title: Redirecting T-Cell Specificity to EGFR Using mRNA to Self-limit Expression of Chimeric Antigen Receptor.
abstract: Potential for on-target, but off-tissue toxicity limits therapeutic application of genetically modified T cells constitutively expressing chimeric antigen receptors (CARs) from tumor-associated antigens expressed in normal tissue, such as epidermal growth factor receptor (EGFR). Curtailing expression of CAR through modification of T cells by in vitro-transcribed mRNA species is one strategy to mitigate such toxicity. We evaluated expression of an EGFR-specific CAR coded from introduced mRNA in human T cells numerically expanded ex vivo to clinically significant numbers through coculture with activating and propagating cells (AaPC) derived from K562 preloaded with anti-CD3 antibody. The density of AaPC could be adjusted to affect phenotype of T cells such that reduced ratio of AaPC resulted in higher proportion of CD8 and central memory T cells that were more conducive to electrotransfer of mRNA than T cells expanded with high ratios of AaPC. RNA-modified CAR T cells produced less cytokine, but demonstrated similar cytolytic capacity as DNA-modified CAR T cells in response to EGFR-expressing glioblastoma cells. Expression of CAR by mRNA transfer was transient and accelerated by stimulation with cytokine and antigen. Loss of CAR abrogated T-cell function in response to tumor and normal cells expressing EGFR. We describe a clinically applicable method to propagate and modify T cells to transiently express EGFR-specific CAR to target EGFR-expressing tumor cells that may be used to limit on-target, off-tissue toxicity to normal tissue.
authors.affiliation: *Division of Pediatrics Departments of ‡Biostatistics ∥Neurosurgery §Stem Cell Transplantation and Cellular Therapy, The University of Texas MD Anderson Cancer Center †The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX.
authors.forename: Hillary G
authors.initials: HG
authors.lastname: Caruso
authors.forename: Hiroki
authors.initials: H
authors.lastname: Torikai
authors.forename: Ling
authors.initials: L
authors.lastname: Zhang
authors.forename: Sourindra
authors.initials: S
authors.lastname: Maiti
authors.forename: Jianliang
authors.initials: J
authors.lastname: Dai
authors.forename: Kim-Anh
authors.initials: KA
authors.lastname: Do
authors.forename: Harjeet
authors.initials: H
authors.lastname: Singh
authors.forename: Helen
authors.initials: H
authors.lastname: Huls
authors.forename: Dean A
authors.initials: DA
authors.lastname: Lee
authors.forename: Richard E
authors.initials: RE
authors.lastname: Champlin
authors.forename: Amy B
authors.initials: AB
authors.lastname: Heimberger
authors.forename: Laurence J N
authors.initials: LJ
authors.lastname: Cooper
mesh_terms: D000951:Antigens, Neoplasm / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000502:physiology*; D019496:Cancer Vaccines / Q000276:immunology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018920:Coculture Techniques; D066246:ErbB Receptors / Q000276:immunology; D005818:Genetic Engineering; D005909:Glioblastoma / Q000276:immunology / Q000628:therapy*; D006801:Humans; D007156:Immunologic Memory; D016219:Immunotherapy, Adoptive / Q000379:methods*; D008213:Lymphocyte Activation; D012333:RNA, Messenger / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D037182:T-Cell Antigen Receptor Specificity; D016176:T-Lymphocyte Subsets / Q000502:physiology*
chemical_list: D000951:Antigens, Neoplasm; D019496:Cancer Vaccines; D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell; C512478:EGFR protein, human; D066246:ErbB Receptors