Designing immune-evasive ucar-T cells to overcome allogeneic barriers and advance off-the-shelf immunotherapy

Abstract Chimeric antigen receptor (CAR) T cell therapy has shown considerable potential for treating refractory autoimmune diseases. However, its current reliance on autologous cells poses significant logistical and scalability challenges. An allogeneic, off-the-shelf CAR-T product offers a promising alternative. A key hurdle in developing such universal CAR-T (UCAR-T) therapies is overcoming host immune rejection, which we address through engineered ‘hypo-immunity’. Here, we report the development of three distinct UCAR-T products incorporating diverse hypo-immunity strategies. These candidates are currently under clinical evaluation in multiple investigator-initiated trials (IITs). Methods: We have developed three UCAR-T cell products, each incorporating a distinct hypo-immunity strategy. All constructs feature TCR knockout to prevent GvHD and co-express a dual-targeting CD19/BCMA CAR.Strategy 1 (HLA-E and CLEC2D knock-in): B2M and CIITA are knocked out to eliminate MHC-I and MHC-II expression, reducing T cell-mediated rejection. HLA-E and CLEC2D are knocked in to inhibit NK cell activation via NKG2A and CD161 pathways, respectively.Strategy 2 (HLA-C retention): HLA-A and HLA-B are knocked out to minimize T cell recognition, while HLA-C and HLA-E are retained to engage inhibitory NK receptors (KIRs and NKG2A), allowing partial HLA compatibility.Strategy 3 (anti-KIR2D CAR): Based on Strategy 1, CLEC2D is replaced with an anti-KIR2D CAR, enabling active depletion of KIR2DL1/2/3-expressing NK cells to counter residual NK-mediated rejection. Results: To assess resistance to NK cell-mediated rejection, UCAR-T cells were co-cultured with allogeneic peripheral blood NK (PBNK) cells. Strategy 3 UCAR-T cells exhibited enhanced survival and expansion relative to Strategy 1. In comparison, the efficacy of Strategy 2 was dependent on HLA-C/KIR compatibility (e.g., HLA-C1/KIR2DL2/3 or HLA-C2/KIR2DL1); under matched conditions, Strategy 2 UCAR-T cells demonstrated survival comparable to non–B2M knockout CAR-T cells. Strategy 1 conferred only short-term protection against PBNK-mediated cytotoxicity. T cell-mediated rejection was evaluated using mixed lymphocyte reaction (MLR) and activation-induced marker (AIM) assays. Strategy 2 UCAR-T cells—but not those from Strategies 1 or 3—elicited strong allogeneic T cell activation, highlighting the need for HLA-C matching to minimize T cell recognition. All three UCAR-T variants exhibited potent and comparable antitumor activity in xenograft models bearing CD19⁺ Nalm6 or BCMA⁺ MM1.S tumors. No treatment-related toxicities were observed in murine safety studies, supporting the preclinical safety of these UCAR-T products. These UCAR-T candidates are currently under evaluation in multiple investigator-initiated trials (IITs). Notably, the Strategy 2-based product is being assessed in trial NCT06941129, with results to be presented at this meeting. Strategy 1- and Strategy 3-based products have also advanced into clinical investigation through separate IITs. Conclusions: Using distinct hypo-immunity strategies, we developed three allogeneic UCAR-T products engineered to evade rejection by host T and NK cells. All candidates demonstrated promising preclinical efficacy in mitigating immune-mediated clearance and have entered clinical evaluation in multiple investigator-initiated trials, with data expected to be published soon.