A radioimmunoconjugate, pharmaceutical composition, preparation and use

Abstract

The present application relates to the field of radioimmunotherapy technology, and particularly relates to a radioimmunoconjugate, a pharmaceutical composition, a preparation and application. The present application designs a radioimmunoconjugate based on a high-affinity and high-specificity anti-Claudin18.2 humanized antibody, and further adds gentisic acid or a pharmaceutically acceptable salt thereof to greatly improve the radiochemical purity and stability of the radioimmunoconjugate. In-vivo and in-vitro experiments show that the radioimmunoconjugate of the present application has excellent tumor targeting specificity, has significant CLDN18.2 positive tumor targeting and treatment effect, has a longer blood clearance half-life in the body, has a more persistent anti-tumor effect, and no obvious bone marrow suppression or liver and kidney toxicity is observed in the treatment window period, showing a good balance of safety and effectiveness.

Description

Technical Field

[0001] This invention relates to the field of radioimmunotherapy technology, and in particular to a radioimmunoconjugate, pharmaceutical composition, formulation, and application. Background Technology

[0002] Claudin18.2 (CLDN18.2) is a tight junction protein specifically expressed in differentiated gastric epithelial cells. It is primarily expressed in gastric mucosal cells (including normal gastric glands, chief cells, parietal cells, and endocrine cells) and Paneth cells of the duodenum, but is generally not expressed in gastric stem cells and other healthy tissues. Under normal circumstances, its antigenic epitope is hidden within tight junctions, making it difficult for antibodies in the blood to recognize. However, in primary malignant tumors such as gastric cancer, with the disruption of cell polarity, the CLDN18.2 antigenic epitope is exposed on the cell surface, becoming a highly specific target for anti-tumor drugs. This characteristic allows antibodies targeting CLDN18.2 to bind highly specifically to tumor cells without harming normal cells; therefore, CLDN18.2 is an ideal target for cancer therapeutics with a low risk of off-target toxicity.

[0003] Currently, monoclonal antibody drugs targeting CLDN18.2 are available in the immunotherapy field, such as Zolbetuximab (IMAB362). These drugs primarily kill tumor cells through antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) mechanisms. However, the efficacy of monoclonal antibodies is highly dependent on the patient's own immune microenvironment. For patients with immune-cold tumors or those who have failed multiple lines of advanced therapy, the effect of antibody therapy alone is often limited. Therefore, developing novel drug forms that can directly kill tumor cells without relying on the host immune system has significant clinical implications.

[0004] Radionuclide conjugates (RDCs) have become a hot topic in cancer treatment in recent years. Although some studies have attempted to label CLDN18.2 antibodies with radionuclides, the following technical bottlenecks still exist in the current technology: 1. Radiation stability issues: Antibody proteins are sensitive to radiation. During the labeling process of high-activity radionuclides, the free radicals generated by the radiodecomposition of water molecules can easily attack the antibody structure, leading to antibody aggregation, breakage, or loss of immune activity, thereby affecting the shelf life and in vivo distribution of the drug.

[0005] 2. Non-specific uptake: Some full-length antibody markers circulate in the body for too long, leading to the risk of bone marrow suppression; or due to the shedding of chelates, free radionuclides accumulate in bones or liver, causing non-target organ toxicity.

[0006] Therefore, screening for humanized antibodies with high affinity and high specificity, and constructing radioimmunoconjugates targeting CLDN18.2 with high stability and low toxicity are technical problems that urgently need to be solved in this field. Summary of the Invention

[0007] The inventors' team previously developed a high-affinity, high-specificity humanized antibody (see CN117567625 A), and therefore attempted to prepare radioimmunoconjugates based on this antibody. However, the inventors found that the success rate of ligation varied significantly when different commonly used chelating linkers were selected to link the antibody and the radionuclide. After screening and verifying a large number of chelating linkers, it was found that only when p-SCN-Bn-DOTA (DOTA-NCS) was used could the radionuclide be efficiently and successfully labeled onto the antibody.

[0008] Based on this, the present invention first provides a radioimmunoconjugate, comprising: a targeting carrier for Claudin 18.2 and a radionuclide; wherein the radionuclide is connected to the targeting carrier for Claudin 18.2 via a chelating linker; The targeting vector for Claudin18.2 is selected from anti-Claudin18.2 monoclonal antibodies or their antigen-binding fragments; the chelating linker is p-SCN-Bn-DOTA.

[0009] In some embodiments, the anti-Claudin18.2 monoclonal antibody or its antigen-binding fragment contains heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3; the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively.

[0010] SEQ ID NO.1: GYTFTSYR SEQ ID NO.2: IDPSYT SEQ ID NO.3: ARLGRGNTLGY SEQ ID NO.4: QSLLNSGNQKNY SEQ ID NO.5: WAS SEQ ID NO.6: QNGYSYPFT In some embodiments, the anti-Claudin18.2 monoclonal antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO.12.

