Accelerating CAR T Cell Production: Researchers at UPenn Develop Novel Lipid Nanoparticle for T Cell Activation and CAR Gene Delivery

Chimeric antigen receptor (CAR) T cell therapy is offering new hope to cancer patients around the world. As commercial CAR T therapies reach more patients, and new CAR T therapies are developed and approved, optimization of the manufacturing process is critical. Here, we discuss recently published research from Metzloff and colleagues at the University of Pennsylvania (UPenn) about a new nonviral method of CAR gene delivery using lipid nanoparticles (LNPs) that shortens the CAR T manufacturing time and may increase safety.

Magnetic Beads and Viral Gene Delivery for CAR T Manufacturing

There are currently seven FDA-approved CAR T cell therapies and hundreds more in development. Six of the approved CAR T therapies treat hematological cancers, and their use for these indications has generated a significant portion of the clinical knowledge base. As the industry builds upon the success of early CAR T therapies, two main areas are in focus: reducing the manufacturing time and cost and creating products with improved safety and efficacy profiles.

All approved CAR T cell therapies are autologous, meaning they are engineered from the patient’s own T cells, typically collected through leukapheresis. In a general CAR T cell manufacturing process, T cells isolated from the patient’s leukapheresis material are activated using CD3 and CD28 antibodies, which are attached to magnetic beads. This activation step is crucial for T cells to express the CAR and proliferate¹. Activation is accomplished by mimicking the interaction that occurs naturally between T cells and antigen presenting cells (APCs) when the body mounts an immune response. APCs are specialized immune cells that stimulate T cells by presenting them with antigens from pathogens or cancer.

The anti-CD3 and anti-CD28 activation antibodies are complexed with magnetic beads so they can be removed after T cell activation, which typically takes 24 hours. Once T cells are activated, researchers deliver the CAR gene. Current FDA-approved CAR T cell processes use viral vectors, such as lentiviruses or retroviruses to infect T cells and deliver the CAR gene. Engineered lentiviruses are effective at gene delivery by inserting the gene directly into the recipient cell’s genome in a process called transduction. This results in the gene being permanently integrated into the recipient cell’s DNA. After transduction, T cells are expanded to reach the dosage required for patient treatment.

Beyond Viral Vectors for CAR Manufacturing

Introducing the CAR gene using viral vectors that integrate with the cell’s genome, like lentiviruses, is effective but has limitations. Lentiviruses have a restricted size capacity for the transgene they can carry, and CAR transgenes can be quite large. Additionally, producing high-quality and high quantities of virus can be challenging and expensive.

Viral vector gene insertion can occur at almost any actively dividing region in the cell’s genome². and these insertions can be permanent. Consequently, CAR T cells typically express significant levels of the CAR for as long as they persist in the recipient’s body. There is currently no straightforward “off-switch” to deactivate CAR T cell function after they are delivered to a patient, although researchers have developed and are testing clever methods to achieve this³. Additionally, some CAR T cells can persist for years in a patient’s body. For CAR T cells targeting B cell malignancies, this long-term persistence can lead to a lasting reduction in functional B cells and immune suppression, even after the cancer has been eliminated⁴. With the field venturing into targeting malignancies beyond hematological cancers, there may be a stronger need to design CAR T cells with a shorter activity profile.

Lipid Nanoparticles for mRNA Gene Delivery

As an alternative to viral vectors, researchers and clinicians have been exploring the use of lipid nanoparticles (LNPs). LNPs were initially developed by Dr. Peter Cullis at the University of British Columbia starting in the 1980s for drug delivery. These small, self-assembling particles are composed of various customizable lipids that can be tailored for specific functions, like the type of payload they carry, how they enter cells, and how long they persist in the body.

Dr. Cullis’ group and others initially focused on using LNPs to deliver small-interfering RNA (siRNA), which can silence the expression of a target gene, like a misfolded protein causing disease. This strategy gained FDA approval in 2018 for treating hereditary polyneuropathy (Onpattro)⁵.

The payloads carried by LNPs can be diverse, but a specific type called “ionizable LNPs” are used to deliver messenger RNA (mRNA) to target cells. Upon entering the cell, the mRNA is released from the LNP and translated into the desired therapeutic protein. The clinical experience with mRNA LNPs is now extensive, as these systems are the basis of FDA approved COVID-19 vaccines^6,7.

