The murine anti-CD19 scFv FMC63 is employed in FDA-approved CAR T cell therapies. To reduce the potential for immunogenicity, Shukla et al. screened a library of fully human anti-CD19 scFvs for expression, functionality (CD19 binding and cytokine production), and tonic signaling. The best binders killed target cells in vitro, controlled tumors in vivo, and responded to low antigen densities. The top fully human scFv bound a different region of CD19 than FMC63, had weaker CD19 affinity (25nM vs. 0.6nM), and demonstrated comparable or superior in vitro tumor killing, CD19-low responses, and in vivo efficacy to FMC63, and is currently being tested in the clinic.
Contributed by Alex Najibi
ABSTRACT: Patients can develop human anti-mouse immune responses against CD19-specific chimeric antigen receptor (CAR) T cells due to the use of a murine CD19-specific single-chain variable fragment to redirect T cells. We screened a yeast display library to identify an array of fully human CD19 single-chain variable fragment binders and performed a series of studies to select the most promising fully human CAR. We observed significant differences in the ability of CARs employing these CD19 binders to be expressed on the cell surface, induce tonic signaling, redirect T-cell function, mediate tumor killing, recognize lower levels of CD19 antigen, and maintain function upon continuous antigen exposure. From this initial analysis, CAR T cells using two binders (42 and 52) were selected for additional studies. Although CAR T cells using both binders controlled tumor growth well in vivo, we advanced a CAR construct using binder 42 for more advanced preclinical testing because of its greater similarity to binders based on the antibody FMC63, which is the murine antibody underlying four FDA-approved CD19-specific CAR T-cell therapies, and ability to robustly respond to tumors expressing lower levels of CD19. We found that this binder uniquely bound CD19 using distinct contact residues than FMC63 and with _40-fold lower affinity. CARs using binder 42 were non-inferior to those using the FMC63 binder in a mouse model of acute lymphoblastic leukemia, indicating that CAR T cells using binder 42 should be considered for clinical use.
Author Info: (1) Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania. Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, Pennsylvania.
Author Info: (1) Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania. Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, Pennsylvania. (2) Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, Pennsylvania. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. (3) Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, Pennsylvania. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. (4) Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, Pennsylvania. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. (5) Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, Pennsylvania. (6) Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, Pennsylvania. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. (7) Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, Pennsylvania. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. (8) Tmunity Therapeutics/Kite Pharma, Philadelphia, Pennsylvania. (9) Tmunity Therapeutics/Kite Pharma, Philadelphia, Pennsylvania. (10) Tmunity Therapeutics/Kite Pharma, Philadelphia, Pennsylvania. (11) Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, Pennsylvania. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. (12) Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, Pennsylvania. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. Parker Institute for Cancer Immunotherapy, University of Pennsylvania, Philadelphia, Pennsylvania. (13) Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, Pennsylvania. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania. (14) Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania. Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, Pennsylvania.
