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Further advances in construct design and manufacturing scalability are still needed.
Autologous chimeric antigen receptor (CAR) T-cell therapies have been shown to be effective for the treatment of many types of cancers. Challenges with scalability and manufacturing associated with autologous cell therapies have led to rapid advancement of allogeneic off-the-shelf cell therapies. However, allogeneic cell therapy products have their own challenges including problems with excessive immune responses and concerns about graft-versus-host disease (GVHD).
T cells are not the only immune cells, however, and there is significant interest in leveraging natural killer (NK) cells in a similar manner. NK cells are lymphocytes that have the ability to target tumor cells, releasing chemokines and cytokines that activate the innate and adaptive immune systems. They can be isolated from a variety of sources and do not have to be patient-specific. Progress is being made in the development of scalable production processes. Clinical trial data from engineered allogeneic NK cells is already in the public domain, additional clinical data continues to emerge from early-generation products, and data read-outs will soon begin for next-generation engineered products.
NK cells are important lines of defense the body has against infectious agents and tumors. They target any cells lacking a major histocompatibility complex (MHC), a group of genes that code for proteins found on the surfaces of cells that are unique to each individual and part of the human leukocyte antigen (HLA) system, according to Heather Stefanski, vice president of medical services at the National Marrow Donor Program (NMDP)/Be The Match. “Each T cell has a unique T cell receptor that recognizes a cognate antigen presented in the MHC complex; the repertoire of human T cells includes millions of unique T cells. Tumor cells can evade the immune system as tumors often do not have antigens that T cells can recognize, and therefore, the T cells do not attack them,” Stefanski says.
NK cells have the intrinsic ability to mediate tumor killing through innate recognition pathways without prior sensitization, comments Ryan Larson, vice president and head of translational science at Umoja Biopharma. “These intrinsic anti-tumor properties can be augmented by engineering NK cells to express synthetic tumor targeting proteins, such as CARs or adapters compatible with tumor-specific monoclonal antibodies that can be administered in combination,” he notes.
Next-generation CAR cell therapies, contends William Rosellini, co-founder, president, and director of CytoImmune, are focused on NK cells because they are one of nature’s best killing machines. “The NK cell has unbelievable tumor-killing activity with significantly reduced safety issues like on-target/off-tumor effects, GVHD, and cytokine release syndrome (CRS),” he explains.
NK cells do not have endogenous T cell receptors, nor do they cause the secretion of interleukin 6 and other cytokines that can cause undesired immune responses, Stefanski notes. As a result, NK cells typically have shown reduced cytokine production and expansion, and thus reduced rates of some of the serious side effects of CAR T-cell therapies, such as CRS and immune effector cell-associated neurotoxicity syndrome (ICANS), according to Larson. In addition, NK cells do not express antigen-targeting receptors that are tuned specifically to their host, thus eliminating the graft-versus-host risk that exists with allogeneic T-cell therapies.
That latter attribute also makes the development of NK-cell therapies less complex than the development of allogeneic CAR T-cell therapies, Larson adds. “For any allogeneic T-cell product, a genetic manipulation must be made to eliminate T-cell receptor expression. This step is not required for NK cells, and thus simplifies their engineering and manufacturing processes, as well as removing a potential safety issue,” he observes.
The ability to produce allogenic products from healthy starting cells is another advantage. “Autologous, or patient-specific, CAR T-cell therapies are produced using cells collected from patients who are very sick. Those T cells are potentially not ideal for a cell therapy product as some may not be good at expressing CAR proteins. In addition, the types of T cells present in each person will vary, and those differences can have a direct influence on the ability to modify those cells and generate an effective therapy,” Stefanski explains.
Overall, concludes Chris Nowers, CEO of ONK Therapeutics, allogeneic NK-cell therapies have the potential to deliver treatments with compelling clinical profiles that are better tolerated and more easily administered, logistically simpler to manufacture, and produced at lower cost. “We believe that through the reduction of costs and a growing accumulation of clinical data demonstrating the positive longer-term benefits, we will be better able to demonstrate measurable value,” he states.
NK cells are present throughout the body and can be harvested from a number of sources. They can be separated from peripheral blood or isolated from umbilical cord blood or the placenta for the development and production of allogenic therapies.Another option involves differentiation of human pluripotent stem cells (iPSCs).NK cells may also be generated from CD34+ stem cells present in bone marrow.
According to Rosellini, NK cells isolated from umbilical cord blood samples have been shown to have a higher cytotoxic capacity and span as much as 30% of white blood cells recovered, while NK cells isolated from peripheral blood tend to be a more mature population exhibiting more refined cytotoxic capabilities with a lower 15% of white blood cells recovered.
