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Cynthia A. Challener, PhD, is a contributing editor to BioPharm International.
Early successes drive the need to overcome safety issues, increase efficacy, and address manufacturing challenges.
Numerous, remarkable results in early clinical trials have driven significant investment in cell therapies, both by large biopharmaceutical companies and startup biotech firms backed by venture capital. Jain PharmaBiotech identified more than 500 companies involved in cell-therapy technology (1).
Adoptive cell transfer (ACT), which uses a patient’s T cells (T lymphocytes) that are harvested and genetically engineered to produce chimeric antigen receptors (CARs) and recognize specific proteins (antigens) on tumor cells, is receiving a good portion of that attention. The CAR-T cells are expanded and then reinfused back into the patient, where they multiply and attack the targeted cancer cells.
There are challenges to ACT using T cells. T cell expansion and the persistence of infused cells can vary significantly from patient to patient, and both directly influence the treatment outcome. Conditioning of the patient in advance of infusion can have an impact, as can the tumor microenvironment. Selection of the most effective, longest-lasting T cells and the right antigen targets is a key focus of research efforts today. On-target, off-tumor toxicity and cytokine release syndrome are important safety issues that must be addressed. Technologies for the manufacture of therapies based on living cells on a commercial scale must also be developed.
T cells are ideal vehicles for immunotherapy because they are central to cell-mediated immunity and are involved in long-term, antigen-specific responses. They have, in fact, been used in the past to treat various viral infections. Once T cells bind to cells expressing the target antigen, they acquire the specific functional properties necessary for eliminating the target cells and generate long-lasting memory T cells that provide a similar response if any target cells reappear. CAR-T cell therapy leverages these natural behaviors of T cells. T cells can also be engineered to express modified T-cell receptors (TCRs) as an alternative type of cell therapy.
Autologous vs. allogeneic
CAR-T cell therapies in clinical development today are largely autologous therapies; the genetically modified cells originate from tissue taken from an individual patient and are returned to that patient once expanded. There is some concern in the industry that the need to perform the same level of extensive quality control and testing on such small-scale product lots as for large-scale production will prevent these treatments from achieving commercial viability. Starting material variability for autologous CAR-T products also presents a challenge; the T cells from each patient differ depending upon the extent of their disease, previous therapies, genetics, and the status of their immune system at the time of cell collection. “These patient-specific issues will always be a challenge to the ability to manufacture a CAR-T product for every patient and to the consistency of performance observed among CAR-T products,” says Derek Jantz, chief scientific officer for gene editing company Precision Biosciences.
Allogeneic cell therapies, in which the T cells are derived from healthy donors that have been screened for desirable characteristics rather than individual patients, have the potential to allow for larger-scale manufacturing and minimize the heterogeneity associated with using raw material from individual patients. “In addition to transduction to introduce the CAR, gene editing using CRISPR/cas9 (EDITAS, Intellia), TALEN (Cellectis), ZFN (Sangamo), or homing endonucleases such as ARCUS (Precision Biosciences) is also necessary to knock out expression of the endogenous T-cell receptor,” says Jantz. “Such gene-edited allogeneic CAR-T cells would have significantly less potential to cause graft-versus-host disease upon adoptive transfer to the patient and can be reproducibly manufactured without the variability inherent in the manufacturing of an autologous CAR-T product,” he adds. These CAR-T products could also be manufactured at large scale and stored frozen, ready to be delivered to the patient when needed.
One downside of allogeneic therapies is the need for the additional gene editing step, which reduces somewhat the economic advantage that may be achieved due to larger-scale manufacturing. In addition, while off-the-shelf allogeneic treatment products would be available for treating patients immediately, given that autologous therapies are now produced in 2-3 weeks, timeliness of delivery is not an issue in most cases, according to Hyam “Hy” Levitsky, executive vice-president and chief scientific officer of Juno Therapeutics. He also notes that the expansion of T cells is limited. “In order to make a quantity of an allogeneic product sufficient to treat large numbers of patients, there is a concern that the extensive expansion needed will rapidly lead to senescence of the allogeneic T cells such that when infused into patients, their ability to further expand would be limited,” he says.
Finally, even though editing the T-cell receptors on allogeneic CAR-T cells will reduce the risk of graft-versus-host disease, they are still foreign cells and prone to be rapidly rejected by the host immune system, which is much less of an issue with autologous therapies. Furthermore, once rejected, there is no possibility of delivering a second dose. The question of how long these cells persist following administration to the patient must be addressed in clinical trials of allogeneic CAR-T products. “Important issues to be determined in clinical trials are whether destruction of allogeneic CAR-T cells occurs in a time frame and to an extent that limits anti-tumor activity and the number of observed complete and durable responses,” Bruce McCreedy, senior vice-president of cell therapy at Precision BioSciences states.
