With Cell Therapy Products, Multiple Safety Issues Call for Multiple Solutions

Cell therapies promise to revolutionize the treatment of devastating diseases ranging from cancer to neurodegeneration. Therapeutically engineered cells can be either autologous (originating from the patient) or allogeneic (originating from donors). Although the cell therapy market is still in its infancy, it is, in the view of many forecasters, poised for exponential growth. For example, Precedence Research anticipates that the cell therapy market will grow from its current $12.59 billion to more than $60.67 billion by 2030.

Before cell therapies reach their “prime time,” many challenges must be overcome, paramount of which is appropriate safety testing. However, because cell therapies are complex and varied, safety testing methods, and the risk assessments they entail, may be complex and varied as well.

The safety testing of cell therapy products was discussed by several presenters at the Process Development for Cell Therapies Summit, an event that was held October 18–20. Several of these presenters have contributed their thoughts to this article. If there is a consensus, it is this: Cell therapy, as a new field, needs novel testing techniques and personnel with the appropriate expertise.

More specific challenges include the development of compendial and noncompendial approaches to assess sterility and purity. Solutions include focusing on process design and control measures early in development. For example, staging closed cellular processes (and avoiding open steps) helps ensure sterility.

Because cell types may differ, companies may need to customize testing. For example, there are fully differentiated cells that have been altered via genome editing, and there are induced pluripotent stem cells (iPSCs) that have been altered via genome editing. (There are also iPSCs that have been generated without the use of genetic material.) To determine how cells of different types should be tested, investigators rely heavily on genetic characterization through karyotyping and plasmid loss.

Safety testing of CRISPR-based platforms must address risks such as potential off-target cuts to the genome and consequent modifications. Additionally, cryopreservation protocols must be developed that are both scalable and comparable. Finally, the terminal processing step of any process is critical for maintaining sterility and preparing samples for batch release.

Multiple safety approaches

There is no “one size fits all” when it comes to ensuring the safety of cell therapy products. Instead, a variety of safety challenges may be encountered. “Challenges faced include having the technical expertise to run the many molecular, cell, and other tests needed to fully characterize and understand the product,” says Jenessa Smith, PhD, director of process development at Arsenal Biosciences. “We are lucky to have talented scientists and leaders that can execute and define the safety of our CITE process, as an example, in a deep way with several orthogonal assays that demonstrate no significant off-target editing.”

CITE is Arsenal’s “CRISPR-based Integration of Transgenes by Electroporation” process. It is designed to enable precise and homogenous manufacturing and to offer greater operating leverage than viral alternatives.

Additional challenges include ensuring sterility and removal of residuals. “We face these challenges by processing all steps possible in a closed manner, moving to a fully closed system as a key goal for our work,” Smith details. “Closing the process by avoiding open steps that use a biosafety cabinet is important so that sterility is maintained with the highest level of control. We also use reagents thoughtfully to mitigate the presence of any adventitious agents and choose reagents with assays available to easily test residuals.”

Smith says that it’s important to understand the various aspects of each product and have tests that accurately assess the functions. “We developed a matrix of potency and safety assays to understand the various functions [we can build into] our product,” she explains. “[Our] novel integrated circuit T cells (ICT cells) incorporate a synthetic circuit that enhances potency, expansion, and tumor microenvironment penetration.”

For example, Arsenal has developed an ICT cell that is capable of conditional chimeric antigen receptor (CAR) activity and other functions. This ICT cell, which is called AB-X, includes a transgene cassette with two functional modules. One is an AND logic gate designed to limit off-tumor toxicity through dual tumor antigen recognition. The other is a dual shRNA-miR to resist tumor microenvironment suppression and improve ICT cell function. (So-called AND biological gates are functional only when one input AND another are present.)

Arsenal plans to evaluate AB-X in clinical trials for treatment of platinum resistant/refractory ovarian cancer. The company also intends to test its novel ICT cells against several other solid tumor indications.

Next-generation iPSCs

Because iPSCs may differentiate into a variety of cell types, they hold many promising therapeutic applications. “Originating from a single donor, iPSC-derived therapies provide the potential for unlimited multiplex or sequential genetic engineering steps,” notes Chris deBorde, PhD, process development engineer, Century Therapeutics. “[These steps may lead to] a defined single-cell-derived product amenable to multiple indications and improved efficacy.”

