by Yelena Bronevetsky, PhD, Director of Product Management, and James Lim, PhD, Chief Scientific Officer, Xcell Biosciences
As more and more labs get involved in the research, development, and manufacture of cell therapies, it is important to consider a key piece of technology that is often overlooked in these pipelines: the humble incubator.
From the earliest phases of cell therapy design to the final phases of preparing treatments, the goal of culturing is to grow cells that will have the greatest potency and impact in their target environment. Mounting scientific evidence has shown the best way to do that is to acclimate cells to the conditions they will encounter in vivo.
Unfortunately, standard cell incubators lack the customization needed to adapt cells to microenvironments within a human body. Parameters that are defining features in these environments, such as oxygen and interstitial pressure levels, cannot be precisely adjusted in regular incubators.
This adaptation, or metabolic conditioning, is especially important as scientists seek to repeat the successes of cell therapies for hematological malignancies in solid tumors. The hostile microenvironments surrounding these tumors are notoriously challenging for cell therapies, with a host of defenses that prevent cells from getting to the tumor while simultaneously weakening the cells that do get through. Cell therapies grown in similar conditions to these tumor microenvironments (TMEs) appear to be more potent and persistent than those grown in standard incubator conditions.
Recent studies have shown that using a more advanced incubator, with built-in customization for oxygen and hyperbaric pressure levels in addition to the parameters included in conventional incubators, allows for metabolic conditioning of cell therapies that better prepare them for in vivo use. Various assays used to measure performance ex vivo indicate that these cell therapies have superior tumor-killing function that persists across multiple rounds of tumor challenge.
Metabolic conditioning
To treat cancer, therapeutic cells must maintain their killing function not just through their journey to the site of a tumor but also in the face of TME-specific suppressive forces including high pressure, low oxygen, and cells that block T cell function.
The typical approach to designing cell therapies is to focus on genetic engineering, with the goal of conferring potency and tumor-killing function to these cells through genetic changes. But evidence suggests that there is a simpler and potentially more effective method: training cells to survive in the harsh conditions they will encounter in the body. It’s comparable to the way elite athletes train, finding an environment that will give them the same challenges they’ll face in competition. Professional mountain climbers train in low-oxygen environments so they can weather high altitudes; the best tennis players might train in scorching heat to prepare for grueling summertime tournaments.
There is plenty of reason to believe that an analogous approach can produce hardier, higher-functioning cell therapies. “Training” the cells means growing them in conditions designed to mimic those in their target destination. This bucks conventional wisdom, which has long pushed the idea that cells should be grown in conditions that make them flourish. If plenty of oxygen and low-pressure levels keep cells dividing as quickly as possible, scientists reasoned, that must be a good way to produce potent cell therapies. Unfortunately, real-world evidence suggests the opposite is true, particularly for solid tumors. Cells grown in these ideal conditions perform poorly in the reality of a hostile TME.
There have been several studies illustrating this problem. To take one, scientists reported strong cancer-killing function for chimeric antigen receptor (CAR) T cells targeting the ROR1 protein.1 In this study, cells were cultured and killing assays were performed at ambient oxygen levels. But when these seemingly high-performing therapeutic cells were infused into humans, their function was nowhere near expectations. In animal studies conducted to understand what happened, it was clear that these CAR T cells were able to reach the tumor site, but when they arrived, cells lacked sufficient tumor-killing activity. Cells that were by all indications primed to fight tumors faltered in real TME conditions.
Conversely, many experiments have now demonstrated the value of metabolic conditioning, and the link between culture conditions and in vivo efficacy, including in solid tumors.2,3 For example, cells that were grown in hypoxic conditions during T cell activation were found to have more pronounced cytotoxic function in vivo.4 Other work has shown that reducing the presence of glucose in culture media yields cells with greater anti-tumor effects.5
Cell culture media formulation has become an area of focus, with the discovery that the specific cytokine composition can affect cell therapy performance. It’s widely accepted that the typical IL-2 containing media boosts cell growth in culture; however, IL-7, IL-15, and IL-21 are all examples of cytokines that limit expansion in the incubator but drive higher persistence and superior potency for cell therapies in the patient.
Clearly, there is good reason to veer away from the conventional approach to growing cell therapies. Already, studies show that toughening up cells during the culture stage might make them scrappier, more resilient tumor fighters in the body.
Case study at Labcorp
More sophisticated incubators now make it possible for scientists to tune all the standard culture parameters while also adjusting oxygen and pressure levels to create customized conditions that prime cells for the in vivo microenvironments where they must function. This new generation of incubators is a natural fit for scientific workflows related to cell therapies, whether that’s designing a therapy, optimizing it, or manufacturing it for use in a patient.
