技术领域Technical Field
本发明涉及一种PD-L1小分子抑制剂及其制备方法和应用，属于生物医药技术领域。The present invention relates to a PD-L1 small molecule inhibitor and a preparation method and application thereof, belonging to the technical field of biomedicine.
背景技术Background Art
癌症，亦称恶性肿瘤，为细胞恶性增生所致，包括黑色素瘤、非小细胞肺癌、肾癌、肝癌、乳腺癌、结肠癌、胰腺癌和前列腺癌等。癌症多采用综合治疗，以外科手术、化疗、放疗这三种传统治疗手段相结合为主，必要时可结合靶向治疗、生物治疗和抗肿瘤免疫治疗等方法。其中，抗肿瘤免疫治疗是通过增强自身免疫功能来抑制或杀伤癌细胞，很大程度上减少了传统治疗手段对肿瘤患者产生的严重副作用。Cancer, also known as malignant tumor, is caused by malignant cell proliferation, including melanoma, non-small cell lung cancer, kidney cancer, liver cancer, breast cancer, colon cancer, pancreatic cancer and prostate cancer. Cancer is often treated with a combination of three traditional treatment methods: surgery, chemotherapy and radiotherapy. When necessary, targeted therapy, biological therapy and anti-tumor immunotherapy can be combined. Among them, anti-tumor immunotherapy inhibits or kills cancer cells by enhancing the body's immune function, which greatly reduces the serious side effects of traditional treatments on cancer patients.
抗肿瘤免疫治疗是通过重新启动并维持肿瘤-免疫循环，恢复机体正常的抗肿瘤免疫反应，从而控制与清除肿瘤的一种癌症治疗方法。免疫检查点程序性细胞死亡受体1(PD-1)是抗肿瘤免疫治疗的主要研究方向。研究发现，肿瘤细胞膜表面过度表达的程序性细胞死亡配体1(PD-L1)会和效应T细胞表面PD-1结合来抑制T细胞对肿瘤细胞的攻击，从而使肿瘤细胞发生免疫逃脱。而PD-1/PD-L1免疫检查点抑制剂能够通过阻断肿瘤细胞表面的PD-L1与T细胞上的PD-1结合，恢复T细胞抗肿瘤免疫反应，进而抑制肿瘤的快速增殖和转移。目前，将免疫检查点抑制剂与其它抗肿瘤方法联合使用可有效改善免疫治疗的低响应率这一现象已得到广泛证实。Anti-tumor immunotherapy is a cancer treatment method that controls and eliminates tumors by restarting and maintaining the tumor-immune cycle and restoring the body's normal anti-tumor immune response. The immune checkpoint programmed cell death receptor 1 (PD-1) is the main research direction of anti-tumor immunotherapy. Studies have found that programmed cell death ligand 1 (PD-L1) overexpressed on the surface of tumor cell membranes will bind to PD-1 on the surface of effector T cells to inhibit T cells from attacking tumor cells, thereby allowing tumor cells to escape immune system. The PD-1/PD-L1 immune checkpoint inhibitor can restore T cell anti-tumor immune response by blocking the binding of PD-L1 on the surface of tumor cells to PD-1 on T cells, thereby inhibiting the rapid proliferation and metastasis of tumors. At present, it has been widely confirmed that the combination of immune checkpoint inhibitors with other anti-tumor methods can effectively improve the low response rate of immunotherapy.
靶向放射性核素治疗(TRT)是利用具有靶向性的载体将治疗性核素运输到肿瘤中聚积衰变释放电离辐射，从而破坏肿瘤组织的一种癌症靶向治疗方法。研究表明，TRT可以在体内通过诱导免疫原性细胞死亡(ICD)、释放肿瘤新抗原和刺激抗肿瘤免疫效应来提高免疫治疗的效果。最近的一项研究报道证实，低剂量辐射可以重塑肿瘤微环境，有利于抗肿瘤免疫。同时，免疫治疗也可能使肿瘤对TRT更加敏感。因此，将免疫检查点抑制剂与TRT联合应用也许能够产生协同作用，进而达到更好的癌症治疗效果。然而，现阶段，同时作为免疫检查点抑制剂和靶向放射性核素治疗药物的PD-L1小分子抑制剂还未有报道。Targeted radionuclide therapy (TRT) is a targeted cancer therapy that uses targeted carriers to transport therapeutic radionuclides into tumors to accumulate and decay and release ionizing radiation, thereby destroying tumor tissue. Studies have shown that TRT can improve the effect of immunotherapy in vivo by inducing immunogenic cell death (ICD), releasing tumor neoantigens, and stimulating anti-tumor immune effects. A recent study report confirmed that low-dose radiation can reshape the tumor microenvironment and facilitate anti-tumor immunity. At the same time, immunotherapy may also make tumors more sensitive to TRT. Therefore, the combination of immune checkpoint inhibitors and TRT may produce a synergistic effect, thereby achieving better cancer treatment effects. However, at this stage, there are no reports of PD-L1 small molecule inhibitors that are both immune checkpoint inhibitors and targeted radionuclide therapy drugs.
发明内容Summary of the invention
为解决上述问题，本发明提供了一种碘-131标记的PD-L1小分子抑制剂，所述碘-131标记的PD-L1小分子抑制剂具有如下所示结构：In order to solve the above problems, the present invention provides an iodine-131 labeled PD-L1 small molecule inhibitor, wherein the iodine-131 labeled PD-L1 small molecule inhibitor has the following structure:
其中，R为Among them, R is
在本发明的一种实施方式中，当R为时，所述碘-131标记的PD-L1小分子抑制剂具有如下所示结构： In one embodiment of the present invention, when R is When the iodine-131 labeled PD-L1 small molecule inhibitor has the following structure:
本发明还提供了一种非放射性的PD-L1小分子抑制剂，所述非放射性的PD-L1小分子抑制剂具有如下所示结构：The present invention also provides a non-radioactive PD-L1 small molecule inhibitor, wherein the non-radioactive PD-L1 small molecule inhibitor has the following structure:
在本发明的一种实施方式中，当R为时，所述非放射性的PD-L1小分子抑制剂具有如下所示结构：In one embodiment of the present invention, when R is When the non-radioactive PD-L1 small molecule inhibitor has the following structure:
本发明还提供了一种制备上述碘-131标记的PD-L1小分子抑制剂的方法，所述方法包括：将化合物2和4-甲氧基(二乙酰氧基碘)苯溶于溶剂中后，于氮气的保护下进行反应，得到化合物3；对化合物3进行131I的放射性核素标记，得到中间体化合物[131I]1；将中间体化合物[131I]1、化合物4和氰基硼氢化钠溶于溶剂中，得到溶解液；在溶解液中加入冰醋酸后进行反应，得到上述碘-131标记的PD-L1小分子抑制剂；The present invention also provides a method for preparing the above-mentioned iodine-131 labeled PD-L1 small molecule inhibitor, the method comprising: dissolving compound 2 and 4-methoxy (diacetoxyiodine) benzene in a solvent, and reacting under the protection of nitrogen to obtain compound 3; labeling compound 3 with131 I radioactive nuclide to obtain an intermediate compound [131 I]1; dissolving the intermediate compound [131 I]1, compound 4 and sodium cyanoborohydride in a solvent to obtain a solution; adding glacial acetic acid to the solution and reacting to obtain the above-mentioned iodine-131 labeled PD-L1 small molecule inhibitor;
所述化合物4为甘氨酸、丝氨酸、谷氨酸、氨甲基膦酸、磺基丙氨酸、精氨酸、络氨酸、天门冬氨酸、脯氨酸、天冬酰胺、三(羟甲基)氨基甲烷(Tris)或氨基葡萄糖；The compound 4 is glycine, serine, glutamic acid, aminomethylphosphonic acid, cysteic acid, arginine, tyrosine, aspartic acid, proline, asparagine, tris(hydroxymethyl)aminomethane (Tris) or glucosamine;
所述化合物2具有如下所示结构：The compound 2 has the following structure:
所述4-甲氧基(二乙酰氧基碘)苯具有如下所示结构：The 4-methoxy(diacetoxyiodo)benzene has the structure shown below:
所述化合物3具有如下所示结构：The compound 3 has the following structure:
所述中间体化合物[131I]1具有如下所示结构：The intermediate compound [131I ]1 has the following structure:
在本发明的一种实施方式中，所述化合物2的制备方法包括：将化合物1、四(三苯基磷)钯和六正丁基二锡溶于溶剂中后，于氮气的保护下进行加热反应，得到化合物2；In one embodiment of the present invention, the preparation method of the compound 2 comprises: dissolving the compound 1, tetrakis(triphenylphosphine)palladium and hexa-n-butylditin in a solvent, and heating the mixture under the protection of nitrogen to obtain the compound 2;
所述化合物1具有如下所示结构：The compound 1 has the following structure:
在本发明的一种实施方式中，所述化合物1的制备方法包括：将化合物5、3-溴甲基苯甲腈和碳酸铯溶于溶剂中进行反应，得到化合物1；In one embodiment of the present invention, the preparation method of compound 1 comprises: dissolving compound 5, 3-bromomethylbenzonitrile and cesium carbonate in a solvent for reaction to obtain compound 1;
所述化合物5具有如下所示结构：The compound 5 has the following structure:
所述3-溴甲基苯甲腈具有如下所示结构：The 3-bromomethylbenzonitrile has the structure shown below:
在本发明的一种实施方式中，所述化合物5的制备方法包括：在冰浴条件下，将化合物7、化合物6和三苯基磷溶于溶剂中，得到溶解液；在冰浴和氮气搅拌下，于溶解液中滴加偶氮二甲酸二异丙酯，得到反应液；将反应液进行反应，得到化合物5；In one embodiment of the present invention, the preparation method of compound 5 comprises: dissolving compound 7, compound 6 and triphenylphosphine in a solvent under ice bath conditions to obtain a solution; adding diisopropyl azodicarboxylate dropwise to the solution under ice bath and nitrogen stirring to obtain a reaction solution; reacting the reaction solution to obtain compound 5;
所述化合物7具有如下所示结构：The compound 7 has the following structure:
所述化合物6具有如下所示结构：The compound 6 has the following structure:
本发明还提供了一种制备上述非放射性的PD-L1小分子抑制剂的方法，所述方法包括：将化合物1、化合物4和氰基硼氢化钠溶于溶剂中，得到溶解液；在溶解液中加入冰醋酸后进行反应，得到上述非放射性的PD-L1小分子抑制剂；The present invention also provides a method for preparing the above-mentioned non-radioactive PD-L1 small molecule inhibitor, the method comprising: dissolving compound 1, compound 4 and sodium cyanoborohydride in a solvent to obtain a dissolving solution; adding glacial acetic acid to the dissolving solution and reacting to obtain the above-mentioned non-radioactive PD-L1 small molecule inhibitor;
所述化合物4为甘氨酸、丝氨酸、谷氨酸、氨甲基膦酸、磺基丙氨酸、精氨酸、络氨酸、天门冬氨酸、脯氨酸、天冬酰胺、三(羟甲基)氨基甲烷(Tris)或氨基葡萄糖；The compound 4 is glycine, serine, glutamic acid, aminomethylphosphonic acid, cysteic acid, arginine, tyrosine, aspartic acid, proline, asparagine, tris(hydroxymethyl)aminomethane (Tris) or glucosamine;
所述化合物1具有如下所示结构：The compound 1 has the following structure:
在本发明的一种实施方式中，所述化合物1的制备方法包括：将化合物5、3-溴甲基苯甲腈和碳酸铯溶于溶剂中进行反应，得到化合物1；In one embodiment of the present invention, the preparation method of compound 1 comprises: dissolving compound 5, 3-bromomethylbenzonitrile and cesium carbonate in a solvent for reaction to obtain compound 1;
所述化合物5具有如下所示结构：The compound 5 has the following structure:
所述3-溴甲基苯甲腈具有如下所示结构：The 3-bromomethylbenzonitrile has the structure shown below:
在本发明的一种实施方式中，所述化合物5的制备方法包括：在冰浴条件下，将化合物7、化合物6和三苯基磷溶于溶剂中，得到溶解液；在冰浴和氮气搅拌下，于溶解液中滴加偶氮二甲酸二异丙酯，得到反应液；将反应液进行反应，得到化合物5；In one embodiment of the present invention, the preparation method of compound 5 comprises: dissolving compound 7, compound 6 and triphenylphosphine in a solvent under ice bath conditions to obtain a solution; adding diisopropyl azodicarboxylate dropwise to the solution under ice bath and nitrogen stirring to obtain a reaction solution; reacting the reaction solution to obtain compound 5;
所述化合物7具有如下所示结构：The compound 7 has the following structure:
所述化合物6具有如下所示结构：The compound 6 has the structure shown below:
本发明还提供了上述碘-131标记的PD-L1小分子抑制剂或上述非放射性的PD-L1小分子抑制剂在制备预防和/或治疗癌症的药物中的应用。The present invention also provides use of the above-mentioned iodine-131 labeled PD-L1 small molecule inhibitor or the above-mentioned non-radioactive PD-L1 small molecule inhibitor in the preparation of a drug for preventing and/or treating cancer.
在本发明的一种实施方式中，药物含有上述碘-131标记的PD-L1小分子抑制剂和上述非放射性的PD-L1小分子抑制剂。In one embodiment of the present invention, the drug contains the above-mentioned iodine-131 labeled PD-L1 small molecule inhibitor and the above-mentioned non-radioactive PD-L1 small molecule inhibitor.
在本发明的一种实施方式中，所述预防和/或治疗癌症包括通过抑制PD-1/PD-L1通路促进肿瘤死亡，和/或，诱导肿瘤细胞发生免疫原性死亡(ICD)。In one embodiment of the present invention, the prevention and/or treatment of cancer includes promoting tumor death by inhibiting the PD-1/PD-L1 pathway, and/or inducing immunogenic death (ICD) of tumor cells.
在本发明的一种实施方式中，所述通过抑制PD-1/PD-L1通路促进肿瘤死亡包括通过与肿瘤中PD-L1特异性结合和/或降低肿瘤中PD-L1的表达量，抑制PD-1/PD-L1相互作用，激活T细胞的抗肿瘤免疫功能，增强T细胞对肿瘤的杀伤能力，进而促进肿瘤死亡；In one embodiment of the present invention, the promoting tumor death by inhibiting the PD-1/PD-L1 pathway includes inhibiting the PD-1/PD-L1 interaction by specifically binding to PD-L1 in the tumor and/or reducing the expression of PD-L1 in the tumor, activating the anti-tumor immune function of T cells, and enhancing the killing ability of T cells against tumors, thereby promoting tumor death;
所述诱导肿瘤细胞发生免疫原性死亡包括通过促进肿瘤细胞释放高迁移率族蛋白1(HMGB1)和钙网蛋白(CRT)，进而诱导肿瘤细胞发生免疫原性死亡。The inducing immunogenic death of tumor cells includes promoting the release of high mobility group protein 1 (HMGB1) and calreticulin (CRT) by tumor cells, thereby inducing immunogenic death of tumor cells.
在本发明的一种实施方式中，所述激活T细胞的抗肿瘤免疫功能包括增加肿瘤组织中CD4+T细胞比例、增加肿瘤组织中CD8+T细胞比例和/或促进T细胞分泌干扰素-γ。In one embodiment of the present invention, the activation of the anti-tumor immune function of T cells includes increasing the proportion of CD4+ T cells in tumor tissue, increasing the proportion of CD8+ T cells in tumor tissue and/or promoting T cells to secrete interferon-γ.
在本发明的一种实施方式中，所述肿瘤包括实体瘤；所述实体瘤包括黑色素瘤、非小细胞肺癌、肾癌、肝癌、乳腺癌、结肠癌、胰腺癌和/或前列腺癌。In one embodiment of the present invention, the tumor comprises a solid tumor; the solid tumor comprises melanoma, non-small cell lung cancer, kidney cancer, liver cancer, breast cancer, colon cancer, pancreatic cancer and/or prostate cancer.
本发明还提供了一种预防和/或治疗癌症的药物，所述药物含有上述碘-131标记的PD-L1小分子抑制剂和/或上述非放射性的PD-L1小分子抑制剂。The present invention also provides a drug for preventing and/or treating cancer, wherein the drug contains the above-mentioned iodine-131 labeled PD-L1 small molecule inhibitor and/or the above-mentioned non-radioactive PD-L1 small molecule inhibitor.
在本发明的一种实施方式中，药物含有上述碘-131标记的PD-L1小分子抑制剂和上述非放射性的PD-L1小分子抑制剂。In one embodiment of the present invention, the drug contains the above-mentioned iodine-131 labeled PD-L1 small molecule inhibitor and the above-mentioned non-radioactive PD-L1 small molecule inhibitor.
在本发明的一种实施方式中，所述预防和/或治疗癌症包括通过抑制PD-1/PD-L1通路促进肿瘤死亡，和/或，诱导肿瘤细胞发生免疫原性死亡。In one embodiment of the present invention, the prevention and/or treatment of cancer includes promoting tumor death by inhibiting the PD-1/PD-L1 pathway, and/or inducing immunogenic death of tumor cells.
在本发明的一种实施方式中，所述通过抑制PD-1/PD-L1通路促进肿瘤死亡包括通过与肿瘤中PD-L1特异性结合和/或降低肿瘤中PD-L1的表达量，抑制PD-1/PD-L1相互作用，激活T细胞的抗肿瘤免疫功能，增强T细胞对肿瘤的杀伤能力，进而促进肿瘤死亡；In one embodiment of the present invention, the promoting tumor death by inhibiting the PD-1/PD-L1 pathway includes inhibiting the PD-1/PD-L1 interaction by specifically binding to PD-L1 in the tumor and/or reducing the expression of PD-L1 in the tumor, activating the anti-tumor immune function of T cells, and enhancing the killing ability of T cells against tumors, thereby promoting tumor death;
所述诱导肿瘤细胞发生免疫原性死亡包括通过促进肿瘤细胞释放高迁移率族蛋白1(HMGB1)和钙网蛋白(CRT)，进而诱导肿瘤细胞发生免疫原性死亡。The inducing immunogenic death of tumor cells includes promoting the release of high mobility group protein 1 (HMGB1) and calreticulin (CRT) by tumor cells, thereby inducing immunogenic death of tumor cells.
在本发明的一种实施方式中，所述激活T细胞的抗肿瘤免疫功能包括增加肿瘤组织中CD4+T细胞比例、增加肿瘤组织中CD8+T细胞比例和/或促进T细胞分泌干扰素-γ。In one embodiment of the present invention, the activation of the anti-tumor immune function of T cells includes increasing the proportion of CD4+ T cells in tumor tissue, increasing the proportion of CD8+ T cells in tumor tissue and/or promoting T cells to secrete interferon-γ.
在本发明的一种实施方式中，所述肿瘤包括实体瘤；所述实体瘤包括黑色素瘤、非小细胞肺癌、肾癌、肝癌、乳腺癌、结肠癌、胰腺癌和/或前列腺癌。In one embodiment of the present invention, the tumor comprises a solid tumor; the solid tumor comprises melanoma, non-small cell lung cancer, kidney cancer, liver cancer, breast cancer, colon cancer, pancreatic cancer and/or prostate cancer.
