The oncology field has long been in need of a breakthrough, and many experts believe chimeric antigen receptor (CAR) T-cell therapies hold the most hope. Just over six years on from the FDA approval of Kymriah and Yescarta, which deliver complete response rates in the 60% to 80% range for B-cell acute lymphoblastic leukaemia and two types of B-cell non-Hodgkin lymphoma, there is now a total of six CAR T cell therapies treating haematological, B-cell malignancies on the market, four of which target cells expressing B lymphocyte antigen CD19, and two targeting cells expressing tumour necrosis factor receptor superfamily member 17 (also known as BCMA or CD269).
With 1,631 active CAR T-cell products currently in the pipeline according to GlobalData’s Drugs database, there is certainly excitement that if the field succeeds in developing CAR T-cell therapies for a wider range of tumours, this could be the cure for cancer the world has been waiting for.
In addition to broadening CAR T’s applications, there is a strong focus on addressing two critical challenges in this space. With costs anywhere between $300,000 to $500,000 per treatment, CAR T-cell therapies are inaccessible to the general population. Secondly, since side effects include cytokine release syndrome (CRS), neurotoxicity, and on-target off-tumour toxicity, the safety concerns are severe enough that current therapies are limited to later treatment lines, with patients dosed at specific treatment centres with expert staff.
Stable versus transient expression
So far, all approved CAR T-cell therapeutics have relied on viral transduction, which results in permanent expression of CAR over time. This ensures a persistent therapeutic effect, meaning today’s CAR T-cell therapies are administered as one-time treatments. But there are challenges with viral transduction. The high cost and time-intensive nature of viral vector production is one concern, and there’s also growing opinion that persistent expression could be contributing to the toxicities associated with CAR T-cell therapies.
“With the stable expression that you get from a viral vector, as the cells proliferate and expand you basically get an uncontrolled amplification of the CAR protein, which can lead to a very rapid and intense onset of the therapeutic activity,” explains Dr Samuel Clarke, R&D director at Precision NanoSystems. “That expansion coupled with the persistent expression creates a very strong response in the body that can lead to this CRS-related toxicity.”
As an alternative to viral vectors, the delivery of CAR-encoding mRNA to the cytoplasm using non-viral approaches offers a non-integrating and transient method of T-cell engineering. In this case, the CAR is not amplified as the cells expand, and therefore a precise dose can be administered to the patient and re-dosing schemes are possible. This could help to address the toxicities seen with today’s CAR T-cell therapies, with evidence growing that transient expression could eventually enable researchers to develop clinically viable therapies for solid tumour targets, as well as improve the safety profiles of conventional haematological therapies and even therapies for B-cell related autoimmune disease.
“As you look to other cancer types and certain autoimmune diseases, it could be advantageous to use RNA transient expression to reach the much wider range of targets beyond CD19,” Dr Clarke explains. “The distribution of new targets can sometimes be heterogenous; you can have target expression on healthy cells as well as diseased cancer cells. Being able to control the duration and persistence of therapeutic expression could help manage any side effects.”
In early 2023, Cartesian Therapeutics dosed its first participant in a Phase IIb randomised controlled trial for Descartes-08 – a first-in-class therapy engineered by mRNA transfection to express anti-BCMA CAR for a defined length of time. Descartes-08 is intended for patients with generalised myasthenia gravis (MG), an autoimmune disorder causing muscle weakness and fatigue. In the previous Phase Ib/2a study, the therapy was well tolerated, and treatments were followed by clinically significant reductions on MG severity scales up to nine months later.
Driving down costs with allogeneic therapies
Meanwhile, with costs being one of the major issues in the CAR T field, there is a significant focus on optimising the current autologous manufacturing processes in order to lower the cost per dose. The allogeneic approach – where T cells are taken from healthy donors, engineered, expanded to large numbers and banked for off-the-shelf treatment of multiple patients – promises to reduce cost per treatment and patient wait times dramatically.
In addition to the introduction of the therapeutic CAR, allogeneic CAR T therapies require more complex multi-step engineering involving CRISPR/Cas9 gene editing to remove proteins such as the TCR receptor associated with graft-versus-host disease. An interesting opportunity for transient expression has emerged here, whereby delivery of RNA encoding for Cas9 nuclease and guide RNA results in transient expression of Cas9 nuclease and temporary formation of a ribonucleoprotein (RNP) complex. This allows for the desired permanent gene editing event to occur while minimising off-target effects of persistent nuclease expression. The process can then be followed by downstream viral transduction for introduction and stable expression of the CAR.
With such benefits to the transient approach in both autologous and allogeneic applications, scientists are keenly investigating the best way to achieve transient expression. A technique called electroporation has been regarded as the most suitable method to date. It works by delivering electrical pulses to the cell, disrupting the membrane and opening transient pores through which foreign nucleic acids such as mRNA can be introduced to the cytoplasm.
“Electroporation works quite well across different types of nucleic acids, and it has become a standard technique in non-viral delivery,” says Dr Clarke. “But the primary challenge that we hear from people in the field is that once you electroporate the cells, there is a strong impact on toxicity. You can imagine that by electrocuting a cell, you’re going to cause some issues, right?”
“High cell death, low cell yield, and altered gene and protein expression profiles are some of the challenges associated with electroporation,” Dr Clarke adds. According to a recent paper by Kitte et al, these may explain the low anti-tumour response of the mRNA-based CAR T-cell therapies that have been tested in clinical studies so far.[i] The paper goes on to compare electroporation with an alternative method of transient T-cell expression: lipid nanoparticles.
