Engineering anti-CD229 CAR T cell selectivity for multiple myeloma

T cells expressing high affinity chimeric antigen receptors (CAR) against CD229 are able to eradicate multiple myeloma (MM) cells, which express high CD229 levels. However, they also target healthy T cells expressing low CD229 levels. Reducing the affinity of the parental CD229 CAR may increase the selectivity of CD229 CAR T cells, sparing T cells while maintaining activity against MM cells. No robust approaches to engineer minimally altered low affinity variants from existing high-affinity antibodies have been developed. We generated a site-saturation mutagenesis library of the parental CD229 antibody targeting both CDR3 domains resulting in 305 variants. Variants were expressed as soluble single-chain variable fragments and screened using high-throughput assays to determine binding and expression. Affinities were determined by biolayer interferometry. A subset of 26 binders was further characterized functionally in CAR format using primary human T cells. Variant CD229 antibodies carrying single amino acid substitutions showed substantially altered expression and binding of recombinant CD229. Affinities of candidate antibodies varied from 150–10,000nM with mainly off-rate driven reductions in affinity. Analyzing the cytotoxic activity of CD229 CAR variants we found that, in contrast to the parental cells, two variant CAR T cells spared normal T cells while maintaining equal cytotoxic activity against MM cells. Using a novel unbiased mutagenesis approach, we show that generation of minimally altered low affinity variants from existing antibodies is feasible and results in binders with substantially reduced affinity. Low affinity variants show increased selectivity eliminating a key liability of CD229 CAR T cells. E.R.V received funding from the American Foundation for Pharmaceutical Education (AFPE, Pre-Doctoral Fellowship) and the ALSAM Foundation (Skaggs Fellowship). D.A. received funding from the Huntsman Cancer Institute (HCI, Translational Scholar Award). T.L. received funding from the National Comprehensive Cancer Network (NCCN, Young Investigator Award).