Method for labeling nano antibody with radionuclide

Abstract

The invention discloses a method for labeling a nano antibody with radionuclide. The method comprises the following steps: (1) reducing the nano antibody by using a reducing agent to obtain a reduction reaction product; (2) coupling the reduction reaction product with a chelating agent, and purifying to obtain a coupling product; (3) carrying out radionuclide labeling on the coupling product, and purifying to obtain a radionuclide labeled nano antibody; wherein the condition of (1) meets any one of the following groups: the reducing agent is TCEP; the ratio of the amount of substance of the reducing agent to the amount of substance of the nano antibody is 10 *-100 *; the temperature is 25-55 DEG C; the reaction time is 1-2 hours; the reducing agent is 2-MEA, the ratio of the amount of substance of the reducing agent to the amount of substance of the nano antibody is 10 *-100 *, and the temperature is 25-55 DEG C; the reaction time is 1-2 hours. The method is better in reduction effect and higher in marking efficiency.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to a method for radionuclide-labeled nanobodies. Background Technology

[0002] Antibodies (Abs), or immunoglobulins (Igs), are serum glycoproteins produced by the body's immune response in response to external antigen stimulation. Since the advent of the first commercially available CD3-targeting antibody, Muromonab, in 1986, monoclonal antibodies (mABs) have been considered among the best bioactive substances for detection and labeling. They possess high affinity and specificity for antigen targets, making them highly suitable for basic research and clinical applications, and are now widely used in research fields such as tumor immunotherapy, virus detection, and vaccine development. With the continuous deepening of research and the development of bioengineering technology, the requirements for antibody properties are constantly increasing. Although monoclonal antibodies have excellent affinity and specificity, their molecular weight is relatively high, around 150 kDa. Their metabolic rate in animals is insufficient to meet the growing research demands, leading to the development of recombinant antibodies based on monoclonal antibodies. Recombinant antibodies aim to maintain affinity and specificity for the target antigen while reducing the antibody molecular weight, resulting in Fab2 fragment antibodies (~111 kDa), mini antibodies (~80 kDa), Fab fragment antibodies (~50 kDa), and so on.

[0003] Nanobodies (NBs) have a significant advantage over other antibody fragments in terms of molecular weight. However, unlike recombinant antibodies, nanobodies originate from naturally occurring heavy-chain only antibodies (HcAbs) that lack a light chain. Structurally, heavy-chain antibodies contain only three domains: the C-terminus consists of two constant domains, homologous to the CH2 and CH3 domains in human antibodies; the N-terminus contains only one domain, VHH, which has antigen-targeting activity. After being isolated using biotechnology, they are called nanobodies.

[0004] Thanks to their unique structure, nanobodies have advantages over widely used monoclonal antibodies and other recombinant antibodies. In summary:

[0005] 1) Nanobodies have small molecular weights, ranging from 12 to 15 kDa, making them the smallest natural antigen-binding proteins. Due to their small size, they are more likely to penetrate into solid tumors and be rapidly metabolized from the body.

[0006] 2) The CDR3 domain of nanobodies is longer than that of conventional antibodies, and has the same or even better affinity and specificity, which is beneficial for further binding to remote epitopes and hidden targets in antigens.

[0007] 3) Nanobodies contain more hydrophilic groups, making them more hydrophilic, soluble, stable, and less prone to aggregation compared to conventional antibodies.

[0008] 4) Nanobodies have shorter sequences and can be cloned and produced on a large scale using bioengineered bacteria such as E. coli, resulting in lower costs than traditional antibodies.

[0009] Compared to other tumor diagnostic methods, in vivo imaging using targeted agents carrying radionuclides offers significant advantages. Compared to pathological tumor diagnostic methods, such as biopsy, exfoliative cytology, and tumor marker testing, PET imaging can obtain global tumor location information and acquire tumor metastasis data that is difficult to obtain using pathological methods. Regarding other imaging methods, such as X-rays and CT scans, PET imaging simultaneously offers lower radiation risk and higher accuracy and resolution. In summary, it is of great significance for tumor detection and diagnosis. Furthermore, the high affinity, low molecular weight, rapid metabolism, and good stability of nanobodies perfectly meet the target agent requirements of PET imaging. Currently, as an emerging antibody molecule, nanobodies still have significant research gaps and require further development. Summary of the Invention

[0010] To address the shortcomings of existing nanobody labeling technologies, which often rely on random labeling of lysine residues, resulting in inconsistent product properties and low labeling efficiency, this invention provides a... 68 A method for Ga-labeled nanobodies.

