VHH chain of human ICOS-targeted nanobody and use thereof

Abstract

A VHH chain of a human ICOS-targeted nanobody and a use thereof, solving the problems of an excessively long in-vivo circulation time of a monoclonal antibody, excessively high liver uptake, poor tissue permeability, non-specific uptake of a monoclonal antibody Fc segment, and decreased imaging result accuracy.

Description

A VHH chain of a human ICOS-targeted nanoantibody and its application Technical Field The present invention belongs to the field of immunotechnology and relates to a VHH chain of a human ICOS-targeted nanoantibody and applications thereof. Background Art Due to the heterogeneity of the tumor microenvironment, the efficacy of immunotherapy varies greatly among different patients. How to accurately predict / evaluate the efficacy of patient treatment is a major issue that needs to be solved. Current nuclear medicine molecular imaging probes used in such research include: Metabolic small molecule probes: In order to achieve accurate in vivo prediction and evaluation of lung cancer immunotherapy, researchers have begun to try to use PET imaging to evaluate people receiving immunotherapy, hoping to establish corresponding diagnostic standards. 18 F-FDG is the most commonly used small molecule probe for PET / CT in clinical practice. 18 F-FDG imaging has been used in the evaluation of the efficacy of immunotherapy for lung cancer. 18 F-FDG showed high uptake, which to some extent realized the in vivo judgment of the efficacy of immunotherapy; however, it has certain limitations, mainly due to two reasons: because tumor cells and activated T cells in the tumor microenvironment are in a state of rapid proliferation, 18 F-FDG showed high uptake, which made it impossible to distinguish the two in PET / CT imaging; immunotherapy drugs can activate T cells in tumor lymph nodes, and the activated T cells proliferate in large numbers, causing 18 High uptake of F-FDG; and tumor lymph node metastasis will also show 18 The high uptake of F-FDG prevents these two distinct biological processes from 18 F-FDG PET imaging can be used for differentiation; in addition, small molecule probes based on nucleic acid metabolism 18 F-AraG is also used in activated T cell imaging studies. 18 F-FDG, which greatly reduces the uptake of tumor cells and increases the uptake of immune cells inside the tumor, but except for activating T cells, 18 F-AraG is still taken up by macrophages, dendritic cells, and B cells, resulting in non-specific imaging. This shows that small molecule metabolic probes cannot accurately reflect the efficacy of immunotherapy due to their lack of specificity. Targeting T cell imaging of different phenotypes: In 2014, Griessinger's team used 64Cu was used to label T cell receptor (TCR)-targeted antibodies, and in vivo tracing of T cells was completed through PET / CT molecular imaging; the Richard team published articles in *PNAS* and *Cancer Research* in 2014 and 2016 respectively, reporting their team's application of 64 Cu and 89 Zr to label CD8-targeted monoclonal antibodies and conduct in vivo imaging studies on CD8 + effector T cells; subsequently, Kristensen et al. reported the work of using CD4 + and CD8 + bimolecular probes to evaluate the efficacy of the Sym021 (human-mouse cross-reactive PD-1 inhibitor) treatment model

