Abstract 552: FGFR4 and CD276 dualtargeting CAR-T cells for treating rhabdomyosarcoma and other solid tumors
Authors: Tian Meijie , Cheuk Adam , Milewski David , Wei Jun S. , Chou Hsien-Chao , Kim Yong Yean , Song Young K. , St. Croix Brad , Ho Mitchell , Khan Javed 
Published: June 2022

Abstract
Background: Chimeric antigen receptor T-cell therapies (CAR-T) have shown success in treating refractory and relapsed leukemia and lymphoma, while they perform poorly in solid tumors due to heterogenous expression of tumor-associated antigens (TAAs), limited T cell persistence and propensity for exhaustion. The receptor tyrosine kinase FGFR4 and immune checkpoint molecule CD276 are highly and heterogeneously expressed in some solid tumors, including Rhabdomyosarcoma (RMS), a most common soft tissue sarcoma of childhood, and human hepatocellular carcinoma (HCC). However, their expression is usually low in normal human tissues. These features make FGFR4 and CD276 promising therapeutic targets for CAR-T therapy for RMS and HCC. We have developed a FGFR4 targeting CAR construct (3A11-BBz) with a CD8 hinge (H) and a transmembrane domain (TM) infused with a 4-1BB intracellular domain (ICD). 3A11-BBz CAR can efficiently eliminate low RMS disease burden in metastatic models, but less effectively for bulky disease in RMS intramuscular (I.M.) xenograft models. Testing of a CD276 targeting CAR T-cells showed significant shrinking of tumors in RMS I.M. xenograft models.
Methods: To improve the CAR-T cells efficacy, we first modified the H/TM and ICD of 3A11-BBz CAR to CD28 (3A11-CD28z). To overcome tumor heterogeneity, we also created Bicistronic CARs (BiCisCARs) combining the complete FGFR4 and CD276 CAR into a single construct allowing co-expression of both constructs on the same T cells. We then tested the efficacy of these CARs in-vitro and in-vivo using intramuscular FP-RMS xenograft (RH30) or HCC intraperitoneal models.
Results and Conclusions: We found either FGFR4 targeting CARs or dual targeting BiCisCARs, showed similar in-vitro cytotoxicity against RMS cells and HCC cells. However, CARs with CD28 ICD released more IL-2 than those with 4-1BB ICD when co-cultured with target cells. In RMS I.M. xenograft model, 3A11-CD28z CAR-T cells shrank and eliminated the tumor in 5/8 mice whereas 3A11-BBz only suppressed tumor growth. Furthermore, 3A11-CD28z BiCisCAR eradicated tumor cells in 8/8 mice, whereas 3A11-BBz BiCisCAR showed very poor efficacy. Moreover, there are more 3A11-CD28z BiCisCAR T-cells persisting in blood and spleen than the other bicistronic or single CAR-T cells, suggesting this BiCisCAR-T cells have prolonged persistence. Therefore, we have developed a potent BiCisCAR dual targeting both FGFR4 and CD276 that overcomes RMS heterogeneity and effectively eliminates tumors in-vivo, which will be developed as a future therapeutic CAR for clinical trials.
Citation Format: Meijie Tian, Adam Cheuk, David Milewski, Jun S. Wei, Hsien-Chao Chou, Yong Yean Kim, Young K. Song, Brad St. Croix, Mitchell Ho, Javed Khan. FGFR4 and CD276 dualtargeting CAR-T cells for treating rhabdomyosarcoma and other solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 552.