Targeting the membrane-proximal C2-set domain of CD33 for improved CAR T cell therapy.

Current CD33-targeted immunotherapies typically recognize the membrane-distal V-set domain of CD33. Here, we show that decreasing the distance between T cell and leukemia cell membrane increases the efficacy of CD33 chimeric antigen receptor (CAR) T cells. We therefore generated and optimized second-generation CAR constructs containing single-chain variable fragments from antibodies raised against the membrane-proximal C2-set domain, which bind CD33 regardless of whether the V-set domain is present (CD33 ^PAN antibodies). CD33 ^PAN CAR T cells resulted in efficient tumor clearance and improved survival of immunodeficient mice bearing human AML cell xenografts and, in an AML model with limited CD33 expression, forced escape of CD33 ^neg leukemia. Compared to CD33 ^V-set CAR T cells, CD33 ^PAN CAR T cells showed greater in vitro and in vivo efficacy against several human AML cell lines with differing levels of CD33 without increased expression of exhaustion markers. CD33 ^PAN moieties were detected at a higher frequency on human leukemic stem cells, and CD33 ^PAN CAR T cells had greater in vitro efficacy against primary human AML cells. Together, our studies demonstrate improved efficacy with CAR T cells binding CD33 close to the cell membrane, providing the rationale to investigate CD33 ^PAN CAR T cells further toward possible clinical application.
Competing Interests: S.F., G.S.L., C.J.T., and R.B.W. filed a patent application related to CD33 antibodies and CD33-directed CAR T cell therapy. S.F. has filed patents on optimizing CAR T cell cytotoxicity, received research laboratory grants from Bristol Myers Squibb, and is a consultant for Prescient Therapeutics. E.L.K. received research support from Juno Therapeutics. H.-P.K. received support as the inaugural recipient of the José Carreras/E. Donnall Thomas Endowed Chair for Cancer Research and the Stephanus Family Endowed Chair for Cell and Gene Therapy, and is or was a consultant to and has or had ownership interests with Rocket Pharma, Homology Medicines, VOR, and Ensoma. C.J.T. received research funding from Juno Therapeutics/BMS and Nektar Therapeutics; serves on advisory boards for Caribou Biosciences, T-CURX, Myeloid Therapeutics, ArsenalBio, Cargo Therapeutics, and Celgene/BMS Cell Therapy; has served on ad hoc advisory boards/consulting (last 12 months) for Nektar Therapeutics, Century Therapeutics, Legend Biotech, Allogene, Sobi, Syncopation Life Sciences, Prescient Therapeutics, Orna Therapeutics, and IGM Biosciences; and holds stock options in Eureka Therapeutics, Caribou Biosciences, Myeloid Therapeutics, ArsenalBio, and Cargo Therapeutics. R.B.W. received laboratory research grants and/or clinical trial support from Aptevo, Celgene/Bristol Myers Squibb, ImmunoGen, Janssen, Jazz, Kite, Kura, Pfizer, and VOR, and has been a consultant to Wugen.
(© 2024 The Author(s).)