一种新型CD3导向的体内基因修饰CAR（GCAR）慢病毒载体（编码嵌合抗原受体及增强细胞适合度的共刺激蛋白）在人源化异种移植模型中的抗肿瘤活性及在非人灵长类动物中的B细胞发育不全

Introduction: An in vivo gene-modifying CAR (GCAR) platform has been developed to mitigate known clinical and logistical challenges with traditional ex-vivo manufactured CAR-T products. The GCAR platform incorporates an NHP/human reactive CD3-targeted lentivector (LVV) with complement resistant fusogen that encodes CD3zeta based CARs and a compact synthetic costimulatory protein (FD) optimized for proliferation, persistence, and cytotoxicity. The ability of GCAR LVVs to elicit potent, specific and persistent cytotoxicity towards target cells was evaluated in vitro and in humanized and non-human primate models. In M. nemestrina , GCAR CD20 CAR was evaluated for B-cell depletion compared to LVVs encoding conventional 41BB CARs without FD. Methods: Anti-CD19 or anti-CD20 LVV's manufactured from 6L and 25L scales were characterized for CD3 reactivity in M. nemestrina and human PBMC in vitro and in vivo . Serum complement sensitivity and activation assays were used to characterize GCAR LVVs (encoding anti-CD20 CAR and FD) against human and NHP sera and CD3 positive lymphocytes. Escalating doses (IV or IN) of purified LVV's were evaluated in the NHPs. B-cells were quantified by flow cytometry and safety by clinical chemistry, hematology, and physical assessments. Results: Exposure of human or nemestrina PBMC to GCAR LVVs resulted in dose and target-dependent cell lysis with rapid tumor clearance in Raji-Luc disseminated PBMC-humanized DKO NSG models. In CD34-humanized models, GCAR LVV generated CAR-T cells with durable B-cell depletion over >8 weeks of observation. Administration of GCAR CD20 LVV (IV or IN) was well tolerated in NHPs at all doses w/o severe CRS or neurologic toxicity. B-cell depletion kinetics post IV dosing of CD20 CARs with FD were dose dependent: minimally active doses demonstrated delayed onset of B cell depletion (from day 21 post dose) vs. B cell aplasia by day 7 at the most active doses. In contrast, a comparable GCAR LVV encoding 41BB CD20 CAR (w/o FD) by IV and IN injection elicited B cell depletion at 7 days post dose with reduced duration. Conclusion: GCAR LVVs generated genetically modified lymphocytes resulting in durable CAR-dependent cell removal across mechanistic, murine, humanized, and NHP immune systems. The incorporation of FD may further improve cellular expansion and persistence, yielding ex vivo -like CAR-T activity profiles without lymphodepletion or cytokine support. The absence of acute and subacute safety signals, low off-target transduction, serum complement resistance, and deep B-cell depletion support the GCAR platform as a potential next-generation opportunity for clinical investigation as a novel gene modifying CAR approach.