[0026] . However, in the solid tumor microenvironment, there are a large number of exhausted T cells that express TCR, CD3, and CD8. Molecular imaging targeting T cell phenotypes cannot distinguish between exhausted T cells and activated T cells. Specific targeting imaging of activated T cells: Whether it is a metabolic small molecule probe or molecular imaging targeting different T cell phenotypes, it is impossible to accurately identify activated T cells in vivo. The crux of the problem is that the selected target or metabolic pathway has relatively low specificity for activated T cells. Technical problems In view of the above deficiencies in the existing technology, the technical problem to be solved by the present invention is to provide a VHH chain of a human ICOS-targeted nanobody and its application. Technical solutions The object of the present invention is to provide a VHH chain of a human ICOS-targeted nanobody and its application in view of the above problems existing in the existing technology. A VHH chain of a human ICOS-targeted nanobody, the amino acid sequence of which is at least one sequence shown in SEQ ID NO: 6-10. The present invention also provides a human ICOS-targeted nanobody, which includes the VHH chain of the above-mentioned human ICOS-targeted nanobody. The present invention also provides a gene sequence, which encodes the VHH chain of the above-mentioned human ICOS-targeted nanobody or encodes the above-mentioned human ICOS-targeted nanobody. In the above-mentioned gene sequence, there is at least one sequence shown in SEQ ID NO: 1-5. The present invention also provides a nucleotide construct, which contains the above-mentioned gene sequence. The present invention also provides a recombinant expression vector, which contains the above-mentioned nucleotide construct. The present invention also provides a recombinant host cell, which contains the above nucleotide construct or the above recombinant expression vector. The present invention also provides a conjugate, which is obtained by conjugating the above-mentioned human ICOS-targeting nanobody and a chelator of a radionuclide. The present invention also provides a radionuclide-labeled substance, which is characterized in that the above conjugate is labeled with a radionuclide. In the above-mentioned radionuclide-labeled substance, the radionuclide includes 68 Ga. The present invention also provides a pharmaceutical composition, which contains the above radionuclide-labeled substance and a pharmaceutically acceptable carrier. The present invention also provides the applications of the above-mentioned human ICOS-targeting nanobody, the above conjugate, the above radionuclide-labeled substance, and the above pharmaceutical composition in drugs, reagents, and detection kits for diagnosing or treating tumors. Beneficial effects Compared with the prior art, the present invention has the following beneficial effects: By independently developing a human ICOS-targeting nanobody, the present invention solves the problems that the construction of an ICOS molecular probe is based on a monoclonal antibody with a molecular weight of about 150,000, resulting in an overly long in vivo circulation time, excessive liver uptake, and poor tissue permeability, and the Fc segment of the monoclonal antibody can bind to Fc receptors on macrophages and dendritic cells, causing non-specific uptake, which greatly reduces the accuracy of imaging results. Description of the drawings Figure 1 shows the construction of a gene library in Example 1, with 150bp and 200bp gene fragments on the left; 330bp gene fragment on the right; Figure 2 shows the verification of c-myc expression by flow cytometry in Example 1; Figure 3 shows the decay exponential wave during the electroporation process in Example 1; Figure 4 shows the verification results of colony PCR in Example 1; Figure 5 shows the verification results of enzyme digestion in Example 1; Figure 6 shows the sequencing results in Example 1; Figure 7 shows the verification of the binding ability of the ICOS nanobody. Embodiments of the present invention The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments. Example 1 S1. Expression of ICOS protein antigen: Prokaryotic expression vector: pET, expression host: BL21(DE3) ORF: ICOS poly his The target protein expression usually uses competent cells of E.coli Strain BL21(DE3). After culturing the successfully identified clone, the recombinant plasmid is extracted. Then the recombinant plasmid is transformed into competent cells of E.coli Strain BL21(DE3), and plasmid extraction and transformation are carried out. (1) The E.coli Strain BL21(DE3) strain containing the recombinant plasmid is inoculated into 5 ml of LB medium containing antibiotics and cultured overnight at 37 °C with 200 rpm. The overnight culture medium is inoculated into 500 ml of LB medium containing antibiotics and cultured until OD600 = 0.4–0.6. (2) IPTG is added to induce the expression of the target protein. The concentration and time of IPTG can be explored. The exploration concentration of IPTG is 0.05 - 1.0 mM, and the induction time is 1 - 5 h. (3) The E. coli cells are collected by centrifugation (16,000 g for 3 min), and the cell pellet is suspended in lysis buffer (100 μl of 50 mM Tris–HCl buffer, pH 8.0, containing 200 mM NaCl, 2% Triton X-100 and 3 mM protease inhibitor PMSF), and then sonicated on ice 20 times (sonicated 3 s with 7 s intervals between each pulse). (4) The sonicated cells are centrifuged (16,000 g for 