[0011] SEQ ID NO.7: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYRMHWVRQAPGQGLEWIGEIDPSNSYTNYNQNFRGRSTLTVDKSTSTAYMELSSLRSEDTAVYYCARLGRGNTLGYWGQGTTVTVSS SEQ ID NO.8: QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYRMHWVRQAPGQGLEWIGEIDPSNSYTNYNQNFRGRSTLTVDKSTSTAYMELSSLRSEDTAVYYCARLGRGNTLGYWGQGTTVTVSS SEQ ID NO.9: QVQLLESGGGLVKPGGSLRLSCAASGYTFTSYRMHWVRQAPGQGLEWIGEIDPSNSYTNYNQNFRGRSTLSVDKAKSSAYLQMNSLRAEDTAVYYCARLGRGNTLGYWGQGTTVTVSS SEQ ID NO.10: DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNGYSYPFTFGQGTRLEIK SEQ ID NO.11: DIQMTQSPSTLSASVGDRVTITCKSSQSLLNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNGYSYPFTFGQGTRLEIK SEQ ID NO.12: DIQMTQSPSSSLSASVGDRVTITCKSSQSLLNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQNGYSYPFTFGQGTRLEIK Both the heavy chain variable region and the light chain variable region mentioned above are human-derived sequences.

[0012] When the anti-Claudin18.2 monoclonal antibody is fully humanized, compared to commonly used murine or chimeric antibodies in the prior art (such as early versions of Zolbetuximab), the fully humanized design significantly reduces the immunogenicity of the heterologous protein and can overcome the human anti-mouse antibody (HAMA) response. This not only avoids the rapid clearance of the drug by the immune system in the body and prolongs the drug's half-life, but also significantly reduces the risk of allergic reactions or the formation of neutralizing antibodies during administration, thus improving the safety of clinical use.

[0013] In some embodiments, the radionuclide is selected from... 177 Lu、 94 Tc, 99m Tc, 90 In、 111 In、 67 Ga、 68 Ga、 86 Y、 90 Y、 151 Tb, 186 Re、 188 Re、 64 Cu、 67 Cu、 55 Co、 57 Co、 43 Sc、 44 Sc、 47 Sc、 225 Ac、 213 Bi、 212 Bi、 212 Pb, 227 Th、 153 Sm、 166 Ho、 152 Gd, 153 Gd, 157 Gd or 166 Dy.

[0014] Preferably, the molar ratio of the chelating linker to the targeting carrier targeting Claudin18.2 is (1~8):1; more preferably, it is (6~8):1.

[0015] Preferably, each chelating linker chelates one radionuclide.

[0016] Preferably, the p-SCN-Bn-DOTA is coupled to an amino group of an anti-Claudin18.2 monoclonal antibody or its antigen-binding fragment via a lysine residue.

[0017] Furthermore, the present invention provides a pharmaceutical composition comprising the aforementioned radioimmunoconjugate.

[0018] Preferably, the pharmaceutical composition further contains other drugs for the diagnosis or treatment of Claudin18.2 positive tumors.

[0019] In some embodiments, the pharmaceutical composition further contains pharmaceutically acceptable excipients.

[0020] In some implementations, the excipients include fillers, excipients, lubricants, wetting agents, diluents, etc.

[0021] To further improve the stability of the radioimmunoconjugate, the present invention, through screening a large number of radiation stabilizers, found that gentisic acid or its pharmaceutically acceptable salt can significantly improve the radiochemical purity (RCP) and stability of the radioimmunoconjugate.

[0022] Based on this, the present invention provides a formulation comprising the aforementioned radioimmunoconjugate and a radiation stabilizer; the radiation stabilizer being gentianic acid or a pharmaceutically acceptable salt thereof.

[0023] Preferably, the formulation is an aqueous formulation.

[0024] Preferably, the pharmaceutically acceptable salt is a gentianate.

[0025] Preferably, the gentic acid salt is selected from sodium gentic acid, potassium gentic acid, calcium gentic acid, magnesium gentic acid, zinc gentic acid, aluminum gentic acid, choline gentic acid, meglumine gentic acid, diethanolamine gentic acid, or triethanolamine gentic acid.

[0026] Preferably, the pH of the formulation is 4.5 to 5.5; and / or, the concentration of the radioimmunoconjugate in the formulation is 1 to 10 mCi / mL; and / or, the concentration of the radiation stabilizer in the formulation is 1 to 6 mg / mL.

[0027] The formulation of the present invention can solve the problems of low response rate of CLDN18.2 targeted drugs in the treatment of solid tumors and insufficient stability of radioimmunoconjugates in the prior art, and provides a guarantee for the clinical transportation and storage of drugs (for example, the radiochemical purity can still be maintained at more than 95% after being placed at 37°C for 168 hours).