In a recent example of this, scientists at Labcorp Drug Development used the AVATAR incubation system from Xcell Biosciences to evaluate the potency and performance of CAR T cells, comparing cells grown in conventional culture to those grown in conditions better approximating that of the TME.6
CD3+ T cells were transduced with a CD19 CAR-encoding lentivirus and cultured for a total of 12 days, at which point CAR expression was measured by flow cytometry and cytotoxic activity was evaluated using a targeted killing assay. Results showed that the populations grown under hypoxic, pressurized conditions exhibited increased CAR expression compared to the cells grown in standard culture conditions, with overall yields of the desired cells as much as double in the hypoxic condition.
Cytotoxicity was assessed in vivo using a mouse model for B cell acute lymphoblastic leukemia. Mice inoculated with NALM6 cells were then dosed with CD19-CAR T cells, and outcomes were followed for five weeks. Animals treated with cells grown in the advanced incubator conditions showed good tumor control, and T cells collected from blood samples indicated strong persistence with the desired phenotype. In a subsequent experiment, CAR T cells were grown under even lower levels of oxygen and co-cultured with tumor cells at very low effector to target (E:T) ratios for an extended time; in this scenario, T cells must perform serial killing in order to control tumor growth. CAR T cells grown in the low oxygen, pressurized conditions exhibited increased cytotoxicity compared to cells grown in regular culture conditions.
But by what mechanism can cells function effectively in a solid tumor microenvironment? Recent research from the Sadelain lab shows that overexpression of the glucose transporter GLUT1 enhances potency and maintains T stem cell-like memory populations in CAR T cells, prolonging the survival of mice bearing glioblastoma tumors.7 Interestingly, GLUT1 expression is controlled by Hypoxia-inducible factor-1 alpha (HIF1a), a master transcriptional regulator that is induced under low oxygen conditions. Induction of HIF1a leads to significant metabolic changes in CAR T cells, promoting the expression of genes associated with glycolysis and mitochondrial respiration. In solid tumor microenvironments where oxygen levels are low, CAR T cells manufactured under hypoxic conditions can revert to glycolysis (away from oxidative phosphorylation) to produce enough energy to divide and kill tumor cells.
Looking ahead
The disappointing past performance of cell therapies against solid tumors underscores the need to seek creative approaches that could improve the potency and persistence of future therapies. For optimal results, those approaches must factor in the harsh conditions of the TME. With mounting evidence from scientific studies of metabolic conditioning and examples such as the Labcorp work described above, there is no question that continued cell therapy investigations should include more sophisticated incubators to help adapt cells to their target environment.
About the authors:
Yelena Bronevetsky is the Director of Product Management at XCellbio. Yelena received her B.A. in Biology from New York University and her PhD in Immunology from the University of California, San Francisco. She also conducted her post-doctoral work at UCSF.
James Lim is the co-founder and Chief Scientific Officer at Xcellbio, and is leading the company’s scientific effort in the development of an advanced cell therapy manufacturing and analytics platform. He received his B.Sc. In Cell Biology from McGill University, and his Ph.D. in Biophysics from The Scripps Research Institute. James conducted his post-doctoral studies at Harvard Medical School and launched Xcellbio while working as a researcher at the Lawrence Berkeley National Laboratory.
References
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2. Zhang Y, Kurupati R, Liu L, Zhou XY, et al. Enhancing CD8+ T Cell Fatty Acid Catabolism within a Metabolically Challenging Tumor Microenvironment Increases the Efficacy of Melanoma Immunotherapy. Cancer Cell. 2017 Sep 11;32(3):377-391.e9. doi: 10.1016/j.ccell.2017.08.004.
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5. Klein Geltink RI, Edwards-Hicks J, Apostolova P, O’Sullivan D, et al. Metabolic conditioning of CD8+ effector T cells for adoptive cell therapy. Nat Metab. 2020 Aug;2(8):703-716. doi: 10.1038/s42255-020-0256-z.
6. Xing Y, Garcia C, Eaker S, Bronevetsky Y, et al. Metabolic Reprogramming Enhances Expansion and Potency of CAR-T cells. Poster #6334 presented at AACR 2024. https://www.xcellbio.com/_files/ugd/7d1633_a906d8bc86584e8e9951aedc38b998c4.pdf
7. Shi Y, Kotchetkov IS, Dobrin A, Hanina SA, Rajasekhar VK, Healey JH, Sadelain M. GLUT1 overexpression enhances CAR T cell metabolic fitness and anti-tumor efficacy. Mol Ther. 2024 Jul 3;32(7):2393-2405. doi: 10.1016/j.ymthe.2024.05.006.