All cell-based immunotherapies inspire hope for a less cancer-plagued future, especially the ones that engineer T cells to detect and destroy cancer cells. Of all the engineered T cells that have been developed, one stands out—at least when it comes to regulatory approvals for clinical use. This is the chimeric antigen receptor (CAR) T cell. CAR T-cell therapeutics have secured regulatory approvals in the United States,1 Europe (including Switzerland), Canada, Australia, and Japan.

Despite this distinction, CAR T cells shared top billing at a recent industry event, the CAR-TCR Summit Europe. The event’s speakers conscientiously covered T-cell therapeutics based on engineered CARs, as well as those based on engineered T-cell receptors (TCRs). Unlike CARs, which recognize proteins that are expressed on a cancer cell’s surface, TCRs recognize peptide fragments derived from intracellular proteins that have been processed for presentation by the major histocompatibility complex.

At the CAR-TCR Summit Europe, which was held last February in London, CARs might have had more salience than TCRs in discussions of manufacturing. After all, just six months before the event, two CAR T-cell therapies were approved by the European Commission (EC).2,3 Tisagenlecleucel (Kymriah®, Novartis) was approved for use in pediatric and young adult patients with B-cell acute lymphoblastic leukemia, as well as for adults with relapsed or refractory diffuse large B-cell lymphoma. And axicabtagene ciloleucel (Yescarta®, Gilead) was approved for adults with relapsed/refractory diffuse large B-cell lymphoma or primary mediastinal large B-cell lymphoma (PMBCL).

Although these CAR T-cell therapies are generating excitement, they also pose practical difficulties. For example, they are costly. Also, regardless of the funds that may be available, demand may greatly exceed supply if CAR T-cell therapies come to be delivered more widely.

If such difficulties are to be resolved, CAR T-cells will have to be produced more efficiently. Some of the efficiency-boosting suggestions discussed at CAR-TCR Summit Europe (and reviewed in this article) might be taken up by CAR T-cell manufacturers such as Novartis and Gilead. In Europe, Novartis is expanding its CAR T-cell manufacturing through its acquisition of CellforCure (a CDMO in Les Ulis, France) and a partnership with the Fraunhofer Institute (in Leipzig, Germany). Gilead is building a new plant in Amsterdam, near the airport, to shorten turnaround times.

True personalization

Today’s approved CAR T-cell therapies represent “a truly new manufacturing paradigm,” one that would be considered innovative anywhere, within or beyond medicine, declared Qasim Rafiq, PhD, senior lecturer in bioprocessing of regenerative, cellular, and gene therapy at University College London. “In many manufacturing industries, there’s some customization, but you don’t, for example, have a truly personalized iPhone that’s designed to fit your hand and body.”

Currently approved CAR T-cell therapies are being made using a patient’s own T cells. Consequently, these therapies are, in a sense, intrinsically personalized. They also acquire another kind of personalization during processing. They are collected intravenously and transported to a manufacturing facility, where they’re engineered with a disarmed virus to express synthetic CARs on their surface—CARs that may be designed to attach to specific antigens, ideally ones that are preferentially expressed on a patient’s tumor cells. In the approved CAR T-cell therapies, the CARs target an antigen on B cells called CD19.

Once the T cells have been engineered, they’re cultured in the laboratory to create hundreds of millions of patient-specific CAR T cells before being returned to the patient. Here, they target and kill cancer cells with surface expression of CD19.4

“Manufacturing individual batches for each patient is true personalization,” noted Rafiq. “In a manufacturing sense, it’s truly unique, and manufacturing science is trying to understand how to do it.” Biopharmaceutical companies, he explained, are more accustomed to large-volume production of drugs with a supply chain and economies of scale.

Manufacturing challenges

“There are incredible examples of how CAR T cells, when targeting CD19, have resulted in Lazarus moments for patients, but these profound anti-tumor responses are limited to a subset of patients that can access these immunotherapies, and one reason is the cost is too high,” explained Laurence J.N. Cooper, PhD, MD, chief executive officer, Ziopharm Oncology. In the United States, treatment with Yescarta is reportedly priced at around $373,000, whereas Kymriah costs a reported $475,000.5,6 Part of the problem, Cooper pointed out, is the cost of using viruses to genetically modify T cells, as the viral reprogramming takes weeks in an incubator or bioreactor.

Currently, commercial CAR T-cell production is also a time-consuming process. The median time from T-cell collection to product delivery of Yescarta, for example, is an estimated 17 days.7 “These viral-vector-based approaches are running into problems to maintain product integrity and at scale to benefit the lives of patients with leukemias and lymphomas,” stated David Connolly, vice president of corporate communications and investor relations at Ziopharm Oncology.