15 min at 4 °C), and a part of the supernatant is mixed with 6xloading buffer and heated at 95 °C for 5 min. SDS-PAGE is used for gel running and Coomassie Brilliant Blue R250 is used for staining to detect whether the protein expression is successful. S2, His Protein Purification Binding buffer: 20 mM sodium phosphate buffer, 0.5 M sodium chloride, 20 - 40 mM imidazole, pH 7.4; Elution buffer: 20 mM sodium phosphate buffer, 0.5 M sodium chloride, 500 mM imidazole, pH 7.4; The sample needs to be filtered before loading, and the buffer needs to be pre-filtered and sonicated to remove air bubbles. (1) Set a low flow rate for the system, remove the plug at the top of the pre-packed column, and connect the column to the connector of the ÄKTA purifier, noting that it should be connected drop by drop to prevent the introduction of air bubbles. (2) Remove the plug at the bottom of the column and connect it to the purifier. (3) Wash away the ethanol with 3 - 5 column volumes of distilled water. (4) Equilibrate the column with 5 column volumes of binding buffer. The recommended flow rate is 1 ml / min (for a 1-ml column) and 5 ml / min (for a 5-ml column). (5) Load the pretreated sample using a sample loop or superloop (the sample must be centrifuged and filtered before loading onto the column). The recommended flow rate during loading is 0.2 - 1 ml / min (for a 1-ml column) and 0.5 - 5 ml / min (for a 5-ml column). (6) Wash with binding buffer for at least 10 to 15 column volumes until the absorption peak reaches a stable baseline or no substances elute in the effluent. The recommended flow rate during washing is 1 - 2 ml / min (for a 1-ml column) and 5 - 10 ml / min (for a 5-ml column). (7) Elute with elution buffer using either a one-step elution or a linear gradient elution (imidazole concentration gradient). The one-step elution is usually 5 column volumes, and the linear elution is usually 10 - 20 column volumes. Maintain a flow rate of 1 - 2 ml / min (for a 1-ml column) and 5 - 10 ml / min (for a 5-ml column) during elution. (8) After elution, wash the column with 3 - 5 column volumes of binding buffer, and then add 20% ethanol. Screw on the plugs at both ends of the column to prevent it from drying out. S3. Construct a yeast surface display library (YSD) This invention selected 3 different loop lengths for each loop based on the loop lengths observed in the VHH domain in the human species. As shown in Figure 1, a gene library was constructed. The VHH gene library was homologously recombined with a vector containing the Aga2 protein for yeast surface display at the N-terminus and the c-myc epitope for detecting full-length VHH at the C-terminus, obtaining 8x10 7 yeast transformants. Among the 25 clones sequenced, 15 matched the library design. 4 (35%) contained frameshifts, and 1 (4%) had improper annealing or unintended homologous recombination in yeast. Therefore, 75% (16 / 26 x (1 - 0.44)) of the transformed cells should display full-length VHH. The expression of VHH was verified by flow cytometry analysis (data shown in Figure 2). This library contains approximately 2 × 10 7 full-length VHH clones. Electroporation transformation steps: (1) Culture EBY100 overnight in YPD medium. Inoculate 100 mL of YPD with 1x10^8 cells (OD = 0.1). Culture at 30 °C and 250 rpm until the cell density reaches 1.3 - 1.5x10 ^7 cells / mL. (2) Incubate at 30 °C and 250 rpm until the cell density reaches 1.3 - 1.5 x 10 ^7 cells / mL, which usually takes 6 - 8 hours. If necessary, inoculate at a lower density and incubate for a longer time, such as overnight, to prepare competent cells. (3) Cool the electroporation cells and E buffer (add 25 ml LiAC to 1.3 OD EBY100 per 50 ml): Centrifuge at 3000 rpm for 3 minutes to remove the supernatant; Resuspend in 25 mL of 100 mM lithium acetate, 10 mM Tris, pH 7.5, 1 mM EDTA; Resuspend the pellet in a solution containing 100 mM lithium acetate, 10 mM Tris (pH 7.5), and 1 mM EDTA; Incubate at 30 °C for 15 - 60 minutes; Incubate at 30 °C for 15 - 60 minutes with gentle shaking (110 rpm); Add 0.096 g DTT to 0.5 mL of 1M Tris, pH 8.0. After filter sterilization, add to EBY100; Incubate at 30 °C and 250 rpm for 15 minutes; Centrifuge at 2500g and 4 °C for 3 minutes. Discard the supernatant; Wash the cells with 25 mL of E buffer (10 mM Tris, pH 7.5, 270 mM sucrose, 1 mM MgCl2); Centrifuge at 2500g and 4 °C for 3 minutes. Discard the supernatant; Resuspend the cells in 1 mL of E buffer. Transfer to a 1.5 mL tube; Centrifuge at 5000g and 4 °C for 1 minute; Discard the supernatant; Resuspend in 1 mL of E buffer; Centrifuge at 5000g and 4 °C for 1 minute. Discard the supernatant; Resuspend the cells to a total volume of 300 uL. (4) Electroporation: Prepare the sample in a microcentrifuge tube: 150 uL of cells + 2 uL of vector (4 ug) + all inserts (10 ug); Prepare the sample in a microcentrifuge tube: 150 uL of cells, 2 uL of vector (4 ug), and all inserts (10 ug); Transfer the mixture to two electroporation cells and incubate on ice for about 5 minutes; Pulse at 25 uF and 0.54 kV (for 2 mm cuvettes); The time constant should be 15 - 45 ms. (Data shown in Figure 3) Add 1 - 2 mL of room temperature YPD to the cells. Transfer to a 14 mL Falcon tube; Incubate at 30 °C and 250 rpm for 1 - 2 hours; Centrifuge at 1300g for 1 minute; Resuspend in 1 mL of SD - CAA. Transfer to a flask with 50 - 1000 mL of SD - CAA. As can be seen from Figure 3, the TC time of this damped exponential wave conforms to the optimal perforation time for yeast