本发明技术方案，具有如下优点：The technical solution of the present invention has the following advantages:
本发明提供了本发明提供了碘-131标记的PD-L1小分子抑制剂[131I]LG-12以及非放射性的PD-L1小分子抑制剂LG-12，碘-131标记的PD-L1小分子抑制剂[131I]LG-12以及非放射性的PD-L1小分子抑制剂LG-12具有以下优势：The present invention provides an iodine-131 labeled PD-L1 small molecule inhibitor [131 I] LG-12 and a non-radioactive PD-L1 small molecule inhibitor LG-12. The iodine-131 labeled PD-L1 small molecule inhibitor [131 I] LG-12 and the non-radioactive PD-L1 small molecule inhibitor LG-12 have the following advantages:
第一，PD-1/PD-L1 TR-FRET实验结果表明，非放射性的PD-L1小分子抑制剂LG-12的EC50值为34.55±3.21nM，可见，LG-12对PD-1/PD-L1相互作用具有较高的抑制活性，能够显著抑制肿瘤生长，表现出较好的抗肿瘤免疫治疗效果；First, the results of the PD-1/PD-L1 TR-FRET experiment showed that the EC50 value of the non-radioactive PD-L1 small molecule inhibitor LG-12 was 34.55±3.21nM. It can be seen that LG-12 has a high inhibitory activity on the PD-1/PD-L1 interaction, can significantly inhibit tumor growth, and exhibits a good anti-tumor immunotherapy effect;
第二，体外稳定性实验结果表明，碘-131标记的PD-L1小分子抑制剂[131I]LG-12在体外12h仍保持稳定，可见，[131I]LG-12具有体外稳定强的优势；Second, the results of in vitro stability experiments showed that the iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12 remained stable in vitro for 12 hours, indicating that [131 I]LG-12 has the advantage of strong in vitro stability;
第三，细胞摄取和生物分布实验结果表明，碘-131标记的PD-L1小分子抑制剂[131I]LG-12在小鼠黑色素瘤细胞B16-F10中的最大摄取值为5.33±0.33％AD，且可被非放射性的PD-L1小分子抑制剂LG-12显著阻断摄取(2.56±0.06％AD)，同时，尾静脉注射1h后，碘-131标记的PD-L1小分子抑制剂[131I]LG-12在B16-F10荷瘤鼠肿瘤部位的摄取值为6.50±1.05％ID/g，可见，[131I]LG-12能够与肿瘤中PD-L1特异性结合；Third, the results of cellular uptake and biodistribution experiments showed that the maximum uptake value of the iodine-131 labeled PD-L1 small molecule inhibitor [131I ]LG-12 in mouse melanoma cells B16-F10 was 5.33±0.33%AD, and the uptake could be significantly blocked by the non-radioactive PD-L1 small molecule inhibitor LG-12 (2.56±0.06%AD). At the same time, 1 hour after tail vein injection, the uptake value of the iodine-131 labeled PD-L1 small molecule inhibitor [131I ]LG-12 in the tumor site of B16-F10 tumor-bearing mice was 6.50±1.05%ID/g. It can be seen that [131I ]LG-12 can specifically bind to PD-L1 in tumors.
第四，体内显像实验结果表明，尾静脉注射30min后，碘-131标记的PD-L1小分子抑制剂[131I]LG-12在B16-F10荷瘤鼠的肿瘤中快速积聚，在B16-F10荷瘤鼠肿瘤中的活性比肌肉中的活性高2.3倍，并且，在被非放射性的PD-L1小分子抑制剂LG-12阻断后，肿瘤部位的摄取明显降低，可见，[131I]LG-12能够与PD-L1靶向特异性结合；Fourth, the results of in vivo imaging experiments showed that 30 minutes after tail vein injection, the iodine-131 labeled PD-L1 small molecule inhibitor [131I ]LG-12 rapidly accumulated in the tumors of B16-F10 tumor-bearing mice, and its activity in the tumors of B16-F10 tumor-bearing mice was 2.3 times higher than that in the muscles. Moreover, after being blocked by the non-radioactive PD-L1 small molecule inhibitor LG-12, the uptake in the tumor site was significantly reduced. It can be seen that [131I ]LG-12 can specifically bind to PD-L1.
第五，T细胞/肿瘤细胞共培养实验结果表明，当非放射性的PD-L1小分子抑制剂LG-12浓度为0.78μM和3.125μM时，与T细胞共培养的B16-F10细胞的存活率分别降至0.66±0.06和0.55±0.03，可见，LG-12能够激活T细胞的抗肿瘤免疫功能，并以剂量依赖的方式增强T细胞对肿瘤的杀伤能力；Fifth, the results of T cell/tumor cell co-culture experiments showed that when the concentration of the non-radioactive PD-L1 small molecule inhibitor LG-12 was 0.78μM and 3.125μM, the survival rate of B16-F10 cells co-cultured with T cells dropped to 0.66±0.06 and 0.55±0.03, respectively. It can be seen that LG-12 can activate the anti-tumor immune function of T cells and enhance the killing ability of T cells against tumors in a dose-dependent manner;
第六，T细胞/肿瘤细胞共培养实验结果表明，当B16-F10/T细胞为1/20时，与浓度分别为0.78μM和3.125μM的非放射性的PD-L1小分子抑制剂LG-12作用后，干扰素-γ的表达量由34.44±1.39pg/100μL分别升高至40.28±1.97pg/100μL和76.33±5.24pg/100μL，当B16-F10/T细胞为1/40时，与浓度分别为0.78μM和3.125μM的非放射性的PD-L1小分子抑制剂LG-12作用后，干扰素-γ的表达量由53.19±2.85pg/100μL分别升高至83.13±3.64pg/100μL和110.26±1.72pg/100μL，可见，LG-12能够通过阻断PD-1/PD-L1信号通路，增加干扰素-γ的分泌，促进CD8+T细胞的活化，进而激活T细胞的抗肿瘤免疫功能，并以剂量依赖的方式增强T细胞对肿瘤的杀伤能力；Sixth, the results of T cell/tumor cell co-culture experiments showed that when the B16-F10/T cell ratio was 1/20, after the non-radioactive PD-L1 small molecule inhibitor LG-12 at concentrations of 0.78 μM and 3.125 μM, the expression of interferon-γ increased from 34.44±1.39 pg/100 μL to 40.28±1.97 pg/100 μL and 76.33±5.24 pg/100 μL, respectively. When the concentration of LG-12 was 1/40, the expression of interferon-γ increased from 53.19±2.85pg/100μL to 83.13±3.64pg/100μL and 110.26±1.72pg/100μL respectively after the non-radioactive PD-L1 small molecule inhibitor LG-12 with concentrations of 0.78μM and 3.125μM. It can be seen that LG-12 can increase the secretion of interferon-γ and promote the activation of CD8+ T cells by blocking the PD-1/PD-L1 signaling pathway, thereby activating the anti-tumor immune function of T cells and enhancing the killing ability of T cells against tumors in a dose-dependent manner.
第七，体内抗肿瘤实验结果表明，腹腔注射非放射性的PD-L1小分子抑制剂LG-12后，所有小鼠在治疗过程中均没有引起明显的体重减轻或死亡，可见，LG-12在所有剂量都是耐受性良好；Seventh, the results of in vivo anti-tumor experiments showed that after intraperitoneal injection of the non-radioactive PD-L1 small molecule inhibitor LG-12, all mice did not cause significant weight loss or death during the treatment process, indicating that LG-12 was well tolerated at all doses;
第八，体内抗肿瘤实验结果表明，腹腔注射非放射性的PD-L1小分子抑制剂LG-12后，小鼠的肿瘤体积和肿瘤重量显著缩小，小鼠肿瘤组织中CD4+T细胞比例和CD8+T细胞比例增加，小鼠肿瘤组织中中PD-L1的表达量减少，并且，小鼠血清中干扰素-γ的表达量显著增加，可见，LG-12具有很好的体内抗肿瘤效果；Eighth, the results of in vivo anti-tumor experiments showed that after intraperitoneal injection of the non-radioactive PD-L1 small molecule inhibitor LG-12, the tumor volume and weight of mice were significantly reduced, the proportion of CD4+ T cells and CD8+ T cells in the tumor tissue of mice increased, the expression of PD-L1 in the tumor tissue of mice decreased, and the expression of interferon-γ in the serum of mice increased significantly. It can be seen that LG-12 has a good in vivo anti-tumor effect;
第九，体外克隆实验结果表明，与碘-131标记的PD-L1小分子抑制剂[131I]LG-12共培养的B16-F10细胞形成集落的数量显著降低，且作用效果远超与Na131I共培养的B16-F10细胞，可见，[131I]LG-12能够显著抑制肿瘤细胞的增殖，具有很好的体外抗肿瘤效果；Ninth, the results of in vitro cloning experiments showed that the number of colonies formed by B16-F10 cells co-cultured with the iodine-131 labeled PD-L1 small molecule inhibitor [131I ]LG-12 was significantly reduced, and the effect was far greater than that of B16-F10 cells co-cultured withNa131I . It can be seen that [131I ]LG-12 can significantly inhibit the proliferation of tumor cells and has a good in vitro anti-tumor effect;
第十，体外放射性药物刺激肿瘤细胞的实验结果表明，被碘-131标记的PD-L1小分子抑制剂[131I]LG-12刺激后，B16-F10细胞外的高迁移率族蛋白1(HMGB1)的表达量显著增加(B16-F10细胞内的HMGB1蛋白的表达量显著降低即表明B16-F10细胞外的HMGB1蛋白的表达量显著增加)，同时，B16-F10细胞表面的钙网蛋白(CRT)表达量显著增加，可见，[131I]LG-12能够通过促进肿瘤细胞释放HMGB1蛋白和CRT蛋白，进而诱导肿瘤细胞发生免疫原性死亡，具有很好的体外抗肿瘤效果；Tenth, the experimental results of in vitro radioactive drug stimulation of tumor cells showed that after stimulation by the iodine-131 labeled PD-L1 small molecule inhibitor [131I ]LG-12, the expression of high mobility group protein 1 (HMGB1) outside B16-F10 cells increased significantly (the expression of HMGB1 protein inside B16-F10 cells decreased significantly, indicating that the expression of HMGB1 protein outside B16-F10 cells increased significantly). At the same time, the expression of calreticulin (CRT) on the surface of B16-F10 cells increased significantly. It can be seen that [131I ]LG-12 can promote the release of HMGB1 protein and CRT protein by tumor cells, thereby inducing immunogenic death of tumor cells, and has a good in vitro anti-tumor effect;
第十一，体内抗肿瘤实验结果表明，腹腔注射碘-131标记的PD-L1小分子抑制剂[131I]LG-12后，所有小鼠在治疗过程中均没有引起明显的体重减轻或死亡，可见，[131I]LG-12对正常组织的毒性较低；Eleventh, the results of in vivo anti-tumor experiments showed that after intraperitoneal injection of iodine-131-labeled PD-L1 small molecule inhibitor [131I ]LG-12, all mice did not cause significant weight loss or death during the treatment process, indicating that [131I ]LG-12 has low toxicity to normal tissues;
第十二，体内抗肿瘤实验结果表明，腹腔注射碘-131标记的PD-L1小分子抑制剂[131I]LG-12后，小鼠肿瘤组织中HMGB1蛋白和CRT蛋白的表达量显著增加，且作用效果远超腹腔注射Na131I的小鼠，可见，[131I]LG-12能够通过促进肿瘤细胞释放HMGB1蛋白和CRT蛋白，进而诱导肿瘤细胞发生免疫原性死亡，具有很好的体内抗肿瘤效果；Twelfth, the results of in vivo anti-tumor experiments showed that after intraperitoneal injection of iodine-131 labeled PD-L1 small molecule inhibitor [131I ]LG-12, the expression of HMGB1 protein and CRT protein in mouse tumor tissues increased significantly, and the effect was far greater than that of mice injected withNa131I intraperitoneally. It can be seen that [131I ]LG-12 can promote the release of HMGB1 protein and CRT protein by tumor cells, thereby inducing immunogenic death of tumor cells, and has a good in vivo anti-tumor effect;
第十三，体内抗肿瘤实验结果表明，先尾静脉注射碘-131标记的PD-L1小分子抑制剂[131I]LG-12再腹腔注射非放射性的PD-L1小分子抑制剂LG-12后，小鼠的肿瘤体积显著缩小，小鼠肿瘤组织中CD4+T细胞比例和CD8+T细胞比例增加，小鼠肿瘤组织中PD-L1的表达量减少，小鼠血清中干扰素-γ的表达量显著增加，且作用效果远超单独腹腔注射非放射性的PD-L1小分子抑制剂LG-12的小鼠，可见，[131I]LG-12和LG-12联用具有协同效应，能够显著抑制肿瘤生长，增强抗肿瘤免疫应答，具有很好的体内抗肿瘤效果。Thirteenth, the results of in vivo anti-tumor experiments showed that after tail vein injection of iodine-131 labeled PD-L1 small molecule inhibitor [131I ]LG-12 and then intraperitoneal injection of non-radioactive PD-L1 small molecule inhibitor LG-12, the tumor volume of mice was significantly reduced, the proportion of CD4+ T cells and CD8+ T cells in the tumor tissue of mice increased, the expression of PD-L1 in the tumor tissue of mice decreased, and the expression of interferon-γ in the serum of mice increased significantly. The effect was far better than that of mice injected with non-radioactive PD-L1 small molecule inhibitor LG-12 alone. It can be seen that the combination of [131I ]LG-12 and LG-12 has a synergistic effect, can significantly inhibit tumor growth, enhance anti-tumor immune response, and has a good in vivo anti-tumor effect.
综上，碘-131标记的PD-L1小分子抑制剂[131I]LG-12以及非放射性的PD-L1小分子抑制剂LG-12均能够有效预防和/或治疗癌症，并且，[131I]LG-12和LG-12联用具有协同效应，这种基于PD-L1小分子抑制剂的核素/免疫联合治疗策略在癌症治疗中具有巨大的潜力，因此，[131I]LG-12和LG-12在制备预防和/或治疗癌症的药物中极具应用前景。In summary, both the iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12 and the non-radioactive PD-L1 small molecule inhibitor LG-12 can effectively prevent and/or treat cancer, and the combination of [131 I]LG-12 and LG-12 has a synergistic effect. This radionuclide/immunotherapy combination strategy based on PD-L1 small molecule inhibitors has great potential in cancer treatment. Therefore, [131 I]LG-12 and LG-12 have great application prospects in the preparation of drugs for the prevention and/or treatment of cancer.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1：非放射性的PD-L1小分子抑制剂LG-12的合成过程。Figure 1: Synthesis process of the non-radioactive PD-L1 small molecule inhibitor LG-12.
图2：标记前体化合物3的合成过程。Figure 2: Synthesis process of labeled precursor compound 3.
图3：非放射性的PD-L1小分子抑制剂LG-12的ESI-MS分析图。Figure 3: ESI-MS analysis of the non-radioactive PD-L1 small molecule inhibitor LG-12.
图4：化合物2的ESI-MS分析图。Figure 4: ESI-MS analysis of compound 2.
图5：标记前体化合物3的ESI-MS分析图。Figure 5: ESI-MS analysis of labeled precursor compound 3.
图6：化合物5的核磁共振氢谱图。Figure 6: H NMR spectrum of compound 5.
图7：化合物5的核磁共振碳谱图。Figure 7: C NMR spectrum of compound 5.
图8：化合物1的核磁共振氢谱图。Figure 8: H NMR spectrum of compound 1.
图9：化合物1的核磁共振碳谱图。Figure 9: C NMR spectrum of compound 1.
图10：非放射性的PD-L1小分子抑制剂LG-12的核磁共振氢谱图。Figure 10: H NMR spectrum of the non-radioactive PD-L1 small molecule inhibitor LG-12.
图11：非放射性的PD-L1小分子抑制剂LG-12的核磁共振碳谱图。Figure 11: Carbon NMR spectrum of the non-radioactive PD-L1 small molecule inhibitor LG-12.
图12：化合物2的核磁共振氢谱图。Figure 12: H NMR spectrum of compound 2.
图13：化合物2的核磁共振碳谱图。Figure 13: C NMR spectrum of compound 2.
图14：化合物3的核磁共振氢谱图。Figure 14: H NMR spectrum of compound 3.
图15：化合物3的核磁共振碳谱图。Figure 15: C NMR spectrum of compound 3.
图16：非放射性的PD-L1小分子抑制剂LG-12对PD-1/PD-L1相互作用的抑制活性。Figure 16: Inhibitory activity of the non-radioactive PD-L1 small molecule inhibitor LG-12 on PD-1/PD-L1 interaction.
图17：中间体化合物[131I]1(a)和碘-131标记的PD-L1小分子抑制剂[131I]LG-12(b)纯化前后的radio-HPLC分析图。Figure 17: Radio-HPLC analysis of the intermediate compound [131I ]1 (a) and the iodine-131 labeled PD-L1 small molecule inhibitor [131I ]LG-12 (b) before and after purification.
图18：中间体化合物[131I]1和碘-131标记的PD-L1小分子抑制剂[131I]LG-12体外稳定性。Figure 18: In vitro stability of the intermediate compound [131 I]1 and the iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12.
图19：碘-131标记的PD-L1小分子抑制剂[131I]LG-12在B16-F10细胞中孵育1、2和4h的摄取情况(***P<0.001)。Figure 19: Uptake of 131I-labeled PD-L1 small molecule inhibitor [131I ]LG-12 in B16-F10 cells after 1, 2, and 4 h of incubation (***P<0.001).
图20：碘-131标记的PD-L1小分子抑制剂[131I]LG-12在B16-F10荷瘤小鼠体内SPECT/CT显像(A)、体内生物分布分析(B)以及肿瘤和肌肉组织的放射自显影分析(C)(***P<0.001)。Figure 20: SPECT/CT imaging (A), in vivo biodistribution analysis (B), and autoradiographic analysis of tumor and muscle tissues (C) of iodine-131-labeled PD-L1 small molecule inhibitor [131 I]LG-12 in B16-F10 tumor-bearing mice (***P<0.001).
图21：B16-F10细胞和T细胞分别与非放射性的PD-L1小分子抑制剂LG-12孵育48h的细胞毒性。Figure 21: Cytotoxicity of B16-F10 cells and T cells incubated with the non-radioactive PD-L1 small molecule inhibitor LG-12 for 48 hours.
图22：不同浓度的非放射性的PD-L1小分子抑制剂LG-12(0、0.78和3.125μM)与B16-F10细胞/T细胞(1/20或1/40)共孵育后的B16-F10细胞的细胞活力(A)以及培养液中干扰素-γ的含量(B)(N.S.：无显著差异、*p<0.05、**p<0.01和***p<0.001)。Figure 22: Cell viability (A) of B16-F10 cells and the content of interferon-γ in the culture medium (B) after co-incubation of B16-F10 cells/T cells (1/20 or 1/40) with different concentrations of non-radioactive PD-L1 small molecule inhibitor LG-12 (0, 0.78 and 3.125 μM) (N.S.: no significant difference, *p<0.05, **p<0.01 and ***p<0.001).
图23：非放射性的PD-L1小分子抑制剂LG-12(0、5mg/kg和20mg/kg)在B16-F10小鼠黑色素瘤模型中的肿瘤抑制作用(对照组n＝4，治疗组n＝5)。图23中，(A)为肿瘤生长曲线；(B)为最终肿瘤重量；(C)为小鼠体重；(D)为剥离肿瘤照片(**p<0.01和***p<0.001)。Figure 23: Tumor inhibitory effect of non-radioactive PD-L1 small molecule inhibitor LG-12 (0, 5 mg/kg and 20 mg/kg) in B16-F10 mouse melanoma model (control group n=4, treatment group n=5). In Figure 23, (A) is the tumor growth curve; (B) is the final tumor weight; (C) is the mouse body weight; (D) is a photo of the peeled tumor (**p<0.01 and ***p<0.001).
