285. Exploring On-Target Hematopoietic Toxicity With Highly Potent, High Affinity FRβ-Specific CAR T Cells for AML

Abstract

On-target, off-tumor recognition of antigen in normal tissues is one of the largest safety concerns in chimeric antigen receptor (CAR) T cell therapy. Careful assessment of target protein expression by flow cytometry or IHC can provide helpful data for prediction of potential toxicity. However, high affinity reagents should be carefully chosen in order to assure the most sensitive and thorough investigation of target antigen levels, which may be low in normal tissues. We are currently optimizing our folate receptor beta (FRβ)-directed CAR T cell platform for acute myeloid leukemia (AML) and have isolated a high affinity antibody single-chain-variable fragment (scFv) for production of CAR T cells. The high affinity (m923, 2.48nM KD) CAR T cells exhibited greatly enhanced reactivity against FRβ(+) AML in vitro and in vivo compared to previously generated lower affinity (m909, 54.3nM KD) FRβ-specific CAR T cells. However, higher affinity reagents pose the potential for increased risk of toxicity directed against normal tissues. To most accurately predict m923 CAR T cell toxicity, we utilized high affinity m923 IgG for analysis of FRβ expression in normal hematopoietic tissues by flow cytometry. We were able to detect high levels of FRβ in peripheral blood monocytes as well as low levels in myeloid-lineage bone marrow progenitor cells which were non-detectable using m909 IgG. Notably, CD34(+) bone marrow hematopoietic stem cells (HSCs) displayed very low expression of FRβ and m923 CAR T cells did not inhibit HSC colony formation in classic CFU assays. We next examined the ability of m923 CAR T cells to target whole bone marrow populations. Upon co-culture, m923 CAR T cells were able to eliminate FRβ(+) myeloid progenitors while sparing neighboring FRβ(-) bone marrow cells. We hypothesized that, because high affinity FRβ-specific CAR T cells did not impact bone marrow HSCs and non-myeloid-committed progenitors, short-term treatment with CAR T cells may allow for tumor clearance, following which any affected myeloid populations could be reconstituted from healthy HSCs. Therefore, we investigated transient expression via mRNA electroporation of the m923 CAR platform. mRNA-CAR T cells retained effective anti-tumor activity. Our results highlight the importance of antibody affinity for assessment of target protein during CAR development. In addition, we report a highly potent, high affinity FRβ-specific CAR T cell platform that does not target HSCs, and, when delivered transiently through mRNA electroporation, could allow for AML tumor destruction while providing a decreased risk for long-term myeloid toxicity.