LNPs offer several advantages as gene delivery systems including:

• Positive safety profile
• Relatively inexpensive manufacturing
• Can be produced in cell-free systems
• Have increased transgene carrying capacity compared to some viral vectors
• Do not need complicated procedures or equipment to deliver genes to cells

Some disadvantages of LNPs include:

• Can be difficult to target specific cell types or tissues
• Infusion reactions and off-target CAR delivery when delivered directly to patients
• Limited endosomal escape leading to inefficient gene delivery
• Regulatory landscape is still being formed

These attributes make LNPs well-suited for CAR T cells development. mRNA encoding of the CAR gene can be delivered by LNPs and transiently expressed by T cells, in contrast to the permanent expression seen with lentivirus transduction. This transient expression using mRNA CAR T cells is being explored for achieving short-term tumor burden reduction with potentially less toxicity, fewer side effects, and a simplified, less expensive manufacturing process⁸.

Activating aLNPs: Combining T cell Activation and Nonviral Gene Delivery

In their article published in Advanced Materials, Metzloff et al. report a non-viral method that simultaneously activates T cells and delivers the CAR gene using activating lipid nanoparticles (aLNPs). Let’s dive deeper into their research.

As mentioned previously, T cell activation during the CAR manufacturing process is typically achieved using anti-CD3 and anti-CD28 antibodies attached to magnetic beads before CAR gene delivery. Removing these magnetic beads (in a process called debeading) is a necessary but time consuming and potentially product-damaging step in finalizing the CAR T cell product for use.

The researchers reasoned that LNPs carrying CAR mRNA could be engineered to express the anti-CD3 and anti-CD28 antibodies on their surface. In this workflow, the single-step LNP would provide both the activation signal and the CAR mRNA, eliminating the traditional two-step process and reducing manufacturing time.

Using clever chemistry, the team at UPenn attached the anti-CD3 and anti-CD28 antibodies to LNPs, creating the novel aLNPs. To demonstrate that aLNPs could deliver mRNA without activation beads, the researchers loaded the aLNPs with mRNA encoding the reporter gene luciferase, which emits light when its substrate luciferin is added. Their experiments showed successful mRNA delivery. The researchers then optimized the ratios of anti-CD3 and anti-CD28 loaded onto the aLNPs by measuring the percent of T cells expression the CAR gene by flow cytometry.

Functional Validation of aLNP-made CAR T Cells

To test the functionality of aLNP-generated CAR T cells, the researchers tested their ability to:

• kill tumor cells in the lab
• proliferate in culture
• express markers associated with an activated phenotype

Flow cytometry was used for each of these experiments. The author’s data demonstrates that their aLNP technology could produce CAR T cells that kill target tumor cells at a range of doses, proliferate creating multiple generations of CAR T cells, and express an activated phenotype.

The researchers further tested their aLNP CAR T cells using a specialized mouse model. For the model, they used NOD scid gamma (NSG) mice, a strain of immunocompromised mice that can be transplanted with human cancer cells. This system allows for researchers to test the efficacy of human CAR T cells to kill human tumor cells in a preclinical, in vivo setting. NSG mice were challenged with human leukemia cells expressing luciferase at a dose designed to mimic a low level of leukemia, matching the low tumor burden scenario for which mRNA CAR T cells might be advantageous and allowing the researchers to track tumors over the course of treatment. Four days after tumor challenge, mice were dosed with either CAR T cells manufactured using lentivirus or using the novel aLNPs. A total of three treatments were delivered, one on Day 0, another on Day 3, and the final dose on Day 6. The aLNP CAR T treatment-controlled tumors as effectively as conventionally manufactured CAR T cells, with tumor cells undetectable by luciferase until Day 34. However, only one dose of conventional CAR T cells was required compared to three doses of aLNP CAR Ts, demonstrating the transient nature of mRNA CAR expression compared to lentivirus transduction. These results provide strong evidence for aLNP technology to produce CAR T cells without magnetic bead activation or virus transduction, albeit with the potential requirement for additional doses.

mRNA CAR T Cells and Beyond

This research study by Metzloff et al. from the University of Pennsylvania describes the development and testing of a novel LNP technology. This technology offers a non-viral approach that simultaneously activate T cells and delivers the CAR gene for transient CAR expression in T cells. This approach has the potential to shorten the manufacturing time, reduce reagent requirements, and simplify the overall manufacturing workflow for mRNA CAR T cells. However, mRNA CAR T cell approaches are still under investigation and their clinical potential remains to be fully determined. Looking forward, LNP technology is a rapidly evolving field, and researchers are continuously exploring new applications for this versatile technology.