Johns Hopkins Medicine scientists have developed what they describe as a simplified version of biodegradable nanoparticles (NPs) that can “educate” the immune system to find and destroy disease-causing cells throughout the body. The targeted, polymer-based nanoparticles encapsulate an mRNA encoding a therapeutic anti-CD19 chimeric antigen receptor (CAR). The nanoparticles are engineered to travel to and stimulate disease-fighting immune T cells, which, in turn, seek out and destroy immune system B cells, the source of diseases such as lupus and cancers of the blood, including leukemia and lymphoma.
The study, the team suggests, advances the field of engineering immune cells within a patient’s own body to combat cancers and autoimmune diseases, including lupus, among other conditions. The team, headed by Jordan Green, PhD, the Herschel L. Seder Professor of Biomedical Engineering at the Johns Hopkins University School of Medicine, reported on development of the nanoparticle technology, including the results of tests in mice, in a paper in Science Advances, titled “Biodegradable targeted polymeric mRNA nanoparticles enable in vivo CD19 CAR T cell generation and lead to B cell depletion.”
Engineered immune cells have been successfully used to treat an array of blood cancers, using chimeric antigen receptor T (CAR T cells). The approach involves engineering a patient’s own T cells in the laboratory to express a coat of receptors on their surface that recognizes and kills cancer cells. However, the process to remove patients’ blood cells and individually engineer them outside of the body is costly and inefficient.
“While chimeric antigen receptor (CAR) T cell therapies have demonstrated therapeutic efficacy against B cell malignancies, widespread implementation of these therapies is hindered by a cumbersome, ex vivo manufacturing process,” the team wrote. “Given the significant logistical complexities and costs associated with ex vivo CAR T cell manufacturing and administration, in vivo generation of CAR T cells has the potential to make these therapies safer and more accessible.”
They further suggest that reprogramming a patient’s own T cells to express a CAR would avoid many of the “roadblocks” in current manufacturing processes, primarily the need for T cell isolation, ex vivo engineering, and expansion. However, the team pointed out, among the challenges to this approach, T cells are “… historically a difficult cell type to transfect and engineer in vivo.”
Green and colleagues now report on their development of a targeted polymeric NP platform for in vivo T cell transfection and CAR T cell generation. These polymeric nanoparticles (PNPs) are composed of a poly(beta-amino ester) (PBAE) polymer that biodegrades in water. “The PBAE used in the tPNP system draws on distinct advantages from both LNPs and PNPs, with both having proven efficacy as mRNA delivery vectors,” the investigators stated.
The nanoparticle surface is decorated with anti-CD3 and anti-CD28 antibody molecules that help the nanoparticles find and stimulate T cells. These nanoparticles encapsulate an anti-CD19 CAR-encoding mRNA and act by instructing T cells to express receptors on their surface that detect cancer and lupus-causing B cells.
Other researchers have recently developed lipid-based nanoparticles (LNPs) with five components. In comparison, the nanoparticles developed by Green et al. have a simpler design that requires only three components. “Of note, rather than necessitating four to five separate biomaterial components to construct next-generation targeted LNPs, these polymeric NPs (PNPs) only require the PBAE polymer, a PEG lipid, and the mRNA cargo,” they wrote. The PBAE NPs can also be kept in long-term frozen storage or lyophilized, which is traditionally not possible for LNPs. “These differences in processing and formulation have large translational implications in the clinic, where clinicians may need to handle and administer NPs under various storage and supply chain conditions, ultimately informing our decision to use a PBAE-based NP.”
Through their newly reported study, the team demonstrated that 24 hours after injecting the new nanoparticles into healthy mice, 95% of the target B cells were depleted in circulating blood, and about 50% of B cells were destroyed in the animal’s spleen. The study showed that after a week, B cells in the blood returned to about 50% their original quantity. “The potent splenic depletion, combined with almost full depletion of all circulating B cells merely 24 hours after a single injection, further highlights the efficacy of the tPNP system,” the scientists stated.
“These experiments were successful using just one dose of the nanoparticles, and an advantage of using an off-the-shelf therapy is the potential for scalable manufacture and broad accessibility, whereas current forms of CAR T therapies are very expensive and time-consuming,” said Green. The authors added, “B cell depletion was observed in a ligand-and CAR mRNA–mediated manner, showing that targeted delivery of the CAR-encoding mRNA is critical for eliciting a therapeutic effect.”
It has taken five years to get to this point of success, noted Green, a biomedical engineer who worked with immunology expert Jonathan Schneck, MD, PhD, to develop the nanoparticles used for the current study. They blended Schneck’s work on developing artificial immune cells that stimulate other immune cells and Green’s research on polymer-based nanoparticles.
Designing nanoparticles that can reach T cells throughout the blood and organs is more difficult than delivering them directly to a localized site, such as the eye, Green commented. When nanoparticles reach T cells, they tend to resist taking up the particles, but even if they do get internalized, the cells often chew them up and spit them out. “This makes sense, because if T cells easily internalize things like viruses, viral programming would take over the immune system, like what happens in HIV,” Green stated.
Green explained that the nanoparticles work in a stepwise way, much like rockets traveling to outer space work in stages to lift off, engage boosters, detach them, and finally deliver cargo. In the case of the nanoparticles, the scientists designed ships for inner space to seek out T cells, stimulate them to activate and multiply, pass through the cell wall into the T cells, and then degrade to deliver an mRNA cargo.
The scientists created a combination blend of two molecules (anti-CD3 and anti-CD28) that help the nanoparticles find and latch onto T cells. They found that the degradable nanoparticles worked just as well as commercially made magnetic beads designed to latch on to T cells for laboratory research purposes, but then were also able to enter the T cells to reengineer them from the inside out. In a previous study, Green and colleagues found that about 10% of the Johns Hopkins-developed nanoparticles successfully escape the cell’s degradation compartments to deliver their sensitive genetic cargo, compared with 1–2% of other nanoparticles that immediately get degraded and ejected from the cell.
In the current study, the scientists saw that the nanoparticles degrade and release their mRNA contents within a few hours in the mice. The researchers said the Johns Hopkins research team aims to continue refining the nanoparticles, tailoring them better to diseased B cells and able to dial up or down the amount of T-cell stimulation. “Ultimately, tPNPs are an innovative polymeric platform that is relatively inexpensive and simple to manufacture,” the authors stated. “Their potency as highly selective gene delivery vectors to T cells rivals or outperforms current LNP and PNP standards … Future investigation of this platform in different B cell–based malignancies spanning both murine and human models of cancer and autoimmune diseases are currently underway.”
