Remarkable progress has been made in the treatment of relapsed/refractory acute lymphocytic leukemia and non-Hodgkin lymphoma with CD19-targeted chimeric antigen receptor (CAR) T cells. In contrast, progress against CD19-negative hematological cancers and solid tumors has been limited. Intensive efforts to optimize cellular therapeutics for better efficacy include provision of cytokine support, countering immuno-suppression, and enhancing cellular delivery to solid tumors, which are all valid strategies. An additional significant hurdle is the lack of sufficient antigen to drive expansion and persistence. We present a strategy where we fuse the CD19 extracellular domain to a binding ligand to redirect CD19 CAR T cells towards other tumors while utilizing CD19 for sustained antigen presentation to promote cellular therapeutic expansion, efficacy and persistence. Then we detail CD19 extracellular domain engineering to make this approach generalizable. We used yeast surface display, saturation and multi-site mutagenesis, fluorescence activated cell sorting (FACS), and deep sequencing to identify mutations that enhanced stability and solubility of properly folded CD19. Mutation combinations identified as beneficial were programmed into a multi-site library which produced variants that were more thermostable, more protease-resistant, and with increased FMC63 anti-CD19 antibody binding as compared to wild-type. We show that the improved CD19 scaffolds recover and improve expression of the fusion proteins, that their affinity is at or above wild-type Kd, and they can redirect CAR T cells to CD19 to kill non-CD19 expressing targets. In addition, we map the epitope of antibody FMC63 against the extracellular CD19 domain.

Redirected CAR T activity via CD19-ligand fusions engineered by deep mutational scanning

2019 Spring National Meeting

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