanobody and Trop2 is fully protected, ensuring the high affinity and high specificity of Trop2 in the body, so that the probe can accurately identify the lesion.

[0050] (2) The probe prepared by the application can realize the non-invasive visualization of Trop2 in vivo, and further realize the non-invasive diagnosis of Trop2 expression positive tumors.

[0051] (3) The probe prepared by the application is a multivalent Trop2 diagnosis and treatment synergistic probe, which has the advantages of simple preparation process, low cost, high specificity, high stability, easy clinical transformation and the like. Compared with the monovalent antibody probe, it has the advantages of reducing the uptake of non-target organs and improving the image quality, and is expected to screen patients with high expression of Trop2, further develop Trop2 specific radioimmunotherapy, and reduce the toxic and side effects of radioimmunotherapy, so as to realize the target specific diagnosis and treatment integration of Trop2 expression positive tumors.

[0052] (4) The preparation process of the probe of the application is simplified and the cost of raw materials is controlled, so that the technology is easier to popularize and apply, and has high commercialization and clinical transformation value. BRIEF DESCRIPTION OF DRAWINGS

[0053] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of the drawings.

[0054] Figure 1 is the SDS-PAGE experimental result diagram of Trop2 specific multivalent nanobody BT4 and TT4 prepared in the present application;

[0055] Figure 2 is the HPLC experimental result diagram of Trop2 specific multivalent nanobody BT4 and TT4 prepared in the present application;

[0056] Figure 3 is the affinity determination result of Trop2 specific multivalent nanobody BT4 and TT4 prepared in the present application and human Trop2 protein;

[0057] Figure 4 is the quality control diagram of probe 68 Ga-labeled nanobody BT4 and TT4 68 Ga]Ga-NOTA-BT4 and 68 Ga]Ga-NOTA-TT4 prepared in the present application;

[0058] Figure 5 is the PET / CT imaging diagram, region of interest (ROI) and in-vitro biodistribution diagram of probe 68 Ga]Ga-NOTA-BT4 prepared in the present application in the unblocked group and the blocked group of T3M4 pancreatic cancer tumor model;

[0059] Figure 6 is the PET / CT imaging diagram, ROI and in-vitro biodistribution diagram of probe 68 Ga]Ga-NOTA-TT4 prepared in the present application in T3M4 pancreatic cancer tumor model;

[0060] Figure 7 is the quality control diagram of probe 18 F]AlF-RESCA-BT4 and 18 F]AlF-RESCA-TT4 prepared in the present application;

[0061] Figure 8 is the imaging result of probe 18 F]AlF-RESCA-TT4 prepared in the present application in a normal volunteer;

[0062] Figure 9 is the comparative probe 18F]AlF-RESCA-T4 imaging results in a normal volunteer;

[0063] Figure 10 is a probe prepared by the present application 18 F]AlF-RESCA-BT4 imaging results in a patient after thyroid cancer surgery. DETAILED DESCRIPTION

[0064] The embodiments of the present application will be described in detail below with reference to the drawings.

[0065] The above examples are merely used to illustrate the embodiments of the present application, and the skilled in the art can easily understand other advantages and effects of the present application from the content disclosed in the present specification. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. The present application can also be implemented or applied through other different specific embodiments, and the details in the present specification can be modified or changed based on different views and applications without departing from the spirit of the present application. It should be noted that the following embodiments and features in the embodiments can be combined with each other without conflict. Based on the embodiments in the present application, all other embodiments obtained by the skilled in the art without creative labor are within the scope of protection of the present application.