图24：非放射性的PD-L1小分子抑制剂LG-12治疗后主要组织(心、肝、脾、肺、肾、肌肉)H&E染色分析(标尺为50μM，n＝4)。Figure 24: H&E staining analysis of major tissues (heart, liver, spleen, lung, kidney, muscle) after treatment with the non-radioactive PD-L1 small molecule inhibitor LG-12 (scale bar: 50 μM, n=4).
图25：B16-F10荷瘤小鼠经非放射性的PD-L1小分子抑制剂LG-12(5mg/kg和20mg/kg)治疗后肿瘤组织的IHC和H&E染色分析(A)和小鼠血清中IFN-γ水平(B)(标尺为50μM，n＝4，*p<0.05和**p<0.01)。Figure 25: IHC and H&E staining analysis of tumor tissues (A) and IFN-γ levels in mouse serum (B) after B16-F10 tumor-bearing mice were treated with the non-radioactive PD-L1 small molecule inhibitor LG-12 (5 mg/kg and 20 mg/kg) (scale bar is 50 μM, n=4, *p<0.05 and **p<0.01).
图26：不同剂量的碘-131标记的PD-L1小分子抑制剂[131I]LG-12(0、0.49、0.98、1.97、3.94和7.89KBq/μL)对B16-F10细胞形成集落的抑制作用。Figure 26: Inhibitory effect of different doses of iodine-131 labeled PD-L1 small molecule inhibitor [131I ]LG-12 (0, 0.49, 0.98, 1.97, 3.94 and 7.89KBq/μL) on colony formation of B16-F10 cells.
图27：碘-131标记的PD-L1小分子抑制剂LG-12诱导肿瘤ICD的研究。图27中，(A)为通过Western Blot测定[131I]LG-12(0和1.85MBq/mL)处理的B16-F10细胞中HMGB1和CRT蛋白表达水平；(B)为[131I]LG-12或Na131I(11.1MBq)在B16-F10荷瘤小鼠模型中的抗肿瘤作用后剥离肿瘤照片；(C)为[131I]LG-12或Na131I(11.1MBq)治疗后B16-F10荷瘤小鼠中HMGB 1和CRT的IHC分析(标尺为50μM，n＝4)。Figure 27: Study on the induction of tumor ICD by LG-12, a small molecule inhibitor of PD-L1 labeled with iodine-131. In Figure 27, (A) is the expression level of HMGB1 and CRT proteins in B16-F10 cells treated with [131I ]LG-12 (0 and 1.85MBq/mL) determined by Western Blot; (B) is a photo of the tumor peeled off after the anti-tumor effect of [131I ]LG-12 orNa131I (11.1MBq) in the B16-F10 tumor-bearing mouse model; (C) is the IHC analysis of HMGB1 and CRT in B16-F10 tumor-bearing mice after treatment with [131I ]LG-12 orNa131I (11.1MBq) (scale bar is 50μM, n=4).
图28：[131I]LG-12/LG-12联合治疗在B16-F10荷瘤小鼠模型(n＝5)中的体内抗肿瘤活性。图28中，(a)为治疗结束剥离肿瘤照片；(b)为肿瘤生长曲线(插入：B组和C组的比较)；(c)为治疗后B16-F10荷瘤小鼠血清中IFN-γ水平；(d)为肿瘤组织IHC分析图(CD4+T细胞、CD8+T细胞和PD-L1)；(e)为肿瘤组织CD4+T细胞、CD8+T细胞和PD-L1表达定量分析(标尺为50μM，n＝4，*p<0.05、***p<0.001)。Figure 28: In vivo antitumor activity of [131I ]LG-12/LG-12 combination therapy in the B16-F10 tumor-bearing mouse model (n=5). In Figure 28, (a) is a photo of the tumor peeled off at the end of treatment; (b) is a tumor growth curve (insert: comparison of group B and group C); (c) is the IFN-γ level in the serum of B16-F10 tumor-bearing mice after treatment; (d) is an IHC analysis of tumor tissue (CD4+ T cells, CD8+ T cells and PD-L1); (e) is a quantitative analysis of the expression of CD4+ T cells, CD8+ T cells and PD-L1 in tumor tissue (scale is 50μM, n=4, *p<0.05, ***p<0.001).
具体实施方式DETAILED DESCRIPTION
提供下述实施例是为了更好地进一步理解本发明，并不局限于所述最佳实施方式，不对本发明的内容和保护范围构成限制，任何人在本发明的启示下或是将本发明与其他现有技术的特征进行组合而得出的任何与本发明相同或相近似的产品，均落在本发明的保护范围之内。The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.
下述实施例中未注明具体实验步骤或条件者，按照本领域内的文献所描述的常规实验步骤的操作或条件即可进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规试剂产品。If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.
下述实施例中涉及的PD-1/PD-L1结合检测试剂盒(目录#72038)购自BPSBioscience公司。在化学表征中，使用四极杆串联质谱仪ZMD4000 LC/MS(美国沃特斯)进行电喷雾电离质谱(ESI-MS)分析，使用配备紫外和放射性检测器的高效液相色谱(HPLC)和放射性-HPLC上的C18色谱柱(250×4.6mm，10μm，Phenomenex)的泵(美国沃特斯)进行色谱分析，使用布鲁克400MHz核磁共振波谱仪(德国布鲁克)分析1H/13C核磁共振波谱。The PD-1/PD-L1 binding detection kit (catalog #72038) involved in the following examples was purchased from BPS Bioscience. In the chemical characterization, electrospray ionization mass spectrometry (ESI-MS) analysis was performed using a quadrupole tandem mass spectrometer ZMD4000 LC/MS (Waters, USA), chromatographic analysis was performed using a high performance liquid chromatography (HPLC) equipped with an ultraviolet and radioactive detector and a C18 column (250×4.6 mm, 10 μm, Phenomenex) on a radio-HPLC pump (Waters, USA), and1 H/13 C nuclear magnetic resonance spectra were analyzed using a Bruker 400 MHz nuclear magnetic resonance spectrometer (Bruker, Germany).
下述实施例中涉及的细胞培养和动物建模过程如下：The cell culture and animal modeling processes involved in the following embodiments are as follows:
肿瘤细胞培养：将小鼠黑色素瘤细胞系B16-F10(购自中国科学院细胞库)以1×106个的接种量平铺于培养皿，使用10mL含1％(v/v)青霉素-链霉素双抗(购自上海碧云天)、10％(v/v)胎牛血清(购自BI)的DMEM培养基(购自BI)，在37℃、5％(v/v)CO2的培养箱培养。当细胞培养至处于对数增长期且生长状态良好时即可用于体外细胞实验。Tumor cell culture: Mouse melanoma cell line B16-F10 (purchased from the Cell Bank of the Chinese Academy of Sciences) was plated in a culture dish at an inoculation volume of 1×106 cells, and cultured in a 37°C, 5% (v/v) CO2 incubator using 10 mL of DMEM medium (purchased from BI) containing 1% (v/v) penicillin-streptomycin double antibody (purchased from Shanghai Biyuntian) and 10% (v/v) fetal bovine serum (purchased from BI). When the cells are cultured to the logarithmic growth phase and the growth state is good, they can be used for in vitro cell experiments.
T细胞培养：BALB/c小白鼠(购自常州卡文斯实验动物公司)处死后从脾脏中提取小鼠T淋巴细胞；在24孔板中以2μL/孔的添加量加入CD3+(使用抗体稀释液稀释至浓度为5μg/mL后使用，CD3+和抗体稀释液均购自BioLegend)，并以2μL/孔的添加量加入CD28(使用抗体稀释液稀释至浓度为5μg/mL后使用，CD28和抗体稀释液均购自BioLegend)后，于4℃孵育24h，得到CD3+/CD28(5μg/mL)抗体包被的24孔板；将提取的T淋巴细胞以3×106个/孔的接种量铺板于CD3+/CD28(5μg/mL)抗体包被的24孔板中，使用500μL/孔含10％(v/v)胎牛血清(购自BI)的1640培养基(购自BI)，在37℃、5％(v/v)CO2的培养箱培养。当T淋巴细胞聚集成团时，即可用于体外细胞实验。T cell culture: BALB/c mice (purchased from Changzhou Cavens Laboratory Animal Company) were killed and mouse T lymphocytes were extracted from the spleen; CD3+ (diluted to a concentration of 5 μg/mL using antibody diluent, both CD3+ and antibody diluent were purchased from BioLegend) were added at 2 μL/well in a 24-well plate, and CD28 (diluted to a concentration of 5 μg/mL using antibody diluent, both CD28 and antibody diluent were purchased from BioLegend) was added at 2 μL/well, and then incubated at 4°C for 24 hours to obtain a 24-well plate coated with CD3+ /CD28 (5 μg/mL) antibodies; the extracted T lymphocytes were plated on CD3+ at an inoculum of 3×106 cells/well. In a 24-well plate coated with /CD28 (5 μg/mL) antibody, 500 μL/well of 1640 medium (purchased from BI) containing 10% (v/v) fetal bovine serum (purchased from BI) was used for culture in an incubator at 37°C and 5% (v/v) CO2. When T lymphocytes aggregate into clusters, they can be used for in vitro cell experiments.
动物建模：取雌性4～5周龄BALB/c小白鼠(购自卡文斯实验动物公司)；将B16-F10细胞种植于小白鼠(接种剂量3×106/只)的右上腋窝；每隔一天监测一次肿瘤直径，肿瘤直径达到50～100mm3时，使用荷瘤小鼠进行进一步体内实验。所有动物研究实验均按照江苏省核医学研究所伦理委员会制定的原则进行。Animal modeling: Female 4-5 week old BALB/c mice (purchased from Cavens Laboratory Animal Company) were selected; B16-F10 cells were implanted in the right upper axilla of mice (inoculation dose 3×106 /mouse); tumor diameter was monitored every other day, and when the tumor diameter reached 50-100 mm3 , tumor-bearing mice were used for further in vivo experiments. All animal research experiments were conducted in accordance with the principles established by the Ethics Committee of Jiangsu Institute of Nuclear Medicine.
实施例1：一种非放射性的PD-L1小分子抑制剂LG-12Example 1: A non-radioactive small molecule PD-L1 inhibitor LG-12
本实施例提供了一种非放射性的PD-L1小分子抑制剂LG-12，所述非放射性的PD-L1小分子抑制剂LG-12具有如下所示结构：This embodiment provides a non-radioactive PD-L1 small molecule inhibitor LG-12, wherein the non-radioactive PD-L1 small molecule inhibitor LG-12 has the following structure:
实施例2：一种制备非放射性的PD-L1小分子抑制剂LG-12的方法Example 2: A method for preparing a non-radioactive PD-L1 small molecule inhibitor LG-12
本实施例提供了实施例1所述的非放射性的PD-L1小分子抑制剂LG-12的制备方法(合成路线见图1)，具体步骤如下：This example provides a method for preparing the non-radioactive PD-L1 small molecule inhibitor LG-12 described in Example 1 (see Figure 1 for the synthesis route), and the specific steps are as follows:
步骤一：参照文献“Dieter Enders；Jeanne Fronert；Tom Bisschops；FlorianBoeck.Asymmetric total synthesis of smyrindiol employing an organocatalyticaldol key step.Beilstein Journal ofOrganic Chemistry.2012,8,1112–1117.”、“LvGaochao；MiaoYinxing；ChenYinfei；Lu Chunmei；Wang Xiuting；Xie Minhao；Qiu Ling；Lin Jianguo.Promising potential of a18F-labelled small-molecular radiotracerto evaluate PD-L1 expression in tumors by PET imaging.Bioorganic Chemistry2021,115,105294.”合成化合物6和化合物7；Step 1: Refer to the literature “Dieter Enders; Jeanne Fronert; Tom Bisschops; Florian Boeck. Lu Chunmei;Wang Xiuting; Compound 7;
所述化合物7具有如下所示结构：The compound 7 has the following structure:
所述化合物6具有如下所示结构：The compound 6 has the structure shown below:
步骤二：在冰浴条件下，将化合物7(2.56g，10mmol)、化合物6(2.64g，10mmol)和三苯基磷(3.9g，15mmol)溶于四氢呋喃(THF，20mL)中，得到溶解液；于冰浴和氮气搅拌下，在溶解液中滴加偶氮二甲酸二异丙酯(2mL，10mmol)，滴加完毕，得到反应液；将反应液于室温(25℃)搅拌(150rpm)反应16h，反应完毕，得到反应产物；将反应产物旋转蒸发除去四氢呋喃得到固体产物；将固体产物先用乙酸乙酯洗涤，再以正己烷/乙酸乙酯的混合液(正己烷：乙酸乙酯＝2：1，v/v)为洗脱剂进行硅胶柱层析纯化，得到白色固体化合物5(3.1g，产率：62％)；Step 2: Under ice bath conditions, compound 7 (2.56 g, 10 mmol), compound 6 (2.64 g, 10 mmol) and triphenylphosphine (3.9 g, 15 mmol) were dissolved in tetrahydrofuran (THF, 20 mL) to obtain a solution; diisopropyl azodicarboxylate (2 mL, 10 mmol) was added dropwise to the solution under ice bath and nitrogen stirring until the addition was complete to obtain a reaction solution; the reaction solution was stirred (150 rpm) at room temperature (25° C.) for 16 h, and the reaction was completed to obtain a reaction product; the reaction product was rotary evaporated to remove tetrahydrofuran to obtain a solid product; the solid product was first washed with ethyl acetate, and then purified by silica gel column chromatography using a mixture of n-hexane/ethyl acetate (n-hexane: ethyl acetate = 2:1, v/v) as an eluent to obtain a white solid compound 5 (3.1 g, yield: 62%);
所述化合物5具有如下所示结构：The compound 5 has the following structure:
所述3-溴甲基苯甲腈具有如下所示结构：The 3-bromomethylbenzonitrile has the structure shown below:
化合物5的氢谱和碳谱数据如下(核磁共振氢谱和碳谱见图6和图7)：The hydrogen spectrum and carbon spectrum data of compound 5 are as follows (see Figures 6 and 7 for hydrogen and carbon nuclear magnetic resonance spectra):
1H NMR(500MHz,DMSO-d6,δ:ppm)δ＝11.17(s,1H),9.99(s,1H),8.05(s,1H),7.53(dd,J＝7.6,1.3,1H),7.28(t,J＝7.6,1H),7.20(dd,J＝7.7,1.4,1H),6.93(d,J＝8.2,1H),6.81-6.72(m,3H),5.28(s,2H),4.29(s,4H),2.24(s,3H).13C NMR(126MHz,DMSO-d6,δ:ppm)δ＝190.1,163.5,162.9,143.4,143.0,142.1,140.3,135.0,134.8,134.2,130.2,127.6,125.9,122.6,118.8,118.2,117.3,101.6,75.7,70.3,68.3,64.6,64.6,22.4,16.5.1 H NMR (500MHz, DMSO-d6 , δ: ppm) δ = 11.17 (s, 1H), 9.99 (s, 1H), 8.05 (s, 1H), 7.53 (dd, J = 7.6, 1.3, 1H) ,7.28(t,J＝7.6,1H),7.20(dd,J＝7.7,1.4,1H),6.93(d,J＝8.2,1H),6.81-6.72(m,3H),5.28(s,2H ),4.29(s,4H),2.24(s,3H).13 C NMR(126MHz,DMSO-d6 ,δ:ppm)δ=190.1,163.5,162.9,143.4,143.0,142.1,140.3,135.0,134.8,134.2,130.2,127.6,125.9,122.6,118.8,118.2,117.3,101.6,75.7, 70.3,68.3,64.6 ,64.6,22.4,16.5.
步骤三：将化合物5(2.5g，5mmol)、3-溴甲基苯甲腈(1.69g，7mmol)和碳酸铯(4.77g，14.6mmol)混合于N,N-二甲基甲酰胺(30mL)中后，于室温(25℃)搅拌(150rpm)反应16h，反应完毕，得到反应产物；将反应产物先加水淬灭，然后用乙酸乙酯萃取，再用无水硫酸钠干燥，接着旋转蒸发浓缩有机相，最后以正己烷/乙酸乙酯的混合液(正己烷：乙酸乙酯＝3：2，v/v)为洗脱剂进行硅胶柱层析纯化，得到白色固体化合物1(2.6g，产率：85％)；Step 3: Compound 5 (2.5 g, 5 mmol), 3-bromomethylbenzonitrile (1.69 g, 7 mmol) and cesium carbonate (4.77 g, 14.6 mmol) were mixed in N,N-dimethylformamide (30 mL), and the mixture was stirred (150 rpm) at room temperature (25°C) for 16 h. After the reaction was completed, a reaction product was obtained; the reaction product was first quenched with water, then extracted with ethyl acetate, and then dried over anhydrous sodium sulfate, and then the organic phase was concentrated by rotary evaporation, and finally purified by silica gel column chromatography using a mixture of n-hexane/ethyl acetate (n-hexane: ethyl acetate = 3:2, v/v) as an eluent to obtain a white solid compound 1 (2.6 g, yield: 85%);
所述化合物1具有如下所示结构：The compound 1 has the following structure:
化合物1的氢谱和碳谱数据如下(核磁共振氢谱和碳谱见图8和图9)：The hydrogen spectrum and carbon spectrum data of compound 1 are as follows (see Figures 8 and 9 for the nuclear magnetic resonance hydrogen spectrum and carbon spectrum):
1H NMR(500MHz,DMSO-d6,δ:ppm)δ＝10.17(s,1H),8.05(s,2H),7.87(dd,J＝20.6,7.8,2H),7.65(t,J＝7.8,1H),7.54(d,J＝7.2,1H),7.27(t,J＝7.6,1H),7.21(d,J＝7.5,1H),7.12(s,1H),6.94(d,J＝8.2,1H),6.81-6.75(m,2H),5.41(d,J＝44.2,4H),4.29(s,4H),2.28(s,3H).13C NMR(126MHz,DMSO-d6,δ:ppm)δ＝187.1,163.1,162.8,162.8,143.4,143.0,142.2,138.6,138.3,134.9,134.8,134.5,132.8,132.4,131.5,130.4,130.3,128.0,126.0,122.6,120.7,119.1,118.2,117.3,112.1,99.8,77.7,70.7,69.7,64.6,36.3,31.2,16.7.1 H NMR (500MHz, DMSO-d6 , δ: ppm) δ = 10.17 (s, 1H), 8.05 (s, 2H), 7.87 (dd, J = 20.6, 7.8, 2H), 7.65 (t, J = 7.8,1H),7.54(d,J＝7.2,1H),7.27(t,J＝7.6,1H),7.21(d,J＝7.5,1H),7.12(s,1H),6.94(d,J ＝8.2,1H),6.81-6.75(m,2H),5.41(d,J＝44.2,4H),4.29(s,4H),2.28(s,3H).13 C NMR (126MHz, DMSO-d6 ,δ:ppm)δ=187.1,163.1,162.8,162.8,143.4,143.0,142.2,138.6,138.3,134.9,134.8,134.5,132.8,132.4,131.5,130.4,130.3,128.0,126.0 ,122.6,120.7,119.1 ,118.2,117.3,112.1,99.8,77.7,70.7,69.7,64.6,36.3,31.2,16.7.