[0028] Furthermore, the present invention provides the use of the radioimmunoconjugate, the pharmaceutical composition, and the formulation in the preparation of diagnostic reagents or pharmaceuticals for the diagnosis or treatment of Claudin18.2 positive tumors.

[0029] Preferably, the Claudin18.2 positive tumor is gastric cancer, gastroesophageal junction adenocarcinoma, pancreatic cancer, or esophageal cancer.

[0030] Furthermore, the present invention provides a method for preparing the radioimmunoconjugate, comprising: The targeting vector targeting Claudin18.2 was coupled with the chelating linker in an alkaline buffer solution to obtain a targeting vector-chelating agent conjugate. The target carrier-chelating agent conjugate is labeled with the radionuclide under weakly acidic conditions to obtain the radioimmunoconjugate.

[0031] Preferably, the pH value of the alkaline buffer solution is 8.5 to 9.5.

[0032] Preferably, the pH value of the weakly acidic condition is 4.5 to 5.5.

[0033] Preferably, the alkaline buffer solution includes Na2HPO4, NaH2PO4 and Na2CO3.

[0034] Preferably, the weakly acidic conditions include an ammonium acetate-hydrochloric acid buffer solution.

[0035] Preferably, the radionuclide is provided in the form of a radionuclide solution for labeling reaction, wherein the activity of the radionuclide in the radionuclide solution is 1 mCi to 10 mCi / mL.

[0036] Preferably, the radioactive nuclide is 177 Lu.

[0037] 177 Lu (lutetium-177) is an ideal therapeutic radionuclide. It has a physical half-life of 6.7 days and emits beta particles with energies of 176 (12.2%), 384 (9.1%), and 497 (78.6%) keV. It exhibits strong killing power against tumor cells but relatively weak penetrating power, thus avoiding damage to surrounding normal cells, making it a promising radionuclide for radiotherapy. Furthermore, 177 Lu can also emit energies of 113 (6.4%) and 208 (11.0%) keV. γ Radiation can be used to trace drugs within the body. Furthermore... 177Lu has a relatively long half-life, which is close to the in vivo half-life of the antibody of the present invention. It has a good match with the antibody of the present invention and can effectively achieve therapeutic effects.

[0038] Preferably, the radioactive nuclide solution is 177 LuCl3 solution.

[0039] Furthermore, the present invention provides a method for preparing the formulation, comprising: (1) The targeting carrier targeting Claudin18.2 and the chelating linker are coupled in an alkaline buffer to obtain a targeting carrier-chelating agent conjugate; (2) The target carrier-chelating agent conjugate is labeled with the radionuclide under weakly acidic conditions; In step (2), gentian acid or its pharmaceutically acceptable salt is added to the labeling reaction system or the product after the reaction.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention designs a radioimmunoconjugate based on a high-affinity, high-specificity humanized anti-Claudin18.2 antibody, and further enhances the radiochemical purity and stability of the radioimmunoconjugate by adding gentianic acid or its pharmaceutically acceptable salt. In vitro and in vivo experiments demonstrate that the radioimmunoconjugate of this invention exhibits excellent tumor-targeting specificity, significant targeting and therapeutic efficacy against CLDN18.2-positive tumors, a long blood clearance half-life in vivo, and a more durable anti-tumor effect. Furthermore, no significant bone marrow suppression or hepatotoxicity was observed during the therapeutic window, demonstrating a good balance between safety and efficacy. Therefore, the radioimmunoconjugate and its formulations provided by this invention have broad application prospects in the diagnosis and treatment of Claudin18.2-positive tumors. Attached Figure Description

[0041] Figure 1 The results are HPLC results of the labeled product after 1 hour of labeling reaction in Example 1 of this invention.

[0042] Figure 2 The results are HPLC results of the labeled product after 2 hours of labeling reaction in Example 1 of this invention.

[0043] Figure 3 The results are HPLC results of the labeled product purified after 2 hours of labeling reaction in Example 1 of this invention.

[0044] Figure 4 The results are HPLC results of the labeled product after purification following a 2-hour labeling reaction in Example 1 of this invention, and after being placed at 37°C for 15 hours.

[0045] Figure 5The results are HPLC results of the formulation in Example 2 of this invention after being placed at 37°C for 120 hours.

[0046] Figure 6 The results are HPLC results of the formulation in Example 3 of this invention after being placed at 37°C for 168 hours.

[0047] Figure 7 It is prepared according to Example 1 of the present invention. 177 Lu-DOTA-h1D6 technology roadmap.

[0048] Figure 8 This is the blood drug concentration change curve of normal KM mice in Example 5 of the present invention.