electroporation, and the transformation efficiency of this transformation constant is the highest in the following library construction. S4. Yeast growth and induction: (1)Day 1: At 5:00 PM, thaw a vial of library cells in hand or inoculate the cells into a 2-L flask with a magnetic stir bar containing 1 L of S(D)CAA P / S on the laboratory bench, and keep at 4°C for a period of time. The cell density should be less than 2.5 x 10 ^6 cells / mL. Incubate overnight at 30°C at 250 rpm. (Start the culture from an OD of 0.1 to 0.2); (2)Day 2: At 8:30 AM, count the cells (hemocytometer) and dilute the cells 10-fold. At a cell density of 2 x 10 ^6 cells per milliliter, seed in pre-warmed S(D)CAA +P / S at 30°C at least 10-fold of the library diversity (if using the original library, use 20-fold for libraries with a diversity less than 2 x 10 ^7 . Inoculate the cells into a 2-L flask with a magnetic stir bar containing 1 L of S(D)CAA P / S. Incubate at 250 rpm at 30°C for 7 hours (ensure it is in the logarithmic growth phase, the size of the original cells is approximately 2 times that of the dividing cells) (after 7 hours, the OD should not exceed 3); After 7 hours of growth, determine the cell density (hemocytometer). Harvest 6 x 10^9 cells in a 50-mL conical centrifuge tube, centrifuge at 2500g for 5 minutes, discard the supernatant, and transfer the cells to 300 mL of pre-warmed S(G)CAA +P / S (galactose yeast nutrient solution containing a final concentration of 2% galactose and 0.1% glucose) at a density of 2 x 10 ^7 / mL, and transfer to a 1-L flask with a magnetic stir bar. Incubate at 250 rpm at 30 (20)°C for at least 16 hours. Store the S(D)CAA culture in the refrigerator until the first round of sorting is completed and the results are verified by colony PCR. Figure 4 shows the results of colony PCR verification. It can be seen from the figure that the positive clone rate of the colonies reaches 100%. Figure 5 shows the results of restriction enzyme digestion verification. It can be seen from the figure that the first-round library construction of the yeast plasmid NL540 was successfully verified by restriction enzyme digestion. Figure 6 shows the sequencing result diagram. It can be seen from the figure that by sequencing from the 5' and 3' ends of this sequence, there are 3 variable regions in the middle, the distribution frequencies of 4 types of bases are the same, and the constant structural regions at both ends are consistent with the domain sequences of VHH, verifying the accuracy of library construction. Among them, SEQ ID NO: 1: CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGTTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTTGAGCGTTGGACCATGGGGTGGTTCCGCCAGACCCCAGGGAAGGAGCGCGAGGGGATCTCATGTATTAGTAGTAGTGATGGTAGCACATACTATGGAGACTCCAAATACTATGGAGACTCCGTTCGGGGCCGATTCACTATTTCCAGAGACAACGCCAAGAACACAGTATATCTGCAAATGAACAACTTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGCCCAAGGGTATAGCGACTATGGATGCTACGGCATGGAGTACTGGGGCAAAGGGACCCTGGTCACCGTCTCCTCAT; S5, Naïve Yeast Library and Culture Conditions Media: Commonly used media include YPD, SDCAA, SGCAA media and their corresponding plates Temperature: Generally, the temperature for yeast surface display is maintained between 28°C and 30°C. These temperatures are ideal for yeast growth and protein expression. pH value: The pH value of the medium should be within an appropriate range, usually between pH 5.5 and 6.5. Controlling the pH value helps with yeast cell growth and protein expression. Antibiotic selection: The yeast strain has an antibiotic resistance marker of AmpiciIIin to maintain the expression of the antibiotic resistance gene and the screening process. S6, Screening Process of ICOS Nanobody The initial yeast library is grown, induced, and subjected to magnetic bead selection to reduce unwanted binders and improve the efficiency of binders to biotinylated antigen. The resulting population is further enriched by FACS to select for high affinity and analyzed and monitored. By extracting plasmids from the enriched population, their sequences are analyzed, and the prevalent sequences are transformed into yeast to characterize monoclonal clones. Once the clones of interest are identified, new libraries are generated by performing error-Prone PCR and transforming yeast by homologous recombination. For nM affinity, high-affinity binders are enriched by FACS selection, and for nM affinity, they are enriched by iterative FACS selection in kinetic competition until the desired affinity is achieved. (1)Magnetic bead screening: The initial library screening was performed using Biotin Binder dynabead (Invitrogen). The ICOS antigen must be biotinylated before sorting. The initial library was induced for more than 16 h at 30 °C under SGCAA medium conditions, and the biotinylated ICOS antigen was used to link the dynabead. Positive magnetic selection: After incubation at 4 °C for 2 h, the bead-bound cells were placed on a magnetic stand, and the unbound cells were transferred to a tube with washed, antigen-coated beads. Incubate at 4 °C for at least 2 h. Negative magnetic selection: Yeast cell growth and induction. The newly induced yeast population should be rich in ICOS / antigen conjugates. To ensure the separation of specific binders from non-specific yeast, increase the stringency of washing and perform negative selection. After two rounds of magnetic bead enrichment, the double-positive yeast was isolated by FACS, and the yeast cells showing full-length VHH clones were screened. (2)VHH mutagenesis