Nuclide-labeled targeted bispecific antibody as well as preparation method and application thereof
The Iodogen method labeling of PD-1/CTLA-4 bispecific antibody [131I]I-AK104 solved the problem of high toxicity and limited efficacy of caldunil in tumor treatment, and achieved efficient and stable nuclide marking, which was suitable for the diagnosis and treatment of advanced tumors.
Patent Information
- Application Number
- CN202510521802.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-24
- Publication Date
- 2025-07-29
AI Technical Summary
In the prior art, Candonilimab, as a PD-1/CTLA-4 bispecific antibody, has a problem of high toxicity risk and limited efficacy in tumor treatment, especially in patients with low PD-L1 expression.
Cadonil was labeled with Iodogen method to prepare nuclide-labeled targeted bispecific antibody [131I]I-AK104. The labeling efficiency and purity were improved by incubating the PD-1/CTLA-4 bispecific antibody with 131I- and iodizing agent and purifying the chromatography column.
The high labeling yield and high radiochemical purity of [131I]I-AK104 are achieved, with good stability, mainly distributed in the blood and blood-rich organs, and are suitable for the clinical diagnosis and treatment of advanced tumors, especially non-small cell lung cancer (NSCLC).
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Figure CN120383676A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a radionuclide-labeled double antibody [ 131 I]I-AK104, and further discloses its preparation method and application. Background Art
[0002] Candonilimab (trade name: Katanil, code name: AK104) is the world's first commercialized PD-1 / CTLA-4 dual ICI and the first approved dual immune checkpoint inhibitor for cancer. Currently, candonilimab is primarily targeted at various malignancies, including lung cancer, liver cancer, gastric cancer, cervical cancer, kidney cancer, esophageal squamous cell carcinoma, and nasopharyngeal carcinoma. In clinical studies of these tumors, it has demonstrated lower toxicity and a better safety profile than either PD-1 or CTLA-4 inhibitors alone, while also demonstrating significant advantages in efficacy.
[0003] AK104 is a bispecific antibody targeting human PD-1 and CTLA-4. It simultaneously blocks both PD-1 and CTLA-4 immunosuppressive pathways: PD-1 inhibition relieves T cell fatigue, restoring their ability to recognize and attack tumor cells; while CTLA-4 inhibition promotes T cell activation and proliferation, enhancing the overall immune response. AK104 blocks the interaction between PD-1 and CTLA-4 and their ligands PD-L1 / PD-L2 and B7.1 / B7.2, thereby disrupting the immunosuppressive effects of the PD-1 and CTLA-4 signaling pathways and promoting tumor-specific T cell activation, ultimately exerting its anti-tumor effects. This dual mechanism of action has enabled canidolimab to demonstrate significant efficacy in patients with varying PD-L1 expression levels, particularly in patients with low PD-L1 expression, where previous PD-1 monoclonal antibodies had limited efficacy. Furthermore, this "killing two birds with one stone" strategy not only improves therapeutic efficacy but also reduces the potential toxicity risks associated with combining PD-1 and CTLA-4 inhibitors. Summary of the invention
[0004] To this end, the technical problem to be solved by the present invention is to provide a radionuclide-labeled targeted bispecific antibody [ 131 I]I-AK104;
[0005] The second technical problem to be solved by the present invention is to provide the above-mentioned radionuclide-labeled double antibody [ 131 I] Preparation method of I-AK104;
[0006] The second technical problem to be solved by the present invention is to provide the above-mentioned radionuclide-labeled double antibody [ 131Application of I]I-AK104 in the field of diagnosing and / or treating tumors, especially NSCLC.
[0007] To solve the above technical problems, a radionuclide-labeled targeting bispecific antibody described in the present invention, the radionuclide-labeled targeting bispecific antibody includes 131 I]I-labeled PD-1 / CTLA-4 bispecific antibody;
[0008] The PD-1 / CTLA-4 bispecific antibody includes Cadonilimab, and its monoclonal antibody abbreviation is AK104. The radionuclide-labeled targeting bispecific antibody is denoted as 131 I]I-Cadonilimab or 131 I]I-AK104.
