We show continuous tumor exposure results in a loss of chimeric antigen receptor (CAR) T cell (CART) endocytic activity due to downregulation of Rab5. Loss of endocytic activity exacerbates the effects of trogocytosis, the bidirectional transfer of tumor target antigens and CARs between malignant cells and CARTs, resulting in CART dysfunction and fratricide. Constitutive expression of Rab5 within the CARTs reduced fratricide by reducing the amount of trogocytosed antigens on the cell surface, while simultaneously enhancing CAR availability through dissociation of CAR from target, recycling unbound CAR back to the plasma membrane, and limiting CAR capture by tumor cells. Rab5-expressing CARTs exhibited superior antitumor activity in both BCMA-CARTs isolated from the bone marrow of treated patients and mesothelin-specific CARTs in a solid tumor model. These studies uncover an unexpected relationship between endocytosis and CART function and suggest that pairing Rab5 with CAR expression could improve the clinical efficacy of CART therapy.
Rab5 improves CAR T cell efficacy via reducing fratricide and maintaining surface CAR levels
https://orcid.org/0000-0001-5704-5064
https://orcid.org/0000-0003-2076-6794
https://orcid.org/0000-0001-9218-4318
https://orcid.org/0000-0003-3009-7686
https://orcid.org/0009-0003-5617-6494
https://orcid.org/0000-0002-6275-5789
https://orcid.org/0000-0003-2098-2251
https://orcid.org/0000-0002-9551-9491
https://orcid.org/0000-0002-1580-9844
https://orcid.org/0000-0003-0939-3843
https://orcid.org/0000-0003-2791-5748
https://orcid.org/0000-0002-1057-576X
Disclosures: M. Gu reported a patent to Augmenting CAR T cell activity pending “WO2025171295A1.” N.C. Sheppard reported grants from ScaleReady; personal fees from BlueWhale Bio, Waypoint Bio,Pan Cancer T, UroGen Pharma, and Able Sciences; and “other” from CARTx Therapeutics; in addition, N.C. Sheppard had a patent to WO2024206569A2 pending. M.C. Milone reported “other” from Verismo Therapeutics and Cabaletta Bio outside the submitted work. A.L. Garfall reported grants from Novartis and NIH/NCI during the conduct of the study; and personal fees from Novartis, Johnson & Johnson, Regeneron, Gracell, AstraZeneca, Pfizer, BMS, MJH Life Sciences, DAVA Oncology, and Curio Science outside the submitted work; in addition, A.L. Garfall had a patent to US11747346B2 licensed “Novartis,” a patent to US20200371091A1 pending, a patent to US20200360431A1 pending, and a patent to WO2024148212A2 pending. J.L. Riley reported grants, personal fees, and “other” from Tmunity/Kite Pharma/Gilead during the conduct of the study; and grants from BlueWhale Bio and “other” from BlueWhale Bio outside the submitted work; in addition, J.L. Riley had a patent to Augmenting car t cell activity pending and a patent to Augmenting car T cell activity licensed “Kite Pharma.” No other disclosures were reported.