and electroporation: To reintroduce diversity into the enriched population of VHH conjugates, the plasmids were isolated from yeast and mutagenized. Yeast plasmids were extracted, and mismatch PCR was performed using 8-oxo-2-deoxyguanosine-5-triphosphate and 2-deoxy-p-nucleoside-5 triphosphate (8-oxo-dGTP and dPTP) as raw materials (shown in Table 1). The amplification of mutagenized VHH (shown in Table 2) was to generate a sufficient number of DNA inserts for yeast transformation. Table 1: Mismatch PCR Table 2: Amplification PCR Using pCT-CON2 as the vector, the amplified gene fragments were mixed and electroporated into wild-type yeast cells. The linearized vector and inserts were introduced into yeast by electroporation transformation. Yeast will naturally perform homologous recombination to recycle the linearized vector and inserts. SEQ ID NO: 6: QVQLVESGGGLVQPGGSLRLSCAASGFTFERWTMGWFRQTPGKEREGISCISSSDGSTYYGDSKYYGDSVRGRFTISRDNAKNTVYLQMNNLKPEDTAVYYCAAAQGYSDYGCYGMEYWGKGTLVTVSS; (3)FACS screening After several rounds of magnetic bead enrichment, FACS can be used to quantitatively screen the yeast-displayed VHH library. The library will be labeled for display by c-myc tag detection and binding to soluble antigen. Using this dual labeling, collection screening is performed. When sorting for high-affinity binding with nM binding affinity, dissociation competition can be used. First, label with a stoichiometric excess of biotinylated antigen, wash, and then compete with unlabeled non-biotinylated antigen. (4)Monoclonal identification and analysis Collect yeast cells with nM affinity, extract yeast plasmids, transfer them into DH5α, amplify and select monoclonal clones, amplify, extract bacterial plasmids, and send them for sequencing. (5)ICOS nanobody expression According to the finally collected sequencing sequences, construct the PET-ICOS sequence. Using the above prokaryotic expression system, the VHH protein can be collected by ultrasonic wave. After cell lysis, according to the purification tag fused to VHH, use the appropriate tag to purify the protein. (6)Flow cytometry verification of the binding ability of ICOS nanobody Group Positive cell and Negative cell. Positive cell (add PMA / INON to stimulate for 48 - 72h 24h after resuscitation of 50 million PBMC). 2. Transfer 5X104 cells per well to a 96-well U-bottom plate for Flow staining. Wash with 200ul PBS and centrifuge at 2000rpm to discard the supernatant. Add 1ul of ICOS nanobody per well (first take a part of each sequence and dilute it to 1ug / ul) and incubate in the dark for 20min. 3. Wash with 200ul PBS and centrifuge at 2000rpm to discard the supernatant, repeat once. 4. Add 1ug of secondary antibody per well and incubate in the dark for 20min. 5. Wash with 200ul PBS and centrifuge at 2000rpm to discard the supernatant, repeat once. 6. Resuspend in facs buffer and load onto the machine (the results are shown in Figure 7). It can be seen from the figure that the binding ability of the ICOS nanobody to activated T cells is significantly higher than that to resting T cells. (7)Human ICOS-targeted nanobody- 68 Ga nuclide labeling. Example 2 The difference from Example 1 is only that the gene sequence is as shown in SEQ ID NO: 2, and the VHH chain sequence of its human ICOS-targeted nanobody is as shown in SEQ ID NO: 7. Example 3 The difference from Example 1 is only that the gene sequence is as shown in SEQ ID NO: 3, and the VHH chain sequence of the human ICOS-targeting nanobody thereof is as shown in SEQ ID NO: 8. Example 4 The difference from Example 1 is only that the gene sequence is as shown in SEQ ID NO: 4, and the VHH chain sequence of the human ICOS-targeting nanobody thereof is as shown in SEQ ID NO: 9. Example 5 The difference from Example 1 is only that the gene sequence is as shown in SEQ ID NO: 5, and the VHH chain sequence of the human ICOS-targeting nanobody thereof is as shown in SEQ ID NO: 10. For the points not exhausted in the numerical values of the technical scope claimed by the present invention in the embodiments herein and the new technical solutions formed by the equivalent replacement of single or multiple technical features in the technical solutions of the embodiments, they are also within the scope claimed by the present invention; at the same time, in all the exemplified or unexemplified embodiments of the present invention, the various parameters in the same embodiment only represent an example of its technical solution (i.e., a feasible solution), and there is no strict cooperation and limitation relationship between the various parameters. Among them, the various parameters can be replaced with each other without violating the axioms and the requirements of the present invention, unless otherwise specially stated. The technical means disclosed in the present invention is not limited to the technical means disclosed in the above technical means, but also includes the technical solutions composed of any combination of the above technical features. The above is the specific implementation manner of the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements are also regarded as the protection scope of the present invention. The specific embodiments described herein are only illustrative of the spirit of the present invention. Those skilled in the art of the present invention can make various modifications or supplements to the described specific embodiments or use similar ways to replace them, but will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