步骤四：将化合物1(617mg，1mmol)、三(羟甲基)氨基甲烷(Tris，242mg，2mmol)和氰基硼氢化钠(360mg，6mmol)溶于N,N-二甲基甲酰胺(DMF，9mL)中，得到溶解液；在溶解液中加入冰醋酸(360μL)后，于室温(25℃)搅拌(150rpm)反应16h，反应完毕，得到反应产物；将反应产物先加水淬灭，然后用乙酸乙酯萃取，再用无水硫酸钠干燥，接着旋转蒸发浓缩有机相，最后以二氯甲烷/甲醇的混合液(二氯甲烷：甲醇＝10：1，v/v)为洗脱剂进行硅胶柱层析纯化，得到白色固体化合物LG-12(223mg，产率：31％)。Step 4: Compound 1 (617 mg, 1 mmol), tris(hydroxymethyl)aminomethane (Tris, 242 mg, 2 mmol) and sodium cyanoborohydride (360 mg, 6 mmol) were dissolved in N,N-dimethylformamide (DMF, 9 mL) to obtain a solution; glacial acetic acid (360 μL) was added to the solution, and the mixture was stirred (150 rpm) at room temperature (25°C) for 16 h. After the reaction was completed, a reaction product was obtained; the reaction product was first quenched with water, then extracted with ethyl acetate, and then dried over anhydrous sodium sulfate, and then the organic phase was concentrated by rotary evaporation, and finally purified by silica gel column chromatography using a mixed solution of dichloromethane/methanol (dichloromethane: methanol = 10:1, v/v) as an eluent to obtain a white solid compound LG-12 (223 mg, yield: 31%).
化合物LG-12的氢谱和碳谱数据如下(核磁共振氢谱和碳谱见图10和图11)：The hydrogen spectrum and carbon spectrum data of compound LG-12 are as follows (see Figures 10 and 11 for hydrogen and carbon nuclear magnetic resonance spectra):
1H NMR(500MHz,DMSO-d6,δ:ppm)δ＝8.03(s,1H),7.92-7.90(m,1H),7.83(d,J＝1.6,2H),7.62(s,1H),7.50(dd,J＝7.6,1.4,1H),7.24(d,J＝7.5,1H),7.21-7.12(m,1H),7.00(s,1H),6.94(d,J＝8.2,1H),6.82-6.72(m,2H),5.33(s,2H),5.25(s,2H),4.29(s,4H,),4.15(s,2H),3.59(s,6H),2.27(s,3H).13C NMR(126MHz,DMSO-d6,δ:ppm)δ＝143.4,143.0,142.1,138.7,135.4,134.8,134.4,132.8,132.2,131.4,130.2,130.1,128.0,125.9,122.6,119.2,118.2,117.3,112.0,99.6,75.6,70.2,69.3,64.6,64.6,16.6.1 H NMR (500MHz, DMSO-d6 , δ: ppm) δ = 8.03 (s, 1H), 7.92-7.90 (m, 1H), 7.83 (d, J = 1.6, 2H), 7.62 (s, 1H) ,7.50(dd,J＝7.6,1.4,1H),7.24(d,J＝7.5,1H),7.21-7.12(m,1H),7.00(s,1H),6.94(d,J＝8.2,1H ),6.82-6.72(m,2H),5.33(s,2H),5.25(s,2H),4.29(s,4H,),4.15(s,2H),3.59(s,6H),2.27(s ,3H).13 C NMR (126MHz, DMSO-d6 , δ: ppm) δ = 143.4, 143.0, 142.1, 138.7, 135.4, 134.8, 134.4, 132.8, 132.2, 131.4, 130.2, 130.1, 128.0, 125.9, 122.6, 119.2, 118.2, 117.3, 11 2.0,99.6,75.6, 70.2,69.3,64.6,64.6,16.6.
化合物LG-12的质谱结果为(ESI-MS见图3)：ESI-MS(m/z):723[M+H]+.The mass spectrum result of compound LG-12 is (ESI-MS see Figure 3): ESI-MS (m/z): 723 [M+H]+ .
实施例3：一种碘-131标记的PD-L1小分子抑制剂[131I]LG-12Example 3: An iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12
本实施例提供了一种碘-131标记的PD-L1小分子抑制剂[131I]LG-12，所述碘-131标记的PD-L1小分子抑制剂[131I]LG-12具有如下所示结构：This embodiment provides an iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12, wherein the iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12 has the following structure:
实施例4：一种制备碘-131标记的PD-L1小分子抑制剂[131I]LG-12的方法Example 4: A method for preparing iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12
本实施例提供了实施例3所述的碘-131标记的PD-L1小分子抑制剂[131I]LG-12的制备方法(合成路线见图2)，具体步骤如下：This example provides a method for preparing the iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12 described in Example 3 (the synthetic route is shown in FIG2 ), and the specific steps are as follows:
步骤一：参照实施例2的方法合成化合物1。Step 1: Compound 1 was synthesized according to the method of Example 2.
步骤二：化合物1(234mg，0.38mmol)、四(三苯基磷)钯(22mg，5mol)和六正丁基二锡(217μL，0.418mmol)溶于干燥二氧六环(5mL)中后，于氮气的保护下，加热至80℃搅拌(150rpm)反应6h，反应完毕，得到反应产物；过滤反应产物，取滤液；将滤液先旋转蒸发除去二氧六环，再以正己烷/乙酸乙酯的混合液(正己烷：乙酸乙酯＝4：1，v/v)为洗脱剂进行硅胶柱层析纯化，得到无色油状物化合物2(271mg，产率：84％)；Step 2: Compound 1 (234 mg, 0.38 mmol), tetrakis(triphenylphosphine)palladium (22 mg, 5 mol) and hexabutylditin (217 μL, 0.418 mmol) were dissolved in dry dioxane (5 mL), and heated to 80° C. under nitrogen protection and stirred (150 rpm) for 6 h. After the reaction was completed, a reaction product was obtained; the reaction product was filtered and the filtrate was taken; the filtrate was first rotary evaporated to remove dioxane, and then purified by silica gel column chromatography using a mixture of n-hexane/ethyl acetate (n-hexane: ethyl acetate = 4:1, v/v) as an eluent to obtain a colorless oil compound 2 (271 mg, yield: 84%);
所述化合物2具有如下所示结构：The compound 2 has the following structure:
化合物2的氢谱和碳谱数据如下(核磁共振氢谱和碳谱见图12和图13)：The hydrogen spectrum and carbon spectrum data of compound 2 are as follows (see Figures 12 and 13 for hydrogen and carbon nuclear magnetic resonance spectra):
1H NMR(500MHz,DMSO-d6,δ:ppm)δ＝10.29(s,1H),8.04(d,J＝1.8,1H),7.91-7.82(m,2H),7.77-7.61(m,2H),7.37(dd,J＝7.4,1.7,1H),7.28-7.16(m,2H),7.02(s,1H),6.93(d,J＝8.2,1H),6.78-6.70(m,2H),5.44(s,2H),5.23(d,J＝4.2,2H),4.28(s,4H),2.23(s,3H),1.44-1.24(m,6H),1.16(h,J＝7.3,6H),0.96-0.81(m,6H),0.76(t,J＝7.3,9H).13CNMR(126MHz,DMSO-d6,δppm)δ＝187.9,169.8,164.1,143.4,143.0,142.3,138.7,137.5,136.9,135.0,134.9,134.8,133.4,132.9,132.6,132.3,132.1,131.5,130.6,130.3,130.3,129.2,126.0,122.4,121.5,119.2,119.1,118.1,117.2,112.1,97.1,70.0,69.3,68.4,64.6,64.6,29.0,27.0,16.4,13.9,9.7.1 H NMR (500MHz, DMSO-d6 , δ: ppm) δ = 10.29 (s, 1H), 8.04 (d, J = 1.8, 1H), 7.91-7.82 (m, 2H), 7.77-7.61 (m, 2H),7.37(dd,J＝7.4,1.7,1H),7.28-7.16(m,2H),7.02(s,1H),6.93(d,J＝8.2,1H ),6.78-6.70(m,2H),5.44(s,2H),5.23(d,J＝4.2,2H),4.28(s,4H),2.23(s,3H),1.44-1.24(m,6H ),1.16(h,J＝7.3,6H),0.96-0.81(m,6H),0.76(t,J＝7.3,9H).13 CNMR(126MHz,DMSO-d6 ,δppm)δ=187.9,169.8,164.1,143.4,143.0,142.3,138.7,137.5,136.9,135.0,134.9,134.8,133.4,132.9,132.6,132.3,132.1,131.5,130.6, 130.3,130.3,129.2,126.0 ,122.4,121.5,119.2,119.1,118.1,117.2,112.1,97.1,70.0,69.3,68.4,64.6,64.6,29.0,27.0,16.4,13.9,9.7.
化合物2的的质谱结果为(ESI-MS见图4)：ESI-MS(m/z):804[M+Na]+.The mass spectrometry results of compound 2 are as follows (ESI-MS see Figure 4): ESI-MS (m/z): 804 [M+Na]+ .
步骤三：将化合物2(390mg，0.5mmol)和4-甲氧基(二乙酰氧基碘)苯(211mg，0.5mmol，CAS：16308-14-8)溶于乙腈(ACN，10mL)中后，在氮气的保护下，于室温(25℃)搅拌(150rpm)反应16h，反应完毕，得到反应产物；将反应产物先旋转蒸发除去乙腈，然后加入过量的乙醚(20mL)析出固体，再离心，最后收集白色固体真空干燥，得到标记前体化合物3(214mg，产率：48％)；Step 3: Compound 2 (390 mg, 0.5 mmol) and 4-methoxy(diacetoxyiodo)benzene (211 mg, 0.5 mmol, CAS: 16308-14-8) were dissolved in acetonitrile (ACN, 10 mL), and the mixture was stirred (150 rpm) at room temperature (25° C.) for 16 h under the protection of nitrogen. After the reaction was completed, a reaction product was obtained; the reaction product was first rotary evaporated to remove acetonitrile, and then an excess of ether (20 mL) was added to precipitate a solid, and then centrifuged. Finally, the white solid was collected and vacuum dried to obtain a labeled precursor compound 3 (214 mg, yield: 48%);
所述4-甲氧基(二乙酰氧基碘)苯具有如下所示结构：The 4-methoxy(diacetoxyiodo)benzene has the structure shown below:
所述标记前体化合物3具有如下所示结构：The labeled precursor compound 3 has the following structure:
标记前体化合物3的氢谱和碳谱数据如下(核磁共振氢谱和碳谱见图14和图15)：The hydrogen spectrum and carbon spectrum data of the labeled precursor compound 3 are as follows (the nuclear magnetic resonance hydrogen spectrum and carbon spectrum are shown in Figures 14 and 15):
1H NMR(500MHz,DMSO-d6,δ:ppm)δ＝10.25(s,1H),8.65(s,1H),8.21(s,1H),7.93(t,J＝1.7,1H),7.90-7.71(m,4H),7.64(t,J＝7.8,1H),7.47(d,J＝8.1,2H),7.39(td,J＝7.3,6.4,2.1,1H),7.35-7.24(m,3H),7.20(s,1H),7.10(d,J＝7.8,2H),7.01-6.87(m,3H),6.87-6.77(m,2H),5.57(s,1H),5.50(s,1H),5.39(d,J＝9.5,2H),4.30(d,J＝1.4,4H),3.74(s,3H),3.28(s,3H),2.25(s,3H).13C NMR(126MHz,DMSO-d6,δ:ppm)δ＝162.0,160.7,157.9,146.2,143.5,143.1,142.4,138.4,138.0,137.2,137.0,135.9,134.9,134.8,134.7,134.7,132.7,132.3,131.6,131.3,130.8,130.4,128.9,128.7,128.5,126.2,125.9,122.6,122.4,119.1,118.2,117.6,117.5,117.4,117.4,112.0,105.5,99.8,98.7,97.1,70.8,70.1,69.4,65.4,64.6,56.1,54.0,21.2,16.7,16.7,15.6.1 H NMR (500MHz, DMSO-d6 , δ: ppm) δ = 10.25 (s, 1H), 8.65 (s, 1H), 8.21 (s, 1H), 7.93 (t, J = 1.7, 1H), 7.90 -7.71(m,4H),7.64(t,J＝7.8,1H),7.47(d,J＝8.1,2H),7.39(td,J＝7.3,6.4,2.1,1H),7.35-7.24(m ,3H ),7.20(s,1H),7.10(d,J＝7.8,2H),7.01-6.87(m,3H),6.87-6.77(m,2H),5.57(s,1H),5.50(s,1H ), 5.39 (d, J = 9.5, 2H), 4.30 (d, J = 1.4, 4H), 3.74 (s, 3H), 3.28 (s, 3H), 2.25 (s, 3H).13 C NMR (126MHz ,DMSO-d6 ,δ:ppm)δ=162.0,160.7,157.9,146.2,143.5,143.1,142.4,138.4,138.0,137.2,137.0,135.9,134.9,134.8,134.7,134.7,132.7,132.3,131.6 ,131.3,130.8,130.4 ,128.9,128 .7,128.5,126.2,125.9,122.6,122.4,119.1,118.2,117.6,117.5,117.4,117.4,112.0,105.5,99.8,98.7,97.1,70.8,70.1,69.4,65.4,64.6, 56.1,54.0,21.2,16.7 ,16.7,15.6.
标记前体化合物3的质谱结果为(ESI-MS见图5)：ESI-MS(m/z):724[M-TsO-]+.The mass spectrum result of the labeled precursor compound 3 is (ESI-MS see Figure 5): ESI-MS (m/z): 724 [M-TsO- ]+ .
步骤四：氮气吹干Na131I溶液(740MBq)，得到白色固体Na131I；在白色固体Na131I中加入使用无水乙腈(500μL)溶解的前体化合物3(1.2mg)后，于90℃反应60min完毕，得到反应产物；将反应产物以正己烷/乙酸乙酯的混合液(正己烷：乙酸乙酯＝3：2，v/v)为洗脱剂进行小型硅胶柱层析纯化，得到中间体化合物[131I]1(500MBq)，使用radio-HPLC分析。Step 4: Dry the Na131 I solution (740 MBq) with nitrogen to obtain white solid Na131 I; add the precursor compound 3 (1.2 mg) dissolved in anhydrous acetonitrile (500 μL) to the white solid Na131 I, and react at 90° C. for 60 min to obtain a reaction product; purify the reaction product by small silica gel column chromatography using a mixture of n-hexane/ethyl acetate (n-hexane:ethyl acetate=3:2, v/v) as an eluent to obtain an intermediate compound [131 I]1 (500 MBq), which was analyzed by radio-HPLC.
所述中间体化合物[131I]1具有如下所示结构：The intermediate compound [131I ]1 has the following structure:
步骤五：氮气吹干中间体化合物[131I]1；将氮气吹干的中间体化合物[131I]1、三(羟甲基)氨基甲烷(Tris，1.2mg，0.01mmol)和氰基硼氢化钠(1.8mg，0.03mmol)溶于N,N-二甲基甲酰胺(DMF，100μL)中，得到溶解液；在溶解液中加入冰醋酸(1.8μL)后，于室温(25℃)搅拌(150rpm)反应10h，反应完毕，得到反应产物；将反应产物以二氯甲烷/甲醇的混合液(二氯甲烷：甲醇＝9：1，v/v)为洗脱剂进行小型硅胶柱层析纯化，得到碘-131标记的PD-L1小分子抑制剂[131I]LG-12，使用radio-HPLC分析。Step 5: Dry the intermediate compound [131I ]1 with nitrogen; dissolve the nitrogen-dried intermediate compound [131I ]1, tris(hydroxymethyl)aminomethane (Tris, 1.2 mg, 0.01 mmol) and sodium cyanoborohydride (1.8 mg, 0.03 mmol) in N,N-dimethylformamide (DMF, 100 μL) to obtain a solution; add glacial acetic acid (1.8 μL) to the solution, stir (150 rpm) at room temperature (25°C) for 10 h, and the reaction is completed to obtain a reaction product; purify the reaction product by small silica gel column chromatography using a mixture of dichloromethane/methanol (dichloromethane: methanol = 9:1, v/v) as an eluent to obtain an iodine-131 labeled PD-L1 small molecule inhibitor [131I ]LG-12, which is analyzed using radio-HPLC.
中间体化合物[131I]1和碘-131标记的PD-L1小分子抑制剂[131I]LG-12纯化前后的radio-HPLC分析结果见图17。如图17所示，标记前体化合物3与Na131I在乙腈中于90℃下反应，得到中间体化合物[131I]1，放射性转化率为70％，纯化后放化纯度大于98％。中间体化合物[131I]1与Tris经还原胺化反应得到碘-131标记的PD-L1小分子抑制剂[131I]LG-12，放射性转化率为50％，纯化后放化纯度为97％。碘-131标记的PD-L1小分子抑制剂[131I]LG-12的总放化产率为7.0±2.4％，摩尔活度为27GBq/μmol。The radio-HPLC analysis results of the intermediate compound [131I ]1 and the iodine-131 labeled PD-L1 small molecule inhibitor [131I ]LG-12 before and after purification are shown in Figure 17. As shown in Figure 17, the labeled precursor compound 3 reacts withNa131I in acetonitrile at 90°C to obtain the intermediate compound [131I ]1, with a radioactive conversion rate of 70% and a radiochemical purity of more than 98% after purification. The intermediate compound [131I ]1 is subjected to a reductive amination reaction with Tris to obtain the iodine-131 labeled PD-L1 small molecule inhibitor [131I ]LG-12, with a radioactive conversion rate of 50% and a radiochemical purity of 97% after purification. The total radiochemical yield of the iodine-131 labeled PD-L1 small molecule inhibitor [131I ]LG-12 is 7.0±2.4%, and the molar activity is 27GBq/μmol.
实施例5：一种非放射性的PD-L1小分子抑制剂LG-2～LG-11和LG-13Example 5: A non-radioactive PD-L1 small molecule inhibitor LG-2 to LG-11 and LG-13
本实施例提供了一种非放射性的PD-L1小分子抑制剂LG-2～LG-11和LG-13，所述非放射性的PD-L1小分子抑制剂LG-2～LG-11和LG-13具有如下所示结构：This embodiment provides a non-radioactive PD-L1 small molecule inhibitor LG-2 to LG-11 and LG-13, wherein the non-radioactive PD-L1 small molecule inhibitor LG-2 to LG-11 and LG-13 have the following structures:
其中，R为Among them, R is
实施例6：一种制备非放射性的PD-L1小分子抑制剂LG-2～LG-11和LG-13的方法Example 6: A method for preparing non-radioactive PD-L1 small molecule inhibitors LG-2 to LG-11 and LG-13
本实施例提供了实施例5所述的非放射性的PD-L1小分子抑制剂LG-2～LG-11和LG-13的制备方法，具体步骤如下：This example provides a method for preparing the non-radioactive PD-L1 small molecule inhibitors LG-2 to LG-11 and LG-13 described in Example 5, and the specific steps are as follows:
在实施例2的基础上，将步骤四的三(羟甲基)氨基甲烷(Tris，242mg，2mmol)分别替换为甘氨酸(250mg，3.3mmol)、丝氨酸(250mg，2.4mmol)、谷氨酸(250mg，1.7mmol)、氨甲基膦酸(250mg，2.3mmol)、磺基丙氨酸(250mg，1.5mmol)、精氨酸(250mg，1.4mmol)、络氨酸(250mg，1.4mmol)、天门冬氨酸(250mg，1.9mmol)、脯氨酸(250mg，1.9mmol)、天冬酰胺(250mg，1.9mmol)、氨基葡萄糖(250mg，1.4mmol)，得到白色固体化合物LG-2(212mg，产率：31％)、LG-3(233mg，产率：33％)、LG-4(245mg，产率：33％)、LG-5(190mg，产率：16％)、LG-6(186mg，产率：24％)、LG-7(260mg，产率：34％)、LG-8(234mg，产率：30％)、LG-9(256mg，产率：36％)、LG-10(289mg，产率：40％)、LG-11(310mg，产率：43％)、LG-13(178mg，产率：23％)。On the basis of Example 2, the tris(hydroxymethyl)aminomethane (Tris, 242 mg, 2 mmol) in step 4 was replaced by glycine (250 mg, 3.3 mmol), serine (250 mg, 2.4 mmol), glutamic acid (250 mg, 1.7 mmol), aminomethylphosphonic acid (250 mg, 2.3 mmol), cysteic acid (250 mg, 1.5 mmol), arginine (250 mg, 1.4 mmol), tyrosine (250 mg, 1.4 mmol), aspartic acid (250 mg, 1.9 mmol), proline (250 mg, 1.9 mmol), asparagine (250 mg, 1.9 mmol). ), glucosamine (250 mg, 1.4 mmol) to obtain white solid compound LG-2 (212 mg, yield: 31%), LG-3 (233 mg, yield: 33%), LG-4 (245 mg, yield: 33%), LG-5 (190 mg, yield: 16%), LG-6 (186 mg, yield: 24%), LG-7 (260 mg, yield: 34%), LG-8 (234 mg, yield: 30%), LG-9 (256 mg, yield: 36%), LG-10 (289 mg, yield: 40%), LG-11 (310 mg, yield: 43%), LG-13 (178 mg, yield: 23%).
