Precision In Vivo CAR-T generation via CLAMP-enabled mRNA delivery: Toward scalable and translatable cell therapy

Abstract

Abstract Background:Chimeric Antigen Receptor (CAR) T cell therapies have transformed the treatment landscape of hematological malignancies and offer curative potential to certain patients. However, autologous CAR-T therapies encounter key challenges, including toxicity from lymphodepletion, complex ex-vivo manufacturing and high cost, all of which limits broader clinical application. In vivo CAR-T therapy is an emerging modality designed to overcome these barriers by enabling direct T cell engineering within patients. Effective in vivo CAR-T therapy requires targeted T cell delivery, durable CAR expression, potent cytotoxic activity, and potential for re-dosing. Toward this goal, we report the development and preclinical evaluation of GT801, a novel anti-CD19 in vivo CAR-T candidate. Methods:GT801 was developed using novel T-cell-targeted lipid nanoparticles (T-LNP) encapsulating chemically modified linear mRNA encoding an anti-CD19 CAR gene. Both LNP formulation and mRNA design were systematically optimized to enhance delivery specificity, promote robust CAR expression, and maximize CAR-T functionality. T-LNPs were surface-engineered with a VHH antibody directed against a T cell-specific target, enabling selective uptake by endogenous T cells. Antibody conjugation to the LNP surface was achieved using CLAMP (Controllable Ligand Attachment Modification and Purification), a proprietary technology that enables site-specific antibody attachment and precise control of ligand density. This approach enhances targeting efficiency while minimizing non-specific uptake. GT801 was thoroughly characterized for purity, identity, potency, and biodistribution. Anti-tumor efficacy and pharmacokinetics/pharmacodynamics (PK/PD) were assessed in vitro and in vivo using humanized NOG mouse models. Results:When combined with optimized mRNA chemistry, the T-LNP platform enables robust and sustained CAR expression in human PBMC, with expression persisting for over 14 days in vitro. In human PBMC-engrafted NOG mice, a single intravenous dose as low as 0.01 mg/kg achieved >95% B cell depletion. The conjugation strategy and stealth-layer design minimized off-target uptake by monocytes, macrophages, and dendritic cells to below 3%. By targeting T cell-specific markers, the system achieved receptor-saturating delivery efficiency across multiple lymphoid tissues at clinically relevant doses, driving >30-fold in vivo expansion of CAR-T cells. Potent antitumor activity was demonstrated in CDX (cell line-derived xenograft) model following a single or multiple dosing. The LNP formulation incorporates proprietary ionizable lipids with favorable PK and safety profiles across multiple species. Serial dosing elicited minimal cytokine release (IL-6, TNF-α), supporting the safety and re-dosing potential of this platform. Conclusion:These findings demonstrate that our T-LNP platform enables efficient, targeted, and sustained in vivo CAR expression with a favorable safety profile and scalable manufacturing process. A clinical batch is currently in production, and a first-in-human investigator-initiated trial (IIT) in B cell malignancies is anticipated to launch in late 2025.

Topics & Concepts