[0009] The present invention also discloses a preparation method of the radionuclide-labeled targeting bispecific antibody, including the step of mixing and labeling the PD-1 / CTLA-4 bispecific antibody Cadonilimab, radionuclide 131 I - and an iodinating agent.
[0010] Specifically, in the preparation method of the radionuclide-labeled targeting bispecific antibody, the iodinating agent includes Iodogen.
[0011] Specifically, the preparation method of the radionuclide-labeled targeting bispecific antibody includes the step of adding a solution containing (Cadonilimab) and 131 I - solution into a reaction tube coated with Iodogen and mixing them, and incubating the reaction at 20 - 30 °C for 10 - 30 min;
[0012] Preferably, the 131 I - solution includes Na 131 I] solution.
[0013] Specifically, in the preparation method of the radionuclide-labeled targeting bispecific antibody, there is no special requirement for the dosage of Cadonilimab, radionuclide 131 I - and the iodinating agent, and they can be added according to the conventional dosage in the art. To ensure the labeling efficiency, it is preferred to add a sufficient amount of radionuclide 131 I - and the iodinating agent for radionuclide labeling. As a preferred scheme, the dosage of Cadonilimab, radionuclide 131 I - can be selected as 1:1.
[0014] Specifically, for the preparation method of the radionuclide-labeled targeted bispecific antibody, the method further includes a step of purifying the labeled product by a chromatography column;
[0015] Preferably, the chromatography column includes a PD10 column.
[0016] Specifically, for the preparation method of the radionuclide-labeled targeted bispecific antibody, the method further includes a step of activating and / or purifying the chromatography column;
[0017] Preferably, the reagent for the activating and / or purifying step includes PBS buffer;
[0018] Preferably, the concentration of the PBS buffer is 0.01 - 0.03 M.
[0019] Specifically, for the preparation method of the radionuclide-labeled targeted bispecific antibody, the method further includes a step of detecting the radioactive labeling yield and radiochemical purity of the labeled product by radiohigh performance liquid chromatography.
[0020] The present invention also discloses the use of the radionuclide-labeled targeted bispecific antibody or the radionuclide-labeled targeted bispecific antibody prepared by the method for preparing an integrated probe for targeted diagnosis and treatment.
[0021] The present invention also discloses the use of the radionuclide-labeled targeted bispecific antibody or the radionuclide-labeled targeted bispecific antibody prepared by the method for preparing a diagnostic agent for tumors, especially advanced tumors;
[0022] Preferably, the tumor includes non-small cell lung cancer (NSCLC).
[0023] The present invention also discloses the use of the radionuclide-labeled targeted bispecific antibody or the radionuclide-labeled targeted bispecific antibody prepared by the method for preparing a drug for treating tumors, especially advanced tumors;
[0024] Preferably, the tumor includes non-small cell lung cancer (NSCLC).
[0025] The radionuclide-labeled targeted bispecific antibody of the present invention 131 uses the radionuclide 131 I] to perform radionuclide labeling on the PD-1 / CTLA-4 bispecific antibody AK104 under clinical development. The
[0026] I-AK104 has a good therapeutic effect on tumors, especially advanced tumors, and has high clinical value for the diagnosis and treatment of NSCLC in particular. 131I]I-AK104 has good stability when incubated in physiological saline and human serum, and its stability in the human body is relatively ideal; the radionuclide-labeled targeting bispecific antibody described in the present invention 131 I]I-AK104 is mainly distributed in the blood and organs rich in blood such as the liver and spleen, and has a high radioactive uptake, which is suitable for clinical application.
