Blog | February 17, 2022

Zen & The Art Of Allogeneic NK Cell Therapy Manufacturing

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By Anna Rose Welch, Editorial & Community Director, Advancing RNA


Over the past year, there have been an increasing number of cell and gene therapy companies journeying beyond the world of T cells into natural killer (NK) cell therapies. Though my previous article with Ken Chrobak, VP of CMC for Wugen, highlighted which learnings could be taken from the autologous manufacturing space and applied to allogeneic therapies, we can’t overlook the layers of complexity that the cell type inherently throws into the manufacturing mix.

For many companies, including Wugen, a large portion of the manufacturing work today is devoted to upstream efforts — particularly expanding and activating the cell of choice. As you can imagine, however, much of the “double double toil and trouble” of finding the best recipe for feeding, growing, and engineering cell lines in the CGT space is kept behind tightly locked doors. This lack of publicly available information has been one of the bigger challenges of developing an NK cell therapy platform, Chrobak admitted. NK cells are one of the “new[er cell types] on the block,” meaning information — particularly manufacturing information — is scarce to nonexistent. In turn, Wugen has had to rely on the extensive research carried out by Washington University in St. Louis (from which it licensed its CAR-T and NK programs), as well as the company’s experience with its allo-CAR-T program, its in-house manufacturing know-how, and collaborations with its partners.

As Chrobak explained, one of the main challenges of any allogeneic cell therapy program, which garners its starting material from healthy donors, is identifying which cell phenotypes generate the most robust clinical responses in patients. For Wugen, the phenotypic research carried out at Washington University in St. Louis was a valuable asset in helping them identify the optimal cell phenotype, and in turn, their “super donors”— or those with the best phenotypes for creating the most effective therapy — from pools of healthy donors.

“The initial investigator-initiated studies for our allogeneic memory NK cell program recruited donors that were relatives of each patient,” Chrobak said. “There were over 50 patients in total — so, over 50 different donors. In correlative studies, the investigators first identified the properties of each donor and, following treatment of each patient, determined the potency and durability of each therapy. The patient’s responses were then correlated back to the phenotypic data to determine which phenotypes generated the best clinical responses in the patient.”

Of course, like many other cell types, a common stumbling block when working with NK cells is determining the most productive expansion strategies while also maintaining their activity level. Employing feeder cells has been one of the most common solutions for generating enough high quality, active cells. However, going the feeder cell route also requires companies to perform an additional purification step. This is why Wugen decided to take another approach, striking up a partnership with HCW Biologics to use its biologics-based expansion method. 

“This complex protein cocktail is our ‘special sauce,’” Chrobak tells me; “It ensures a more robust and safer manufacturing system because we don’t need to purify out any feeder cells. Instead, we’re able to activate and expand our memory NK cells in a straight-forward, robust manner.”

Beyond expansion, identifying the most appropriate cryopreservation method to work with NK cells has been another challenge. Regardless of cell type, cryopreservation has been flagged as a big hurdle for companies across the board — particularly in the allogeneic cell therapy space. (In fact, as one executive in the allogeneic industry argued in this article, cryopreservation technology for CGTs is an area ripe for more innovative solutions.) Cryopreserving the material puts the cells through a fundamental state-change which can jeopardize the health and stability of the cells.

When exploring cryopreservation for a new product/cell type, CGT companies aren’t necessarily starting from a blank slate; they can take what they’ve learned from working with another type of cell (most likely T cells) and optimize these processes for the new cell. However, in Chrobak’s experience, these previous learnings will not always neatly translate over to a new cell type. For example, Chrobak and his team learned that many of the commercially available (primarily T-cell specific) cryopreservation medias were not a suitable match for NK cells, and, at the time, there were no commercially available NK-specific cryopreservation agents. As such, Chrobak and his team had to implement practices from the academic world and in-house trial and error.

All this hard work resulted in a process with which Wugen can maintain the pre-freeze activity for a high percentage of its cells. The phrase “it’s a loss we can live with” is a phrase I’ve heard time and again in the CGT space — and it’s a mindset Chrobak embraces as well when evaluating the minimal cell loss following the cryopreservation process. However, he remains optimistic that the percentage lost to cryopreservation will continue to decrease overtime. As more investment occurs in the NK cell realm, he anticipates media companies will, likewise, rise to the occasion with a variety of NK-specific products. Similarly, there are steps companies can take in-house to better understand and optimize the freezing process itself.

“Most people use equipment like controlled rate freezers, so they can control the rate of freezing,” he said. “But a lot of us rely on the default programs, which may not be the best route to take for the product. So, optimizing exactly how you freeze your products will be a critical step toward advancing and improving cryopreservation of NK cells in the future.” 

In part 3, Chrobak and I will continue our discussion on the technical infrastructure and mindset necessary to turn an academic manufacturing process into a commercially viable process that can also span continents. Stay tuned!