The lymphoma-derived NK-92 cell line is one established cell line designed to be easy to maintain and expand, as well as easily genetically modified using both viral and non-viral methods. It does require irradiation prior to clinical use, however, which can impact activity.
All of these different sources have been used to produce CAR NK-cell therapies undergoing clinical development. ONK Therapeutics, for instance, uses umbilical cord blood provided by Anthony Nolan Cell & Gene Therapy Services for development of its engineered NK-cell therapies. “We believe that cord blood offers a widely available source of NK cells that has been clinically validated and provides cells with a potential attractive phenotypic makeup,” Nowers observes.
Larson’s perspective is that we don’t know yet whether the source will affect the application. “It is likely that some combination of source, CMC [chemistry, manufacturing, and controls] processes, engineering/synthetic biology, and tumor-targeting approach (e.g., CAR) will determine the best therapeutic applications,” he states.
As with CAR T-cell therapies, once the NK cells for development of the therapy have been isolated, they must be expanded and genetically modified to express the CAR protein. The methods for cell expansion and cell engineering are similar for NK cells and T-cells, with some specialization accruing to each cell type, according to Larson. In most cases, large-scale expansion using cytokines and feeder cells is required to produce sufficient NK cells for clinical applications, notes Nowers. This approach facilitates efficient engineering.
Rosellini adds that retroviral, lentiviral, or non-viral delivery of genetic material is used, with viral-vector gene delivery systems currently considered the most effective. ONK Therapeutics is an exception. The company uses a combination of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) editing and transposition of transgenes employing a non-viral TcBuster transposon approach (Bio-Techne) to produce large numbers of edited CAR-NK cells, according to Nowers.
Larson believes that the method that gives the largest scale possible will be the best to commercialize, as long as it can accommodate some engineering to make sure that the cells will perform well, including in the tumor microenvironment (TME) for solid tumors. He does note, however, that there are schools of thought that think it best to use minimally expanded cells.
While the theoretical unlimited expansion capacity of iPSCs makes them an attractive option for the production of CAR NK-cell therapies, Larson also points out there have been challenges to translating this feature to downstream yield of relevant immune cell progenitor populations in an efficient and scalable format. “Solutions for scaling a directed differentiation manufacturing process will realize the full value of iPSC as a starting material from a CMC perspective-in terms of simplicity, reducing cost, and patient access,” he remarks.
In this vein, Umoja recently entered a collaboration with TreeFrog Therapeutics to develop CAR NK-cell therapies (1). Umoja’s iPSCs are engineered with a synthetic rapamycin-activated cytokine receptor (RACR) to drive differentiation to, and expansion of innate cytotoxic lymphoid cells, including but not limited to natural killer (NK) cells in the absence of exogenous cytokines and feeder cells. TreeFrog’s proprietary C-Stem technology relies on the high-throughput encapsulation (>1000 capsules/second) of iPSCs within biomimetic alginate shells that promote in vivo-like exponential growth and protect cells from external stress.
One of the less-desirable attributes of CAR NK-cell therapies when compared to CAR T-cell therapies is their lack of persistence in the body. NK cells have a much shorter half-life on the order of weeks. Administration of cytokines such as interleukin 2 to patients can prolong NK cell lifetimes, but Stefanski notes this approach often comes with harmful side effects.
The need for lymphodepletion is another important issue associated with allogeneic CAR NK-cell therapies. Prior to administration, patients receive a short course of chemotherapy to kill their existing T cells as a means for increasing persistence. “Lymphodepletion adds a veneer of immune system damage that provides short-term benefits in enabling engraftment of therapeutic cells but is detrimental to immune system function and may contribute to recurrences or other problems in the long term,” Larson says.
It is important to employ strategies to improve drug exposure through engineering strategies and be able to dose more than once at high dose levels. “Scalable manufacturing solves a lot of problems,” Larson states. Developing NK cells that secrete their own cytokines, such as interleukin 15, is also an approach to improving NK persistence, according to Nowers. He notes that this strategy can be synergistic with the knockout of the cytokine inducible SH2-containing (CISH) protein.
The development of stealthy NK cells that are edited to evade the immune system is another strategy being pursued by several companies, according to Nowers. ONK Therapeutics is also looking to increase the ability of its NK cells to target specific tumors by using cells that express high levels of favorable chemokine receptors such as CXCR4 and CXCR3 and E-selectin ligands. “These features ensure a higher likelihood that the NK cells will traffic to the site of the tumor within their natural lifetimes,” says Nowers.
