Lipid nanoparticle delivery of synthetic antigens to sensitize solid tumors to CAR-mediated cytotoxicity

Introduction:: Chimeric antigen receptor (CAR) T cell therapies demonstrate tremendous clinical potential in treating hematological malignancies, with some patients experiencing regression or complete remission of their disease. These new “living drugs” promise to fundamentally alter the way we treat liquid tumors. However, several barriers limit their widespread clinical implementation for solid tumors. The scarcity of tumor-associated antigens (TAAs) and heterogenous antigen expression between patients represents a significant impediment in engineering CAR constructs that are widely applicable across different cancers. This challenge of antigen selection renders cancers such as triple-negative breast cancer (TNBC), which altogether lack targetable tumor antigens, unresponsive to conventional CAR T cell therapies. Additionally, the tendency of TAAs to mutate and escape immune cell surveillance further underscores the need for improved strategies. To address these challenges, we designed genetically encoded synthetic antigens comprised of an extracellular camelid RSV-F VHH domain for sustained expression on the tumor. Here, we use lipid nanoparticles (LNPs) to transfect tumor cells with synthetic antigen mRNA and sensitize solid tumors to recognition and elimination by engineered αVHH CAR T cells regardless of their expression of an endogenous target antigen.


Materials and Methods:: Splenocytes isolated from Pmel-1 mice were transduced with a retroviral vector containing an EF1α driven αVHH CAR fused to CD28 and CD3ζ signaling domains. cKK-E12, DOPE, cholesterol and PEG14-2000 were combined to prepare mRNA-loaded LNPs (Figure 1a). E0771 cells were transfected with 0-100ng of cKK-E12 particles and cocultured with αVHH CAR T cells at a 1:1 effector-to-target ratio. 24 hours later, αVHH CAR killing was assessed by a lactate dehydrogenase (LDH) assay (Figure 1b). 5.0 x 10⁵ E0771 cells were engrafted in C57BL/6J mice. 18 days later, mice received an intratumoral injection of saline or 5ug of LNP encapsulating Fluc or VHH mRNA. After 6 hours, 5.0 x 10⁶ αVHH CAR T cells were adoptively transferred into mice originally receiving Fluc or VHH LNP. The mice received the same treatment 7 days post initial ACT and tumor volumes and survival were tracked for the duration of the study (Figure 1c, 1d).

Results, Conclusions, and Discussions:: We observed a significant and dose-dependent increase in killing when VHH-transfected E0771 target cells were co-cultured with α-VHH CAR T cells as compared to wildtype T cells (p< 0.0001, Figure 1b). Additionally, we found that E0771 tumor bearing mice treated with VHH LNP and αVHH CAR T cells experienced a significant delay in tumor growth as compared to both saline and Fluc LNP controls (p=0.0007, Figure 1c). Mice in the saline and the Fluc LNP cohorts all reached euthanasia criteria within 30 days of the initial ACT. In contrast, mice receiving two treatments of VHH LNP and αVHH CAR T cells exhibited improved survival, with one mouse experiencing complete tumor clearance (p=0.0051, Figure 1d).

We demonstrate a strategy to overcome heterogenous antigen expression and associated toxicities to sensitize solid tumors to CAR-mediated cytotoxicity. Here, we use lipid nanoparticles to deliver mRNA encoding for synthetic antigens to tumors to enable their elimination by engineered α-VHH CAR T cells. Looking forward, this platform has the potential to broaden access to CAR T cell therapies by allowing clinicians to treat heterogeneous tumors or those lacking a targetable tumor-associated antigen.