Better cell selection
“Over the past ten years there has been a tremendous increase in our understanding of how T cells function and how the immune system regulates itself. This knowledge has now enabled us to develop highly effective T-cell-based immunotherapies,” Levitsky observes. He adds that one of the key challenges is to identify and select the most effective T-cell subsets to develop into efficacious therapies. “From an efficacy perspective, the main challenges continue to be expansion and persistence of CAR-T cells following administration to the patient and activity of the CAR-T cells within the tumor microenvironment where numerous immunosuppressive factors are at work,” agrees Jantz.
Some companies are moving toward manufacturing schemes in which a defined mix of CD4+ and CD8+ CAR-T cells with naïve and memory phenotypes (i.e., not terminally differentiated and exhausted cells that do not expand well and persist following administration) are represented in the final product.
Strep-tag technology (2) developed by researchers at the Fred Hutchinson Cancer Research Center, Technical University of Munich, and San Raffaele Scientific Institute in Milan looks like a promising approach to the problem. The small protein tag can be used to separate out T cells carrying a CAR protein to yield highly pure samples that can then be expanded to provide more potent therapies with high regenerative potential in less time than is needed for mixed cell samples. In addition, the researchers have shown that by using a special antibody that binds the Strep-tag, engineered cells can be rapidly and repeatedly expanded. The Strep-tag when used in combination with a different antibody may also serve as a “kill switch” if cytokine release syndrome (CRS) or other toxic events occur. Once infused into patients, T cells with the Strep-tag can also be tracked using a fluorescent antibody specific for the tag.
Juno Therapeutics, which funded the work at the Hutchinson Center, has an exclusive license to the tag technology for uses related to oncology (as well as a non-exclusive license for other purposes). “We have a significant program focused on the development of technology for the physical selection of specific cells in order to generate defined cell products. These investments are allowing us to select and steer cells at the early manufacturing stage,” Levitsky states.
Another approach involves the engineering of “armored” CAR-T cells that are genetically modified to express a pro-inflammatory cytokine (e.g., interleukin 12, IL-12) in addition to the CAR. The localized secretion of IL-12 recruits help from other immune cells and supports the activity of CAR-T cells within the immunosuppressive tumor microenvironment, according to McCreedy. In addition, replacement of the murine scFv (tumor targeting portion of the CAR that is exposed on the outer surface of the cell) with human sequences that do not negatively impact the binding affinity of the scFv is expected to reduce the frequency of patient immune responses directed against CAR-T cells and hopefully improve their persistence. Gene-editing technologies are also being employed to genetically modify CAR-T cells in ways that render them more capable of trafficking to tumor sites and make them less susceptible to immunosuppression within the tumor microenvironment.
Manufacturing of CAR-T cell therapies involves multiple steps, including collection of the raw material, separation of the T cells, transduction with a viral vector (typically gammaretrovirus or lentivirus) to introduce the CAR receptor and other genetic modifications, expansion of the engineered cells, cryopreservation, and eventual infusion into the patient. While effective small-scale bioprocessing methods have been developed to meet the product needs for early-phase clinical trials, because these treatments are based on living cells (and thus the cells must be isolated as the product, not a recombinant protein), larger-scale manufacturing presents unique challenges. “An incredibly high level of organization and standardization of processes are both essential,” Levitsky notes.
In a poster presented at the American Society of Hematology Annual Meeting in December 2015, Novartis reported on how it has successfully transferred cell processing technology from the University of Pennsylvania to the company’s cell manufacturing center in Morris Plains, NJ (3). Novartis was the first healthcare company to initiate Phase II CAR-T therapy trials in the United States, Europe, Canada, and Australia, and the manufacturing facility now supports their global clinical trial program, according to a company spokesperson.
Commercial-scale cell-therapy production processes must be designed as cost-effective, closed manufacturing systems that are flexible, yet meet cGMP manufacturing requirements, and allow the use of simple techniques for cell recovery on a large scale. Cell expansion is particularly challenging at larger scales because cell culture must be achieved while maintaining the phenotype and function of the cells. At small scale, 2D culture processes are widely used and understood. They are not suitable, however, for the production of trillions of cells, which may be the typical lot size for allogeneic therapies. For autologous treatments, however, planar technologies using adherent 2D culture flasks, multilayer vessels, or multiplate bioreactors may be sufficient. Advances in the automation of these systems can be advantageous as well.