[0049] Figure 9 This is a SPECT / CT imaging result of the formulation in tumor-bearing nude mice in Example 6 of the present invention; this image is a fusion of CT and SPECT, in which: the white area is mainly the area where bones and radiopharmaceuticals accumulate, the brighter the white area, the greater the bone density or the more radiopharmaceuticals accumulate, except for some noise in the image.

[0050] Figure 10 This is in embodiment 7 of the present invention. 177 Biodistribution map of Lu-DOTA-h1D6 in tumor-bearing nude mice.

[0051] Figure 11 This is in embodiment 7 of the present invention. 177 The ratio of Lu-DOTA-h1D6 uptake in tumor tissue to uptake in major organs / tissues in tumor-bearing nude mice.

[0052] Figure 12 These are H&E staining images of the major organs of tumor-bearing nude mice in the control group and the drug-treated group in Example 8 of this invention.

[0053] Figure 13 This is a graph showing the changes in tumor volume in nude mice bearing tumors in Example 9 of this invention.

[0054] Figure 14 These are in vitro tumor photographs of mice in the control group and the drug administration group in Example 9 of this invention.

[0055] Figure 15 This is a comparison chart of tumor weight between the control group and the treatment group in Example 9 of the present invention.

[0056] Figure 16 This is the HPLC result of the labeled product after 1 hour of labeling reaction in Comparative Example 1 of this invention.

[0057] Figure 17 This is the HPLC result of the labeled product after 4 hours of labeling reaction in Comparative Example 1 of this invention.

[0058] Figure 18The results are HPLC results of the formulation of Comparative Example 2 of this invention after being placed at 37°C for 2 hours. Detailed Implementation

[0059] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0060] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0061] The h1D6 antibody used in the following examples is a human IgG1 type anti-Claudin18.2 monoclonal antibody, purchased from the Institute of Pharmaceutical Biotechnology, Peking Union Medical College. Its amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.10, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.13, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.14.

[0062] SEQ ID NO.13: AKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDLLGGPSVFIFPPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQIS WFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEVLHNHLTTKTISRSLGK SEQ ID NO.14: RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC BGC823 in the following embodiments CLDN18.2 Tumor-bearing nude mouse models were prepared according to the following method.

[0063] BGC823-CLDN18.2 cells (disclosed in CN119792570A) were revived and cultured in DMEM medium containing 10% fetal bovine serum (GibcoBRL Inc.), 2 mM glutamine, 100 μg / mL streptomycin, and 100 U / mL penicillin in a 37°C incubator with 5% CO2. When the cells reached 80% confluence, they were passaged using trypsin at a ratio of 1:2 to 1:3. The specific steps were as follows: The 0.25% trypsin-EDTA digestion solution was preheated to room temperature. The culture medium in the flask was discarded. 3-5 mL of PBS buffer was added to the flask, and the solution was gently washed and discarded. 1-2 mL of preheated trypsin was added to the flask, and the cells were incubated at room temperature until they became rounded. Then, 3 mL of complete culture medium was added to terminate the digestion. Gently pipette the cells off the flask wall to remove them completely. Then collect the cell suspension, centrifuge at 1000 rpm for 3-5 min, discard the supernatant, resuspend the cells in complete culture medium, and passage them.

[0064] After expanding BGC823-CLDN18.2 cells to the target number, the cells were collected and processed at a ratio of 5 × 10⁻⁶ cells / year. 6 0.2 mL / mouse was injected into the right forelimb axilla of BALB / c nude mice (18-22 g). The long diameter a and the short diameter b perpendicular to it were measured every 3-4 days, and V was calculated using the formula V = a × b. 2 The tumor volume was calculated by multiplying by 0.5. Approximately 10-20 days after inoculation, the average tumor volume grew to 100-300 mm. 3 size.

[0065] Example 1 This embodiment provides a radioimmunoconjugate ( 177 Lu-DOTA-h1D6, including a targeting vector (h1D6 antibody) targeting Claudin18.2 and a radionuclide ( 177 Lu); the radionuclide is linked to the amino group of the h1D6 antibody via the lysine residue of the chelating linker p-SCN-Bn-DOTA (DOTA-NCS); the molar ratio of the chelating linker to the antibody is 6.5:1, and each chelating linker chelates one radionuclide.

[0066] The preparation method of the radioimmunoconjugate in this embodiment is as follows, and the technical route diagram is shown below. Figure 7 As shown: (1) Add Na2CO3 solution to 100 μL of 0.02M Na2HPO4-NaH2PO4 buffer solution to make pH=9, and the molar ratio of antibody (2mg / mL) to DOTA-NCS is 1:20. Incubate at 37℃ for 2h to obtain the conjugated product.