Although some aspects of safety testing are similar to those for other cell-based products, there also are important differences. “Strategies for adventitious agent testing, which is heavily dependent on process raw materials and residuals, may be unique to iPSC therapies given the nascency of the field, distinct manufacturing processes, and particular raw material needs and vendors,” deBorde explains. “Additionally, iPSC and other genome edited cell therapies rely heavily on genetic characterization through karyotyping and plasmid loss. When any number of edits are made to the genome of a cell, it is critical to ensure that these edits do not cause unwanted mutations or off-target effects, particularly as iPSCs are further differentiated into immune effector cells.”

According to deBorde, Century Therapeutics has developed quality control strategies for its iPSC-derived therapies that employ well-characterized compendial and noncompendial procedures, including sterility, endotoxin, and mycoplasma tests as well as a risk-based approach to adventitious testing at different points in the manufacturing process. The company also employs genetic characterization with PCR, flow cytometry, karyotyping, and genomic sequencing to ensure a highly pure and genetically stable cell therapy product.

However, iPSC therapies offer several safety advantages. “In contrast to other allogeneic cell therapies that use multiple healthy donors for their cell source, iPSC-derived therapies can utilize a single master cell bank,” deBorde notes. “This eliminates heterogeneity from the starting material and allows for a deeply characterized master cell bank to feed an entire product for its lifetime. Also, because iPSCs can have their genome edited sequentially, a single clone can be selected and then expanded that incorporates all of the specific edits targeted for that product. As a self-renewable resource, iPSCs will provide an unlimited supply of material for future products.”

The company is developing genetically engineered iPSC-derived natural killer and T-cell product candidates to specifically target hematologic and solid tumor cancers.

Engineered B-cell medicines

“B cells represent a game changer for medicine,” states Mark Lalli, PhD, associate director of process development at Be Biopharma. “[They promise to have a] profound patient impact across therapeutic areas.” The company is creating B cells that can produce specific therapeutic proteins for disease treatment. Lalli elaborates, “Be Bio’s platform is made possible through precise engineering and a convergence of gene editing, B-cell biology, and cell therapy manufacturing and development.”

Safety considerations are similar to those for other cell-based therapies. Lalli explains, “With respect to safety from a manufacturing perspective, it is important to focus on process design and control to ensure sterility and identity of the final product.” He also emphasizes that the final processing step of any process is critical for maintaining sterility and preparing samples for batch release. For example, cryopreservation protocols must be developed that are both scalable and comparable to ensure sterility and quality. Additionally, automating fill and finish technologies can reduce contamination as well as time and cost.

Lalli adds, “Both off-the-shelf and autologous cell therapies require standard safety tests, such as sterility, mycoplasma, and endotoxin tests, whereas graft-versus-host disease considerations generally apply only to off-the-shelf therapies.”

The company is advancing both autologous and allogeneic B-cell medicine platforms across multiple therapeutic areas with an initial focus on rare disease and oncology. The potential resulting B-cell products would be single-dose biologic protein medicines that produce an endogenous and continuous therapeutic infusion at constant levels.

Making the cut safely

Artisan Bio is employing nonviral engineering to design scalable, cost-effective, and reliable processes for cell production, according to Nick Timmins, PhD, the company’s chief development officer. He explains, “Our STAR-CRISPR platform had been developed from the outset to employ ribonucleoproteins. In simple terms: the nuclease, the gRNAs, and the DNA repair template (for knock-in) are combined in solution, and then various transfection methods can be used to deliver these components to the nucleus of cells. Our proprietary nucleases are engineered to enhance the efficiency with which ribonucleoprotein complexes transit across the nuclear membrane.”

Safety concerns include potential off-target cuts to the genome and consequent modifications. Similarly, on-target cutting could potentially result in other unwanted outcomes (for example, template copy errors or structural changes). “It is important to note that these are outcomes that might occur,” Timmins advises. “Careful target choice and system design can be used to minimize the probability of occurrence, and appropriate analytics can be deployed to detect any such outcomes should they occur.”

The company is deploying a suite of molecular assays early in development to detect not only off-target edits, but also unintended outcomes of both off- and on-target edits. These assays are coupled to data-driven approaches for assessing the hazard potential of any such outcomes.

“We leverage the massive quantities of data obtained in databases such as COSMIC and ClinVar/GenVar to evaluate what the functional and/or clinical consequences of a potential unwanted outcome might be,” Timmins elaborates. “All of this occurs very early in development during selection of optimal gRNAs before substantial costs accrue and at a time when changes are more easily made. Functional cell-based assays provide an additional opportunity to identify unintended outcomes through altered or aberrant behavior, prior to testing preclinical animal models and staging appropriately designed clinical trials.”