[0027] The radionuclide-labeled targeting bispecific antibody described in the present invention 131 I]I-AK104 is labeled by the Iodogen method. The radioactive labeling yield of [131I]I-AK104 is greater than 80%, and the radiochemical purity (RCP) > 98%. Moreover, the 131 I]As a classic diagnostic and therapeutic radionuclide, it has rich sources and low prices, and the cost of the entire labeling method is relatively low. Brief Description of the Drawings
[0028] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention in combination with the drawings, wherein,
[0029] Figure 1 is the principle schematic and labeling result of the labeling method described in the present invention;
[0030] Figure 2 is the HPLC chromatogram of AK104;
[0031] Figure 3 is the 131 I]I-AK104 HPLC chromatogram;
[0032] Figure 4 is the 131 I]I-AK104 TLC chromatogram;
[0033] Figure 5 is the [131I] I-AK104 effect on tumor treatment. Detailed Embodiments
[0034] As Figure 1 shown in the method schematic diagram in (A) below, in the following examples of the present invention, the PD-1 / CTLA-4 bispecific antibody AK104 is labeled with a radionuclide by the Iodogen iodination method, preferably 131 I]labeling. As an exemplary implementation method, in the following examples of the present invention, the iodination labeling method uses Iodogen as the iodinating agent, and by mixing the solution containing AK104 and Na 131The 131 I] solution is incubated in a reaction tube coated with Iodogen to achieve
[0035] Example 1
[0036] The activated PD10 column (Cytiva, UK) is rinsed with 0.02 M phosphate buffer (PBS). After draining, PBS is added for purification. 100 μL of AK104 solution (Akeso, China) and 100 μL of Na 131 I] solution (Xinke, China) are added to the reaction tube coated with Iodogen. The reaction is carried out at room temperature for 20 min, and then the reaction solution is added to the purified PD10 column. The labeled product is collected with a 1.5 mL EP tube.
[0037] In this example, the HPLC chromatogram of the said AK104 is as shown in Figure 2 the appendix, and the HPLC chromatogram of the labeled product 131 I]I-AK104 is as shown in Figure 3 the appendix, and the TLC chromatogram of the labeled product 131 I]I-AK104 is as shown in Figure 4 the appendix. It can be seen that the labeling method of the present invention can achieve the radionuclide labeling of AK104.
[0038] Example 2
[0039] The activated PD10 column (Cytiva, UK) is rinsed with 0.01 M phosphate buffer (PBS). After draining, PBS is added for purification. An appropriate amount of AK104 solution (Akeso, China) and a sufficient amount of Na 131 I] solution (Xinke, China) are added to the reaction tube coated with Iodogen. The reaction is carried out at room temperature for 30 min, and then the reaction solution is added to the purified PD10 column. The labeled product is collected with a 1.5 mL EP tube.
[0040] Example 3
[0041] The activated PD10 column (Cytiva, UK) is rinsed with 0.03 M phosphate buffer (PBS). After draining, PBS is added for purification. An appropriate amount of AK104 solution (Akeso, China) and a sufficient amount of Na 131 I] solution (Xinke, China) are added to the reaction tube coated with Iodogen. The reaction is carried out at room temperature for 10 min, and then the reaction solution is added to the purified PD10 column. The labeled product is collected with a 1.5 mL EP tube.
[0042] Comparative Example 1
[0043] The labeling method described in this comparative example is the same as that in Example 1, except that the iodination method is the direct labeling Chloramine-T method.
[0044] Activate the PD10 column. First, rinse it with 20 mL of pure water, and then rinse it with 20 mL of 0.02 M PBS after completion. Keep it for later use after the rinsing. The experiment is carried out in 1 tube for labeling. In each Iodogen tube, add 105 μL of AK104 solution, 105 μL of 0.02 M PBS, and 40 μL of CH-T solution (5 mg / mL). Add 105 μL of [131I]NaI with a total activity of 144 MBq. Place it in a constant temperature reactor and react gently and evenly for 7 minutes at room temperature with a rotation speed of 800 RPM.
[0045] After the reaction, add 80 μL of Na2S2O5 solution (5 mg / mL), continue to keep at room temperature, mix gently, and react for 7 minutes with a rotation speed of 800 RPM.
[0046] After the reaction, let it stand for 4 minutes. Drain the 0.02 M PBS in the PD10 column, add all the reaction solution, and drain until it is level with the packing material. Add 0.02 M PBS to the PD10 column with a volume of 2.5 mL - the volume of all the reaction solution, and drain until it is level with the packing material. Add 0.02 M PBS for purification, collect the solution with a 1.5 mL EP tube, collect one tube for every 10 drops, and collect a total of 10 tubes.
[0047] Measure the radioactivity of each tube and calculate the yield of the radioactive probe.
