Chimeric antigen receptor (CAR)-T cell therapy has shown remarkable clinical efficacy in relapsed or refractory hematologic malignancies, such as leukemia and lymphoma. However, current CAR-T therapies rely on a labor-intensive ex vivo manufacturing process involving T cell isolation, activation, genetic modification, expansion, and reinfusion. This process is not only time-consuming and costly but also limits timely and scalable patient-specific application. To address these challenges, in vivo CAR-T programming through direct delivery of CAR-encoding gene into immune cells has emerged as a promising alternative.
Among non-viral gene delivery platforms, lipid nanoparticles (LNPs) encapsulating messenger RNA (mRNA@LNP) have shown considerable potential due to their scalability, transient expression kinetics, and favorable safety profile. Nevertheless, conventional LNPs suffer from poor cell-type specificity, resulting in off-target transfection and unintended immunogenicity. While antibody-mediated targeting offers a viable solution, existing chemical conjugation methods are often complex, rely on toxic reagents, and suffer from instability due to PEG detachment.
To overcome these limitations, we developed a chemical-free targeting strategy using a recombinant fusion of apolipoprotein A1 (ApoA1) with antibodies, termed “Grab Antibody” (GrAb). Owing to the intrinsic lipid-binding affinity of ApoA1, GrAb spontaneously forms a stable and functional protein corona on the LNP surface, enabling correct antibody orientation without compromising LNP integrity.
In this study, we engineered a CD3-targeted GrAb by fusing ApoA1 to the Fc domain of the humanized anti-CD3 antibody. CD3 is abundantly expressed on T cells and mediates efficient receptor-driven endocytosis. We demonstrate that CD3-GrAb-LNPs selectively deliver CAR-encoding mRNA into T cells, resulting in robust CAR expression, preserved viability, and potent cytotoxicity against CD19+ leukemia cells. This platform offers a clinically translatable approach for in vivo generation of CAR-T cells, with potential to overcome the logistical and economic barriers of conventional CAR-T therapy.