While the use of cell therapies to treat hematological malignancies has been hailed as a clinical breakthrough, this promising new class of therapies has been far less effective in treating solid tumors. Since the vast majority of cancers involve solid tumors, there is a pressing need to address current issues and make cell therapies a good option for far more patients.

Perhaps the greatest challenge facing cell therapies for this purpose lies in the immunosuppressive mechanisms found in the tumor microenvironment. This is harsh biological territory, with high interstitial pressure, reduced oxygen tension, and a barrage of immunosuppressive proteins. Studies show that therapeutic cells exposed to this environment suffer from depletion, exhaustion, and mitochondrial dysfunction.

Thus, there is a critical need for manufacturing processes to generate more potent cell populations, both by producing higher proportions of antigen-targeted cells (for example, chimeric antigen receptor T cells, or CAR T cells) and by boosting the cytotoxic potential of those cells.

New evidence indicates that cell therapies can be “rewired” metabolically to overcome these challenges, leading to treatments that could be far more effective against a range of solid tumors. The idea is simple: instead of growing therapeutic cells in conventional culture conditions designed to keep cells as happy as possible, why not acclimate them under conditions mimicking that of the tumor microenvironment they will have to face in vivo?

The cells that survive and expand in vitro under these harsh conditions should be far more likely to remain effective when they reach the tumor microenvironment. Results are already showing that metabolic rewiring can create higher-yielding, effective cell therapies.

Feasible approach

This approach is feasible for any scientist developing cell therapies. For the work described below, teams at Xcell Biosciences and Labcorp used the AVATAR system to compare the performance and effectiveness of CAR T cells grown in conventional culture conditions versus conditions more closely mirroring the tumor microenvironment.

Cells were prepared according to established cell therapy manufacturing protocols and cultured under a range of O2 and pressure levels. We first assessed three different culture environments: standard culture conditions with no change to oxygen or pressure in a conventional CO2 incubator (20.5% O2 with 0 pounds per square inch (PSI)); the AVATAR incubator set to pressure and oxygen levels replicating the arterial vasculature system at 15% O2 with pressure at 2 PSI; and the AVATAR incubator at 15% O2 and pressure at 5 PSI. All incubation conditions were maintained at 5% CO2 and 37°C.

CD3 T cells were thawed alongside soluble anti-CD3 and cultured for two days. All three cell populations were then transduced with a lentivirus designed for CD19 CAR expression and returned to their incubator conditions to grow for 10 more days. CD19 CAR expression was evaluated with flow cytometry, and cytotoxic activity was assessed with a targeted killing assay.

These in vitro experiments demonstrated that culturing with adjusted O2 and pressure levels had no detrimental effect on the therapeutic cells, and these cells actually outperformed cells grown in standard incubator conditions (Figure 1). For example, we found that both cell populations grown with modified oxygen and pressure settings had higher proportions of CAR T cells following lentiviral transduction compared to the conventional incubator population. Cells transduced in the conventional CO2 incubator conditions yielded 10% to 20% CD19 CAR T cells, while those transduced in high-pressure conditions yielded as much as 40% of the desired therapeutic cells.

Figure 1. Generating CD19 CART cells under pressurized conditions in the AVATAR system results in higher CAR expression and overall yield.

By the end of the expansion process, cell yield was more than twice as high in the two groups expanded at 15% O2 and at 2 or 5 PSI as compared to the standard incubator cells.
Potency tested

The potency of CD19 CAR T cells manufactured in the three culture environments was next tested in vitro with a targeted NALM6 cytotoxicity assay. For this work, we co-cultured T cells with NALM6, CD19-expressing target cells, for 48 hours, and measured lysis rates by flow cytometry. As expected, CD19 CAR–specific killing was exhibited in a dose-dependent manner, and cells grown in the AVATAR system showed no impairments to their cytotoxic function.

Cytotoxicity was confirmed in a mouse model of B-cell acute lymphoblastic leukemia. CD19 CAR T cells were administered to mice that had been inoculated with NALM6 cells intravenously. For five weeks, we regularly measured tumor growth with bioluminescence imaging, and we collected blood samples to identify circulating T cells and assess their phenotype using flow cytometry.

Results showed that mice treated with the two AVATAR cell populations had good tumor control outcomes, confirming that the modified culture conditions do not impair cancer-fighting ability in vivo (Figure 2). We also examined blood samples for T cells using Labcorp’s Custom Expanded Persistence T Memory Panel. The animals exhibited persistence of the therapeutic cells in relevant organs, and the CAR T cells maintained their phenotype with strong central memory and effector memory populations.

Figure 2. In vivo NALM6 challenged of CD19 CAR T cells cultured in the AVATAR system.

Finally, we performed experiments on CD19 CAR T cells grown in conditions of even more acute hypoxia, with conditions of 5%, 10%, and 15% oxygen, while maintaining 5 PSI, and compared them to cells grown in the conventional CO2 incubator. We again evaluated the cells’ potency in vitro with a targeted NALM6 cytotoxic assay, but importantly, tested the ability of CD19 CAR T cells to kill for extended time periods at low effector-to-target cell ratios (Figure 3).

Figure 3. In vitro NALM6 challenge of CD19 CAR T cells cultured in the AVATAR system at lower O 2 levels. [Xcell Biosciences]
In these circumstances, T cells must engage in serial killing activity to manage the large numbers of tumor cells. Encouragingly, cells grown under reduced O2 and high pressure had specific cytotoxic function that outperformed that of cells grown under standard conditions in both acute killing (measured at 24 hours) and extended killing (measured at 72 hours) activity, especially under low effector-to-target ratios (1:16, 1:32).

The improved cytotoxic function of CD19 CAR T cells grown under lower oxygen conditions is currently being confirmed in further in vivo experiments. While this work is being performed with a mouse model for hematological cancer, follow-up studies using this same approach for solid tumors are already underway.

All of this work was completed with well-established protocols for cell therapy development, with no special techniques required for the cells expanded in the AVATAR system. In the future, a more automated workflow will be enabled with the upcoming AVATAR Foundry system, which has been designed specifically for the manufacture of cell therapies. This would allow clinical research teams to incorporate hyperbaric pressure and oxygen control into the manufacturing process more easily, accelerating the process of discovering the optimal environmental conditions to enhance the potency and yield of each therapy candidate.

The GMP-compliant AVATAR Foundry system is currently available to scientists through a beta access program. To see more of this data, view a poster (“Metabolic reprogramming enhances expansion and potency of CAR-T cells”) that Xcell Biosciences and Labcorp scientists presented at the American Association for Cancer Research Annual Meeting in 2024.

James Lim, PhD, is a co-founder and the CSO of Xcell Biosciences. Scott Wise, MS, is executive director of preclinical oncology at Labcorp.

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