[0066] Example 1

[0067] The embodiment provides a preparation method of Trop2 specific multivalent nanobody BT4 and TT4. The amino acid sequence of the nanobody BT4 is shown as SEQ ID No. 4 (QVQLVESGGGTVGAGGSLRLSCVVSGLPYERYCVAWFRQGPGKEREGVARILSDGTTSYSDSVKGRFTISKDNAKNTLYLQMNSLKSEDTATYYCAAEAFRPFTPSDGDCTTVLGIDYWGKGTLVTVSSGGGGSGGGGSGGGSQVQLVESGGGTVGAGGSLRLSCVVSGLPYERYCVAWFRQGPGKEREGVARILSDGTTSYSDSVKGRFTISKDNAKNTLYLQMNSLKSEDTATYYCAAEAFRPFTPSDGDCTTVLGIDYWGKGTLVTVSSGGGGSGGGGSGGGSLLQG), and the gene sequence is shown as SEQ ID No. 5. The amino acid sequence of the nanobody TT4 is shown as SEQ ID No. 6 (QVQLVESGGGTVGAGGSLRLSCVVSGLPYERYCVAWFRQGPGKEREGVARILSDGTTSYSDSVKGRFTISKDNAKNTLYLQMNSLKSEDTATYYCAAEAFRPFTPSDGDCTTVLGIDYWGKGTLVTVSSGGGGSGGGGSGGGSQVQLVESGGGTVGAGGSLRLSCVVSGLPYERYCVAWFRQGPGKEREGVARILSDGTTSYSDSVKGRFTISKDNAKNTLYLQMNSLKSEDTATYYCAAEAFRPFTPSDGDCTTVLGIDYWGKGTLVTVSSGGGGSGGGGSGGGSQVQLVESGGGTVGAGGSLRLSCVVSGLPYERYCVAWFRQGPGKEREGVARILSDGTTSYSDSVKGRFTISKDNAKNTLYLQMNSLKSEDTATYYCAAEAFRPFTPSDGDCTTVLGIDYWGKGTLVTVSSGGGGSGGGGSGGGSLLQG), and the gene sequence is shown as SEQ ID No. 7.

[0068] The specific preparation steps are as follows:

[0069] 1) Construct DNA sequence based on the gene sequence shown in SEQ ID No. 5 and SEQ ID No. 7, and then use high-fidelity PCR polymerase (PV2 DNA Polymerase) for gene amplification. Specifically, first dilute the primers (primer sequences and specific information are shown in Table 1) to 10 pmol / ul with deionized water; then add dNTPs (Shengong B500055-0250, 25 mM diluted to 10 mmol / ul), PV2 DNA Polymerase (Hunan Zhongsheng Quanpei 500ul) and reaction buffer (Hunan Zhongsheng Quanpei 2ml) in the reaction system, and obtain the enriched target gene fragment through multiple cycles of pre-denaturation, denaturation-annealing-extension in the PCR instrument. The specific reaction parameters are shown in Table 2. The reaction conditions are as follows: 95°C for 3 minutes, 95°C for 25 seconds, 60°C for 20 seconds, 25 cycles, 72°C for 60 seconds, 72°C for 1 minute, 10°C permanently. In Table 2, reaction system 1 is as follows: primer: 1.0ul each; DNA polymerase: 1.0ul; 5X buffer: 10ul; 10mM dNTP: 1ul; ddH2O: make up to 50ul. Reaction system 2 is as follows: primer: 1.5ul each; one round of product: 4.0ul; DNA polymerase: 1.0ul; 5X buffer: 10ul; 10mM dNTP: 1ul; ddH2O: make up to 50ul.

[0070] Table 1

[0071]

[0072] Table 2

[0073]

[0074] 2) Then, prepare the following reaction system (as shown in Table 3) in a 96-well PCR reaction plate to carry out gene recombination and connection, and then obtain the recombined and connected gene.