[0048] Experimental Example
[0049] 1. Detection of labeling yield and purity
[0050] Respectively collect the labeled products in Example 1 and Comparative Example 1 above, and use radiometric High Performance Liquid Chromatography (radiometric HPLC) to detect the radioactive labeling yield and radiochemical purity (RCP) of the labeled products under each scheme. The results are shown in Table 1 below.
[0051] Table 1 Radioactive labeling yield and radiochemical purity
[0052] Number Yield of radioactive label / % Radiochemical purity / % Example 1 80 98 Comparative Example 1 60 95
[0053] It can be seen that the present invention is based on the Iodogen method for AK104 radioactive labeling, and the labeled product 131The radiolabeling yield of I]I-AK104 is higher than 80%, and the radiochemical purity (RCP) > 98%. The labeling method of the present invention not only has a high labeling rate but also an ideal labeling purity of the product, and has better clinical applicability.
[0054] 2. In vivo stability
[0055] In this example, the labeled product 131 I]I-AK104 prepared by the method in Example 1 was used for in vivo stability experiments.
[0056] The 131 I]I-AK104 (37 MBq, 1 mL) prepared in Example 1 was mixed with 1 mL of human serum filtrate and incubated at 37 °C. Its stability was measured by radioactive HPLC or TLC at 2, 6, 12, 24, 48, and 72 h.
[0057] As shown in the result of Figure 1 (B) in the appendix, the HPLC chromatogram indicates that 131 I]I-AK104 has good stability in saline and human serum incubation. A small amount of degradation occurs after 12 h, and the RCP remains stable above 95% within 24 h. The saline group decreases after 24 h and remains at about 90% at 72 h.
[0058] It can be seen that the 131 I]I-AK104 prepared by the present invention has good stability in the in vivo environment and has the prospect of clinical translation.
[0059] 3. In vivo distribution
[0060] In this example, the labeled product 131 I]I-AK104 prepared by the method in Example 1 was used for in vivo distribution and radioactivity tests.
[0061] The 131 I]I-AK104 (0.37 MBq, 100 μL) prepared in Example 1 was injected into the tail vein of ICR mice (6 weeks old, male). Then, euthanasia was performed at 4, 24, 96, 168, 336, and 504 h (n = 3).
[0062] The brain, liver, spleen, lung, heart, kidney, stomach, small intestine, large intestine, muscle, fat, bone, testis, bladder, and pancreas were collected. After the organs were collected, they were washed twice with deionized water and dried, placed in a pre-weighed test tube, weighed again, and the sample weight was calculated.
[0063] Blood was collected through the abdominal aorta, and immediately after collection, 100 μL was quantified and weighed into a specified centrifuge tube. The samples were measured on the day of collection, and the radioactivity count CPM values of all blood samples and tissue samples were measured using a gamma counter.
[0064] As shown in Figure 1 the results of (C)-(D) in the appendix, the labeled product prepared by the present invention 131 [[131I]]I-AK104 was mainly distributed in the blood (C) and organs rich in blood such as the liver and spleen (D), and the radioactive uptakes were 5.56±0.47% ID / g, 1.8±0.71% ID / g, and 1.4±0.46% ID / g respectively. By 24 hours, the radioactive uptakes in the blood and various organs were significantly reduced, and by 336 hours, they were gradually cleared and approached the background.
[0065] It can be seen that the radionuclide-labeled targeting bispecific antibody 131 [[131I]]I-AK104 prepared by the present invention has good stability in incubation with normal saline and human serum, and its stability in the human body is relatively ideal; the radionuclide-labeled targeting bispecific antibody 131 [[131I]]I-AK104 is mainly distributed in the blood and organs rich in blood such as the liver and spleen, and has a relatively high radioactive uptake, which is suitable for clinical applications.
[0066] 4. NSCLC tumor treatment
[0067] In this example, the labeled product 131 [[131I]]I-AK104 prepared by the method in Example 1 was used for NSCLC tumor treatment experiments.
