Abstract 1784: FGFR4 and CD276 dual targeting CAR T cells demonstrate synergistic antitumor activity in childhood rhabdomyosarcoma
Authors: Tian Meijie , Wei Jun S. , Cheuk Adam , Milewski David , Zhang Zhongmei , Kim Yong Yuan , Liu Can , Badr Sherif , Kelly Michael C. , Wu Jerry T. , Rahmy Abdelrahman , Chou Hsien-Chao , Wen Xinyu , Khan Javed 
Published: April 2023

Background Chimeric antigen receptor T cells (CAR T) are potential therapies for rhabdosarcoma (RMS), the most common soft tissue sarcoma in children, where patients with relapsed or refractory disease have a dismal cure rate, and effective therapies are urgently needed. RMS tumors express high levels of the cell surface receptors FGFR4 and CD276, and both are direct targets of the PAX3-FOXO1 chimeric oncogene. However, CAR T have shown poor performance in solid tumors due to T cell exhaustion, limited persistence, and heterogeneous expression of target antigens. We hypothesize that engineering CAR constructs targeting FGFR4 and CD276 will enhance their activity and dual targeting will overcome the heterogenous target expression to effectively eliminate RMS tumors.

Methods We modified and tested different hinge and transmembrane domains (HTM) or intracellular domain (ICD) of FGFR4 CAR constructs and developed multiple dual targeting Bicistronic CARs (BiCisCARs) against FGFR4 and CD276. We tested these CARs in aggressive mouse models and performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) of tumor infiltrating CAR T cells to functionally characterize these constructs.

Results FGFR4 targeting CAR construct with CD8HTM and 4-1BB ICD could eliminate low burden RMS but was less effective against orthotopic tumors. Replacing CD8HTM with CD28HTM improved the efficacy in a moderate burden RMS model but did not show any benefit against a more aggressive RMS559 orthotopic model. Further modification using the CD28 ICD significantly enhanced the activity in a large burden RMS model but was unable to eradicate all tumors. Similarly, CAR T cells targeting CD276 alone, showed significant anti-tumor activity in moderate burden RMS models but could not eliminate all tumors. The BiCisCAR with FGFR4.CD28HTM.CD28ICD and CD276.CD8HTM.4-1BBICD was the most potent construct, eradicating 100% tumors in all tested orthotopic models, and those with heterogenous expression of target antigens. CITE-seq and flow cytometry assays demonstrated that this BiCisCAR showed the most significant tumor infiltration, and persistence, with limited exhaustion. This BiCisCAR exhibited a synergistic effect on cytokine production and anti-tumor activity, compared to single targeting CARs. Moreover, biochemical characterization revealed that the combined use of both CD28 and 4-1BB ICDs in the BiCisCAR resulted in the activation of three TCR downstream signaling pathways including AKT, Erk1/2 and p65.

Conclusions and Future DirectionsThus, we have developed a potent BiCisCAR with dual targeting of FGFR4 and CD276 that shows optimal biochemical activity, persistence, and limited exhaustion, and addresses heterogenous expression of target antigens. This BiCisCAR will be further developed for future clinical trials in patients with high-risk RMS.

Citation Format: Meijie Tian, Jun S. Wei, Adam Cheuk, David Milewski, Zhongmei Zhang, Yong Yuan Kim, Can Liu, Sherif Badr, Michael C. Kelly, Jerry T. Wu, Abdelrahman Rahmy, Hsien-Chao Chou, Xinyu Wen, Javed Khan. FGFR4 and CD276 dual targeting CAR T cells demonstrate synergistic antitumor activity in childhood rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1784.