[0075] Table 3

[0076]

[0077] 3) The recombinant ligation gene was then transformed into competent *E. coli*. Competent cells were thawed in a refrigerator and placed in a 4°C freezer for 8 minutes with the recombinant ligation product. The competent cells were then heat-shocked at 42°C for 90 seconds on a PCR instrument heating plate. After the heat shock, the competent cells were removed, and the PCR instrument was turned off. The competent cells were then placed in a 4°C freezer for another 4 minutes. All the competent cell culture was collected and spread onto solid culture dishes, which were then incubated for 8 hours. Eight single colonies were selected from the solid culture dishes and screened using colony screening PCR. The primer sequences for bacterial detection were as follows: BI-CMV-F CGCAAATGGGCGGTAGGCGTG (SEQ ID NO. 50); BI-SEQ-RAGCGTAAAAGGAGCAACATAGT (SEQ ID NO. 51). The reaction system used for bacterial detection PCR was reaction system 1 from step 1), and the reaction conditions were the same as in step 1). The PCR product, purified by bacterial testing, and the linearized expression vector were centrifuged in a microplate centrifuge for 3 seconds, and then placed in a PCR instrument and reacted at 50°C for 20 minutes to complete seamless cloning.

[0078] 4) After confirming that the sequence is 100% correct through nucleic acid sequencing, the strain containing the correct plasmid is cultured in a large-scale manner. This process uses a modified alkaline lysis method combined with column chromatography purification to ensure that high-purity, high-concentration plasmid DNA is obtained for cell transfection.

[0079] 5) Collect 100 mL of the bacterial culture from step 4) after overnight incubation and centrifuge to obtain a bacterial pellet. Resuspend the pellet in Buffer P1, lyse it with Buffer P2 (alkaline), and neutralize it with Buffer P3 (acidic). At this point, chromosomal DNA and proteins form a white flocculent precipitate, while plasmid DNA remains in the supernatant. After filtration, the supernatant is loaded onto a pre-equilibrated chromatography column, where the plasmid DNA specifically binds to the silica gel membrane. Impurities are then removed by washing with QC Buffer, and finally, the purified plasmid DNA is eluted with a low-salt Elution Buffer.

[0080] 6) The eluent was concentrated by isopropanol precipitation and rinsed with 75% ethanol, then dissolved in sterile deionized water. The final plasmid concentrations were determined by NanoDrop: the BT4 plasmid concentration was 1650.63 ng / μL, and the TT4 plasmid concentration was 1324.33 ng / μL, which fully met the transfection requirements.

[0081] 7) Subsequently, CHO (Chinese hamster ovary) cells were selected as the expression system. First, CHO cells were resuscitated and passaged in a shaker at 37°C and 8% CO2 to maintain their optimal growth state.

[0082] 8) When CHO cells reach the appropriate density and viability, electrotransfection is performed. The CHO cells are mixed with the prepared plasmid DNA in an electrotransfection cup, and an electric pulse of a specific voltage is applied to form temporary pores on the cell membrane, so that the plasmid DNA can enter the cytoplasm of the cells. The transfected cells are cultured at 37°C, 120 rpm, and 200 ml. The entire expression cycle lasts for 6 days.

[0083] 9) Antibody purification uses Protein A affinity chromatography, which is based on the principle that Protein A protein can specifically bind to the Fc segment of the antibody. The culture supernatant obtained in step 8) is used as a sample and loaded onto an Amsphere A3 Protein A chromatography column previously equilibrated with PBS. The antibody is specifically bound to the filler, while host cell proteins, nucleic acids and other impurities are eluted (flow-through) with the mobile phase. Then, PBS is used for washing to further remove weakly non-specifically bound impurities. Finally, sodium acetate buffer at pH 3.4 is used for elution, and high-purity antibodies are dissociated from the filler. The elution peak is immediately collected and neutralized to maintain the stability of the antibody. The purified antibody solution is further dialyzed and replaced into a PBS (pH 7.2-7.4) buffer system to remove residual salt and placed in the optimal storage environment.