[0011] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0012] This invention provides a method for labeling nanobodies with radionuclides, the method comprising the following steps:

[0013] (1) The nanobody was reduced using a reducing agent to obtain the reduction reaction product;

[0014] (2) The reduction reaction product is coupled with a chelating agent and purified to obtain the coupled product;

[0015] (3) The conjugated product is radiolabeled and purified to obtain radiolabeled nanobodies;

[0016] Wherein, the condition in (1) satisfies any one of the following sets:

[0017] The reducing agent is TCEP; the ratio of the reducing agent to the nanobody material is (10-100):1; and / or, the temperature is 25-55℃; the reaction time is 1-2 h;

[0018] The reducing agent is 2-MEA, and the ratio of the reducing agent to the nanobody material is (10-100):1; and / or, the temperature is 25-55℃; the reaction time is 1-2 h.

[0019] In some embodiments, in step (2), the molar ratio of the reduction reaction product to the chelating agent is 1:(10-100); and / or, the coupling conditions include reacting at 25-55°C for 30-120 minutes.

[0020] In some preferred embodiments, the condition of (1) satisfies any one of the following sets:

[0021] The reducing agent is TCEP, and the ratio of its amount to the nanobody material is 10:1. The temperature is 25℃, 37℃, or 55℃; the reaction time is 1 h, 1.5 h, or 2 h.

[0022] The reducing agent is 2-MEA, and the ratio of its amount to the nanobody material is 20:1. The temperature is 25℃, 37℃, or 55℃, and the reaction time is 1 h, 1.5 h, or 2 h.

[0023] And / or, in (2), the molar ratio of the reduction reaction product to the chelating agent is 1:10; and / or, the coupling conditions include: reacting at 37°C for 90 minutes.

[0024] In some preferred embodiments, (1) and (2) are carried out simultaneously in one system; the molar ratio of nanoantibody, reducing agent and chelating agent in the system is 1:(10-100):(1-100); and / or the reaction conditions of the system include: reacting at 25-55°C for 30-120 minutes.

[0025] In some preferred embodiments, the molar ratio of nanobody, reducing agent, and chelating agent in the system is 1:10:10; and / or, the reaction conditions of the system include: reacting at 37°C for 90 minutes.

[0026] In some further preferred embodiments, the concentration of the nanobody is 444 μM.

[0027] In some further preferred embodiments, the chelating agent is selected from one or more of Mal-NOTA, Mal-DOTA, Mal-NODAGA, NHS-DOTA, Mal-AAZTA, NHS-AAZTA, and Mal-DFO.

[0028] In some preferred embodiments, the chelating agent is selected from one or more of Mal-DOTA, Mal-NOTA, or Mal-DFO.

[0029] In some specific embodiments, the chelating agent is Mal-NOTA.

[0030] In some implementations, the purification in (2) includes the following steps:

[0031] Purification was performed using HiTrap™ Desalting columns, ultrafiltration tubes, or PD10 columns.

[0032] In some preferred embodiments, purification is performed using a HiTrap™ Desalting column.

[0033] In some preferred embodiments, the mobile phase is 0.9% sodium chloride injection, the flow rate is 2.0 mL / min, the elution time is 30 min, and the eluent during the period from 2.6 min to 3.6 min is collected based on the ultraviolet absorption peak to obtain the coupling product.

[0034] In some embodiments, the system of (3) comprises the coupling product, the radionuclide, and a sodium acetate solution;

[0035] The reaction conditions for the system include: reacting at 25-50℃ and 0-1000rpm for 5-60 minutes.

[0036] In some preferred embodiments, the radionuclide is selected from... 68 Ga、 64 Cu、 177 Lu、 90 Y、 111 In、 44 Sc、 225 One or more of Ac.

[0037] In some specific implementations, the radionuclide is 68 Ga.

[0038] In some preferred embodiments, the system comprises the coupling product, 0.4 mL 68 GaCl3 elution solution and 0.1 mL of 3M sodium acetate solution at pH 4.5;

[0039] The reaction conditions for the system include: reacting at 37°C and 650 rpm for 15 minutes.

