Enhancing Chimeric Antigen Receptor T Cell Anti-tumor Function through Advanced Media Design

Abstract

Effective chimeric antigen receptor (CAR)-T cell therapy is dependent on optimal cell culture methods conducive to the activation and expansion of T cells ex vivo , as well as infection with CAR. Media formulations used in CAR-T cell manufacturing have not been optimized for gene delivery, cell expansion, and overall potency. Bioactive components and derivatives that support the generation of functionally-competent T cell progeny with long-lasting persistence are largely undefined. Current media formulations rely on fetal bovine serum (FBS) or human serum (HS), which suffer from a lack of consistency or supply issues. We recognize that components of blood cellular fractions that are absent in serum may have therapeutic value. Here we investigate whether a concentrated growth factor extract, purified from human transfusion grade whole blood fractions, and marketed as PhysiologixTM xeno-free (XF) hGFC (Phx), supports CAR-T cell expansion and function. We show that Phx supports T cell proliferation in clinical and research-grade media. We also show that Phx treatment enhances lentiviral-mediated gene expression across a wide range of multiplicity of infections (MOIs). We compared the ability of anti-GD-2 CAR-T cells expanded ex vivo in medium conditioned with either Phx or HS to clear tumor burden in a human xenograft model of neuroblastoma. We show that T cells expanded in Phx have superior engraftment and potency in vivo , as well as CAR-induced cytolytic activity in vitro . Metabolomic profiling revealed several factors unique to Phx that may have relevance for CAR-T cell preclinical discovery, process development, and manufacturing. In particular, we show that carnosine, a biogenic amine modestly enriched in Phx relative to HS, enhances lentiviral gene delivery in activated T cells. By limiting extracellular acidification, carnosine enhances the metabolic fitness of T cells, shifting their metabolic profile from an acidic, stressed state toward an oxidative, energetic state. These findings are very informative regarding potential derivatives to include in medium customized for gene delivery and overall potency for T cell adoptive immunotherapies.

Tinkering under the Hood: Metabolic Optimisation of CAR-T Cell Therapy

“…Enhancing CAR-T cell memory is key to improving their persistence and anti-tumour activity and increasing their therapeutic capacity. CAR-T cells are currently created, expanded, and maintained in media formulation containing excessive nutrient levels such as carbohydrates and amino acids [ 4 , 9 ]. In particular, two supraphysiological nutrients are glucose and glutamine, which are essential fuels for rapidly proliferating, activated immune cells [ 13 , 40 ].…”

“…Efforts have been made to move away from the reliance on human serum in media used for CAR-T cell manufacturing with the identification of a concentrated growth factor derived from human transfusion grade whole blood fractions, known as PhysiologixTM xeno-free (XF) hGFC (Phx) [ 9 ]. Phx, in comparison to human serum, has been demonstrated to enrich carnosine levels that enhance downstream T cell proliferation and peripheral blood abundance in both clinical and research grade media.…”

“…Phx, in comparison to human serum, has been demonstrated to enrich carnosine levels that enhance downstream T cell proliferation and peripheral blood abundance in both clinical and research grade media. Furthermore, the authors demonstrate that carnosine inclusion in media formulations are able to neutralise extracellular protons (H + ) derived from lactate (via aerobic glycolysis), promoting a metabolic transition from glycolysis towards an oxidative phenotype in CAR-T cells, thus supporting its inclusion in media formulation [ 9 ]. Interestingly, previous methods that curtail T cell glycolysis levels ex vivo promote anti-tumour activity, highlighting the potential importance of including carnosine in CAR-T cell clinical culture methods [ 41 ].…”

“…Furthermore, the aforementioned treatments are accompanied with different degrees of aggressivity that is dependent on age [ 8 ]. Whilst, previous strategies for enhancing CAR-T cell therapy have focussed on enhancing T cell receptor (TCR)-related and co-stimulatory pathways, recently consideration is being given to optimising nutrient culturing conditions and strategically enhancing metabolism to better equip CAR-T cells in the TME [ 9 ].…”

Citius, Altius, Fortius: Performance in a Bottle for CAR T-Cells

“…Increasing evidence suggests that subtle adjustments in medium formulation can have a dramatic impact on T cell bioactivity and anti-tumor function in several preclinical models of cancer. In a recent article, we provide direct evidence that standard medium formulations are suboptimal, and introduce a serum-free, concentrated, platelet extract as a superior alternative to human serum in clinical-grade medium for CAR T-cells [ 2 ].…”