[0084] 10) The NanoDrop is used to measure the absorbance value at 280 nm to calculate the protein concentration. The final concentration of BT4 is 5.96 mg / mL, and that of TT4 is 6.85 mg / mL. SDS-PAGE results show that the purity of BT4 and TT4 is greater than 95% ( Figure 1 ), and HPLC results show that the purity of BT4 and TT4 is greater than 93% ( Figure 2 ). The limulus test method shows that the endotoxin content of BT4 and TT4 is 0-1 EU / mg. The affinity is determined by Biacore, and the affinity of BT4 and TT4 to human recombinant Trop2 protein is extremely high, which is 19.37 pM and 11.55 pM ( Figure 3 ). Compared with T4 monovalent nanobodies, the affinity of BT4 and TT4 to human recombinant Trop2 protein is increased by 964.53 pM and 954.35 pM, respectively. Therefore, the affinity of the multivalent nanobodies prepared in the present application is significantly improved.

[0085] Example 2

[0086] The present embodiment provides a method for preparing a 68 Ga-labeled multivalent nanobody probe 68 Ga]Ga-NOTA-BT4 and 68 Ga]Ga-NOTA-TT4. The specific steps are as follows:

[0087] 1) p -SCN-Bn-NOTA modified BT4 and TT4 preparation of intermediates NOTA-BT4, NOTA-TT4

[0088] Dissolve 1 mg of BT4 and TT4 in 1 mL of phosphate buffer (PBS) respectively, adjust the pH of the nanobody solution to 9.0-10 with 0.1 mL of 0.1 M sodium carbonate (Na2CO3, PH = 11.4) buffer, and the reaction system volume is 1.1 mL. Add 0.1 mL of freshly dissolved dimethyl sulfoxide (DMSO) to the reaction system, and the molar ratio of SCN-Bn-NOTA to nanobody is 10:1. p -SCN-Bn-NOTA and nanobody molar ratio is 10:1, add freshly dissolved dimethyl sulfoxide (DMSO) to the reaction system, and the molar ratio of SCN-Bn-NOTA to nanobody is 10:1. p -SCN-Bn-NOTA (CAS Number: 147597-66-8; Macrocyclics) is added to the above nanobody solution. The reaction system is placed at room temperature for 2h, and then the purified NOTA-BT4, NOTA-TT4 is purified with a pre-equilibrated PD-10 desalting column (GE Healthcare) using PBS as the mobile phase. p -SCN-Bn-NOTA modified nanobody, collect NOTA-BT4, NOTA-TT4; then concentrate with an ultrafiltration tube (Merck Millipore) with a cutoff value of 10 KDa, and determine the concentration of NOTA-BT4, NOTA-TT4 with NanoDrop, and store at -80°C for future use.

[0089] 2) 68 Ga labeling of NOTA-BT4 and NOTA-TT4 preparation 68 Ga]Ga-NOTA-BT4, 68 Ga]Ga-NOTA-TT4

[0090] Elute the germanium gallium generator (Eckert & Ziegler Radiopharma Inc) with 4 mL of 0.05 M hydrochloric acid solution (HCl), and collect the same volume of activity of about 370-555 MBq of 68 Ga eluate; take the middle section with the highest activity 68 Ga eluate 2 mL, add 0.1 mL of 1 M sodium acetate solution (NaoAc) to adjust the 68 Ga eluate pH to 4.0-4.5; take 100-200 μg of NOTA-BT4, NOTA-TT4 ready for coupling each to 68 Ga eluate, the reaction system volume is <2.5 mL; place the reaction system in a constant temperature oscillator at room temperature for 10 minutes; after the labeling reaction is completed, separate the free 68 Ga, and purify the final product68 Ga]Ga-NOTA-BT4, 68 Ga]Ga-NOTA-TT4.

[0091] 3) 68 Ga]Ga-NOTA-BT4, 68 Ga]Ga-NOTA-TT4quality control

[0092] 10 µL of [ 68 Ga]Ga-NOTA-BT4, 68 Ga]Ga-NOTA-TT4were spotted on silica gel plates, and 0.1 M sodium citrate solution (pH = 5) was used as the mobile phase. Radio-TLC (Eckert & Ziegler Radiopharma Inc) was used to determine the radiochemical purity (RCP) of the probes. The results showed that the radiochemical purity of the probes prepared in the present application was greater than 95% ( 68 Ga]Ga-NOTA-BT4, 68 Ga]Ga-NOTA-TT4. Figure 4 ).