[0040] In some implementations, the purification in (3) includes the following steps:

[0041] Purification was performed using a HiTrap™ Desalting column, ultrafiltration tube, or PD10 column, with PD10 column being preferred; the mobile phase was 0.9% sodium chloride injection or 1× PBS, with 1× PBS being preferred.

[0042] In some preferred embodiments, the purification of the radiolabeled product further includes filtering the most radioactive fraction using a 0.22µm filter.

[0043] In some preferred embodiments, the radiolabeled product is quality controlled by HPLC and iTLC before and after purification.

[0044] In some embodiments, the C-terminus of the nanobody contains an amino acid sequence with a Cys group.

[0045] In some preferred embodiments, the nanobody comprises CDR1, CDR2 and CDR3; wherein the amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 3.

[0046] In some preferred embodiments, the amino acid sequence of the nanobody is shown in SEQ ID NO: 4.

[0047] A second aspect of the present invention provides a radionuclide-labeled nanobody, which is prepared by the method described in the first aspect.

[0048] A third aspect of the present invention provides a radionuclide-labeled nanobody, the radionuclide-labeled nanobody comprising the chelating agent as described in the method of the first aspect; and / or the radionuclide as described in the method of the first aspect; and / or the nanobody as described in the method of the first aspect.

[0049] In some preferred embodiments, the chelating agent is coupled to the nanobody via the Cys group at the C-terminus of the nanobody.

[0050] A fourth aspect of the present invention provides a diagnostic or pharmaceutical composition comprising a nanobody as described in the second or third aspect, and a pharmaceutically acceptable carrier or excipient.

[0051] A fifth aspect of the present invention provides a kit comprising nanobodies as described in the second or third aspect, or diagnostic or pharmaceutical compositions as described in the fourth aspect.

[0052] The sixth aspect of the present invention provides the use of nanobodies as described in the second or third aspect, diagnostic compositions or pharmaceutical compositions as described in the fourth aspect, or kits as described in the fifth aspect, in the preparation of targeted molecular imaging diagnostic and / or targeted therapeutic agents for diseases.

[0053] In some preferred embodiments, the disease is a tumor.

[0054] In some preferred embodiments, the tumor is a PSMA-positive tumor.

[0055] In some preferred embodiments, the tumor is colon cancer, esophageal cancer, thyroid cancer, lung cancer, prostate cancer, renal cell carcinoma, or brain tumor.

[0056] In some specific implementations, the tumor is metastatic castration-resistant prostate cancer.

[0057] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0058] The reagents and raw materials used in this invention are all commercially available.

[0059] The positive and progressive effects of this invention are as follows:

[0060] 1. The isotope nanobody labeling method of the present invention has better reduction effect, higher labeling efficiency, and optimizes the final recovery rate of radioactive products.

[0061] 2. This invention uses novel nanobodies, which are different from traditional antibodies. They have advantages such as rapid metabolism, small molecular weight, strong affinity, good stability, and low cost, and are promising as targeting agents in the diagnosis and treatment of targeted radiation-induced tumors. Attached Figure Description

[0062] Figure 1 The image shows the UV absorption peaks after purification using the desalting column employed in this invention.

[0063] Figure 2 After purification and filtration for this invention 68 HPLC quality control results of Ga-NOTA-Nb products.

[0064] Figure 3 The image shows the HPLC results of the Nota-Nb product after 60 minutes of reaction with the reducing agent 2-MEA of this invention.

[0065] Figure 4 The image shows the HPLC results of the Nota-Nb product after 60 minutes of reaction using the reducing agent TCEP of this invention.

[0066] Figure 5This is a graph showing the SDS-PAGE gel running results of the nanobody used in this invention.

[0067] Figure 6 This is a graph showing the HPLC quality control results of the batch of nanobody production used in this invention.

[0068] Figure 7 After purification and filtration for this invention 68 HPLC quality control results of Ga-DFO-Nb products.

[0069] Figure 8 After purification and filtration for this invention 68 HPLC quality control results of Ga-DOTA-Nb products. Detailed Implementation

[0070] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the procedures described herein, including those related to molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional procedures widely used in their respective fields. To better understand this invention, definitions and explanations of related terms are provided below: In this invention, the term "antibody" generally refers to a protein containing a portion that binds to an antigen, and optionally, a scaffold or backbone portion that allows the antigen-binding portion to adopt a conformation that promotes antibody-antigen binding. Typically, it may include a variable region (VL) of the antibody light chain, a variable region (VH) of the antibody heavy chain, or both. For example, a "heavy chain antibody" in this invention does not contain a VL region but only a VH region. The VH or VL regions may be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are scattered within more conserved regions called framework regions (FRs). Each VH or VL may consist of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. Examples of antibodies include, but are not limited to, full-length antibodies, heavy chain antibodies (HCAbs), antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, and / or dAbs), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, provided they exhibit the desired antigen-binding activity.