[0067] (2) After the coupling reaction, the PD MiniTrap G-25 column was treated with 0.01M Na2HPO4-NaH2PO4 buffer solution. Then the coupling product was purified using the treated PD MiniTrap G-25 column to remove unbound DOTA-NCS. The purified product was then obtained by centrifugation using a 50K molecular weight ultrafiltration centrifuge tube. (3) Replace the Na2HPO4-NaH2PO4 buffer system with CH3COONH4 buffer (0.25M, adjusted to pH=5 with 0.1M hydrochloric acid), and add it to the coupling purified product. 177 LuCl3 solution (4–8 mCi / mL) was reacted at 37°C for 1 and 2 hours, respectively, and samples were taken for analysis. After labeling, the radioimmunoconjugate was purified using a PD MiniTrap G-25 column. 177 Lu-DOTA-h1D6 solution.

[0068] The HPLC (radio-HPLC) results of the labeled reaction at 1 h, 2 h, and the purified radioimmunoconjugate are as follows: Figures 1-3 As shown, the labeling rate was approximately 72.9% after one hour of reaction and approximately 79.2% after two hours of reaction. After purification, 177 The radiochemical purity of Lu-DOTA-h1D6 can reach over 95%. The HPLC results of the purified radioimmunoconjugate after incubation at 37°C for 15 hours are as follows. Figure 4 As shown.

[0069] Example 2 This embodiment provides a formulation containing the radioimmunoconjugate prepared in Example 1. 177 Lu-DOTA-h1D6 and sodium gentianate.

[0070] The formulation has a pH of 5.0; the concentration of the radioimmunoconjugate in the formulation is 7 mCi / mL; and the concentration of the radiation stabilizer in the formulation is 1 mg / mL.

[0071] The preparation method of the formulation in this embodiment differs from that in Example 1 only in that: In step (3), add to the coupling purified product177 Simultaneously, 25 µL (6 mg / ml) of sodium gentianate solution was added to LuCl3 solution, and the mixture was reacted at 37°C for 2 h. The resulting radioimmunoconjugate was then purified using a PD MiniTrap G-25 column. 177 Lu-DOTA-h1D6 solution, in radioimmunoconjugate 177 Sodium gentianate was added to the Lu-DOTA-h1D6 solution to a final concentration of 1 mg / ml to obtain the formulation.

[0072] The HPLC results of the formulation in this embodiment after being placed at 37°C for 120 hours are as follows: Figure 5 As shown.

[0073] Example 3 This embodiment provides a formulation containing the radioimmunoconjugate prepared in Example 1. 177 Lu-DOTA-h1D6 and gentic acid.

[0074] The formulation has a pH of 5.0; the concentration of the radioimmunoconjugate in the formulation is 7 mCi / mL; and the concentration of the radiation stabilizer in the formulation is 1 mg / mL.

[0075] The preparation method differs from Example 2 only in that sodium gentioate is replaced with gentic acid.

[0076] The HPLC results of the formulation in this embodiment after being placed at 37°C for 168 hours are as follows: Figure 6 As shown.

[0077] Figures 1-6 In the HPLC results, the peak on the left with a retention time of approximately 10.3 min is the target product peak, and the peak on the right with a retention time of approximately 14.4 min is the [other product]. 177 Radioactive peaks of Lu ions.

[0078] Example 4 This embodiment tests the lipid-water partition coefficient of the formulation from Example 3. The steps are as follows: 2.9 mL of PBS (0.1 mol / L, pH=7.4) and 3 mL of n-octanol were added to a 15 mL centrifuge tube, followed by 100 μL (approximately 7.4 MBq) of the formulation. The mixture was vortexed at room temperature for 2 min to ensure thorough mixing, and then centrifuged at 4500 r / min for 5 min. After the mixture was allowed to settle and separate into layers, three 500 μL samples were taken from each of the organic and aqueous phases. The radioactivity count per minute (CPM) of each sample was measured using an automated gamma-ray counter, and calculated according to the formula log D. pH=7.4 = log(CPM 正辛醇 / CPM PBSCalculate its lipid-water partition coefficient at pH=7.4.

[0079] The results are shown in Table 1.

[0080] Table 1

[0081] The results showed that the formulation in Example 3 had an average lipid-water partition coefficient of -3.16 in octanol and PBS, indicating that it was more water-soluble than lipid-soluble.

[0082] Example 5 This study investigates the pharmacokinetic analysis of the formulation from Example 3 in normal KM mice.