实施例7：一种碘-131标记的PD-L1小分子抑制剂[131I]LG-2～[131I]LG-11和[131I]LG-13Example 7: Iodine-131 labeled PD-L1 small molecule inhibitors [131 I]LG-2 to [131 I]LG-11 and [131 I]LG-13
本实施例提供了一种碘-131标记的PD-L1小分子抑制剂[131I]LG-2～[131I]LG-11和[131I]LG-13，所述碘-131标记的PD-L1小分子抑制剂[131I]LG-2～[131I]LG-11和[131I]LG-13具有如下所示结构：This embodiment provides an iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-2 to [131 I]LG-11 and [131 I]LG-13. The iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-2 to [131 I]LG-11 and [131 I]LG-13 have the following structures:
其中，R为Among them, R is
实验例1：非放射性的PD-L1小分子抑制剂LG-2～LG-13的TR-FRET实验Experimental Example 1: TR-FRET experiment of non-radioactive PD-L1 small molecule inhibitors LG-2 to LG-13
本实验例提供了实施例1的非放射性的PD-L1小分子抑制剂LG-2～LG-13的TR-FRET实验，具体过程如下：This experimental example provides a TR-FRET experiment of the non-radioactive PD-L1 small molecule inhibitors LG-2 to LG-13 of Example 1. The specific process is as follows:
使用PD-1/PD-L1结合检测试剂盒通过PD-1/PD-L1 TR-FRET实验分别检测实施例1和实施例5的非放射性的PD-L1小分子抑制剂LG-2～LG-13(待测化合物)对PD-1/PD-L1结合的抑制作用。根据试剂盒说明书进行实验。实验组：将5μL不同浓度待测化合物(100、25、6.25、1.5625、0.390625、0.097656、0.024414、0.006104、0.001526和0.000381μM)和5μLPD-L1-生物素(11μg/mL)混合后，在室温(25℃)下孵育10min，得到孵育液；在孵育液中加入5μLPD-1-Eu(0.2μg/mL)和5μL染料标记物混合，得到混合物。阳性组：将5μLPD-L1-生物素、5μL纯水、5μLPD-1-Eu和5μL染料标记物混合，得到混合物。阴性组：将5μL缓冲液(1×)、5μL纯水、5μLPD-1-Eu和5μL染料标记物混合，得到混合物。将三组混合物在室温(25℃)下避光孵育90min(384孔板)后，在分子器件仪器(PerkinElmer EnVision)上读取荧光强度，并以320nm为激发波长，分别在620nm和665nm发射波长处测量吸光度；使用比值(665nm吸光度/620nm吸光度)和抑制率％＝(阳性比值-样品比值)/(阳性比值-阴性比值)×100进行数据分析，分析结果见图16和表1。The PD-1/PD-L1 binding detection kit was used to detect the inhibitory effects of the non-radioactive PD-L1 small molecule inhibitors LG-2 to LG-13 (test compounds) of Example 1 and Example 5 on PD-1/PD-L1 binding by PD-1/PD-L1 TR-FRET experiment. The experiment was performed according to the instructions of the kit. Experimental group: 5 μL of different concentrations of test compounds (100, 25, 6.25, 1.5625, 0.390625, 0.097656, 0.024414, 0.006104, 0.001526 and 0.000381 μM) and 5 μL PD-L1-biotin (11 μg/mL) were mixed and incubated at room temperature (25°C) for 10 minutes to obtain an incubation solution; 5 μL PD-1-Eu (0.2 μg/mL) and 5 μL dye marker were added to the incubation solution to obtain a mixture. Positive group: 5 μL PD-L1-biotin, 5 μL pure water, 5 μL PD-1-Eu and 5 μL dye marker were mixed to obtain a mixture. Negative group: 5 μL buffer (1×), 5 μL pure water, 5 μL PD-1-Eu and 5 μL dye marker were mixed to obtain a mixture. After the three groups of mixtures were incubated at room temperature (25°C) in the dark for 90 min (384-well plate), the fluorescence intensity was read on a molecular device instrument (PerkinElmer EnVision), and the excitation wavelength was 320 nm, and the absorbance was measured at the emission wavelengths of 620 nm and 665 nm, respectively; the ratio (665 nm absorbance/620 nm absorbance) and inhibition rate % = (positive ratio-sample ratio)/(positive ratio-negative ratio) × 100 were used for data analysis, and the analysis results are shown in Figure 16 and Table 1.
PD-1/PD-L1 TR-FRET实验结果表明(图16和表1)，非放射性的PD-L1小分子抑制剂LG-2的EC50值为837±12.3nM、LG-3的EC50值为237±5.56nM、LG-4的EC50值为188.34±4.43nM、LG-5的EC50值为387±7.21nM、LG-6的EC50值为429±4.98nM、LG-7的EC50值为252±3.78nM、LG-8的EC50值为67.83±0.75μM、LG-9的EC50值为168±11.87nM、LG-10的EC50值为2.253±0.05μM、LG-11的EC50值为1.756±0.04μM、LG-12的EC50值为34.55±3.21nM、LG-13的EC50值为50.23±2.89nM，可见，LG-12对PD-1/PD-L1相互作用具有较高的抑制活性，能够显著抑制肿瘤生长，表现出较好的抗肿瘤免疫治疗效果。The results of PD-1/PD-L1 TR-FRET experiments showed (Figure 16 and Table 1) that the EC50 value of the non-radioactive PD-L1 small molecule inhibitor LG-2 was 837±12.3 nM, the EC50 value of LG-3 was 237±5.56 nM, the EC50 value of LG-4 was 188.34±4.43 nM, the EC50 value of LG-5 was 387±7.21 nM, the EC50 value of LG-6 was 429±4.98 nM, the EC50 value of LG-7 was 252±3.78 nM, the EC50 value of LG-8 was 67.83±0.75 μM, the EC50 value of LG-9 was 168±11.87 nM, the EC50 value of LG-10 was 2.253±0.05 μM, and the EC 50 value of LG-11 was 2. The EC50 value of LG-12 was 1.756±0.04μM, the EC50 value of LG-12 was 34.55±3.21nM, and the EC50 value of LG-13 was 50.23±2.89nM. It can be seen that LG-12 has a high inhibitory activity on the PD-1/PD-L1 interaction, can significantly inhibit tumor growth, and exhibits a good anti-tumor immunotherapy effect.
表1不同小分子抑制剂的EC50值Table 1 EC50 values of different small molecule inhibitors
实验例2：碘-131标记的PD-L1小分子抑制剂[131I]LG-12的体外稳定性实验Experimental Example 2: In vitro stability study of iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12
本实验例提供了实施例3的碘-131标记的PD-L1小分子抑制剂[131I]LG-12的体外稳定性实验，具体过程如下：This experimental example provides an in vitro stability experiment of the iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12 of Example 3. The specific process is as follows:
实验一：将实施例4制得的中间体化合物[131I]1(370KBq)加入N,N-二甲基甲酰胺(450μL)溶液中，得到混合液；将混合液在37℃孵育36h；孵育结束后，取孵育液使用Radio-HPLC进行放射性HPLC分析考察样品的体外稳定性，分析结果见图18。Experiment 1: The intermediate compound [131I ]1 (370KBq) prepared in Example 4 was added to N,N-dimethylformamide (450μL) solution to obtain a mixed solution; the mixed solution was incubated at 37°C for 36h; after the incubation, the incubation solution was taken and radio-HPLC analysis was performed to examine the in vitro stability of the sample. The analysis results are shown in Figure 18.
实验二：将实施例3中的碘-131标记的PD-L1小分子抑制剂[131I]LG-12(370KBq)加入PBS缓冲液(pH＝7.4，0.01M，450μL)中，得到混合液；将混合液在37℃孵育4h和12h；孵育结束后，取孵育液使用Radio-HPLC进行放射性HPLC分析考察样品的体外稳定性，分析结果见图18。Experiment 2: The iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12 (370KBq) in Example 3 was added to PBS buffer (pH=7.4, 0.01M, 450μL) to obtain a mixed solution; the mixed solution was incubated at 37°C for 4h and 12h; after the incubation, the incubation solution was taken and radio-HPLC analysis was performed to examine the in vitro stability of the sample. The analysis results are shown in Figure 18.
由图18可知，中间体化合物[131I]1在DMF中36h仍稳定，可以满足下一步反应的要求；碘-131标记的PD-L1小分子抑制剂[131I]LG-12在PBS缓冲液中孵育12h也仍保持稳定，表明中间体和产物均具有良好的体外稳定性。As shown in Figure 18, the intermediate compound [131I ]1 is still stable in DMF for 36 hours and can meet the requirements of the next reaction; the iodine-131 labeled PD-L1 small molecule inhibitor [131I ]LG-12 is also still stable after incubation in PBS buffer for 12 hours, indicating that both the intermediate and the product have good in vitro stability.
实验例3：碘-131标记的PD-L1小分子抑制剂[131I]LG-12的细胞摄取和生物分布实验Experimental Example 3: Cellular uptake and biodistribution experiment of iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12
本实验例提供了实施例3的碘-131标记的PD-L1小分子抑制剂[131I]LG-12的细胞摄取和生物分布实验，具体过程如下：This experimental example provides a cellular uptake and biodistribution experiment of the iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12 of Example 3. The specific process is as follows:
将B16-F10细胞以2.6×105/孔的接种量接种至添加有含1％(v/v)青霉素-链霉素双抗、10％(v/v)胎牛血清的DMEM培养基(1500μL)的六孔板中后，于37℃、5％(v/v)CO2的培养箱中孵育16h；孵育16h后，将六孔板中的孔分为两组，两组分别为阻断组和非阻断组，每组各3个平行组。其中，阻断组：吸出培养基，在阻断组的孔中先加入LG-12(50μM，1mL，溶剂为DMEM培养基)，于37℃、5％CO2培养箱孵育30min进行阻断，然后加入[131I]LG-12(3.7×10-2MBq，200μL，溶剂为DMEM培养基)，于37℃、5％(v/v)CO2的培养箱分别孵育1、2、4h，再用PBS缓冲液润洗两次孔中的B16-F10细胞，最后加0.3MNaOH裂解B16-F10细胞10min，得到裂解液。非阻断组：吸出培养基，在非阻断组的孔中直接加入1mLDMEM培养基和[131I]LG-12(3.7×10-2MBq，200μL，溶剂为DMEM培养基)，于37℃、5％(v/v)CO2的培养箱分别孵育1、2、4h，再用PBS缓冲液润洗两次孔中的B16-F10细胞，最后加0.3M NaOH裂解B16-F10细胞10min，得到裂解液。收集裂解液，用γ计数器(1470Wizard,Perkins Elmer)检测细胞内的放射性，检测结果见图19。B16-F10 cells were inoculated at a density of 2.6×105 /well into a six-well plate supplemented with DMEM medium (1500 μL) containing 1% (v/v) penicillin-streptomycin dual antibody and 10% (v/v) fetal bovine serum, and incubated in an incubator at 37°C and 5% (v/v) CO2 for 16 h. After incubation for 16 h, the wells in the six-well plate were divided into two groups, namely a blocking group and a non-blocking group, with 3 parallel groups in each group. Among them, blocking group: aspirate the culture medium, first add LG-12 (50μM, 1mL, solvent is DMEM culture medium) to the wells of the blocking group, incubate in a 37℃, 5%CO2 incubator for 30min for blocking, then add [131I ]LG-12 (3.7×10-2MBq , 200μL, solvent is DMEM culture medium), incubate in a 37℃, 5% (v/v)CO2 incubator for 1, 2, and 4h, respectively, then rinse the B16-F10 cells in the wells twice with PBS buffer, and finally add 0.3MNaOH to lyse the B16-F10 cells for 10min to obtain the lysate. Non-blocking group: aspirate the culture medium, add 1 mL of DMEM culture medium and [131I ]LG-12 (3.7×10-2 MBq, 200 μL, solvent is DMEM culture medium) directly to the wells of the non-blocking group, incubate in a 37°C, 5% (v/v)CO2 incubator for 1, 2, and 4 h, respectively, rinse the B16-F10 cells in the wells twice with PBS buffer, and finally add 0.3 M NaOH to lyse the B16-F10 cells for 10 min to obtain lysate. Collect the lysate, and use a γ counter (1470 Wizard, Perkins Elmer) to detect the radioactivity in the cells. The detection results are shown in Figure 19.
由图19可知，孵育1h时，[131I]LG-12的细胞摄取值为3.93±0.15％AD，经LG-12阻断后降至1.46±0.03％AD；孵育4h时，[131I]LG-12的细胞摄取值增加到5.33±0.33％AD，经LG-12阻断后显著降低至2.56±0.06％AD。此结果表明LG-12可以与肿瘤细胞中的PD-L1特异性结合。As shown in Figure 19, after incubation for 1 hour, the cell uptake value of [131I ]LG-12 was 3.93±0.15%AD, which was reduced to 1.46±0.03%AD after LG-12 blocking; after incubation for 4 hours, the cell uptake value of [131I ]LG-12 increased to 5.33±0.33%AD, which was significantly reduced to 2.56±0.06%AD after LG-12 blocking. This result indicates that LG-12 can specifically bind to PD-L1 in tumor cells.
实验例4：碘-131标记的PD-L1小分子抑制剂[131I]LG-12的体内显像实验Experimental Example 4: In vivo imaging experiment of iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12
本实验例提供了实施例3的碘-131标记的PD-L1小分子抑制剂[131I]LG-12的体内显像实验，具体过程如下：This experimental example provides an in vivo imaging experiment of the iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12 of Example 3. The specific process is as follows:
SPECT/CT显像实验：用含有2％(v/v)异氟烷的氧气以2L/min的流速麻醉荷瘤小鼠(n＝1)后，将荷瘤小鼠的四肢与尾巴固定，并对荷瘤小鼠通过尾静脉注射碘-131标记的PD-L1小分子抑制剂[131I]LG-12(11.1MBq，溶解在100μL生理盐水中)；注射结束后，通过30min静态扫描获得荷瘤小鼠全身SPECT/CT成像，成像结果见图20中的A。SPECT/CT imaging experiment: After anesthetizing tumor-bearing mice (n=1) with oxygen containing 2% (v/v) isoflurane at a flow rate of 2L/min, the limbs and tail of the tumor-bearing mice were fixed, and the tumor-bearing mice were injected with iodine-131-labeled PD-L1 small molecule inhibitor [131I ]LG-12 (11.1MBq, dissolved in 100μL saline) through the tail vein; after the injection, whole-body SPECT/CT imaging of the tumor-bearing mice was obtained by 30min static scanning. The imaging results are shown in A in Figure 20.
体内生物分布分析实验：用含有2％(v/v)异氟烷的氧气以2L/min的流速麻醉荷瘤小鼠(n＝4)后，将荷瘤小鼠的四肢与尾巴固定，并对荷瘤小鼠通过尾静脉注射碘-131标记的PD-L1小分子抑制剂[131I]LG-12(5MBq，溶解在100μL生理盐水中)，并取同体积的[131I]LG-12作为衰减校正对照；注射1h后，处死小鼠并解剖，取肿瘤及主要脏器(心、肝、脾、肺、肾、胃、肠、骨、肌肉、脑)部位进行称重；称重后，用γ计数器(1470Wizard,Perkins Elmer)测量样品的放射性，检测结果见图20中的B。[131I]LG-12的生物分布情况以每克组织注射剂量的百分比(％ID/g)表示。In vivo biodistribution analysis experiment: After anesthetizing tumor-bearing mice (n=4) with oxygen containing 2% (v/v) isoflurane at a flow rate of 2L/min, the limbs and tails of the tumor-bearing mice were fixed, and the tumor-bearing mice were injected with iodine-131-labeled PD-L1 small molecule inhibitor [131I ]LG-12 (5MBq, dissolved in 100μL normal saline) through the tail vein, and the same volume of [131I ]LG-12 was taken as an attenuation correction control; 1h after injection, the mice were killed and dissected, and the tumors and major organs (heart, liver, spleen, lung, kidney, stomach, intestine, bone, muscle, brain) were weighed; after weighing, the radioactivity of the samples was measured with a γ counter (1470Wizard, Perkins Elmer), and the test results are shown in B in Figure 20. The biodistribution of [131I ]LG-12 is expressed as the percentage of injected dose per gram of tissue (%ID/g).
肿瘤和肌肉组织的放射自显影分析实验：用含有2％(v/v)异氟烷的氧气以2L/min的流速麻醉荷瘤小鼠(n＝3)后，将荷瘤小鼠的四肢与尾巴固定，并对荷瘤小鼠通过尾静脉注射碘-131标记的PD-L1小分子抑制剂[131I]LG-12(5MBq，溶解在100μL生理盐水中)；注射1h后，处死小鼠并解剖，剥离皮下肿瘤组织和腿部肌肉，先用PBS缓冲液清洗1次，然后使用冷冻包埋剂在-25℃下包埋，再使用冷冻切片机(CM1950，SLEE/MNT)将其制成组织切片(30μm)于载玻片，接着载玻片在荧光屏上平铺4h，并用Cyclone Plus储磷屏成像系统(C431200，PerkinElmer)扫描荧光屏以获取图像，最后使用OptiQuant软件对图像进行处理分析，分析结果见图20中的C。Autoradiographic analysis of tumor and muscle tissues: After anesthetizing tumor-bearing mice (n=3) with oxygen containing 2% (v/v) isoflurane at a flow rate of 2 L/min, the limbs and tails of the tumor-bearing mice were fixed, and the tumor-bearing mice were injected with iodine-131-labeled PD-L1 small molecule inhibitor [131I ]LG-12 (5 MBq, dissolved in 100 μL saline) through the tail vein; 1 hour after the injection, the mice were killed and dissected, and the subcutaneous tumor tissue and leg muscles were peeled off, washed once with PBS buffer, and then embedded in a cryoembedding agent at -25°C, and then made into tissue sections (30 μm) on slides using a freezing microtome (CM1950, SLEE/MNT), and then the slides were spread on a fluorescent screen for 4 hours and Cyclone The Plus phosphor storage screen imaging system (C431200, PerkinElmer) was used to scan the phosphor screen to acquire images, and finally the images were processed and analyzed using OptiQuant software. The analysis results are shown in C of FIG20 .