[0071] The term "nanobody" in this invention refers to a variable region consisting only of an antibody heavy chain. A single-domain antibody consisting of only one heavy chain variable region is constructed, which is the smallest antigen-binding fragment with full function.

[0072] The term "pharmaceuticalally acceptable excipient" refers to a component in a pharmaceutical composition that, apart from the active ingredient, is non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, and / or preservatives.

[0073] In this art, antibody CDRs can be defined using various methods, such as the Kabat definition rule based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, 5th Edition, National Institutes of Health, Bethesda, Maryland (1991)) and the Chothia definition rule based on the location of structural loop regions (see, Al-Lazikani et al., JMol Biol 273:927-48, 1997). This invention can also use a Combined definition rule incorporating both the Kabat and Chothia definitions to determine amino acid residues in variable domain sequences and full-length antibody sequences. In this invention, the sequences determined according to the Kabat definition rule are shown in the table below.

[0074] The antibody CDR definition method of this invention (see http: / / bioinf.org.uk / abs / ).

[0075] Kabat Chothia Combined HCDR1 H31--H35 H26--H32 H26-H35 HCDR2 H50--H65 H52--H56 H50-H65 HCDR3 H95--H102 H95--H102 H95-H102

[0076] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0077] Example 1: Optimization of conditions for nanobody reducing agent

[0078] The CDR sequence of the nanobody YNS002 used in this invention is shown below:

[0079] CDR1: INTMH (SEQ ID NO: 1)

[0080] CDR2: TIFIDLNTIVTDSVKG (SEQ ID NO: 2)

[0081] CDR3: DVSGYGRA (SEQ ID NO: 3)

[0082] The amino acid sequence of YNS002 containing the Cys group is shown below:

[0083] EVQLVESGGGLVQPGGSLRLSCAASRTDRNINTMHWYRQAPGKGREWVGTIFIDLNTIVTDSVKGRFTISRDNAKNTLYLQMNTLRAEDTAVYYCAADVSGYGRAWGQGTTVSSHHHHHHGGGGSGGC (SEQ ID NO: 4)

[0084] The nanobody YNS002 used in this invention also has the following properties:

[0085] Molecular weight (kDa): 14.01; Isoelectric point: 8.69; Extinction coefficient: 1.73.

[0086] The SDS-PAGE gel and HPLC spectra of the nanobodies of this invention are as follows: Figure 5 and Figure 6 As shown.

[0087] In the nanobody reduction step, this invention compares the reduction effects of two commonly used disulfide bond reducing agents, TCEP and 2-MEA, under different reduction times (0 min, 30 min, 60 min), different temperatures (25℃, 37℃, 50℃), and different ratios of the agent to the nanobody (1X, 10X, 20X). All other experimental conditions are kept consistent, and the reduction effect is verified by HPLC.

[0088] Neither reducing agent successfully reduced the dimer under any conditions at 0 min and 30 min time points; after 1 h of reaction at 20X and 37℃, 2-MEA showed a dimer content of 10.5% and a monomer content of 87.5%. Figure 3 ); TCEP was completely reduced after reacting at 10X and 37℃ for 1 hour, with a dimer content of 0% and a monomer content of 99.4%. Figure 4 Therefore, in this comparison, the present invention determined the reduction conditions to be TCEP (10X, 37°C, 60 min).

[0089] Example 2 Screening of chelating agents

[0090] This invention screened three metal chelating agents: Mal-NOTA, Mal-DOTA, and Mal-DFO, all using TCEP as a reducing agent and employing... 68 Ga is a radioactive nuclide. 68 Ga-DFO-Nb and 68 The HPLC quality control chromatogram of Ga-DOTA-Nb is as follows: Figure 7 and Figure 8 As shown.

[0091] In the metal chelating agent screening experiment, since the radiochemical purity of the nanobody-labeled products of Mal-DFO and Mal-DOTA was lower than that of Mal-NOTA, NOA was used as the preferred metal chelating agent in subsequent experiments.