[0093] Example 3

[0094] This example is to diagnose pancreatic cancer by [ 68 Ga]Ga-NOTA-BT4, 68 Ga]Ga-NOTA-TT4immuno-PET imaging. The specific steps are as follows:

[0095] 1) Subcutaneous pancreatic cancer model construction: select Trop2 high expression cell line T3M4 (human pancreatic cancer cells); 2 x 10 6 number of T3M4 cells were suspended in PBS and Matrigel (Corning) at a ratio of 1:1 and injected into the right shoulder of 4-5 week old Balb / c nude mice to establish a subcutaneous pancreatic cancer model.

[0096] 2) The small animal PET / CT imaging acquisition involved in this example was completed using an IRIS small animal PET / CT scanner (Inviscan Imaging Systems). The closed group was co-injected with 200 µg of BT4 when injected with [ 68 Ga]Ga-NOTA-BT4, 68 Ga]Ga-NOTA-BT4, 68[Ga]Ga-NOTA-TT4 was used to anesthetize mice one hour after injection with isoflurane mixed with oxygen (2% concentration). The mice, now in a deep anesthetized state, were placed in a supine position on a PET / CT scanning table, and PET and CT images were continuously acquired. Image reconstruction was performed using the software built into the IRIS system. Regions of interest (ROIs) such as the heart and major organs (liver, lungs, kidneys, and muscles) were delineated on the reconstructed PET images using the OsiriX Lite image processing workstation (Pixmeo SARL). The radioactive uptake values ​​of important organs were calculated in %ID / g (percent of injected dose per gram). Figure 5 Display closed and open groups [ 68 Ga]Ga-NOTA-BT4 PET / CT imaging results. Figure 5 The top left image shows the PET / CT imaging results of the unblocked group, which reveals the Trop2-specific multivalent nanobody probe. 68 Ga]Ga-NOTA-BT4 has high uptake in tumor tissues that highly express Trop2, and also high non-specific uptake in major excretory (kidney) and metabolic (liver) tissues; Figure 5 The top right image shows the PET / CT imaging effect of the closed group, which shows that the uptake of the tumor site is significantly reduced. Figure 5 The bottom left image shows the analysis by drawing ROIs. 68 Distribution of Ga-NOTA-BT4 unblocked and blocked groups in vivo. Figure 5 The lower right figure shows the results of the in vitro biodistribution experiment, which further reveals the distribution of the probe in major tissues and organs in vivo, indicating that [ 68 The Ga-NOTA-BT4 probe can non-invasively visualize Trop2 expression and has high specificity. Similarly, [ 68 PET / CT imaging results of Ga]Ga-NOTA-TT4 in mice with pancreatic cancer are as follows: Figure 6 As shown, significant uptake at the tumor site can be observed. Figure 6 Top left figure), subsequent quantitative ROI analysis ( Figure 6 (Top right image) and in vitro distribution experiment ( Figure 6 The following diagram proves [ 68 Ga]Ga-NOTA-TT4's targeting capability for Trop2.

[0097] Example 4

[0098] This embodiment provides a 18 F-labeled Trop2-specific nanobody probes [ 18F] AlF-RESCA-BT4 and 18 F] Preparation method of AlF-RESCA-TT4. The specific steps are as follows:

[0099] 1) (±)-H3RESCA-TFP modified BT4, TT4 preparation of intermediates RESCA-BT4 and RESCA-TT4

[0100] Dissolve 1 mg of BT4, TT4 in 0.05M NaHCO3 solution (pH=8.6) to obtain a nanobody solution. Add (±)-H3RESCA-TFP freshly dissolved in dimethyl sulfoxide (DMSO) to the above nanobody solution at a molar ratio of (±)-H3RESCA-TFP: nanobody = 12:1. Place the reaction system at room temperature for 2h, then use 0.1M CH3COONH4 solution (pH=4.6) as the mobile phase, and purify the (±)-H3RESCA-TFP modified nanobody with a pre-equilibrated PD-10 desalting column (GE Healthcare). Collect RESCA-BT4 and RESCA-TT4; then use an ultrafiltration tube with a cutoff value of 10 KDa (Merck Millipore) for concentration, and determine the concentration of RESCA-BT4 and RESCA-TT4 with a NanoDrop. Store them at -80℃ for standby.

