肿瘤浸润CD8^(+)T细胞SPECT/CT显像预测抗PD-1免疫治疗疗效的小鼠实验

SPECT/CT imaging of tumor-infiltrating CD8^(+)T cell to predict the efficacy of anti-PD-1 immunotherapy in mice

目的制备^(99)Tc^(m)-联肼尼克酰胺(HYNIC)-αCD8/Fab(^(99)Tc^(m)-αCD8/Fab)探针,并探讨其用于SPECT/CT显像以预测抗程序性细胞死亡蛋白-1(PD-1)免疫治疗疗效的价值。方法合成前体HYNIC-αCD8/Fab和IRDye800-αCD8/Fab(Dye-αCD8/Fab);以SnCl2为还原剂,进行^(99)Tc^(m)与前体HYNIC-αCD8/Fab的标记反应,制备^(99)Tc^(m)-αCD8/Fab探针,分析其标记率、放化纯及稳定性,并探究其与体外淋巴细胞结合的特异性。建立BALB/c小鼠CT26结直肠肿瘤模型,通过SPECT/CT显像分析^(99)Tc^(m)-αCD8/Fab的体内特异性结合能力;对荷瘤小鼠行抗PD-1治疗,然后行近红外荧光成像及SPECT/CT显像,检测肿瘤CD8+T细胞浸润状况,并用HE染色和免疫荧光检测验证;经抗PD-1治疗后的荷瘤小鼠尾静脉给予抗CD8抗体,分析其损耗抗PD-1治疗疗效的情况。采用双因素方差分析进行组间差异比较。结果^(99)Tc^(m)-αCD8/Fab的标记率为90%,放化纯为95%,于PBS和胎牛血清(FBS)中放置720 min,放化纯仍达80%以上,稳定性良好;细胞结合实验显示,^(99)Tc^(m)-αCD8/Fab与小鼠脾CD8+T细胞受体和人外周血CD8+T细胞受体结合值分别为(10.30±0.81)和(1.78±0.61)百分加入放射性剂量(%AD)/106个细胞,过量冷αCD8抗体可导致结合值降低至(1.59±0.25)%AD/106个细胞(F=10.07,P<0.001)。SPECT/CT显像示,^(99)Tc^(m)-αCD8/Fab在小鼠CT26结直肠肿瘤模型中具有良好的肿瘤摄取。近红外荧光成像示,对抗PD-1免疫治疗响应组小鼠肿瘤荧光强度为(8.9±1.1)%,明显高于非响应组的(7.1±0.8)%(F=4.69,P=0.024),SPECT/CT显像同样显示响应组肿瘤摄取较高;HE和免疫荧光结果表明,响应组肿瘤和淋巴结组织中淋巴细胞浸润比例明显高于非响应组。此外,CD8+T细胞损耗显著削弱了抗PD-1治疗疗效。结论成功制备了^(99)Tc^(m)-αCD8/Fab,该探针能对肿瘤浸润CD8+T细胞清晰显像;CD8靶向SPECT显像对临床预测和评估免疫治疗疗效具有潜在的应用价值。

Objective To prepare^(99)Tc^(m)-hydrazinonicotinamide(HYNIC)-αCD8/Fab(^(99)Tc^(m)-αCD8/Fab),and explore the predictive value of^(99)Tc^(m)-αCD8/Fab SPECT/CT imaging for the efficacy of anti-programmed death-1(PD-1)immunotherapy.Methods TheαCD8/Fab was modified with HYNIC-N-hydroxysuccinimide(NHS)and IRDye800-NHS to form HYNIC-αCD8/Fab and IRDye800-αCD8/Fab(Dye-αCD8/Fab),respectively.^(99)Tc^(m)-αCD8/Fab was prepared in sodium bicarbonate buffer(pH=8.5),with SnCl2 being used as the reducing agent.The labeling yield and radiochemical purity of^(99)Tc^(m)-αCD8/Fab and its stability in PBS and fetal bovine serum(FBS)were tested in vitro.The mouse spleen and human peripheral blood lymphocytes were isolated for cell-specific binding and blocking experiments of^(99)Tc^(m)-αCD8/Fab in vitro.SPECT/CT imaging was used to analyze the specific binding ability of the^(99)Tc^(m)-αCD8/Fab probe in CT26 colon cancer mouse models(BALB/c).The near infrared fluorescence imaging and SPECT/CT imaging were performed to detect the intra-tumoral CD8+T cell infiltration after anti-PD-1 therapy in tumor bearing mice,and the results were further verified by HE and immunofluorescence staining.CD8+T cell depletion study was performed to determine the role of CD8+T cells in the tumor responses to anti-PD-1 therapy.Two-way analysis of variance was used to compare the data difference.Results The labeling yield of^(99)Tc^(m)-αCD8/Fab was 90%with a high radiochemical purity(95%)and good stability in vitro(radiochemical purity still more than 80%after 720 min in PBS and FBS).^(99)Tc^(m)-αCD8/Fab could specifically bind to mouse CD8+T cells((10.30±0.81)percent added radioactive dose(%AD)/106 cells),compared with the binding ability in human peripheral blood lymphocytes group and CD8 antibody blocking group((1.78±0.61)and(1.59±0.25)%AD/106 cells;F=10.07,P<0.001).SPECT/CT imaging showed that^(99)Tc^(m)-αCD8/Fab had markedly higher tumor uptake in the CT26 colon cancer mouse models.Near-infrared fluorescence imaging showed that the tumor uptake of^(99)Tc^(m)-αCD8/Fab in the responsive group was significantly higher than in the nonresponsive group after anti-PD-1 treatment((8.9±1.1)%vs(7.1±0.8)%;F=4.69,P=0.024),and SPECT/CT imaging found the similar result.HE and immunofluorescence staining of tumor and lymph nodes showed that the proportion of lymphocyte infiltration was higher in the responsive group.Furthermore,CD8+T cell depletion significantly reversed the therapeutic effect of anti-PD-1 immunotherapy in tumor-bearing mice.Conclusions In this study,^(99)Tc^(m)-αCD8/Fab was successfully obtained.CD8-specific SPECT imaging could sensitively visualize the tumor-infiltrating CD8+T cells,suggesting the potential application value to predict and evaluate the efficacy of immunotherapy in the clinical settings.