Going virus free

Ziopharm Oncology is pioneering a nonviral approach to CAR T-cell therapy. Specifically, the company is advancing Sleeping Beauty, a transposon/transposase-based system that uses DNA plasmids and a process called electroporation to transfer genes into T cells. According to Cooper, DNA plasmids are cheaper than the lentivirus constructs used in viral-based CAR T-cell manufacturing, and Sleeping Beauty can modify T cells “on the spot” without needing to wait for virally modified T cells to grow in the production facility.

sleeping Beauty system
For the production of chimeric antigen receptor (CAR) T cells, a faster and potentially less expensive alternative to viral-mediated gene transfer is the Sleeping Beauty system, which relies on electroporation to deliver a transposon/transposase construct. In this image, from Ziopharm Oncology, a Sleeping Beauty procedure enables T cells to co-express a CD19-specific CAR and membrane-bound interleukin-15.

To shorten the time from genetic modification to infusion to two days or less, the company genetically tethers a cytokine called interleukin 15 (IL-15) to the T-cell membrane during the CAR T-cell production process. This is referred to as membrane bound IL-15 (mbIL-15). IL-15, Cooper explained, is a superior “go” signal that encourages T cells to continue multiplying while avoiding becoming exhausted. By providing a recombinant supply of mbIL-15, the CAR T cells may avoid the need to rely on natural production of this cytokine to continue functioning.

“In the CAR T-cell world, the targeting of CD19 could be expedited by Sleeping Beauty,” Connolly asserted. “Our goal is to undertake the gene transfer into the T cells and put them back into the patient within two days.”

By including IL-15 on the surface of T cells expressing a CD19-specific CAR, the company believes it can encourage T cells taken directly from the blood stream to be very rapidly reprogrammed and then returned to propagate in the body to target cancer in two days or less. This approach may reduce the numbers needed to be infused for successful CAR T-cell therapy, which may avoid toxicity issues that have complicated the management of some patients receiving commercial T-cell products. In addition, this approach can avoid the need for lymphodepletion where a patient’s existing T cells are “carpet bombed” to avoid them competing with CAR T cells for naturally circulating IL-15.

Ziopharm Oncology has already undertaken two clinical trials to establish proof-of-concept for the Sleeping Beauty technology. The company plans to begin a Phase I trial at the University of Texas MD Anderson Cancer Center in the second half of 2019, infusing CD19-specific CAR T cells produced in two days or less via gene transfer.

Targeting multiple antigens

Ziopharm Oncology indicates that its approach is to use genetic engineering to solve manufacturing problems. Marker Therapeutics is also developing a new therapy that aims to reduce the manufacturing challenges of producing large quantities of CAR T cells. The company’s Multi Tumor Associated Antigen Approach (MultiTAA) dispenses with the need to create a synthetic CAR molecule to be genetically engineered into a patient’s own T cells.

Marker Therapeutics
In today’s leading cell-based therapies, T cells are prepared by genetic engineering to recognize a single portion (or epitope) of a single tumor-associated antigen. An alternative approach has been developed by Marker Therapeutics. This approach, called MultiTAA, exposes T cells to a blend of tumor-associated antigens and cytokines in vitro, bypassing the need for genetic modification.

According to Juan F. Vera, MD, chief product development officer of Marker Therapeutics and assistant professor at Baylor College of Medicine, the MultiTAA involves exposing T cells to synthetic peptides and eliciting an immune response. These peptides represent hundreds of antigen epitopes expressed by cancer cells. Exposure to them allows the T cells to target multiple types of tumor cells once they’re reintroduced into the patient.

MultiTAA relies on the natural capability of T cells to target cancer, rather than a synthetic CAR. As a result, MultiTAA reduces the risk of T-cell exhaustion—the loss of T cell function and their physical depletion over time. “People have started to realize that engineering T cells can accelerate T-cell aging or exhaustion,” Vera said. “Some of these things are consequences of in vitro manipulation, whereas our approach is a more natural way of amplifying tumor-specific T cells.”

By reducing T-cell exhaustion, Marker Therapeutics can infuse fewer T cells into the patient, Vera noted. He also contended that the company’s process can restore natural immunity against cancer and cause fewer side effects than do CARs. “We use very small cell doses, and patients aren’t lymphodepleted,” noted Vera. “And once cells are administered, we don’t see toxicity—patients can be monitored for a few hours and go home.”

The company’s technology has been tested in seven different clinical protocols in multiple settings, including protocols for blood malignancies and bone marrow cancer, and it has been used to treat 78 patients so far.

