Abstract We previously described a chimeric killer immunoglobulin (KIR)-like receptor (cKIR) approach to modifying T cell antigenic specificity that mimics the multichain design of natural KIR immunoreceptors (Wang E et al. Cancer Immunol Res 2014). T cells modified to express a cKIR with the natural ITAM-containing adaptor, DAP12 that associates with activating KIRs, show significantly enhanced anti-tumor activity acrossmultiple solid tumor models when compared to traditional CD3z-based chimeric antigen receptors (CARs). However, unlike TCR, where assembly with ITAM-containing CD3 family members is required for plasma membrane expression, natural and chimeric KIR exhibit membrane expression in the absence of DAP12. We show that cKIR engineeredT cells also produce IFN-g and retain cytotoxic activity in the absence of DAP12, revealing previously unrecognized DAP12-independent signaling pathways. This is associated with phosphorylation of ERK, JNK and STAT3 at early time points (30-60 mins) following cKIR engagement with target antigen. Although the cytoplasmic domain of KIR2DS2 used in cKIR is short and lacks canonical signaling motifs, we hypothesized that this domain plays a functional role in T cells by recruiting alternative adaptors to promote noncanonical signaling in the absence of DAP12. Immunoprecipitation of cKIR in T cells lacking DAP12 identified GRB2 as an associated adaptor protein. CRISPR- mediated GRB2 knockout selectively abolished ERK activation, establishing GRB2 as a crucial link to the Ras/MAPK pathway in cKIR-activated T cells. Structure-functionanalysis of the KIR2DS2 cytoplasmic tail, containing one tyrosine, one threonine, and four serine residues, revealed that progressive truncation of these residues had minimal effect on membrane expression, but increasingly impaired cytotoxicity, cytokine production, proliferation, and downstream signaling with a 25-fold loss of IFN-g andcytotoxicity observed with the truncation of C-terminal to serine residue 3. Collectively, these results suggest that DAP12-independent signaling by cKIR, mediated through GRB2 and likely other yet-to-be-identified adaptors, contribute to diverse signaling in T cells and may contribute to the enhanced anti-tumor function observed with KIR-based CAR T cells compared with conventional CAR designs. Citation Format: Qian Zhang, Kevin Zhou, Zhongze Sun, Chen Zhang, Marta Chebotar, Michael C. Milone. Understanding DAP12-independent antitumor activation in KIR-based CAR T cells abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6596.
Zhang et al. (Fri,) studied this question.