[0068] Four groups of C57BL / 6J tumor-bearing mice, with 5 mice in each group, were respectively injected with 0.1 mL of PBS, [131I] NaI (11.1 MBq, 0.1 mL), AK104 (0.4 mg, 0.1 mL), [131I] [[131I]]-AK104 (equivalent to 11.1 MBq of [131I] NaI and 0.4 mg of AK104, 0.1 mL) via the tail vein. The above experimental groups were given equal doses on the 1st day and the 7th day respectively. The general conditions and tumor growth of the mice were observed, the tumor volume and body weight of the mice were measured every 3 days, and the tumor growth curve and body weight curve were plotted.
[0069] In this example, the efficacy of [131I] [[131I]]-AK104 in tumor treatment was evaluated in LLC tumor-bearing mice. As Figure 5 shown in the results, the tumor growth in the [[131I]]I-AK104 group slowed down, and the tumor volume was the smallest among all groups at the end of the treatment. The other groups included the PBS group (control group), AK104 group (monoclonal antibody group),131 Group I.
[0070] It can be seen that the labeled product 131 II-AK104 prepared by the present invention can be used for treating advanced tumors and has the prospect of clinical transformation.
[0071] In summary, for the radionuclide-labeled targeting bispecific antibody of the present invention, the PD-1 / CTLA-4 bispecific antibody AK104 under clinical development is radiolabeled with a radionuclide 131 I, and the 131 II-AK104 has a good therapeutic effect on tumors, especially advanced tumors, and has high clinical value for the diagnosis and treatment of NSCLC in particular.
[0072] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.
Claims
1. A radionuclide-labeled targeted bispecific antibody, characterized in that, The nuclide-labeled targeted bispecific antibody includes 131 I]-labeled PD-1 / CTLA-4 bispecific antibody; The PD-1 / CTLA-4 bispecific antibody includes cadonilimab.
2. A method for preparing a radionuclide-labeled targeting bispecific antibody as described in claim 1, characterized in that, Comprising the step of mixing and labeling the PD-1 / CTLA-4 bispecific antibody cadonilim, a radionuclide 131 I - and an iodinating agent.
3. The preparation method of the radionuclide-labeled targeting bispecific antibody according to claim 2, wherein, The iodinating agent includes Iodogen.
4. The preparation method of the radionuclide-labeled targeting bispecific antibody according to claim 3, characterized in that, Including the step of adding the solution containing the Cadonilimab into a reaction tube coated with Iodogen and 131 I - mixing the solutions, and incubating the reaction at 20 - 30 °C for 10 - 30 min; Preferably, the 131 I - solution includes Na 131 I] solution.
5. The preparation method of the radionuclide-labeled targeting bispecific antibody according to any one of claims 2 to 4, characterized in that, The method further includes a step of purifying the labeled product by a chromatography column; Preferably, the chromatography column includes a PD10 column.
6. The preparation method of the radionuclide-labeled targeting bispecific antibody according to claim 5, wherein, The method further includes a step of activating and / or purifying the chromatography column; Preferably, the reagent for the activation and / or purification step includes a PBS buffer solution; Preferably, the concentration of the PBS buffer solution is 0.01 - 0.03 M.
7. The preparation method of the radionuclide-labeled targeting bispecific antibody according to any one of claims 2-6, characterized in that, The method further includes a step of detecting the radioactive labeling yield and radiochemical purity of the labeled product by radio-high performance liquid chromatography.
8. Use of the radionuclide-labeled targeted bispecific antibody according to claim 1 or the radionuclide-labeled targeted bispecific antibody prepared by the method according to any one of claims 2 - 7 for preparing an integrated probe for targeted diagnosis and treatment.
9. Use of the radionuclide-labeled targeted bispecific antibody according to claim 1 or the radionuclide-labeled targeted bispecific antibody prepared by the method according to any one of claims 2 - 7 for preparing a tumor diagnostic agent; Preferably, the tumor includes non-small cell lung cancer (NSCLC).
10. Use of the radionuclide-labeled targeted bispecific antibody according to claim 1 or the radionuclide-labeled targeted bispecific antibody prepared by the method according to any one of claims 2 - 7 for preparing a drug for treating tumors; Preferably, the tumor includes non-small cell lung cancer (NSCLC).