Leucine zipper-based sorting system enables generation of multi-functional CAR T cells

Resistance to chimeric antigen receptor (CAR) T cell therapy develops through multiple mechanisms including antigen-loss escape and tumor-induced immune suppression. Expression of multiple CARs may overcome multi-antigen-loss escape. Similarly, expression of switch receptors that convert inhibitory immune checkpoint signals into positive costimulatory signals may enhance CAR T cell activity in the tumor microenvironment. Engineering multiple features into one cell product, however, is limited by transgene packaging constraints of current vector systems. Here, we describe a leucine zipper-based cell sorting methodology that enables selective single-step immunomagnetic purification of cells co-transduced with two vectors, designed to potentially double the number of incorporated transgenes. This “Zip-sorting” system facilitated generation of T cells simultaneously expressing up to four CARs and co-expressing up to three switch receptors. These multi-CAR multi-Switch receptor arrays enabled T cells to eliminate antigenically heterogeneous syngeneic leukemia populations co-expressing multiple inhibitory ligands. Zip-sorted multi-CAR multi-Switch receptor T cells represent a potent therapeutic strategy to overcome multiple mechanisms of CAR T cell resistance. ### Competing Interest Statement S.E. James, L. Jahn., and M.R.M. van den Brink are co-inventors on patent applications related to this work. J. U. Peled reports research funding, intellectual property fees, and travel reimbursement from Seres Therapeutics, and consulting fees from DaVolterra, CSL Behring, and from MaaT Pharma. He serves on an Advisory board of and holds equity in Postbiotics Plus Research. He has filed intellectual property applications related to the microbiome (reference numbers #62/843,849, #62/977,908, and #15/756,845). S.A. Vardhana is an advisor for Immunai and has received consulting fees from Koch Disruptive Technologies. C.A. Klebanoff is on the scientific and/or clinical advisory boards for Achilles Therapeutics, Aleta BioTherapeutics, Bellicum Pharmaceuticals, Catamaran Bio, Obsidian Therapeutics, and T-knife; has consulted for Bristol Myers Squibb, Decheng Capital, PACT Pharma, and Roche/Genentech; and has provisional patents related to the Fas DNR described in this manuscript. M.R.M. van den Brink has received research support and stock options from Seres Therapeutics and stock options from Notch Therapeutics and Pluto Therapeutics; he has received royalties from Wolters Kluwer; has consulted, received honorarium from or participated in advisory boards for Seres Therapeutics, Vor Biopharma, Rheos Medicines, Frazier Healthcare Partners, Nektar Therapeutics, Notch Therapeutics, Ceramedix, Lygenesis, Pluto Therapeutics, GlaskoSmithKline, Da Volterra, Thymofox, Garuda, Novartis (Spouse), Synthekine (Spouse), Beigene (Spouse), Kite (Spouse); he has IP Licensing with Seres Therapeutics and Juno Therapeutics; and holds a fiduciary role on the Foundation Board of DKMS (a nonprofit organization). MSKCC has institutional financial interests relative to Seres Therapeutics.