[0101] 2) 18 F Labeling of RESCA-BT4, RESCA-TT4 preparation 18 F] AlF-RESCA-BT4 and 18 F] AlF-RESCA-TT4

[0102] Add 500μL 18 F solution (about 100 mCi) to a QMA column (Waters GmbH, Germany), use 500μL of normal saline to flush the QMA column and collect 18 F solution, add 16μL of 2mM aluminum chloride solution (pH 4.4-4.6) to it, and stand at room temperature for 5 minutes. Add 400μg of RESCA-BT4, RESCA-TT4 ready for coupling to the reaction system, add 800μL of 0.1M CH3COONH4 solution (pH=4.6), and place the reaction system in a constant temperature oscillator at room temperature for 12 minutes. After the labeling reaction is completed, use normal saline as the mobile phase, and separate the free 18 F again with a pre-equilibrated PD-10 desalting column to purify the end product 18 F] AlF-RESCA-BT4 and 18F]AlF-RESCA-TT4; The unattenuated corrected radiochemical yield (RCY) obtained according to the above steps is >50%.

[0103] 3) [ 18 F]AlF-RESCA-BT4 and [ 18 F]AlF-RESCA-TT4 Quality Control

[0104] Take 10 µL [ 18 F]AlF-RESCA-BT4 and [ 18 F]AlF-RESCA-TT4 was spotted onto a silica gel plate, and physiological saline was used as the mobile phase. The radiochemical purity (RCP) of the probe was determined using radio-TLC (Eckert & Ziegler Radiopharma Inc). Figure 7 As shown, freshly prepared [ 18 F]AlF-RESCA-BT4 and [ 18 The RCP of F]AlF-RESCA-TT4 is greater than 99%.

[0105] Example 5

[0106] This embodiment is [ 18 ImmunoPET imaging of F]AlF-RESCA-TT4 in normal volunteers. The specific steps are as follows:

[0107] PET / CT imaging was performed using a Total-body PET / CT (uExplorer, United Imaging Healthcare) scanner. Normal volunteers received 3.7-5.55 MBq / kg of [ [ ] via intravenous injection in the elbow vein. 18 F]AlF-RESCA-TT4 was used, and images were acquired 1 hour after injection. Normal volunteers were placed in a supine position with their arms raised above their heads. During the scan, patients maintained stable breathing to minimize image fusion errors. The acquisition time was 10 minutes. PET / CT images were evaluated by two nuclear medicine physicians. Specific nanobody probes ([ 18F]AlF-RESCA-TT4)uptake or CT image prompted tumor area as the region of interest (ROI), according to the CT delineation of the region of interest and by the following formula to calculate the maximum standardized uptake value (SUVmax) of ROI: SUV = [local interest area average radioactivity (MBq / mL)] / [injected radioactivity (MBq) / body weight (g)]. In addition, the same method as the present application is used to prepare[ 18 F]AlF-RESCA-T4 probe, and the immune PET imaging of the present embodiment is carried out for comparison. Figure 8 The immune PET imaging results of the present embodiment can be seen[ 18 F]AlF-RESCA-TT4 is mainly excreted through the urinary system, so there is higher uptake of the imaging agent in the kidneys, bladder and other urinary organs, and different degrees of uptake of the imaging agent are also observed in the parotid gland, submaxillary gland, thyroid gland and pancreas. Compared with the immune PET imaging results of the[ 18 F]AlF-RESCA-T4 probe Figure 9 , the uptake of the imaging agent in the parotid gland and submaxillary gland is significantly reduced, with a reduction of about 8-10 times in the parotid gland. The above results show that the[ 18 F]AlF-RESCA-BT4 probe has good safety and potential effect of reducing the uptake of non-target organs.