[0083] Using KM mice (n=3) as a model, the formulation of Example 3 with an activity of approximately 370 KBq was administered via a single intravenous injection via the tail vein. Blood samples were then collected at predetermined time points to determine their radioactivity levels, in order to quantitatively assess the pharmacokinetic characteristics of the drug in the blood. The results are as follows: Figure 8 As shown. The results show 177 The metabolism of Lu-DOTA-h1D6 in the blood exhibits a typical biphasic elimination pattern: (1) Initial rapid decline phase (0–2h): After tail vein administration, the drug concentration rapidly declines in the initial phase (0–2h), dropping from a high value of nearly 40 ID% / g to about 15 ID% / g. This phase mainly reflects the rapid distribution of the drug from the central compartment to the peripheral compartment, which is consistent with the characteristic of rapid diffusion of the drug to the whole body tissues after intravenous administration; (2) Late-stage slow elimination phase (after approximately 4 hours): After approximately 4 hours, the rate of decrease in drug concentration slows significantly, the curve flattens, and the slow elimination phase begins. At this time, the drug concentration remains at a low level and decreases slowly over time until it approaches the baseline. This phase mainly reflects the slow elimination process of the drug in the body.

[0084] The entire curve exhibits a typical biphasic change: rapid distribution followed by slow elimination, consistent with a two-compartment pharmacokinetic model. The distribution phase half-life in vivo is 39 min, and the elimination phase half-life is 866 min. These results indicate that the drug has a long blood clearance half-life in vivo, providing kinetic assurance for its sufficient accumulation at the target lesion and contributing to a more sustained antitumor effect.

[0085] Example 6 This embodiment studies 177 Lu-DOTA-h1D6 in BGC823 CLDN18.2 Imaging in a tumor-bearing nude mouse model.

[0086] In BGC823 tumor-bearing nude mice with high expression of CLDN18.2 (BGC823... CLDN18.2 In the (n=3) model, mice were subjected to Micro-SPECT / CT imaging after intravenous injection of approximately 3.7 MBq of the formulation from Example 3. The results are as follows... Figure 9 As shown.

[0087] 177 Lu-DOTA-h1D6 exhibited significant radiopharmaceutical uptake in tumors of tumor-bearing nude mice. Furthermore, high levels of radioactivity were also observed in the liver, spleen, and stomach of the mice. (In BGC823) CLDN18.2 In the model, tumor uptake reached a high value (approximately 30% ID% / g) at 24h, maintained a high uptake level between 24 and 240h, and reached a maximum value (approximately 42 ID% / g) at 96h.

[0088] Example 7 This embodiment studies 177 Lu-DOTA-h1D6 in BGC823 CLDN18.2 Biodistribution in tumor-bearing nude mouse models. Details are as follows: Using BGC823 CLDN18.2 In vivo biodistribution studies were conducted using a tumor-bearing nude mouse model (n=3). Each mouse was injected once via tail vein with the formulation of Example 3, with an activity of approximately 370 KBq. The mice were then sacrificed and dissected at predetermined time points, and major tissues and organs were collected to quantitatively determine the radiopharmaceutical uptake levels in each sample.

[0089] The results are as follows Figure 10 As shown, for BGC823 CLDN18.2 Intravenous administration to tumor-bearing nude mice 177 Following Lu-DOTA-h1D6 administration, the drug effectively recognized and targeted tumor-associated antigens. Tumor uptake was 14.59 ± 2.23 %ID / g at 4 hours post-administration, peaking at 47.73 ± 14.90 %ID / g at 72 hours, and maintaining high specific accumulation throughout the 24-240 hour observation period (uptake still reached 13.65 ± 3.62 %ID / g at 240 hours). Simultaneously, the radioactivity in the blood showed a rapid clearance trend, with uptake decreasing rapidly from 38.38 ± 13.16 %ID / g at 4 hours to 0.74 ± 0.32 %ID / g at 240 hours. This kinetic characteristic of high tumor enrichment and rapid blood clearance resulted in a tumor-to-blood uptake ratio of approximately 18.4 in the later stages, providing a favorable safety window for targeted internal irradiation therapy with this radiopharmaceutical.

[0090] Regarding systemic distribution and metabolic excretion in non-target organs, the drug is primarily cleared via the reticuloendothelial system (liver and spleen) and the urinary system (kidneys). In the early post-administration period (4 to 24 hours), the liver, spleen, lungs, and kidneys exhibit high physiological uptake, followed by a stepwise decrease in uptake values ​​over time. The kidneys show the fastest clearance rate (decreasing to 1.37 ± 0.38 %ID / g at 240 hours), while the liver and spleen exhibit some radioactive retention characteristics. Gastric tissue shows a certain increase in physiological uptake within 4 to 72 hours, consistent with the reported basal expression of CLDN18.2 in normal gastric mucosa (PMID: 38503876). Uptake in background tissues such as the brain, bones, and muscles remains consistently at extremely low levels below 5 %ID / g. Furthermore, as... Figure 11 As shown, the target-to-non-target tissue ratio (T / NT) of the drug in key dose-limiting organs (liver, spleen, and kidney) continuously increased over time. At 72 hours post-administration, the tumor uptake ratios to those in the liver, spleen, and kidney reached approximately 4.02, 4.95, and 6.63, respectively. These results indicate that the drug effectively limits the radiation exposure risk to non-target tissues while exerting its targeted internal radiation killing effect, demonstrating good potential for clinical translation.