如图20中的A所示，B16-F10荷瘤小鼠尾静脉注射[131I]LG-12(11.1MBq)30min后，通过SPECT/CT显像图观察到[131I]LG-12在肿瘤部位快速聚积。进一步研究了[131I]LG-12在B16-F10荷瘤小鼠体内的分布。如图20中的B所示，[131I]LG-12在肿瘤组织中的摄取为6.50±1.05％ID/g，明显高于大多数正常组织，表明[131I]LG-12具有体内靶向PD-L1的能力，但在血液、肝脏、心脏和肠道等非靶组织中摄取也较高，这可能是由于[131I]LG-12的亲脂性较高所导致。如图20中的C所示，[131I]LG-12在肿瘤中的活性比肌肉中的活性高2.3倍，进一步证明了[131I]LG-12对PD-L1的靶向性。As shown in Figure 20A, 30 minutes after the tail vein injection of [131I ]LG-12 (11.1MBq) into B16-F10 tumor-bearing mice, [131I ]LG-12 rapidly accumulated at the tumor site by SPECT/CT imaging. The distribution of [131I ]LG-12 in B16-F10 tumor-bearing mice was further studied. As shown in Figure 20B, the uptake of [131I ]LG-12 in tumor tissue was 6.50±1.05%ID/g, which was significantly higher than that in most normal tissues, indicating that [131I ]LG-12 has the ability to target PD-L1 in vivo, but the uptake in non-target tissues such as blood, liver, heart and intestine is also high, which may be due to the high lipophilicity of [131I ]LG-12. As shown in Figure 20C, the activity of [131I ]LG-12 in tumors was 2.3 times higher than that in muscles, further demonstrating the targeting of [131I ]LG-12 to PD-L1.
实验例5：非放射性的PD-L1小分子抑制剂LG-12的生物相容性实验Experimental Example 5: Biocompatibility experiment of non-radioactive PD-L1 small molecule inhibitor LG-12
本实验例提供了实施例1的非放射性的PD-L1小分子抑制剂LG-12的生物相容性实验，具体过程如下：This experimental example provides a biocompatibility experiment of the non-radioactive PD-L1 small molecule inhibitor LG-12 in Example 1, and the specific process is as follows:
通过MTT实验评估实施例1的非放射性的PD-L1小分子抑制剂LG-12的生物相容性。将B16-F10细胞以1×104/孔的接种量接种至添加有含1％(v/v)青霉素-链霉素双抗、10％(v/v)胎牛血清的DMEM培养基(100μL)的96孔板中后，于37℃、5％(v/v)CO2的培养箱中培养16h；培养16h后，吸出培养基，在孔中加入不同浓度(0、1.5625、3.125、6.25、12.5、25、50μM)的LG-12(100μL，溶剂为含1％青霉素-链霉素双抗、10％胎牛血清的DMEM培养基)，于37℃、5％CO2培养箱孵育24h；孵育24h后，吸出培养基，将MTT(使用PBS缓冲液稀释至浓度为5mg/mL后使用，MTT购自上海碧云天，PBS缓冲液购自上海碧云天)以20μL/孔的添加量加入孔中，于37℃、5％CO2培养箱孵育4h；孵育4h后，吸出MTT，将DMSO以150μL/孔的添加量加入孔中，摇晃10min；摇晃结束后，使用酶标仪在490nm处检测样品的吸光度(MD/M5e,VEDENG)，并根据公式：存活率＝样品孔OD值/参比孔OD值(参比孔即加入浓度0μM LG-12的孔)，计算B16-F10肿瘤细胞和T细胞的存活率，实验结果见图21。The biocompatibility of the non-radioactive PD-L1 small molecule inhibitor LG-12 of Example 1 was evaluated by MTT experiment. B16-F10 cells were inoculated at 1×104 /well into a 96-well plate supplemented with DMEM medium (100 μL) containing 1% (v/v) penicillin-streptomycin dual antibody and 10% (v/v) fetal bovine serum, and then cultured in an incubator at 37°C and 5% (v/v) CO2 for 16 h; after 16 h of culture, the medium was aspirated, and different concentrations (0, 1.5625, 3.125, 6.25, 12.5, 25, 50 μM) of LG-12 (100 μL, the solvent is DMEM medium containing 1% penicillin-streptomycin dual antibody and 10% fetal bovine serum) were added to the wells, and the plates were incubated at 37°C and 5% CO 2 for 16 h.2 incubator for 24 hours; after incubation for 24 hours, the culture medium was aspirated, and MTT (used after dilution with PBS buffer to a concentration of 5 mg/mL, MTT purchased from Shanghai Biyuntian, PBS buffer purchased from Shanghai Biyuntian) was added to the wells at an amount of 20 μL/well, and incubated in a 37°C, 5%CO2 incubator for 4 hours; after incubation for 4 hours, MTT was aspirated, and DMSO was added to the wells at an amount of 150 μL/well, and shaken for 10 minutes; after shaking, the absorbance of the sample was detected at 490 nm using an enzyme reader (MD/M5e, VEDENG), and the survival rate of B16-F10 tumor cells and T cells was calculated according to the formula: survival rate = sample well OD value/reference well OD value (reference well is the well added with a concentration of 0 μM LG-12), and the experimental results are shown in Figure 21.
如图21所示，LG-12对B16-F10肿瘤细胞和T细胞均有一定的细胞毒作用，其半抑制浓度IC50值分别为28.81μM和15.36μM；当LG-12浓度为3.13μM时，B16-F10肿瘤细胞和T细胞的存活率分别达到96％和80％以上。因此，后续体内实验均控制LG-12的浓度等于或低于3.13μM。As shown in Figure 21, LG-12 has a certain cytotoxic effect on B16-F10 tumor cells and T cells, and its half-inhibitory concentration IC50 values are 28.81μM and 15.36μM, respectively; when the concentration of LG-12 is 3.13μM, the survival rates of B16-F10 tumor cells and T cells reach 96% and 80%, respectively. Therefore, the subsequent in vivo experiments all controlled the concentration of LG-12 to be equal to or lower than 3.13μM.
实验例6：非放射性的PD-L1小分子抑制剂LG-12的T细胞/肿瘤细胞共培养实验Experimental Example 6: T cell/tumor cell co-culture experiment with the non-radioactive PD-L1 small molecule inhibitor LG-12
本实验例提供了实施例1的非放射性的PD-L1小分子抑制剂LG-12的T细胞/肿瘤细胞共培养实验，具体过程如下：This experimental example provides a T cell/tumor cell co-culture experiment of the non-radioactive PD-L1 small molecule inhibitor LG-12 of Example 1, and the specific process is as follows:
在96孔板中以0.5μL/孔的添加量加入CD3+(使用抗体稀释液稀释至浓度为5μg/mL后使用，CD3+和抗体稀释液均购自BioLegend)，并以0.5μL/孔的添加量加入CD28(使用抗体稀释液稀释至浓度为5μg/mL后使用，CD28和抗体稀释液均购自BioLegend)后，于4℃孵育24h，得到CD3+/CD28(5μg/mL)抗体包被的96孔板；将96孔板中的孔分为两组，两组分别为互作组和非互作组，每组各3个平行组。其中，互作组：先在互作组的孔中加入B16-F10肿瘤细胞(5×103个细胞/孔，100μL，溶剂为含1％青霉素-链霉素双抗、10％胎牛血清的DMEM培养基)，于37℃、5％(v/v)CO2的培养箱培养24h，再在互作组的孔中加入不同浓度(0、0.78和3.125μM)的LG-12(50μL，溶剂为含1％青霉素-链霉素双抗、10％胎牛血清的DMEM培养基)和不同浓度(1×105个细胞/孔和2×105个细胞/孔)的T淋巴细胞(50μL，溶剂为含1％青霉素-链霉素双抗、10％胎牛血清的DMEM培养基)，于37℃、5％CO2培养箱孵育24h。非互作组：直接在非互作组的孔中加入100μL含1％(v/v)青霉素-链霉素双抗、10％(v/v)胎牛血清的DMEM培养基和不同浓度(0、0.78和3.125μM)的LG-12(50μL，溶剂为含1％青霉素-链霉素双抗、10％胎牛血清的DMEM培养基)和不同浓度(1×105个细胞/孔和2×105个细胞/孔)的T淋巴细胞(50μL，溶剂为含1％青霉素-链霉素双抗、10％胎牛血清的DMEM培养基)，于37℃、5％CO2培养箱孵育24h。孵育24h后，收集上清液(100μL)，使用小鼠干扰素-γELISA试剂盒(KE10001，proteintech)检测IFN-γ含量，检测结果见图22中的B。将CCK-8试剂(购自上海碧云天生物技术有限公司)以10μL/孔的添加量加入96孔板剩余100μL体系中，于37℃、5％CO2培养箱孵育4h；孵育4h后，使用酶标仪(MD/M5e，VEDENG)在450nm处检测ODs值，并根据公式：存活率＝样品孔OD值/参比孔OD值(参比孔即加入浓度0μM LG-12的孔)，计算B16-F10肿瘤细胞的存活率，实验结果见图22中的A。CD3+ (diluted to a concentration of 5 μg/mL using antibody diluent, both CD3+ and antibody diluent were purchased from BioLegend) was added at 0.5 μL/well to a 96-well plate, and CD28 (diluted to a concentration of 5 μg/mL using antibody diluent, both CD28 and antibody diluent were purchased from BioLegend) was added at 0.5 μL/well, and then incubated at 4°C for 24 h to obtain a 96-well plate coated with CD3+ /CD28 (5 μg/mL) antibodies; the wells in the 96-well plate were divided into two groups, an interaction group and a non-interaction group, with 3 parallel groups in each group. Among them, interaction group: B16-F10 tumor cells (5×103 cells/well, 100 μL, the solvent is DMEM culture medium containing 1% penicillin-streptomycin double antibody and 10% fetal bovine serum) were first added to the wells of the interaction group, and cultured in an incubator at 37°C and 5% (v/v) CO2 for 24 h. Then, different concentrations (0, 0.78 and 3.125 μM) of LG-12 (50 μL, the solvent is DMEM culture medium containing 1% penicillin-streptomycin double antibody and 10% fetal bovine serum) and different concentrations (1×105 cells/well and 2×105 cells/well) of T lymphocytes (50 μL, the solvent is DMEM culture medium containing 1% penicillin-streptomycin double antibody and 10% fetal bovine serum) were added to the wells of the interaction group, and incubated in an incubator at 37°C and 5% CO2 for 24 h. Non-interaction group: 100 μL of DMEM medium containing 1% (v/v) penicillin-streptomycin double antibody and 10% (v/v) fetal bovine serum and different concentrations (0, 0.78 and 3.125 μM) of LG-12 (50 μL, the solvent is DMEM medium containing 1% penicillin-streptomycin double antibody and 10% fetal bovine serum) and different concentrations (1×105 cells/well and 2×105 cells/well) of T lymphocytes (50 μL, the solvent is DMEM medium containing 1% penicillin-streptomycin double antibody and 10% fetal bovine serum) were directly added to the wells of the non-interaction group, and incubated for 24 hours at 37°C and 5% CO2 incubator. After incubation for 24 hours, the supernatant (100 μL) was collected and the IFN-γ content was detected using a mouse interferon-γ ELISA kit (KE10001, proteintech). The test results are shown in B in Figure 22. CCK-8 reagent (purchased from Shanghai Biotech Biotechnology Co., Ltd.) was added to the remaining 100 μL system of the 96-well plate at an amount of 10 μL/well, and incubated in a 37°C, 5%CO2 incubator for 4 hours; after incubation for 4 hours, the ODs value was detected at 450 nm using a microplate reader (MD/M5e, VEDENG), and the survival rate of B16-F10 tumor cells was calculated according to the formula: Survival rate = sample well OD value/reference well OD value (reference well is the well with 0 μM LG-12 added). The experimental results are shown in A in Figure 22.
如图22中的A所示，LG-12以剂量依赖的方式增强PD-1/PD-L1介导的T细胞激活抑制机制，其中，B16-F10细胞与T细胞按1：20比例共培养时，经LG-12(0.78μM和3.125μM)处理后，B16-F10细胞的细胞存活率分别为0.79±0.13和0.69±0.02，当B16-F10与T细胞比例为1：40时(不加LG-12)，B16-F10细胞的细胞存活率由1：20时的0.90±0.06降至0.70±0.12，这表明增加T细胞的数量可以增加对肿瘤细胞的杀伤作用；当LG-12浓度为0.78μM和3.125μM时，B16-F10细胞存活率分别降至0.66±0.06和0.55±0.03，这表明LG-12能激活T细胞的抗肿瘤免疫功能。As shown in Figure 22A, LG-12 enhanced the PD-1/PD-L1-mediated T cell activation inhibition mechanism in a dose-dependent manner. When B16-F10 cells were co-cultured with T cells at a ratio of 1:20, the cell viability of B16-F10 cells after treatment with LG-12 (0.78 μM and 3.125 μM) was 0.79±0.13 and 0.69±0.02, respectively. When the ratio of B16-F10 to T cells was 1:40 ( Without LG-12, the cell survival rate of B16-F10 cells decreased from 0.90±0.06 at 1:20 to 0.70±0.12, indicating that increasing the number of T cells can increase the killing effect on tumor cells; when the concentration of LG-12 was 0.78μM and 3.125μM, the survival rate of B16-F10 cells decreased to 0.66±0.06 and 0.55±0.03, respectively, indicating that LG-12 can activate the anti-tumor immune function of T cells.
为了进一步探讨LG-12通过阻断PD-1/PD-L1相互作用来激活抗肿瘤免疫治疗的机制，继续研究了CD8+T细胞毒性功能标志物干扰素-γ的表达水平。如图22中的B，用LG-12处理后，干扰素-γ的分泌呈剂量依赖性增强，其中，与浓度为0.78μM(B16-F10/T细胞＝1/20)的LG-12作用后，干扰素-γ的表达量由34.44±1.39pg/100μL略升至40.28±1.97pg/100μL，与浓度为3.125μM(B16-F10/T细胞＝1/20)的LG-12孵育后，干扰素-γ的表达量由34.44±1.39pg/100μL显著升高至76.33±5.24pg/100μL；当B16-F10/T细胞达到1/40时，LG-12(0.78μM和3.125μM)诱导干扰素-γ的表达从83.13±3.64pg/100μL增加到110.26±1.72pg/100μL，干扰素-γ水平始终随着T细胞比例的增加而升高。以上结果表明，LG-12可通过阻断PD-1/PD-L1信号通路，增加干扰素-γ的分泌，促进CD8+T细胞的活化。In order to further explore the mechanism by which LG-12 activates anti-tumor immunotherapy by blocking the interaction between PD-1/PD-L1, the expression level of interferon-γ, a functional marker of CD8+T cell toxicity, was further studied. As shown in Figure 22B, after treatment with LG-12, the secretion of interferon-γ increased in a dose-dependent manner. After the action of LG-12 at a concentration of 0.78 μM (B16-F10/T cell = 1/20), the expression of interferon-γ increased slightly from 34.44±1.39 pg/100 μL to 40.28±1.97 pg/100 μL. After incubation with LG-12 at a concentration of 3.125 μM (B16-F10/T cell = 1/20), the expression of interferon-γ increased slightly from 34.44±1.39 pg/100 μL to 40.28±1.97 pg/100 μL. The expression of interferon-γ increased significantly from 34.44±1.39pg/100μL to 76.33±5.24pg/100μL; when B16-F10/T cells reached 1/40, LG-12 (0.78μM and 3.125μM) induced the expression of interferon-γ from 83.13±3.64pg/100μL to 110.26±1.72pg/100μL, and the level of interferon-γ always increased with the increase of T cell proportion. The above results show that LG-12 can increase the secretion of interferon-γ and promote the activation of CD8+T cells by blocking the PD-1/PD-L1 signaling pathway.