CAR T cells as mass-produced goods

Marker Therapeutics is also trialing MultiTAA therapies with donor cells that match the composition of transplant patients’ bone marrow. “We manufacture cells from the donor of the bone marrow graft,” Vera pointed out. “After transplantation, we administer the product to the patients.”

The technology to manufacture MultiTAA T cells from a donor graft could be extended in the future to an off-the-shelf allogeneic T-cell therapy from donor cells without the need for gene editing, he argued. The development of allogeneic cells from donors is also an approach being explored in CAR T-cell therapies today, but—as Vera cautioned—it has many challenges because of the need for complex and unproven methods of gene editing to avoid cell rejection.

According to Rafiq, allogeneic CAR T-cell therapies are attractive to biopharma companies because they “make more business sense in terms of existing manufacturing and business models.” Unlike current personalized CAR T-cell therapies, in which personalization is partly a matter of using the patient’s own cells, the allogenic CAR T-cell therapies fit the standard biopharma model, which is based on using a universal or master cell to generate made-to-order therapeutics at large scales for administration to multiple patients.

However, these are not proven technologies, unlike the ones for generating therapeutics such as Yescarta or Kymriah. “For the next 5–10 years, the clear indication is toward viral approaches,” he insisted.

Adopting automation

With CAR T-cell therapies now reaching the marketplace, a major trend is improving manufacturing efficiency through automation. “One of the big challenges we’re trying to address for a personalized therapeutic is how do you manufacture multiple patient samples in parallel,” said Rafiq.

Automation is not just replacing people with robots, he argued, but also automating data capture, and using this information to move a sample to the next process step. AUTOSTEM, for example, is a project to create a fully automated production line for human mesenchymal stem cells,8 with the aim of reducing costs, decreasing human error and contamination risks, and improving process control. The lessons learned could be transferred to CAR T-cell production.

“We need to learn from the masters of supply chains, such as Amazon, where the customer making a purchase triggers a chain reaction that is—in many instances—completely automated,” he added.

CliniMACS Prodigy
With the CliniMACS Prodigy, a GMP-compliant instrument produced by Miltenyi Biotec, an automated workflow may be used to produce genetically engineered T cells. The instrument is a closed system that reduces contamination risks by eliminating unnecessary operator transfers between multiple devices.

Automating process monitoring and control can reduce patient-to-patient variability in product quality, he continued. “We’ve proven with multiple donor stem and now T cells, that—if you control raw materials, define the cell culture media and reagents, and monitor and control key parameters, such as temperature—you can significantly reduce variations compared to a serum-based uncontrolled process.”

Adrian Bot, PhD, vice president of translation medicine at Kite, a Gilead company, also spoke at CAR-TCR Summit Europe about optimizing the manufacturing process of Yescarta through “automation of processes such as fill-finish, which is the final stage in the manufacturing process and involves dispensing appropriate volumes in the product bag, addition of cryopreservatives and sealing the bag.”

Miltenyi Biotec lab
Miltenyi Biotec asserts that its CliniMACS Prodigy is a functionally closed and automated system that can be used to manufacture various cell types. The system is designed to accomplish cell enrichment or depletion, centrifugation, and cultivation using a single-use tubing set composed of bags, fluidic paths, centrifugation chamber, columns, and
a temperature control element.

Michael Papadimitrious, PhD, product manager for engineered T cells, Miltenyi Biotec, meanwhile, spoke about tailored automated solutions for producing CAR T cells, including a flow cytometer and cell manufacturing platform. The platform can produce CAR T cells in approximately 12 days or less, and the company hopes that future developments will allow for larger cell volumes and a reduction in manufacturing time.


1. McConaghie A. Novartis and FDA hail ‘historic’ Kymriah, first ever approved CAR-T. Pharmaphorum August 30, 2017.
2. European Medicines Agency [press release]. First two CAR-T cell medicines recommended for approval in the European Union. June 29, 2018.
3. Yescarta become first EU-approved CAR T therapies. Pharmafile August 28, 2018.
4.National Cancer Institute [press release]. CAR T Cells: Engineering Patients’ Immune Cells to Treat Their Cancers. December 14, 2017.
5. CAR-T: how will these $400k therapies be adapted for Europe? Pharm. Technol. July 10, 2018.
6. Beasley D. U.S. Medicare sets outpatient rate for Yescarta reimbursement. Reuters April 5, 2018.
7. U.S. Food and Drug Administration. Summary Basis for Regulatory Action: Yescarta. October 18, 2017.

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