[0091] Example 8 This embodiment studies 177 Lu-DOTA-h1D6 in BGC823 CLDN18.2 Effects on major organs in tumor-bearing nude mouse models. Details are as follows: BGC823 CLDN18.2 Tumor-bearing nude mice were randomly divided into a treatment group and a control group (n=6). Mice in the treatment group received a single injection via tail vein of the formulation from Example 3 with an activity of approximately 18.5 MBq, while mice in the control group received a single injection of an equal volume of 1X PBS solution. Mice were sacrificed on day 14 post-treatment, and tissues from the heart, liver, spleen, lungs, kidneys, stomach, large intestine, and small intestine were collected. These tissues were subsequently fixed, embedded, sectioned, and stained with H&E for histopathological evaluation.

[0092] H&E staining is shown in [link to H&E staining]. Figure 12 The results showed that, compared with the PBS injection group, 177 Lu-DOTA-h1D6 vs BGC823 CLDN18.2The heart, liver, spleen, lungs, kidneys, stomach, large intestine, and small intestine of tumor-bearing rats showed no obvious damage. Heart: Myocardial fibers were neatly arranged with clearly visible striations, without breakage or dissolution. No inflammatory cell infiltration was observed in the interstitium. Liver: Hepatocytes were regularly shaped with homogeneous cytoplasm, and no fatty degeneration or necrosis foci were observed. Spleen: The white and red pulp were clearly demarcated, the lymphoid follicle structure was intact, no abnormal cell aggregation was observed in the splenic sinuses, and no fibrosis was observed in the splenic trabeculae. Lungs: The alveolar structure was intact, the septa were not thickened, and there was no exudate or hemorrhage in the alveolar cavities. The bronchial mucosal epithelium was intact, with no peribronchial inflammatory infiltration, and no vascular congestion or embolism. Kidneys: The glomerular capillary loops were normal in shape, the basement membrane was not thickened, and Bowman's capsule was not dilated. The renal tubular epithelial cells were tightly arranged without degeneration or cast formation, and no fibrosis was observed in the interstitium. Stomach: The mucosal glands were regularly arranged, the chief cells and parietal cells were normal in shape, and there was no erosion or ulceration. Submucosal vessels showed no congestion, and the smooth muscle layer showed no rupture. In the large intestine, the intestinal glands were intact, goblet cells were evenly distributed, and there was no proliferation of lymphoid follicles in the submucosal layer. The muscular nerve plexus structure was clear, with no inflammatory cell infiltration. In the small intestine, the villi structure was intact, the crypts were normal in morphology, and there was no epithelial cell shedding. Pai's nodes were visible, the muscularis mucosa showed no thickening, and the serosa was smooth.

[0093] Example 9 This embodiment studies 177 Lu-DOTA-h1D6 in BGC823 CLDN18.2 The therapeutic effects in tumor-bearing nude mouse models are detailed below: BGC823 CLDN18.2 Tumor-bearing nude mice were randomly divided into a treatment group and a control group (n=6). Mice in the treatment group received a single injection via tail vein of the formulation from Example 3 with an activity of approximately 18.5 MBq, while mice in the control group received a single injection of an equal volume of PBS solution. Tumor volume changes in each group were monitored periodically after administration. Mice were sacrificed on day 14 post-administration, and the tumor tissue was completely dissected, weighed, and photographed in vitro.

[0094] Tumor volume as Figure 13 As shown in the image, the tumor tissue is photographed as follows. Figure 14 As shown, the tumor weight results are as follows: Figure 15 As shown in the figure. The results indicated that by the end of the observation period, the average tumor volume in the control group was 1012.39 ± 354.00 mm. 3 The average tumor volume in the treatment group was effectively controlled at 160.82 ± 63.64 mm. 3 The differences between the two groups were statistically significant (P<0.001). Regarding the weight of ex vivo tumors, the average tumor weight in the treatment group was 101.95±28.88 mg, significantly lower than that in the control group (582.42±171.59 mg), with a statistically significant difference (P<0.001). These results indicate that... 177Lu-DOTA-h1D6 significantly inhibited tumor growth in mice. Regarding apparent safety, the average body weight of mice in both the treatment and control groups was 20 g at the end of treatment, with no significant difference between the two groups before and after treatment. Furthermore, there were no significant changes in daily activity, food intake, or water consumption during the experiment, and no acute systemic apparent toxicity was observed.