实验例7：非放射性的PD-L1小分子抑制剂LG-12的体内抗肿瘤实验Experimental Example 7: In vivo anti-tumor experiment of non-radioactive PD-L1 small molecule inhibitor LG-12
本实验例提供了实施例1的非放射性的PD-L1小分子抑制剂LG-12的体内抗肿瘤实验，具体过程如下：This experimental example provides an in vivo anti-tumor experiment of the non-radioactive PD-L1 small molecule inhibitor LG-12 of Example 1, and the specific process is as follows:
为了评估LG-12的抗肿瘤活性，BALB/c小白鼠接种B16-F10细胞5天后，将所得荷瘤小鼠随机分成三组，三组分别为空白组、低剂量治疗组、高剂量治疗组(n＝5)；其中，低剂量治疗组(B组)和高剂量治疗组(C组)小鼠分别按5mg/kg和20mg/kg的剂量连续2周隔天(实验第1、3、5、7、9、11天)腹腔注射实施例1的非放射性的PD-L1小分子抑制剂LG-12(溶解在100μL生理盐水中)，空白组(A组)小鼠注射等量生理盐水。实验过程中，每隔一天测量一次肿瘤大小和小鼠体重，并根据公式：肿瘤体积＝1/2(长度×宽度2)，计算肿瘤体积，检测结果见图23。实验结束后(即实验第14天)处死荷瘤小鼠。荷瘤小鼠处死前进行摘眼球取全血，血样在室温(25℃)下静置30min后，1000r/min离心5min，收集血清，使用小鼠IFN-γELISA试剂盒(KE10001，proteintech)检测IFN-γ含量，检测结果见图25中的B。荷瘤小鼠处死后，剥离心、肝、脾、肺、肾、肌肉、肿瘤于4％(w/v，g/100mL)多聚甲醛溶液中固定24h，固定后，在15％(w/v，g/100mL)蔗糖溶液和30％(w/v，g/100mL)蔗糖溶液中分别脱水24h，脱水后的组织先使用冷冻包埋剂在-25℃下包埋，然后使用冷冻切片机(CM1950，LEICA)将其制成组织切片(6μm)于载玻片；取载玻片先使用固定液(购自无锡市江原实业技贸有限公司)固定10min，再用苏木素染色液(购自上海碧云天生物技术有限公司)染色10min和伊红染色液(购自上海碧云天生物技术有限公司)染色1min，最后脱水、透明、封片，H&E染色分析结果见图24和图25中的A。取荷瘤小鼠肿瘤组织在4％(w/v，g/100mL)多聚甲醛中固定24h，固定后，用正丁醇配制不同浓度乙醇进行脱水，依次在50％(v/v)乙醇中脱水2h、80％乙醇中脱水3h、65％乙醇中脱水1.5h、50％乙醇中脱水1h、30％乙醇中脱水4h、10％乙醇中脱水4h、纯正丁醇中脱水24h，脱水后的肿瘤组织先在65℃下浸蜡4h，再进行石蜡包埋并切片(6μm)；载玻片脱蜡后，先使用柠檬酸钠抗原修复液(购自BBI公司)进行抗原修复，再使用5％(v/v)山羊血清封闭，封闭后的载玻片先分别与相应一抗(CD4+、CD8+、PD-L1、CRT和HMGB1抗体，各抗体均使用抗体稀释液稀释500倍后作为工作液使用，CD4+和CD8+抗体购自proteintech，PD-L1、CRT和HMGB1抗体购自abcam，抗体稀释液均购自BioLegend)工作液于25℃孵育90min，然后与二抗(CD4+、PD-L1使用山羊抗鼠二抗，CD8+、CRT、HMGB1使用山羊抗兔二抗，抗体使用抗体稀释液稀释至浓度为4000倍后作为工作液使用，各抗体均购自R&DSystem，抗体稀释液均购自BioLegend)工作液25℃孵育30min，再用DAB显色液(购自温州迈新生物技术开发有限公司)显色，接着用苏木素(购自上海碧云天生物科技有限公司)复染，最后脱水、透明、封片，免疫组织化学IHC分析结果见图25中的A。In order to evaluate the anti-tumor activity of LG-12, BALB/c mice were inoculated with B16-F10 cells for 5 days, and the resulting tumor-bearing mice were randomly divided into three groups, namely, a blank group, a low-dose treatment group, and a high-dose treatment group (n=5); among them, the mice in the low-dose treatment group (Group B) and the high-dose treatment group (Group C) were intraperitoneally injected with the non-radioactive PD-L1 small molecule inhibitor LG-12 (dissolved in 100 μL of normal saline) of Example 1 at a dose of 5 mg/kg and 20 mg/kg every other day for 2 consecutive weeks (experimental days 1, 3, 5, 7, 9, and 11), and the blank group (Group A) mice were injected with an equal amount of normal saline. During the experiment, the tumor size and mouse weight were measured every other day, and the tumor volume was calculated according to the formula: tumor volume = 1/2 (length × width2 ), and the test results are shown in Figure 23. Tumor-bearing mice were killed after the end of the experiment (i.e., on the 14th day of the experiment). Before killing the tumor-bearing mice, their eyes were removed and whole blood was collected. The blood samples were allowed to stand at room temperature (25°C) for 30 minutes and then centrifuged at 1000 rpm for 5 minutes to collect serum. The IFN-γ content was detected using a mouse IFN-γ ELISA kit (KE10001, proteintech). The test results are shown in B in Figure 25. After tumor-bearing mice were killed, the heart, liver, spleen, lung, kidney, muscle, and tumor were removed and fixed in 4% (w/v, g/100 mL) paraformaldehyde solution for 24 h. After fixation, they were dehydrated in 15% (w/v, g/100 mL) sucrose solution and 30% (w/v, g/100 mL) sucrose solution for 24 h, respectively. The dehydrated tissues were first embedded in a freezing embedding agent at -25°C, and then made into tissue sections (6 μm) on slides using a freezing microtome (CM1950, LEICA); the slides were first fixed with a fixative (purchased from Wuxi Jiangyuan Industrial Technology and Trade Co., Ltd.) for 10 min, then stained with a hematoxylin staining solution (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) for 10 min and an eosin staining solution (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) for 1 min, and finally dehydrated, transparent, and sealed. The results of H&E staining analysis are shown in Figures 24 and 25 A. Tumor tissues of tumor-bearing mice were fixed in 4% (w/v, g/100 mL) paraformaldehyde for 24 h. After fixation, different concentrations of ethanol were prepared with n-butanol for dehydration, and the tissues were dehydrated in 50% (v/v) ethanol for 2 h, 80% ethanol for 3 h, 65% ethanol for 1.5 h, 50% ethanol for 1 h, 30% ethanol for 4 h, 10% ethanol for 4 h, and pure n-butanol for 24 h. The dehydrated tumor tissues were first immersed in wax at 65°C for 4 h, then embedded in paraffin and sliced (6 μm). After the slides were dewaxed, they were first repaired with sodium citrate antigen repair solution (purchased from BBI Company), and then blocked with 5% (v/v) goat serum. The blocked slides were first incubated with the corresponding primary antibodies (CD4+ , CD8+ , PD-L1, CRT and HMGB1 antibodies, each antibody was diluted 500 times with antibody diluent and used as working solution, CD4+ and CD8+ antibodies were purchased from proteintech, PD-L1, CRT and HMGB1 antibodies were purchased from abcam, and antibody diluents were purchased from BioLegend) working solution was incubated at 25°C for 90 min, and then incubated with secondary antibody (CD4+ and PD-L1 used goat anti-mouse secondary antibody, CD8+ , CRT, HMGB1 used goat anti-rabbit secondary antibody, the antibodies were diluted to a concentration of 4000 times with antibody diluent and used as working solution, each antibody was purchased from R&D System, and antibody diluent was purchased from BioLegend) working solution at 25°C for 30 min, and then colored with DAB color developing solution (purchased from Wenzhou Maixin Biotechnology Development Co., Ltd.), followed by counterstaining with hematoxylin (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.), and finally dehydrated, transparent, and sealed. The results of immunohistochemistry IHC analysis are shown in Figure 25 A.
如图23中的A～图23中的C所示，所有小鼠在治疗过程中均没有引起明显的体重减轻或死亡，表明LG-12所有剂量都是耐受性良好，H&E染色分析进一步证实了此观点(图24)；同时，治疗7天后，相对于空白组，两组治疗组小鼠的肿瘤体积都显著缩小，治疗14天后，低剂量(5mg/kg)治疗组小鼠肿瘤体积(37±7mm3)和重量(0.046±0.01g)明显小于空白组(体积1303±203mm3和重量1.88±0.63g)，而高剂量(20mg/kg)组的抗肿瘤效果相较低剂量组较差，其肿瘤体积和重量分别为201±44mm3和0.44±0.22g，这说明基于小分子抑制剂LG-12的抗肿瘤免疫治疗并不是剂量越高越好，具体机制还需要进一步深入研究。As shown in Figure 23A to Figure 23C, all mice did not cause obvious weight loss or death during the treatment, indicating that all doses of LG-12 were well tolerated, and H&E staining analysis further confirmed this view (Figure 24); at the same time, after 7 days of treatment, the tumor volume of mice in the two treatment groups was significantly reduced compared with the blank group. After 14 days of treatment, the tumor volume (37±7mm3) and weight (0.046±0.01g) of mice in the low-dose (5mg/kg) treatment group were significantly smaller than those in the blank group (volume 1303±203mm3 and weight 1.88±0.63g), while the anti-tumor effect of the high-dose (20mg/kg) group was worse than that of the low-dose group, with a tumor volume and weight of 201±44mm3 and 0.44±0.22g, respectively. This shows that anti-tumor immunotherapy based on the small molecule inhibitor LG-12 is not the higher the dose, the better, and the specific mechanism needs further in-depth study.
治疗结束后，取小鼠肿瘤组织进行免疫组化分析，观察肿瘤中浸润性淋巴细胞(TILs)和PD-L1表达的差异。结果表明，LG-12在低剂量下显著增加了CD4+T细胞和CD8+T细胞的浸润水平，PD-L1的表达水平与空白组相比显著降低，同时，治疗后肿瘤组织H&E染色可见明显的细胞凋亡，可见，LG-12在低剂量下具有良好的体内抗肿瘤作用。同样，高剂量组小鼠肿瘤组织中CD4+、CD8+T细胞含量较高，PD-L1表达降低，但总体低于低剂量组，与抗肿瘤结果一致(图25中的A)。After the treatment, the mouse tumor tissues were taken for immunohistochemical analysis to observe the differences in the expression of infiltrating lymphocytes (TILs) and PD-L1 in the tumor. The results showed that LG-12 significantly increased the infiltration level of CD4+ T cells and CD8+ T cells at low doses, and the expression level of PD-L1 was significantly reduced compared with the blank group. At the same time, H&E staining of tumor tissues after treatment showed obvious cell apoptosis. It can be seen that LG-12 has a good in vivo anti-tumor effect at low doses. Similarly, the content of CD4+ and CD8+ T cells in the tumor tissues of mice in the high-dose group was higher, and the expression of PD-L1 was reduced, but overall it was lower than that in the low-dose group, which is consistent with the anti-tumor results (A in Figure 25).
使用ELISA试剂盒检测小鼠血清中干扰素-γ的表达。结果表明，低剂量治疗组小鼠中干扰素-γ表达为153±54(pg/100mL)，而高剂量治疗组小鼠血清中干扰素-γ表达为72±23(pg/100mL)，这与抗肿瘤和免疫组化的结果一致(图25中的B)。The expression of interferon-γ in mouse serum was detected using an ELISA kit. The results showed that the expression of interferon-γ in the low-dose treatment group mice was 153±54 (pg/100mL), while the expression of interferon-γ in the high-dose treatment group mice serum was 72±23 (pg/100mL), which was consistent with the results of anti-tumor and immunohistochemistry (B in Figure 25).
实验例8：碘-131标记的PD-L1小分子抑制剂[131I]LG-12的体外克隆实验Experimental Example 8: In vitro cloning experiment of iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12
本实验例提供了实施例3的碘-131标记的PD-L1小分子抑制剂[131I]LG-12的体外克隆实验，具体过程如下：This experimental example provides an in vitro cloning experiment of the iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12 of Example 3. The specific process is as follows:
克隆形成实验：克隆形成实验用来评估单个肿瘤细胞经受辐射处理后增殖并形成克隆的能力。将B16-F10细胞以1.2×103/孔的接种量接种至添加有含1％(v/v)青霉素-链霉素双抗、10％(v/v)胎牛血清的DMEM培养基(1mL)的24孔板中后，于37℃、5％(v/v)CO2的培养箱中培养16h；培养16h后，吸出培养基，在孔中加入不同浓度(0、0.148、0.296、0.592、1.184和2.368MBq)的[131I]LG-12(1mL，溶剂为含1％青霉素-链霉素双抗、10％胎牛血清的DMEM培养基)，于37℃、5％CO2培养箱孵育10h；孵育10h后，更换新鲜培养基，于37℃、5％(v/v)的CO2培养箱培养9天；孵育9天后，先用PBS缓冲液润洗两次孔中的B16-F10细胞，然后用甲醇固定20min，再用结晶紫(购自上海碧云天生物科技有限公司)染色15min，接着用流水洗去结晶紫，最后37℃烘干后拍照，拍照结果见图26。Clonogenic assay: Clonogenic assay is used to evaluate the ability of single tumor cells to proliferate and form clones after radiation treatment. B16-F10 cells were seeded at 1.2×103 /well in a 24-well plate supplemented with DMEM medium (1 mL) containing 1% (v/v) penicillin-streptomycin and 10% (v/v) fetal bovine serum, and then cultured in a 37°C, 5% (v/v) CO2 incubator for 16 h. After 16 h of culture, the medium was aspirated, and different concentrations (0, 0.148, 0.296, 0.592, 1.184, and 2.368 MBq) of [131 I]LG-12 (1 mL, the solvent was DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum) were added to the wells, and then incubated in a 37°C, 5% (v/v) CO2 incubator for 10 h. After 10 h of incubation, the medium was replaced with fresh medium, and the wells were incubated in a 37°C, 5% (v/v) CO 2 incubator for 10 h.2 incubator for 9 days; after incubation for 9 days, the B16-F10 cells in the wells were rinsed twice with PBS buffer, then fixed with methanol for 20 minutes, and then stained with crystal violet (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) for 15 minutes, and then washed away with running water. Finally, the cells were dried at 37°C and photographed. The photographic results are shown in Figure 26.
Western Blot实验：为了测试[131I]LG-12是否能在肿瘤中诱导ICD，通过[131I]LG-12处理肿瘤细胞来寻找ICD的特征。ICD过程中的主要特征之一是损伤相关分子模式(DAMPs)，如高迁移率族蛋白1(HMGB1)和钙网蛋白(CRT)。配置RIPA裂解缓冲液(购自上海碧云天生物科技有限公司)和苯甲基磺酰氟PMSF(购自上海碧云天生物科技有限公司)的混合液(RIPA裂解缓冲液：苯甲基磺酰氟PMSF＝100：1，v/v)；将B16-F10细胞以1.5×105个细胞的接种量平铺于添加有含1％(v/v)青霉素-链霉素双抗、10％(v/v)胎牛血清的DMEM培养基(1mL)的小培养皿中后，于37℃、5％(v/v)CO2的培养箱中培养16h；培养16h后，吸出培养基，在孔中加入[131I]LG-12(1.85MBq)(1mL，溶剂为含1％青霉素-链霉素双抗、10％胎牛血清的DMEM培养基)，于37℃、5％CO2培养箱孵育24h；孵育24h后，吸出培养基，加200μL混合液裂解B16-F10细胞10min，得到裂解液，并使用BCA蛋白检测试剂盒对裂解液中的蛋白浓度进行定量；将裂解液低温离心(4℃、12000r/min离心15min)，取上清液；将上清液中蛋白变性，冷却后低温离心，取上清液样品上样、电泳(80V、30min；120V、90min)；通过转膜(300mA，90min)将蛋白质样品转移到0.22μm聚偏二氟乙烯(PVDF)膜上后，先用5％(w/v，g/100mL)脱脂奶粉封闭1h，然后分别与相应一抗(钙网蛋白CRT、HMGB-1、鼠源PD-L1和β-Actin抗体，CRT抗体使用抗体稀释液稀释1000倍后作为工作液使用、HMGB1抗体使用抗体稀释液稀释10000倍后作为工作液使用、PD-L1抗体使用抗体稀释液稀释500倍后作为工作液使用，各抗体均购自abcam，抗体稀释液均购自BioLegend)在4℃下孵育24h，再与二抗(CRT和HMGB1使用山羊抗兔二抗、PD-L1使用山羊抗大鼠二抗，抗体使用抗体稀释液稀释至浓度为4000倍后作为工作液使用，抗兔或抗鼠购自R&D System，抗体稀释液均购自BioLegend)工作液在室温(25℃)下孵育1h，最后通过ECL试剂盒进行化学发光成像仪的膜显影，显影结果见图27中的A。Western Blot Experiment: To test whether [131I ]LG-12 can induce ICD in tumors, tumor cells were treated with [131I ]LG-12 to find the characteristics of ICD. One of the main features of the ICD process is damage-associated molecular patterns (DAMPs), such as high-mobility group box 1 (HMGB1) and calreticulin (CRT). A mixture of RIPA lysis buffer (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) and phenylmethylsulfonyl fluoride (PMSF) (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) was prepared (RIPA lysis buffer: phenylmethylsulfonyl fluoride (PMSF) = 100:1, v/v); B16-F10 cells were plated at a density of 1.5 × 105 cells in a small culture dish supplemented with DMEM medium (1 mL) containing 1% (v/v) penicillin-streptomycin and 10% (v/v) fetal bovine serum, and cultured in an incubator at 37°C and 5% (v/v) CO2 for 16 h; after culturing for 16 h, the culture medium was aspirated, and [131 I]LG-12 (1.85 MBq) (1 mL, the solvent is DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum) was added to the wells, and the wells were incubated at 37°C and 5% CO 2 for 16 h.2 incubator for 24 hours; after 24 hours of incubation, the culture medium was aspirated, and 200 μL of the mixture was added to lyse the B16-F10 cells for 10 minutes to obtain a lysate, and the protein concentration in the lysate was quantified using a BCA protein detection kit; the lysate was centrifuged at low temperature (4°C, 12000r/min for 15 minutes) to obtain the supernatant; the protein in the supernatant was denatured, cooled and centrifuged at low temperature, and the supernatant sample was loaded and electrophoresed (80V, 30min; 120V, 90min); the protein sample was transferred to a 0.22μm polyvinylidene fluoride (PVDF) membrane by membrane transfer (300mA, 90min), and then blocked with 5% (w/v, g/100mL) skimmed milk powder for 1 hour, and then Then, they were incubated with the corresponding primary antibodies (calreticulin CRT, HMGB-1, mouse PD-L1 and β-Actin antibodies, CRT antibody was diluted 1000 times with antibody diluent as working solution, HMGB1 antibody was diluted 10000 times with antibody diluent as working solution, PD-L1 antibody was diluted 500 times with antibody diluent as working solution, each antibody was purchased from abcam, and antibody diluent was purchased from BioLegend) at 4°C for 24 hours, and then incubated with secondary antibody (CRT and HMGB1 used goat anti-rabbit secondary antibody, PD-L1 used goat anti-rat secondary antibody, antibodies were diluted to a concentration of 4000 times with antibody diluent as working solution, anti-rabbit or anti-mouse was purchased from R&D System, and antibody diluent was purchased from BioLegend) working solution at room temperature (25°C) for 1 hour, and finally the membrane was developed by chemiluminescence imager using ECL kit, and the development results are shown in A in Figure 27.
如图26所示，[131I]LG-12对B16-F10细胞增殖的抑制作用是呈剂量依赖关系，且抑制作用明显强于Na131I组。这一结果可能是由于[131I]LG-12对PD-L1的靶向性。As shown in Figure 26, the inhibitory effect of [131I ]LG-12 on B16-F10 cell proliferation was dose-dependent, and the inhibitory effect was significantly stronger than that of theNa131I group. This result may be due to the targeting of [131I ]LG-12 to PD-L1.
如图27中的A所示，B16-F10细胞经[131I]LG-12(1.85MBq/mL)作用48h后，细胞内HMGB1的表达量下降了2.7倍，表明细胞外HMGB1的表达增加，同时肿瘤细胞表面CRT的表达量增加了1.2倍，表明[131I]LG-12可诱导肿瘤细胞发生ICD。As shown in Figure 27A, after B16-F10 cells were treated with [131I ]LG-12 (1.85MBq/mL) for 48 hours, the expression of intracellular HMGB1 decreased by 2.7 times, indicating that the expression of extracellular HMGB1 increased. At the same time, the expression of CRT on the surface of tumor cells increased by 1.2 times, indicating that [131I ]LG-12 can induce ICD in tumor cells.
