(588a) A Low Affinity Humanized CD-19 Targeted Chimeric Antigen Receptor Efficiently Controls B-Cell Malignancy

Chimeric antigen receptor (CAR) engineered T cells have energized the field of cancer immunotherapy with their proven ability to treat hematologic malignancies in the clinic and emerging efficacy in treating other diseases. A CAR is a synthetic receptor that directs T cell activation toward an individual biomarker that a T cell would not ordinarily recognize. The antigen recognition domain that governs this specificity is currently under intense study as molecular parameters including binding affinity, stability, and immunogenicity risk dictate the ability of a CAR T cell therapy to perform. The current gold standard of antigen recognition domains is the CD19-targeted scFv FMC63, which has strong binding affinity, modest stability, and a known immunogenicity risk in humans due to its murine character. This work aims to develop humanized FMC63 variants with decreased immunogenicity risk while maintaining similar efficacy when applied to CAR antigen recognition. Multiple protein engineering strategies were applied to humanize FMC63 in silico yielding two lead candidate scFvs. Yeast surface display techniques were employed to characterize expression, binding affinity, and thermal stability of the FMC63 variants, revealing on the order of 1,000-fold weaker binding affinity but 6-7°C improvements in thermal stability of the humanized variants relative to parental. CARs utilizing these scFv variants demonstrated specific activity against CD19+ tumor cells and equivalent degranulation and cytokine production in vitro. Tumor rechallenge experiments in vitro reveal a binding affinity dependence for CAR T cells to proliferate in the presence of antigen. One CAR using a humanized FMC63 variant performed equivalently to the parental against CD19+ Raji and NALM6 models in vivo, demonstrating that a wide range of affinity levels can yield therapeutic impact with a CAR T cell therapy. Ongoing experiments explore the ability of these low affinity CARs to recognize CD19-low Raji variants, further illuminating the affinity-avidity interplay in CAR T cell activation.