Other challenges for CAR NK-cell therapies, according to Stefanski, are similar to those faced by CAR T-cell therapies: difficulty infiltrating the TME of solid tumors, the use of lentiviral vectors for delivery of genetic material, and the need to know the target antigen. In addition, although CRS is avoided by using NK cells, other off-targets are possible that must be avoided. There are also complexities associated with garnering regulatory approval for cell therapies, Rosellini adds.
As next-generation treatments, the development of CAR NK-cell therapies is not as far along as that of CAR T-cell therapies. Most programs are preclinical to early-phase clinical, including both Phase I and II trials. Cancer is the focus, with the greatest number of candidates targeting blood cancers including various leukemias, lymphomas, and multiple myeloma, but there are trials underway in solid tumors such as glioblastoma, non-small cell lung cancer, head and neck squamous cell carcinoma, and prostate cancer.
Rosellini points out that academic research groups are hard at work understanding how CAR-NK cells could be used on other diseases too. “Similar to cancer cells, we could train NK cells to target other malfunctioning cells in inflammatory diseases,” he notes. Stefanski suggests there is a potential for genetically modified NK cells to fight viruses that infect patients following stem-cell transplants and to treat autoimmune diseases driven by malfunctioning B cells. “Theoretically, CAR NK-cell therapies could be used to treat any disease that involves proliferation of abnormal cells,” she concludes.
The CAR NK-cell therapy field is still quite new. Additional clinical data from Affimed and Fate and readouts for some of the hypo-immune cells from Century are expected during the next 6–12 months, according to Nowers.
Stefanski anticipates seeing the number of Phase II trials continuing to grow. Within the next year, she believes at least one hematological malignancy candidate may advance to Phase III. Five years from now, she hopes there will be at least one CAR NK-cell therapy on the market.
CAR NK-cell therapies, Stefanski also suggests given their limited persistence, may have the potential to get patients into remission and in a position to undergo stem-cell transplants they wouldn’t be eligible for otherwise. The limited persistence of NK cells may also be an advantage from a safety point of view, according to Nowers, because it could lead to reduced risk of prolonged on-target, off-tumor toxicity.
Improvements in manufacturing technologies are also anticipated, whether using NK cells isolated from umbilical cord blood or differentiated from iPSCs. “Enhanced manufacturing procedures and the increasing use of automated closed platforms both have the potential to significantly reduce cost of goods,” Nowers notes.
More advanced cell modifications are also being explored. Approaches that include multiple receptors and different ligands on the NK cells have shown potential. “Researchers are beginning to figure out what is best for a given application, because solutions that work well for one type of cancer aren’t necessarily optimal for others,” Stefanski says. As information is gained about the current targets and how existing CAR NK-cell therapy candidates behave clinically, Rosellini believes that much will also be learned about how to improve their safety and potency.
For instance, Larson observes that organizations including Umoja will introduce second-generation approaches that avoid lymphodepletion, include engineering strategies to enhance engraftment and pharmacokinetic profiles, and enable the production of larger numbers of doses with higher cell numbers per dose. “As these advances are layered on, I expect we will see better and better clinical results,” he states.
Similarly, companies that are exploring gene editing to benefit the cytotoxic potential, metabolic health, and durability of engineered cells in-vivo will demonstrate enhanced persistence and metabolism, improved tumor homing, reduced exhaustion and immune invasion, and maximization of antigen-independent killing, according to Nowers.
Longer follow up and more mature data from ongoing studies should give greater confidence in the durability of CAR-NK approaches as well as in their safety, says Nowers.“The breadth of ongoing research and an increased move to better tolerated, logistically simpler allogeneic NK cell-therapy platforms will allow evolution toward a more traditional treatment model for advanced cell therapies. Simultaneously, the use of big data and artificial intelligence to direct selection of the best patient candidates will lead to better efficacy, greater patient benefits, and increased understanding,” he contends.
The end result will potentially be significantly reduced list price costs combined with a growing clinical database demonstrating the positive longer-term benefits of CAR NK-cell therapies, according to Nowers. “Stronger demonstration of value will help support the willingness to pay of payors and subsequently drive broader patient access and adoption,” he reiterates.
1. Umoja Biopharma, “Umoja Biopharma and TreeFrog Therapeutics Announce Collaboration to Address Current Challenges Facing Ex Vivo Allogeneic Therapies in Immuno-Oncology,” Press Release, June 10, 2022.
Cynthia A. Challener, PhD is a contributing editor to Pharmaceutical Technology.
Vol. 46, No. 8
When referring to this article, please cite it as C. Challener, “Progress in the Development of CAR NK-Cell Therapies,” Pharmaceutical Technology 46 (8) 2022.