[0095] In summary, in vivo biodistribution and pharmacodynamic experiments demonstrate that the radioimmunoconjugate of this invention can specifically and highly absorb CLDN18.2-positive gastric cancer tissue, exhibiting an extremely high tumor / background ratio (T / B Ratio). In BGC823 tumor-bearing nude mice (BGC823) with high CLDN18.2 expression... CLDN18.2 In this model, a single dose significantly inhibited tumor growth, even causing complete regression of some tumors, and no significant bone marrow suppression or hepatotoxicity was observed within the treatment window, demonstrating a good balance between safety and efficacy. Pharmacokinetic studies showed that suitable hydrophilicity (Log D≈-3.16) helps reduce non-specific hepatic uptake, thereby reducing radiation dose to non-target organs and improving the signal-to-noise ratio of imaging and the precision of treatment. The drug has a long blood clearance half-life in vivo, providing kinetic assurance for its sufficient accumulation in the target lesion and helping to maintain a more durable anti-tumor effect.

[0096] Comparative Example 1 This comparative example provides a radioimmunoconjugate, differing from Example 1 only in that the chelating linker uses DOTA-NHS-ester. When prepared according to the method of Example 1, the labeling rate was found to be only 1% after one hour of reaction and only 16% after four hours. See Figure 16 and 17 In the HPLC results, the peak on the left with a retention time of approximately 10 min is the target product peak, and the peak on the right with a retention time of approximately 14.4 min is... 177 Radioactive peaks of Lu ions.

[0097] Comparative Example 2 This comparative example provides a formulation that differs from Example 2 only in that sodium gentioate is replaced with 1 mg / ml of vitamin C.

[0098] The results showed that the radiochemical purity of the formulation in this comparative example decreased to approximately 88% after being placed at 37°C for 2 hours, indicating poor stability. (See...) Figure 18 In the HPLC results, the peak on the left with a retention time of approximately 10 min is the target product peak, and the peak on the right with a retention time of approximately 14.4 min is... 177 Radioactive peaks of Lu ions.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A radioimmunoconjugate, characterized in that, include: A targeting vector for Claudin 18.2 and a radionuclide; the radionuclide is connected to the targeting vector for Claudin 18.2 via a chelating linker; The targeting vector for Claudin18.2 is selected from anti-Claudin18.2 monoclonal antibodies or their antigen-binding fragments; the chelating linker is p-SCN-Bn-DOTA.

2. The radioimmunoconjugate according to claim 1, characterized in that, The anti-Claudin18.2 monoclonal antibody or its antigen-binding fragment contains heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3; the amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively.

3. The radioimmunoconjugate according to claim 2, characterized in that, The anti-Claudin18.2 monoclonal antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7, SEQ ID NO.8 or SEQ ID NO.9; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.10, SEQ ID NO.11 or SEQ ID NO.

12.

4. The radioimmunoconjugate according to any one of claims 1 to 3, characterized in that, The antigen-binding fragment is selected from any one of Fab, Fab', F(ab')2, Fd, Fv, and dAb.

5. The radioimmunoconjugate according to any one of claims 1 to 3, characterized in that, The radionuclides are selected from 177 Lu、 94 Tc, 99m Tc, 90 In、 111 In、 67 Ga、 68 Ga、 86 Y、 90 Y、 151 Tb, 186 Re、 188 Re、 64 Cu、 67 Cu、 55 Co、 57 Co、 43 Sc、 44 Sc、 47 Sc、 225 Ac、 213 Bi、 212 Bi、 212 Pb, 227 Th、 153 Sm、 166 Ho、 152 Gd, 153 Gd, 157 Gd or 166 Dy.

6. A pharmaceutical composition, characterized in that, It comprises the radioimmunoconjugate according to any one of claims 1 to 5.

7. A formulation, characterized in that, It comprises the radioimmunoconjugate according to any one of claims 1 to 5 and a radiation stabilizer; the radiation stabilizer is gentianic acid or a pharmaceutically acceptable salt thereof.

8. The use of the radioimmunoconjugate of any one of claims 1 to 5, the pharmaceutical composition of claim 6, and the formulation of claim 7 in the preparation of diagnostic reagents or pharmaceutical products for the diagnosis or treatment of Claudin 18.2 positive tumors.

9. A method for preparing the radioimmunoconjugate according to any one of claims 1 to 5, characterized in that, include: The targeting vector targeting Claudin18.2 was coupled with the chelating linker in an alkaline buffer solution to obtain a targeting vector-chelating agent conjugate. The target carrier-chelating agent conjugate is labeled with the radionuclide under weakly acidic conditions to obtain the radioimmunoconjugate.

10. The method for preparing the formulation according to claim 7, characterized in that, include: (1) The targeting carrier targeting Claudin18.2 and the chelating linker are coupled in an alkaline buffer to obtain a targeting carrier-chelating agent conjugate; (2) The target carrier-chelating agent conjugate is labeled with the radionuclide under weakly acidic conditions to obtain the formulation; In step (2), gentian acid or its pharmaceutically acceptable salt is added to the labeling reaction system or the product after the reaction.

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