实验例9：碘-131标记的PD-L1小分子抑制剂[131I]LG-12的体内抗肿瘤实验Experimental Example 9: In vivo antitumor experiment of iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12
本实验例提供了实施例3的碘-131标记的PD-L1小分子抑制剂[131I]LG-12的体内抗肿瘤实验，具体过程如下：This experimental example provides an in vivo anti-tumor experiment of the iodine-131 labeled PD-L1 small molecule inhibitor [131 I]LG-12 of Example 3. The specific process is as follows:
为了评估[131I]LG-12靶向放射性核素治疗(TRT)诱导的免疫原性细胞死亡，BALB/c小白鼠接种B16-F10细胞5天后，将所得荷瘤小鼠随机分成三组，三组分别为空白组、[131I]LG-12组、Na131I组(n＝4)；其中，[131I]LG-12组和Na131I组小鼠分别尾静脉注射[131I]LG-12(11.1MBq，溶解在100μL生理盐水中)和Na131I(11.1MBq，溶解在100μL生理盐水中)，空白组小鼠注射等量生理盐水。注射用药治疗48h后处死部分荷瘤小鼠。荷瘤小鼠处死后，取荷瘤小鼠肿瘤组织在4％(w/v，g/100mL)多聚甲醛中固定24h，固定后，用正丁醇配制不同浓度乙醇进行脱水，依次在50％(v/v)乙醇中脱水2h、80％乙醇中脱水3h、65％乙醇中脱水1.5h、50％乙醇中脱水1h、30％乙醇中脱水4h、10％乙醇中脱水4h、纯正丁醇中脱水24h，脱水后的肿瘤组织先在65℃下浸蜡4h，再进行石蜡包埋并切片(6μm)；载玻片脱蜡后，先使用柠檬酸钠抗原修复液(购自BBI公司)进行抗原修复，再使用5％(v/v)山羊血清封闭，封闭后的载玻片先分别与相应一抗(CD4+、CD8+、PD-L1、CRT和HMGB1抗体，各抗体均使用抗体稀释液稀释500倍后作为工作液使用，CD4+和CD8+抗体均购自proteintech，PD-L1、CRT和HMGB1抗体均购自abcam，抗体稀释液均购自BioLegend)工作液于25℃孵育90min，然后与二抗(CD4+、PD-L1使用山羊抗鼠二抗，CD8+、CRT、HMGB1使用山羊抗兔二抗，抗体使用抗体稀释液稀释至浓度为4000倍后作为工作液使用，各抗体购自R&D System，抗体稀释液均购自BioLegend)工作液25℃孵育30min，再用DAB显色液(购自温州迈新生物技术开发有限公司)显色，接着用苏木素(购自上海碧云天生物科技有限公司)复染，最后脱水、透明、封片，免疫组织化学IHC分析结果见图27中的C。To evaluate the immunogenic cell death induced by [131 I]LG-12 targeted radionuclide therapy (TRT), BALB/c mice were inoculated with B16-F10 cells for 5 days, and the resulting tumor-bearing mice were randomly divided into three groups: a blank group, a [131 I]LG-12 group, and a Na131 I group (n=4). The mice in the [131 I]LG-12 group and the Na131 I group were injected with [131 I]LG-12 (11.1 MBq, dissolved in 100 μL saline) and Na131 I (11.1 MBq, dissolved in 100 μL saline) through the tail vein, respectively, and the mice in the blank group were injected with an equal amount of saline. Some tumor-bearing mice were killed 48 hours after the injection treatment. After tumor-bearing mice were killed, tumor tissues of tumor-bearing mice were fixed in 4% (w/v, g/100 mL) paraformaldehyde for 24 h. After fixation, different concentrations of ethanol were prepared with n-butanol for dehydration, and dehydrated in 50% (v/v) ethanol for 2 h, 80% ethanol for 3 h, 65% ethanol for 1.5 h, 50% ethanol for 1 h, 30% ethanol for 4 h, 10% ethanol for 4 h, and pure n-butanol for 24 h. The dehydrated tumor tissues were first immersed in wax at 65°C for 4 h, then embedded in paraffin and sliced (6 μm). After the slides were dewaxed, they were first repaired with sodium citrate antigen repair solution (purchased from BBI Company), and then blocked with 5% (v/v) goat serum. The blocked slides were first incubated with the corresponding primary antibodies (CD4+ , CD8+ , PD-L1, CRT and HMGB1 antibodies, each antibody was diluted 500 times with antibody diluent and used as working solution, CD4+ and CD8+ antibodies were purchased from proteintech, PD-L1, CRT and HMGB1 antibodies were purchased from abcam, and antibody diluents were purchased from BioLegend) working solution was incubated at 25°C for 90 min, and then incubated with secondary antibody (CD4+ and PD-L1 used goat anti-mouse secondary antibody, CD8+ , CRT, HMGB1 used goat anti-rabbit secondary antibody, antibodies were diluted to a concentration of 4000 times with antibody diluent and used as working solution, each antibody was purchased from R&D System, and antibody diluents were purchased from BioLegend) working solution at 25°C for 30 min, and then colored with DAB color developing solution (purchased from Wenzhou Maixin Biotechnology Development Co., Ltd.), followed by counterstaining with hematoxylin (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.), and finally dehydrated, transparent, and sealed. The results of immunohistochemistry IHC analysis are shown in Figure 27C.
如图27中的B所示，与Na131I治疗组和空白组相比，注射一次[131I]LG-12可以缓慢抑制肿瘤的生长。As shown in FIG. 27B , a single injection of [131 I]LG-12 could slowly inhibit tumor growth compared with the Na131 I-treated group and the blank group.
第一次注射用药治疗48h后检测三组荷瘤鼠肿瘤组织中HMGB1蛋白和CRT蛋白的分泌表达。结果表明，Na131I组HMGB 1和CRT分泌水平略有上升，但经[131I]LG-12治疗后，肿瘤中两种蛋白分泌水平约为空白组的2.5倍，可见，[131I]LG-12在体内外均可诱发ICD(图27中的C)。The secretion expression of HMGB1 protein and CRT protein in the tumor tissues of the three groups of tumor-bearing mice was detected 48 hours after the first injection of the drug. The results showed that the secretion levels of HMGB1 and CRT in the Na131 I group increased slightly, but after treatment with [131 I] LG-12, the secretion levels of the two proteins in the tumor were about 2.5 times that of the blank group. It can be seen that [131 I] LG-12 can induce ICD in vitro and in vivo (C in Figure 27).
实验例10：[131I]LG-12和LG-12联合用药的体内抗肿瘤实验Experimental Example 10: In vivo anti-tumor experiment of [131I ]LG-12 and LG-12 combination therapy
本实验例提供了[131I]LG-12和LG-12联合用药的体内抗肿瘤实验，具体过程如下：This experimental example provides an in vivo anti-tumor experiment of [131I ]LG-12 and LG-12 combined with other drugs. The specific process is as follows:
[131I]LG-12可诱导肿瘤细胞表达DMAPs，激活肿瘤特异性细胞毒性T淋巴细胞(CTLs)杀伤肿瘤细胞。为了探究[131I]LG-12和LG-12联合使用是否会改善对肿瘤的免疫治疗效果，研究了[131I]LG-12与LG-12联用对B16-F10荷瘤小鼠的体内抗肿瘤作用。BALB/c小白鼠接种B16-F10细胞5天后，将所得荷瘤小鼠随机分成三组，三组分别为生理盐水组(A组)、LG-12组(B组)和[131I]LG-12与LG-12联合用药组(C组)(n＝5)；其中，[131I]LG-12与LG-12联合用药组小鼠于实验第一天通过尾静脉注射[131I]LG-12(11.1MBq，溶解在100μL生理盐水中)，其余两组小鼠注射等量生理盐水，LG-12组和[131I]LG-12与LG-12联合用药组小鼠按5mg/kg的剂量分别于实验第1、3、6、9、11天腹腔注射LG-12(溶解在100μL生理盐水中)，生理盐水组小鼠注射等量生理盐水。实验过程中，每隔一天测量一次肿瘤大小和小鼠体重，并根据公式：肿瘤体积＝1/2(长度×宽度2)，计算肿瘤体积，检测结果见图28中的a～图28中的b。实验结束后(即实验第16天)处死荷瘤小鼠。荷瘤小鼠处死前进行摘眼球取全血，血样在室温(25℃)下静置30min后，1000r/min离心5min，收集血清，使用小鼠IFN-γELISA试剂盒(KE10001，proteintech)检测IFN-γ含量，检测结果见图28中的c。荷瘤小鼠处死后，取荷瘤小鼠肿瘤组织在4％(w/v，g/100mL)多聚甲醛中固定24h，固定后，用正丁醇配制不同浓度乙醇进行脱水，依次在50％(v/v)乙醇中脱水2h、80％乙醇中脱水3h、65％乙醇中脱水1.5h、50％乙醇中脱水1h、30％乙醇中脱水4h、10％乙醇中脱水4h、纯正丁醇中脱水24h，脱水后的肿瘤组织先在65℃下浸蜡4h，再进行石蜡包埋并切片(6μm)；载玻片脱蜡后，先使用柠檬酸钠抗原修复液(购自BBI公司)进行抗原修复，再使用5％(v/v)山羊血清封闭，封闭后的载玻片先分别与相应一抗(CD4+、CD8+、PD-L1、CRT和HMGB1抗体，各抗体均使用抗体稀释液稀释500倍后作为工作液使用，CD4+和CD8+抗体均购自proteintech，PD-L1、CRT和HMGB1抗体均购自abcam，抗体稀释液均购自BioLegend)工作液于25℃孵育90min，然后与二抗(CD4+、PD-L1使用山羊抗鼠二抗，CD8+、CRT、HMGB1使用山羊抗兔二抗，抗体使用抗体稀释液稀释至浓度为4000倍后作为工作液使用，各抗体均购自R&D System，抗体稀释液均购自BioLegend)工作液25℃孵育30min，再用DAB显色液(购自温州迈新生物技术开发有限公司)显色，接着用苏木素(购自上海碧云天生物科技有限公司)复染，最后脱水、透明、封片，免疫组织化学IHC分析结果见图28中的d～图28中的e。[131I ]LG-12 can induce tumor cells to express DMAPs and activate tumor-specific cytotoxic T lymphocytes (CTLs) to kill tumor cells. To investigate whether the combined use of [131I ]LG-12 and LG-12 would improve the immunotherapy effect on tumors, the in vivo antitumor effect of [131I ]LG-12 combined with LG-12 on B16-F10 tumor-bearing mice was studied. Five days after BALB/c mice were inoculated with B16-F10 cells, the tumor-bearing mice were randomly divided into three groups, namely, normal saline group (group A), LG-12 group (group B), and [131I ]LG-12 and LG-12 combination group (group C) (n=5); among them, mice in the [131I ]LG-12 and LG-12 combination group were injected with [131I ]LG-12 (11.1MBq, dissolved in 100μL normal saline) through the tail vein on the first day of the experiment, and mice in the other two groups were injected with an equal amount of normal saline. Mice in the LG-12 group and the [131I ]LG-12 and LG-12 combination group were injected with LG-12 (dissolved in 100μL normal saline) intraperitoneally at a dose of 5mg/kg on the 1st, 3rd, 6th, 9th, and 11th days of the experiment, respectively, and mice in the normal saline group were injected with an equal amount of normal saline. During the experiment, the tumor size and mouse weight were measured every other day, and the tumor volume was calculated according to the formula: tumor volume = 1/2 (length × width2 ), and the test results are shown in Figure 28 a to Figure 28 b. After the experiment (i.e., the 16th day of the experiment), the tumor-bearing mice were killed. Before the tumor-bearing mice were killed, the eyeballs were removed to collect whole blood. After the blood samples were allowed to stand at room temperature (25°C) for 30 minutes, they were centrifuged at 1000r/min for 5 minutes, and serum was collected. The IFN-γ content was detected using a mouse IFN-γ ELISA kit (KE10001, proteintech), and the test results are shown in Figure 28 c. After tumor-bearing mice were killed, tumor tissues of tumor-bearing mice were fixed in 4% (w/v, g/100 mL) paraformaldehyde for 24 h. After fixation, different concentrations of ethanol were prepared with n-butanol for dehydration, and dehydrated in 50% (v/v) ethanol for 2 h, 80% ethanol for 3 h, 65% ethanol for 1.5 h, 50% ethanol for 1 h, 30% ethanol for 4 h, 10% ethanol for 4 h, and pure n-butanol for 24 h. The dehydrated tumor tissues were first immersed in wax at 65°C for 4 h, then embedded in paraffin and sliced (6 μm). After the slides were dewaxed, they were first repaired with sodium citrate antigen repair solution (purchased from BBI Company), and then blocked with 5% (v/v) goat serum. The blocked slides were first incubated with the corresponding primary antibodies (CD4+ , CD8+ , PD-L1, CRT and HMGB1 antibodies, each antibody was diluted 500 times with antibody diluent and used as working solution, CD4+ and CD8+ antibodies were purchased from proteintech, PD-L1, CRT and HMGB1 antibodies were purchased from abcam, and antibody diluent was purchased from BioLegend) working solution was incubated at 25°C for 90 min, and then incubated with secondary antibodies (goat anti-mouse secondary antibodies were used for CD4+ and PD-L1, goat anti-rabbit secondary antibodies were used for CD8+ , CRT, HMGB1, and antibodies were diluted to a concentration of 4000 times with antibody diluent and used as working solution, and each antibody was purchased from R&D System, antibody diluents were purchased from BioLegend) working solution was incubated at 25°C for 30 min, then color was developed with DAB color developing solution (purchased from Wenzhou Maixin Biotechnology Development Co., Ltd.), followed by counterstaining with hematoxylin (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.), and finally dehydrated, transparent, and sealed. The results of immunohistochemistry IHC analysis are shown in Figure 28d to Figure 28e.
如图图28中的a～图28中的b所示，生理盐水对照组，肿瘤体积快速增长，治疗结束时，肿瘤体积为1498.3mm3，而LG-12(5mg/kg)治疗显著地延缓了肿瘤的生长(66.7mm3)，这与实验例6的结果一致；联合治疗组中，荷瘤小鼠肿瘤体积得到有效抑制(27mm3)，与LG-12单独治疗相比有显著差异，表明[131I]LG-12与LG-12联合治疗取得良好的放射免疫治疗效果。As shown in Figure 28a to Figure 28b, in the normal saline control group, the tumor volume increased rapidly. At the end of treatment, the tumor volume was 1498.3mm3 , while LG-12 (5 mg/kg) treatment significantly delayed tumor growth (66.7mm3 ), which is consistent with the results of Experimental Example 6; in the combined treatment group, the tumor volume of tumor-bearing mice was effectively inhibited (27mm3 ), which was significantly different from that of LG-12 alone, indicating that the combined treatment of [131I ]LG-12 and LG-12 achieved a good radioimmunotherapy effect.
随后对治疗16天后的肿瘤组织中CD4+T细胞、CD8+T细胞比例和PD-L1水平进行检测，进一步探讨放射免疫治疗的抗肿瘤作用。LG-12治疗组中CD4+T细胞和CD8+T细胞的百分比分别是对照组的1.9倍和2倍，而联合治疗组CD4+T和CD8+T细胞相对于对照组上调了2.5倍(图28中的d～图28中的e)。结果表明，[131I]LG-12可提高TILs含量，从而增强抗肿瘤免疫应答作用。此外，联合治疗组肿瘤组织中PD-L1表达水平较对照组下调60％，较LG-12治疗组下调51％(图28中的d～图28中的e)。此外，联合治疗组干扰素-γ的分泌量(285±30pg/100mL)也明显高于LG-12组(177±31pg/100mL)，这也是联合治疗对肿瘤生长抑制作用更强的原因之一(图28中的c)。Subsequently, the proportion of CD4+T cells, CD8+T cells and PD-L1 levels in tumor tissues after 16 days of treatment were detected to further explore the anti-tumor effect of radioimmunotherapy. The percentages of CD4+ T cells and CD8+ T cells in the LG-12 treatment group were 1.9 times and 2 times that of the control group, respectively, while the CD4+T and CD8+T cells in the combined treatment group were increased by 2.5 times compared with the control group (Figure 28d to Figure 28e). The results showed that [131I ]LG-12 can increase the content of TILs, thereby enhancing the anti-tumor immune response. In addition, the expression level of PD-L1 in tumor tissues in the combined treatment group was downregulated by 60% compared with the control group and downregulated by 51% compared with the LG-12 treatment group (Figure 28d to Figure 28e). In addition, the secretion of interferon-γ in the combined treatment group (285±30pg/100mL) was also significantly higher than that in the LG-12 group (177±31pg/100mL), which is one of the reasons why the combined treatment had a stronger inhibitory effect on tumor growth (c in Figure 28).
综上，本发明在LG-12中引入碘-131合成放射性药物[131I]LG-12以通过放射/免疫联合治疗实现抗肿瘤作用的协同增强。其中，由于[131I]LG-12和LG-12在化学结构上是相同的，表现出相同的生物学特性，因此，[131I]LG-12/LG-12将是靶向放射治疗联合免疫治疗抗肿瘤的理想药物。In summary, the present invention introduces iodine-131 into LG-12 to synthesize the radiopharmaceutical [131I ]LG-12 to achieve synergistic enhancement of anti-tumor effects through combined radiotherapy/immunotherapy. Among them, since [131I ]LG-12 and LG-12 are identical in chemical structure and exhibit the same biological properties, [131I ]LG-12/LG-12 will be an ideal drug for targeted radiotherapy combined with immunotherapy against tumors.
细胞摄取和生物分布研究结果表明，[131I]LG-12对PD-L1具有较高的靶向特异性。TR-FRET结合试验证实LG-12对PD-1/PD-L1相互作用具有较高的抑制性。体内外研究结果表明，LG-12通过抑制PD-1/PD-L1通路，具有增强T细胞反应和激活抗肿瘤免疫作用的潜力，并且，LG-12增加CD4+和CD8+T细胞的浸润水平，降低肿瘤组织中PD-L1的表达，刺激IFN-γ的分泌。The results of cellular uptake and biodistribution studies showed that [131I ]LG-12 has high targeting specificity for PD-L1. TR-FRET binding assays confirmed that LG-12 has high inhibitory activity against PD-1/PD-L1 interaction. In vitro and in vivo studies showed that LG-12 has the potential to enhance T cell responses and activate anti-tumor immunity by inhibiting the PD-1/PD-L1 pathway. In addition, LG-12 increased the infiltration level of CD4+ and CD8+ T cells, reduced the expression of PD-L1 in tumor tissues, and stimulated the secretion of IFN-γ.
与LG-12单药治疗相比，单次注射[131I]LG-12虽然没有显著的抑制肿瘤生长，但是可以通过重塑免疫微环境来提高其抗肿瘤疗效。体内外研究表明，[131I]LG-12可诱导肿瘤细胞ICD来释放HMGB 1蛋白和CRT蛋白，增强肿瘤免疫原性。可见，[131I]LG-12和LG-12的联合应用与抗肿瘤免疫治疗具有协同作用。这一策略上调了肿瘤浸润CTL的比例，导致肿瘤中PD-L1水平降低，从而产生了良好的抗肿瘤效果。Compared with LG-12 monotherapy, a single injection of [131I ]LG-12 did not significantly inhibit tumor growth, but it could improve its anti-tumor efficacy by reshaping the immune microenvironment. In vitro and in vivo studies have shown that [131I ]LG-12 can induce tumor cell ICD to release HMGB1 protein and CRT protein, enhancing tumor immunogenicity. It can be seen that the combined use of [131I ]LG-12 and LG-12 has a synergistic effect with anti-tumor immunotherapy. This strategy upregulates the proportion of tumor-infiltrating CTLs, resulting in a decrease in PD-L1 levels in tumors, thereby producing a good anti-tumor effect.
因此，[131I]LG-12和LG-12联合增强抗肿瘤免疫应答。一方面，[131I]LG-12在肿瘤中诱导ICD，触发DMAPs的释放。DAMPs刺激未成熟树突状细胞的成熟，提高树突状细胞识别肿瘤和呈递抗原的能力。这些过程激活了肿瘤特异性CTLs，增加了IFN-γ的分泌，从而增强了肿瘤的免疫原性。另一方面，LG-12阻断PD-1/PD-L1信号通路，阻止肿瘤细胞的免疫逃逸。总的来说，该策略通过重塑肿瘤免疫微环境来增强抗肿瘤免疫应答，包括增强肿瘤抗原的呈递，提高对抗肿瘤免疫应答的敏感性，以及增加肿瘤浸润免疫细胞的比例。Therefore, [131I ]LG-12 and LG-12 combined enhance antitumor immune responses. On the one hand, [131I ]LG-12 induces ICD in tumors and triggers the release of DAMPs. DAMPs stimulate the maturation of immature dendritic cells and improve the ability of dendritic cells to recognize tumors and present antigens. These processes activate tumor-specific CTLs and increase the secretion of IFN-γ, thereby enhancing the immunogenicity of tumors. On the other hand, LG-12 blocks the PD-1/PD-L1 signaling pathway and prevents the immune escape of tumor cells. Overall, this strategy enhances antitumor immune responses by reshaping the tumor immune microenvironment, including enhancing the presentation of tumor antigens, increasing sensitivity to antitumor immune responses, and increasing the proportion of tumor-infiltrating immune cells.
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.
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