Spatial transcriptomics data of RM9-hSTEAP1 tumor tissues treated with CAR-T cells

Immunosuppressive microenvironments, the lack of immune infiltration, and antigen heterogeneity pose challenges for chimeric antigen receptor (CAR)-T cell therapies applied to solid tumors. Previously, CAR-T cells were armored with immunostimulatory molecules, such as interleukin-12 (IL-12), to overcome this issue, but faced high toxicity. Here, we show that collagen-binding domain-fused IL-12 (CBD-IL-12) secreted from CAR-T cells to target human six transmembrane epithelial antigen of the prostate 1 (STEAP1) is retained within murine prostate tumors. This leads to high intratumoral interferon-? levels, without hepatotoxicity and infiltration of T cells into non-target organs compared with unmodified IL-12. Both innate and adaptive immune compartments are activated and recognize diverse tumor antigens after CBD-IL-12-armored CAR-T cell treatment. Combination of CBD-IL-12-armored CAR-T cells and immune checkpoint inhibitors eradicated large tumors in an established prostate cancer mouse model. Additionally, human CBD-IL-12-armored CAR-T cells showed potent anti-tumor efficacy in an 22Rv1 xenograft while reducing circulating IL-12 levels compared with unmodified IL-12-armored CAR-T cells. CBD-fusion to potent payloads of CAR-T therapy may remove obstacles to their clinical translation towards elimination of solid tumors. Overall design: NanoString GeoMx Digital Spatial Profiler (DSP) whole transcriptome analysis (WTA) was performed on RM9-hSTEAP1 tumor samples collected at 10 days after CAR-T cell treatment. PFA-fixed, paraffin embedded tumor blocks were sectioned, and Twelve regions of interest (ROI) were chosen across four different tumor sections in each treated group followed by library preparation and sequencing. A tumor sample in unarmored CAR-T treatment group was not used because the tissue section was damaged.

免疫抑制微环境、免疫浸润缺失以及抗原异质性，给实体瘤领域的嵌合抗原受体（chimeric antigen receptor, CAR）T细胞疗法带来了诸多挑战。此前已有研究通过共表达免疫刺激分子（如白细胞介素-12（interleukin-12, IL-12)）对CAR-T细胞进行武装以克服该难题，但往往伴随严重的毒性反应。本研究证实，由CAR-T细胞分泌并靶向人前列腺六跨膜上皮抗原1（six transmembrane epithelial antigen of the prostate 1, STEAP1）的胶原结合结构域融合型IL-12（collagen-binding domain-fused IL-12, CBD-IL-12），可在小鼠前列腺肿瘤组织中滞留。与未修饰的IL-12相比，该疗法可使肿瘤内干扰素-γ（interferon-γ, IFN-γ）水平显著升高，且不会引发肝毒性，同时未出现T细胞向非靶器官浸润的情况。经CBD-IL-12武装的CAR-T细胞治疗后，先天免疫与适应性免疫组分均被激活，且可识别多种肿瘤抗原。将CBD-IL-12武装的CAR-T细胞与免疫检查点抑制剂联合使用，可在已构建的前列腺癌小鼠模型中完全清除大型肿瘤。此外，与未修饰IL-12武装的CAR-T细胞相比，人源CBD-IL-12武装的CAR-T细胞在22Rv1异种移植瘤模型中展现出更强的抗肿瘤活性，同时可降低循环IL-12水平。将胶原结合结构域融合至CAR-T疗法的强效效应分子中，或可破除实体瘤临床转化的诸多障碍。
本研究的实验设计如下：对CAR-T细胞治疗10天后采集的RM9-hSTEAP1肿瘤样本，进行NanoString GeoMx数字空间分析仪（Digital Spatial Profiler, DSP）全转录组分析（whole transcriptome analysis, WTA）。将多聚甲醛（paraformaldehyde, PFA）固定、石蜡包埋的肿瘤组织块进行切片，在每个处理组的4张不同肿瘤切片中选取12个感兴趣区域（region of interest, ROI），随后进行文库制备与测序。未武装CAR-T治疗组的1例肿瘤样本因组织切片受损，未纳入后续分析。