[0092] Example 3: Reduction and Coupling of NOTA-Nb

[0093] (1) Add 10 times the amount of reducing agent TCEP (10mM, about 1μmol) and chelating agent Mal-NOTA (20mM, about 1μmol) to 250 μL of dimer Nb with a concentration of 444 μM (total amount of substance 0.111μmol) and mix thoroughly.

[0094] (2) The reduction and coupling were completed after incubating the mixture at 37 °C and 650 rpm for 90 min;

[0095] (3) The product after reductive coupling was purified by desalting column (HiTrap™ Desalting, 3×5 mL). The mobile phase was 0.9% sodium chloride injection, the flow rate was 2.0 mL / min, and the elution time was 30 min. The eluent was collected at specific time points based on the UV absorption peak to obtain NOA-Nb, such as... Figure 1 As shown.

[0096] Example 4: Optimization of Purification Techniques

[0097] Given the low protein recovery rate of nanobodies, three protein purification methods—HiTrap™ Desalting column, ultrafiltration tube, and PD10 column—were compared. 1X PBS was used as the elution mobile phase for all methods, and all operation steps except purification were kept consistent. The concentration of the recovered product was measured using a UV spectrophotometer.

[0098] During the purification steps, 1 mg of NOTA-Nb was added, and approximately 0.4 mg was recovered using the HiTrap™ Desalting column, representing a recovery rate of about 40%. 3K (Millipore) ultrafiltration tubes were used, and the buffer was changed after centrifugation. To ensure purification efficiency, the process was repeated 2-3 times. The single ultrafiltration recovery rate was high, approaching 50-60%. However, after repeated operations, the overall recovery rate significantly decreased to approximately 30%. The PD10 column showed a product recovery rate of approximately 50%, with even higher purification recovery.

[0099] Example 5 68 The Ga-NOTA-Nb tag

[0100] (1) Add 0.1 mL of sodium acetate (3M, pH 4.5) and 0.4 mL of sodium acetate to the purified 0.4 mg NOTA-Nb. 68The mixture was incubated with GaCl3 eluent (5 mCi) at 37 °C and 650 rpm for 15 minutes.

[0101] (2) Radiolabeled Nb was purified in a PD-10 column (Cytiva, 17085101) with fresh 0.2M PBS;

[0102] (3) Filter the most radioactive portion using a 0.22µm filter;

[0103] (4) Quality control via HPLC, such as Figure 2 As shown. The RCP of the purified product is 100%.

Claims

1. A method of radionuclide labeling a nanobody, characterized in that, The method comprises the following steps: (1) reducing the nanobody by using a reducing agent to obtain a reduction reaction product; (2) coupling the reduction reaction product with a chelating agent, and obtaining a coupling product after purification; (3) labeling the coupling product with a radionuclide, and obtaining a radionuclide-labeled nanobody after purification; In the (1), the conditions satisfy any one of the following groups: The reducing agent is TCEP; the ratio of the amount of substance of the reducing agent to the nanobody is (10-100) : 1; and / or, the temperature is 25-55℃; and the reaction time is 1-2 h; The reducing agent is 2-MEA, and the ratio of the amount of substance of the reducing agent to the nanobody is (10-100) : 1; and / or, the temperature is 25-55℃; and the reaction time is 1-2 h.

2. The method of claim 1, wherein, In the (2), the ratio of the amount of substance of the reduction reaction product to the chelating agent is 1:(10-100); and / or, the coupling conditions comprise: reacting at 25-55℃ for 30-120 minutes; Preferably, the conditions of the (1) satisfy any one of the following groups: The reducing agent is TCEP, and the ratio of the amount of substance of the reducing agent to the nanobody is 10:1, the temperature is 25℃, 37℃ or 55℃; and the reaction time is 1 h, 1.5 h or 2 h; The reducing agent is 2-MEA, and the ratio of the amount of substance of the reducing agent to the nanobody is 20:1, the temperature is 25℃, 37℃ or 55℃; and the reaction time is 1 h, 1.5 h or 2 h; And / or, in the (2), the ratio of the amount of substance of the reduction reaction product to the chelating agent is 1:10; and / or, the coupling conditions comprise: reacting at 37℃ for 90 minutes; More preferably, the (1) and (2) are carried out simultaneously in a system; the ratio of the amount of substance of the nanobody, the reducing agent and the chelating agent in the system is 1:(10-100):(1-100); and / or, the reaction conditions of the system comprise: reacting at 25-55℃ for 30-120 minutes; preferably, the ratio of the amount of substance of the nanobody, the reducing agent and the chelating agent in the system is 1:10:10; and / or, the reaction conditions of the system comprise: reacting at 37℃ for 90 minutes; further more preferably, the concentration of the nanobody is 444 μM; Further preferably, the chelating agent is selected from one or more of Mal-NOTA, Mal-DOTA, Mal-NODAGA, NHS-DOTA, Mal-AAZTA, NHS-AAZTA, and Mal-DFO; preferably, the chelating agent is selected from one or more of Mal-DOTA, Mal-NOTA or Mal-DFO, for example, Mal-NOTA.