The LNP opportunity
Like electroporation, lipid nanoparticles (LNPs) can mediate the delivery of mRNA to the T-cell cytoplasm. The cells then translate the mRNA into therapeutic proteins to destroy cancer cells or into nucleases for gene editing. The persistence of this transient expression is typically between five and 14 days, depending on a range of tuneable factors such as the dose of LNP, the quality of the RNA and the proliferation rate of the CAR T-cells. There could also be potential to prolong expression duration by using circular RNA, which was found by to be “generally more stable and could be less immunogenic after purification” compared to unmodified mRNA.[ii]
A few significant advantages of LNP-mediated RNA delivery make this approach an attractive alternative to electroporation. Since cellular uptake is mediated by endocytosis – a natural cellular process – Kitte et al found that LNPs are “less harmful to T cells”.
Further, in another recent study by Vavassori et al, nuclease RNA delivery using LNPs was compared against electroporation in an ex vivo gene editing application in both T cells and hematopoietic stem cells.[iii] The LNPs almost completely avoided the occurrence of cell death and significantly improved cell growth, improving tolerance to the procedure and yielding a greater number of edited cells than electroporation.
There is another advantage with LNP from a manufacturing perspective. During the electroporation workflow, cells are typically concentrated for treatment by centrifugation, whereas LNPs are pipetted directly into the cell culture medium. Bypassing the centrifugation step is particularly advantageous when scaling up the cell culture process to large batch sizes for clinical trials or approved therapies.
CAR T-cells and other cell therapies engineered using mRNA LNPs are now emerging in early research programmes and preclinical drug pipelines. For example, in one recent proof of concept, researchers at the University of Pennsylvania demonstrated the promise of an LNP platform to effectively deliver Foxp4 mRNA to immunosuppressive T cells for autoimmune disease therapies.[iv]
In the gene editing space, Intellia Therapeutics has presented preclinical data supporting the development of its NTLA-6001 candidate – an allogeneic CAR T therapy engineered with LNPs, targeting CD30-expressing hematologic cancers. The data proved the LNP-mediated T cells’ ability to expand while retaining high viability in in vitro and in vivo models. The T cells also avoided immune recognition by host cells and were protected from mediation by natural killer cells.[v]
Getting started with LNPs
As the opportunity for transient expression with mRNA continues to develop in the CAR T field, interest in LNPs are expected to grow. LNPs are a highly scalable technology suitable for clinical translation. Their ability to deliver mRNA safely and effectively is well established after the development and deployment of the mRNA-LNP vaccines for COVID-19.
For those compelled by the advantages of LNPs, developing an optimal ionizable lipid mix need not be complicated, since researchers can utilise off-the-shelf lipid nanoparticle reagent kits pre-optimised for the delivery of mRNA into human T-cells, such as the GenVoy-ILM™ T Cell Kit for mRNA, along with LNP manufacturing instruments, such as the NanoAssemblr™ Spark™ and Ignite™, from Precision NanoSystems.
Overall, there is strong emerging potential for LNPs in the CAR T-cell and wider cell therapy field, with the hope that they could enable the development of safer, more cost-effective, next generation cell therapies that could be adopted in earlier lines of treatment and across a wider range of cancers and autoimmune diseases.
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[i] Kitte R, Rabel M, Geczy R, Park S, Fricke S, Köhl U, Tretbar US, Lipid Nanoparticles (LNPs) outperform Electroporation in mRNA-based CAR T cell Engineering, Molecular Therapy: Methods & Clinical Development (2023), doi: https://doi.org/10.1016/j.omtm.2023.101139
[ii] Liu X, Zhang Y, Zhou S, Dain L, Mei L, Zhu G. Circular RNA: An emerging frontier in RNA therapeutic targets, RNA therapeutics, and mRNA vaccines. J Control Release. 2022 Aug;348:84-94. doi: 10.1016/j.jconrel.2022.05.043. Epub 2022 Jun 2. PMID: 35649485; PMCID: PMC9644292.
[iii] Valentina Vavassori, Samuele Ferrari, Stefano Beretta, Claudia Asperti, Luisa Albano, Andrea Annoni, Chiara Gaddoni, Angelica Varesi, Monica Soldi, Alessandro Cuomo, Tiziana Bonaldi, Marina Radrizzani, Ivan Merelli, Luigi Naldini; Lipid nanoparticles allow efficient and harmless ex vivo gene editing of human hematopoietic cells. Blood 2023; 142 (9): 812–826. doi: 10.1182/blood.2022019333
[iv] Ajay S. Thatte, Alex G. Hamilton, Benjamin E. Nachod, Alvin K. Mukalel, Margaret M. Billingsley, Rohan Palanki, Kelsey L. Swingle, Michael J. Mitchell; mRNA Lipid Nanoparticles for Ex Vivo Engineering of Immunosuppressive T Cells for Autoimmunity Therapies. Nano Lett. 2023, 23, 22, 10179–10188. https://doi.org/10.1021/acs.nanolett.3c02573
[v] Intellia Therapeutics Presents Preclinical Data Demonstrating Advancements in its CRISPR-Engineered Allogeneic Platform at the 2022 Keystone Symposia’s Precision Genome Engineering Conference. https://ir.intelliatx.com/news-releases/news-release-details/intellia-therapeutics-presents-preclinical-data-demonstrating-0