Abstract 3796: CAR-T cells secreting anti-PD1/anti-CTLA4 bispecific antibody exhibit superior antitumor efficacy for ccRCC treatment
Authors: Nithyassree Murugan, Yufei Wang, Paul Fallon, Matthew R. Chang, Gabriella Kastrunes, Christian D. Coherd, Wayne A. Marasco
Journal: Cancer Research (2025)

Abstract Clear cell renal cell carcinoma (ccRCC) has been reported to have high presentation of tumor associated antigens (TAA) carbonic anhydrase IX (CAIX) and cluster of differentiation 70 (CD70) regardless of stage of tumor. Single-cell RNA sequencing (scRNA-seq) studies of patient samples have shown that advanced ccRCC patients have enriched populations of exhausted CD8+ T cells, which present immune checkpoint (IC) molecules such as PD1 and CTLA4 as potential therapeutic targets. Anti-PD1 and anti-CTLA4 act independently to reinvigorate T cells, with the former directly blocking effector cell exhaustion and the latter preventing Treg-mediated negative regulation. The combination therapy of anti-PD1 Nivolumab and anti-CTLA4 Ipilimumab exhibits synergistic effects and has superior therapeutic effectiveness to mono-therapy in advanced ccRCC treatment. A bispecific antibody (BsAb) has advantages compared to monospecific antibody, including a superior efficacy, reduced production cost, and less systemic side effects. The P4B3-IgGH3-E1A8 is an anti-PD1/anti-CTLA4 tetravalent, bispecific antibody, the four scFvs linked by the IgG3 hinge. The BsAb is capable of binding to both CTLA4 and PD1 on the surface of T cells preventing ligand binding to CD80 and PD-L1 respectively. In effect, P4B3-IgGH3-E1A8 prevents IC mediated T cell exhaustion and exhibited superior inflammatory cytokine interleukin-2 (IL-2) secretion from PBMCs compared to an equimolar cocktail of its monospecific counterparts and also Ipilimumab and Nivolumab. We designed the dual-targeted fine-tuned immune restoring (DFIR) chimeric antigen receptor (CAR)-T to simultaneously target CAIX and CD70 on the surface of tumor cells and also secrete P4B3-IgGH3-E1A8 as a payload. As the use of CAR-T cells acquired by label-based sorting approaches is not advised for infusion into patients, we isolated DFIR CAR-T cells using the Vision-Sort platform that utilizes high-dimensional morphological profiling and AI to enable label-free cell sorting and unbiased single cell profiling. CAR positive T cells were utilized for the downstream assays. Using an MSD multiplex quantification assay, we showed that DFIR CAR-T cells secreted the bispecific antibody in culture, inhibited growth of Skrc-59 ccRCC tumor cells in-vitro and retained greater levels of cytotoxic activity in a multi-challenge assay. P4B3-IgGH3-E1A8 secreting CAR-T exhibited greater tumor eradication in humanized NSG-SGM3 mice when compared to the CAR-T with an irrelevant payload. In all, we report DFIR CAR-T cell secreting anti-PD1/anti-CTLA4 bispecific antibody has potential to achieve ccRCC cures. Citation Format: Nithyassree Murugan, Yufei Wang, Paul Fallon, Matthew R. Chang, Gabriella Kastrunes, Christian D. Coherd, Wayne A. Marasco. CAR-T cells secreting anti-PD1/anti-CTLA4 bispecific antibody exhibit superior antitumor efficacy for ccRCC treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3796.