Abstract Chimeric antigen receptors (CARs) targeting CD19 demonstrate remarkable clinical outcomes in patients (pts) with B-cell malignancies. Importantly, all four FDA-approved CD3ζ-based CAR T cell therapies use the same FMC63-derived single-chain variable fragment (scFv) to target CD19+ tumors. Although response rates are high in diffuse large B cell lymphoma, only ~40% of pts achieve durable, long-term remissions. Improved CAR T cell therapies are therefore needed. We have previously shown that a novel killer immunoglobulin receptor (KIR)-based CAR expressed in T cells as a multichain receptor with DAP12 (KIR-CAR T) has enhanced anti-tumor activity in several preclinical solid tumor models suggesting that this platform may show better activity in lymphoma. Some recent studies report that lower-affinity CD19 binders compared to the benchmark FMC63-scFv yield equivalent or increased anti-tumor activity of CAR T in pts, raising the question of optimal affinity for a KIR-based CAR. In this study, we aimed to evaluate the relationship of affinity, functional avidity, and in vivo potency of CD19 binders in a KIR-based CAR T cell. Using a highly-diverse 40-billion member canine scFv phage display library, we generated 19 unique human CD19-specific binders that compete with FMC63 binder supporting a similar binding epitope. Different KIR-CAR T cells were generated, and relative affinities were compared via binding to a recombinant (rec) CD19 protein. We found that although CAR receptor expression was similarly expressed, the concentration of soluble rec CD19 protein required to saturate the surface CAR varied more than 2-log among the different scFv binders suggesting a range of affinities relative to FMC63. We selected several scFvs with lower, and equivalent CD19 binding MFI compared to the FMC63-scFv and found that all scFvs could mediate similarly robust in vitro potency. In vivo anti-tumor efficacy was next assessed using the NSG xenograft mouse model of B-cell leukemia NALM6 cells. Interestingly, despite their comparable in vitro functions, some binders with relatively similar binding MFI failed to control the tumors. In contrast, two binders with an ~30-fold apparent affinity relative to FMC63 exhibited robust anti-tumor activity comparable to the benchmark scFv. These preliminary data suggest that the in vivo functional activity of chimeric KIR-based immunoreceptors is insensitive to binder affinity over a wide range of affinities. The failure of some scFvs with comparable affinity and binding epitope suggest that other, yet to be characterized attributes of the binder may be critical for optimal function. Further characterization of these binders is being conducted to understand the relationship between affinity, functional avidity, antigen density, and in vivo potency. This study paves the way for developing an improved CD19-specific KIR-CAR T cell therapy for treating pts with B-cell malignancies. Citation Format: Jun Xu, Selene Nunez-Cruz, John M. Leferovich, Gayathri Gulendran, Chune Zhang, Nora D. Yucel, Megan C. Blair, William S. Stanley, Laura A. Johnson, Don L. Siegel, Michael C. Milone. Evaluating the relationship of affinity, functional avidity, and in vivo potency in KIR-CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6332.