Claims

1. A VHH chain of a human ICOS-targeting nanobody, characterized in that, At least one sequence as shown in SEQ ID NO: 6-10.

2. A human ICOS-targeting nanobody, characterized in that, Comprising the VHH chain of the human ICOS-targeting nanobody according to claim 1.

3. A gene sequence, characterized in that, Encoding the VHH chain of the human ICOS-targeting nanobody according to claim 1 or encoding the human ICOS-targeting nanobody according to claim 2.

4. A gene sequence according to claim 3, wherein Having at least one sequence as shown in SEQ ID NO: 1-5.

5. A conjugate, characterized in that, Obtained by conjugating the human ICOS-targeting nanobody according to claim 2 with a chelator of a radionuclide.

6. A radionuclide marker, characterized in that, Obtained by labeling the conjugate according to claim 2 with a radionuclide.

7. The radioactive nuclide marker according to claim 6, wherein, The radionuclides include 68 Ga.

8. A pharmaceutical composition, characterized in that, Containing the radionuclide label according to claim 3 and a pharmaceutically acceptable carrier.

9. Use of the human ICOS-targeting nanobody according to claim 2, the conjugate according to claim 5, the radionuclide label according to claim 6, and the pharmaceutical composition according to claim 8 in drugs, reagents, and detection kits for diagnosing or treating tumors.

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