[0108] Example 6

[0109] The present embodiment is the immune PET imaging of[ 18 F]AlF-RESCA-BT4 in postoperative patients with thyroid cancer. The specific steps are the same as in Example 5. Figure 10 The patient is a case of total resection of papillary thyroid cancer, as shown in the figure, the[ 18 F]AlF-RESCA-BT4 probe can clearly show the residual primary lesion in the right thyroid bed and the lymph node lesion in the right supraclavicular metastasis of the patient, and it can be noted that the uptake of the imaging agent in the parotid gland of the patient is low, significantly improving the target-to-background ratio of the image.

[0110] The above examples are only to illustrate the technical concept and characteristics of the present application, the purpose is to enable those skilled in the art to understand the content of the present application and to implement it, and it cannot limit the protection scope of the present application. Any equivalent changes or modifications made in accordance with the essence of the present application should be covered within the protection scope of the present application.

Claims

1. A multivalent Trop2 diagnostic probe, characterized in that, The probe comprises a tumor targeting group, a radionuclide and a bifunctional chelator; the tumor targeting group is a Trop2 specific multivalent nanobody; the radionuclide is Ga-68 or F-18; The Trop2 specific multivalent nanobody is composed of a T4 monovalent nanobody, a linker and a tail tag; The amino acid sequence of the T4 monovalent nanobody is shown as SEQ ID NO. 1; the number of T4 monovalent nanobodies is n, and n is equal to 2 or 3; The amino acid sequence of the linker is shown as SEQ ID NO. 2; the number of linkers is n-1; The amino acid sequence of the tail tag is shown as SEQ ID NO.

3.

2. The multivalent Trop2 diagnostic probe of claim 1, wherein, The amino acid sequence of the Trop2 specific multivalent nanobody is shown as SEQ ID NO. 4 or 6.

3. The multivalent Trop2 diagnostic probe of claim 2, wherein, The nucleotide sequence encoding the Trop2 specific multivalent nanobody is shown as SEQ ID NO. 5 or 7.

4. The multivalent Trop2 diagnostic probe of claim 1, wherein, The tumor targeted by the tumor targeting gene is a Trop2 expressing tumor.

5. The multivalent Trop2 diagnostic probe of claim 4, wherein, The Trop2 expressing tumor is a high Trop2 expressing malignant tumor; the high Trop2 expressing malignant tumor includes at least one of urothelial carcinoma, nasopharyngeal carcinoma, lymphoma, multiple myeloma, breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, prostate cancer, cervical cancer, head and neck cancer, lung cancer, oral cancer, throat cancer, esophageal cancer, bladder cancer, uterine cancer, ovarian cancer, brain glioma, glioblastoma, thyroid cancer, liver cancer, kidney cancer, skin cancer, melanoma, and penile cancer.

6. The multivalent Trop2 diagnostic probe of claim 1, wherein, the bifunctional chelator is selected from (±)-H3RESCA-TFP, (±) H3RESCA-Mal, NOTA, MAA-NOTA, p -SCN-Bn-NOTA, p -SCN-Bn-DFO, p -SCN-NODA, MAA-GA-NODA, MAA-DOTA, DOTA-NHS, p -SCN-Bn-DOTA, iEDTA, p -SCN-Bn-DTPA, Iso -SGMIB or Boc2-SGMTB.

7. A method of preparing a multivalent Trop2 diagnostic probe according to any one of claims 1-6, characterized in that, The preparation method comprises the following steps: (1) modifying the tumor targeting group with a bifunctional chelator to obtain a conjugated tumor targeting group; (2) labeling the conjugated tumor targeting group with a radionuclide to obtain the probe.

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