A rapidly emerging immunotherapy approach, adoptive cell transfer (ACT), collects and uses a patient’s own immune cells to treat their cancer. One methodology, Chimeric Antigen Receptor T-Cell (CAR T-cell) therapy, has advanced the furthest in clinical development. Various B-cell malignancies can be addressed with CAR-modified T cells by targeting CD19, a protein that is expressed on the vast majority of B-cell cancers.

Despite manufacturing and cost hurdles, the CAR-T market is experiencing rapid growth and investment, with over $20 billion in recent M&A activity, according to BioInformant, and is expected to yield a CAGR of 32.5% through 2028, notes Coherent Market Insights. Safety remains the fundamental challenge.

A key player in large-scale PCR-based production and chemical modification of DNA, Applied DNA Sciences has developed capabilities to manufacture and chemically modify larger gene-sized amplicons.

“Plasmids and viral vectors are currently used for delivery of cellular therapies. Plasmid DNA carries a host of potential issues including contamination with non-target DNA from both the plasmid and bacteria as well as incorporation of the plasmid’s gene expression control elements. In addition, plasmids are associated with genes for antibiotic resistance, which are often exchanged between bacteria, and are considered to embody a serious threat to global health. Endotoxins are also a concern. There are plenty of reasons not to use DNA sourced from plasmids. Viral vectors offer efficient transfection and expression of plasmid DNA and carry their own risks,” said James Hayward, Ph.D., Sc.D., president and CEO, Applied DNA Sciences.

Since 2017, the FDA has approved two CAR T-based therapies targeting relapsed and refractory acute lymphoblastic leukemia (ALL) and adult B-cell non-Hodgkin lymphoma, expanding options for individuals who are unresponsive to standard treatments. However, despite their efficacy these therapies remain extremely complex to manufacture and are often cost prohibitive.

For example, the registrational trials for the two currently FDA-approved CAR-T therapies, Novartis’ Kymriah and Kite Pharma’s Yescarta, demonstrate that up to 31% of intended patients did not receive treatment primarily due to underlying disease complications, which evolved during the manufacturing process, or from manufacturing failures (Allogene Therapeutics, Form S-1, filed September 14, 2018).

Non-Viral, Plasmid-Free

Applied DNA Sciences’ approach is to begin pre-clinical development of a non-viral, plasmid-free (NVPF) CAR T manufacturing platform thereby offering a disruptive, inexpensive and safer alternative to established viral vector and plasmid platforms.

The NVPF CAR-T manufacturing platform uses linear DNA that is temporarily expressed without incorporation into the genome. Transient expression may suffice for the effectiveness of genetic vaccines or genetic immunotherapies.

“PCR is probably the best understood analytical method for the study of DNA and is used in diagnostics. We have taken the technology to a previously unthinkable scale, and run very large batches in continuous flow modalities. The inputs are well defined and you can perform chemical modification throughout the process. All in all it results in a product with extraordinarily high purity. Contamination issues go away,” explained Dr. Hayward. “Placing a PCR device operated under cGMP in close proximity to the patient would streamline the CAR T-manufacturing process, reduce costs, and resultantly have an impact on patient mortality and morbidity. PCR is a very transparent method, and massively simpler then producing viral vector or plasmids. The goal is to make CAR-T therapies more accessible and affordable to all applicable patients.”

Elements of linear DNA resident in the nucleus are capable of being transcribed and translated without the unintended consequences of recombinant therapies. During the PCR process, the two ends of linear DNA can be protected so they are less susceptible to exonucleases. It may also be possible to control expression over variable time courses.

Applied DNA Sciences’ wholly-owned subsidiary LineaRx, has initiated preclinical development of the NVPF platform and also signed an exclusive North American licensing agreement with iCell Gene Therapeutics, whereby LineaRx will utilize its NVPF platform to develop, manufacture, and commercialize LinCART19, a CAR T-drug candidate, for non-viral delivery.

LinCART19 CAR-T therapy targets the CD19 B-cell surface protein, and has shown 100% remission of ALL in the three patients who have participated to date in Chinese human clinical studies run by iCell. The virally transfected anti-CD19 ACT has yet to begin clinical trials in the U.S. or Europe.

In addition to LinCART19, LineaRx has an agreement with Takis and Evvivax to jointly develop linear DNA expression vectors for two of Takis/Evvivax’s anticancer vaccine candidates. Linear DNA amplicons carrying the DNA sequences for Takis/Evvivax vaccine candidates will be delivered to preclinical animal models using Takis/Evvivax’s proprietary electroporation technology to study antigen-specific immune responses.

The collaboration has already shown promise of yielding immunity in mice that were DNA-vaccinated against the human protein telomerase, which is over-expressed in more than 85% of all cancers. These results demonstrate the potential breadth of the platform to produce various biotherapeutics, including gene and cellular therapies as well as vaccines, according to Dr. Hayward.

“NVPF is a disruptive platform that offers the opportunity to significantly improve upon a world-changing therapy. The improvements we can make may lessen the pressure to find allogenic therapies. We may be able to deliver autologous therapies without the risks associated with large-scale virus manufacture and the long delay of ‘vein to vein.’ Our NVPF approach and PCR devices can redefine bench to bedside therapy; it keeps the supply chain concise and on site,” he concluded.

The NVPF manufacturing platform is available to CAR-T developers worldwide to generate new drugs from CAR-T cells, including the use of custom epitopes and neoantigens. The hope is to turn personalized cancer therapy from a dream into reality.

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