Suspension on microcarriers using 3D culture in typical bioreactors is the most likely way forward for the large-scale expansion of allogeneic CAR-T cells. The challenge is to choose a microcarrier with the appropriate surface characteristics and to establish the optimum microcarrier concentration, cell seeding density, media, and shear conditions for each cell system. The use of microcarriers is attractive at large scale because they provide greater surface area to volume for higher cell densities, and because the expansion can be performed in traditional bioreactors, control of various process parameters is possible.
Harvesting of the cells from the microcarriers is typically achieved via treatment with an enzyme, although some microcarriers are being developed that allow non-enzymatic removal. Once harvested, a volume reduction step is performed, followed by product filling. Development of effective methods for the reduction of larger volumes (< 5-10 liters) is a focus area for many companies, with tangential flow filtration (TFF) and single-use fluidized-bed centrifugation two technologies of interest.
In fact, disposable systems are highly preferred for CAR-T and other cell-therapy production processes due to the need for low-cost, flexible, closed systems that minimize contamination. McCreedy notes that several systems are in development that can separate desired cells (e.g., via elutriation or magnetic beads coated with antibodies), electroporate and/or transduce cells, wash, resuspend, and culture large numbers of cells, including removal of spent media and addition of fresh media that is designed to stimulate the proliferation and expansion of CAR-T cells with specific desired phenotypes. “Such instrumentation to automate the process and minimize the space required in a manufacturing facility should have a positive impact by increasing the consistency and reducing the cost of GMP manufacture of cell therapies,” he states.
The logistics involved in autologous CAR-T cell therapies are often raised as an important issue, but Levitsky believes they are an engineering problem that is not without technical challenges, but certainly not the biggest challenge facing developers of these next-generation treatments. Eventually, he believes it may even be possible to have CAR-T cell therapies produced at the hospital using automated instrumentation that can perform all of the necessary steps. “Such a solution is not out of the realm of possibility; there is nothing to indicate it can’t be done,” Levitsky asserts.
It is also important to note that CAR-T products require the separate manufacture of viral vectors for delivery of the CAR transgene in addition to cell expansion and harvesting. “Challenges associated with process development and validation include establishment of transduction conditions that reproducibly result in an acceptable percentage of T cells that express the CAR at defined levels on the T cell surface,” says McCreedy.
Product Characterization and Release Testing Issues
Product characterization and release testing present additional challenges to GMP manufacturing of CAR-T products. The creation of master cell banks from customized cell lines that express specific ligands and/or reporter molecules for use in expanding CAR-T cells in culture and for use in characterizing the potency and specificity for release of GMP-manufactured CAR-T products are beginning to make their way into the manufacturing process, which should help to provide additional consistency in the process, according to McCreedy.
Because CAR-T products are considered to be both cellular and gene therapy by FDA and the European Medicines Agency, genetic stability studies are required in addition to traditional stability upon storage documentation. The required test for replication-competent lentivirus is particularly onerous due to the cost and time required. Alternatives for delivery of genetic data that avoid the use of viral vectors are in development, such as the introduction of the CAR as a transiently expressing messenger RNA (mRNA), plasmid DNA transfection, and the use of transposable elements (transposons) to replace existing genes with new ones, according to Levitsky. He also believes it is possible that as the field matures and experience with CAR-T cell therapies increases, there will eventually no longer be a need for the test.
More on cell therapy:
Advancing cell therapy safetyCell therapy growth and pains: Investment, collaboration, and controversyRole of contract manufacturing in cell therapy development and manufacturing
1. Jain PharmaBiotech, Cell Therapy-- Technologies, Markets, and Companies (March 2016), accessed April 12, 2016.
2. R. Tompa, Crafting a Better T Cell for Immunotherapy (Feb. 22, 2016), accessed April 18, 2016.
3. J.A. Boyd, et al., Successful Translation of Chimeric Antigen Receptor (CAR) Targeting CD19 (CTL019) Cell Processing Technology from Academia to Industry, Poster, American Society of Hematology 57th Annual Meeting (Orlando, FL, December 2015).
Article DetailsBioPharm International
Vol. 29, No. 5
Citation: When referring to this article, please cite it as C. Challener, "Realizing the Potential of CAR-T Cell Therapies," BioPharm International 29 (5) 2016.