3. The method of claim 1 or 2, wherein, The purification in the (2) comprises the following steps: Purification was performed using HiTrap TM Desalting columns, ultrafiltration tubes or PD10 columns. Preferably, the HiTrap TM Desalting column for purification; More preferably, the mobile phase is 0.9% sodium chloride injection, the flow rate is 2.0 mL / min, and the elution is performed for 30 min, and the eluate of 2.6 min-3.6 min is collected according to the ultraviolet absorption peak to obtain the coupling product.

4. The method according to any one of claims 1 to 3, characterized in that, The system of (3) comprises the coupling product, the radionuclide and the sodium acetate solution; The reaction condition of the system comprises: reaction at 25-50℃, 0-1000 rpm for 5-60 min; Preferably, the radionuclide is selected from one or more of 68 Ga, 64 Cu, 177 Lu, 90 Y, 111 In, 44 Sc, 225 Ac, for example is 68 Ga; More preferably, the system comprises the coupling product, 0.4 mL 68 GaCl3eluate with 0.1 mL of a sodium acetate solution at a concentration of 3 M at a pH of 4.5; The reaction condition of the system comprises: reaction at 37℃, 650 rpm for 15 min.

5. The method according to any one of claims 1 to 4, characterized in that, The purification in (3) comprises the following steps: Using HiTrap TM Purification was performed using a desalting column, ultrafiltration tube, or PD10 column, with PD10 column being preferred; the mobile phase was 0.9% sodium chloride injection or 1× PBS, with 1× PBS being preferred. Preferably, the purification of the radiolabeled product further comprises filtering the highest radioactivity part using a 0.22 µm filter; More preferably, the radiolabeled product is quality controlled by HPLC and iTLC before and after purification.

6. The method according to any one of claims 1 to 5, wherein, The C-terminal of the nanobody comprises an amino acid sequence with a Cys group; Preferably, the nanobody comprises CDR1, CDR2 and CDR3; wherein the amino acid sequence of CDR1 is shown as SEQ ID NO: 1, the amino acid sequence of CDR2 is shown as SEQ ID NO: 2, and the amino acid sequence of CDR3 is shown as SEQ ID NO: 3; More preferably, the amino acid sequence of the nanobody is shown as SEQ ID NO:

4.

7. A radionuclide labeled nanobody, characterized in that, Prepared by the method of any one of claims 1-6.

8. A radionuclide labeled nanobody, characterized in that, The radionuclide-labeled nanobody comprises the chelator in the method of claim 2; and / or the radionuclide in the method of claim 4; and / or the nanobody in the method of claim 4. Preferably, the chelator is coupled to the nanobody through the Cys group of the C-terminal of the nanobody.

9. A diagnostic agent composition or a pharmaceutical composition, comprising the nanobody of claim 7 or 8, and a pharmaceutically acceptable carrier or excipient.

10. A kit, comprising the nanobody of claim 7 or 8, or the diagnostic agent composition or the pharmaceutical composition of claim 9.

11. Use of the nanobody of claim 7 or 8, the diagnostic agent composition or the pharmaceutical composition of claim 9, or the kit of claim 10 in the preparation of a targeted molecular imaging diagnostic and / or a targeted therapeutic preparation for a disease. Preferably, the disease is a tumor. More preferably, the tumor is a tumor positive for PSMA expression; preferably colon cancer, esophageal cancer, thyroid cancer, lung cancer, prostate cancer, renal cell carcinoma or brain tumor, such as metastatic castration-resistant prostate cancer.

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