Research in mRNA chimeric antigen receptor T cell (mRNA CAR T) therapy at Penn

At the Perelman School of Medicine and the Abramson Cancer Center, a team of researchers is illuminating an auspicious mRNA platform for chimeric antigen receptor (CAR) T cell engineering.

Viral engineered CAR T cell therapy can be a life saving modality in the hematological malignancies. However, nearly 70% of adults receiving CAR T therapy have serious immediate and long-term adverse reactions requiring further intervention, including IL-6 blockers and IV immune globulin infusion. These measures and the events that prompt them are among the concerns for investigators exploring CAR T therapy in other diseases and cancer types.

CAR T cell therapy involves the collection and genetic reprogramming of T cells to express a specific CAR against tumor antigens that, once reintroduced into patients, will (in favorable cases) induce an immune response. The genes encoding the engineered receptors are typically introduced into T cells through transduction involving a lentiviral vector.

Viral transduction is a two-edged sword, however, in that it induces continuous and indefinite CAR expression once infused, increasing the risk of direct attack on normal tissues with shared expression of the targeted antigen and provoking long-term adverse effects. Such on-target off-tumor toxicity is especially problematic in solid tumor cancers treated with conventional CAR T therapy. In addition, the individualized ex vivo T cell engineering process for viral transduction is extensive and costly.

Collectively, these factors have motivated investigations into alternative production strategies to generate safer, less expensive CAR T cells. Among the most promising of these efforts is the application of nonviral messenger RNA (mRNA) technology.

An innovation that owes much to foundational research at the Perelman School of Medicine, mRNA-induced CAR expression has been found to be transient, lessening the risks associated with long-term CAR T cell activity, and can be engineered to allow for modulated, dose-dependent CAR expression, assets that offer the opportunity to optimize CAR T cell potency and safety.

mRNA delivery is key

mRNA strands are large, rapidly degradable and negatively charged, all of which present challenges to their delivery into the cytosol of CAR T cells.

To produce mRNA CAR T cells at present, the method most commonly used is electroporation, which uses electric pulses to permeabilize cell membranes for mRNA delivery. Although successful, electroporation does not guarantee consistent membrane disruption across cells and carries the risks of cell content loss, altered gene expression and elevated cytotoxicity.

Lipid nanoparticles (LNPs) offer an alternative to electroporation in mRNA delivery. Used in the mRNA COVID-19 vaccines, LNPs are made up of four distinct lipid components.

One of these, ionizable lipid, allows the LNP to shift from a neutral to a positive charge, facilitating intracellular delivery; others contribute the shell of the lipid particle structure, and bind to and stabilize the mRNA particle. The ratios of the component lipids can be altered to generate unique combinations with specific characteristics (e.g, size, mRNA concentration, pH) for mRNA delivery.

For all of these advantages, however, no one has yet determined which of the many variations on LNP design offers the greatest potency and viability with the least cytotoxicity for mRNA delivery to CAR T cells.

Thus at the Abramson Cancer Center and the Perelman School of Medicine, researchers examined 28 formulations of varied composition in two sequential “libraries” for efficacy of T cell delivery and safety. After a series of assessments, one of the 28, B10, was found best able to generate functional CAR T cells with low cytotoxicity, achieving the most potency at each tested dose and the highest mRNA delivery without decreasing viability.

When used to deliver CAR mRNA to primary human T cells, B10 LNPs demonstrated CAR expression comparable to electroporation with less cytotoxicity. Moreover, in a co-culture assay with acute lymphoblastic leukemia (ALL) cells, the B10 LNP-generated CAR T cells were able to induce the same potent cancer cell killing as electroporation and lentiviral-generated CAR T cells, confirming B10 LNPs as a promising delivery platform for CAR T cell engineering.

While, according to the authors, future work should explore the mechanisms by which this altered composition enhances delivery, the optimized B10 LNP platform has the potential for application in a broad range of T cell engineering platforms. Furthermore, the B10 formulation could inform future work optimizing LNPs with various ionizable lipid components to enhance delivery to other immune cells.

The full report of this investigation is available at NANO Letters.