Mitochondrial Biomass As a Measure of Fitness for T Cells Expressing Chimeric Antigen Receptors
Authors: Jena Bipulendu , Rushworth David , McNamara George T , Cooper Laurence JN 
Published: October 2019

Abstract
Anti-tumor efficacy of genetically modified T cells depends on in vivo expansion and durable persistence of infused cells. Multiple variables including the structure of the CAR and characteristics of the recipient impact the anti-tumor effect of CAR+ T cells. However, a code for an optimal CAR design that would deliver clinically relevant result is yet to emerge. Here we propose a new measure of "fitness" for CAR+ T cells based on mitochondrial biomass that is quantifiable and could be translated to clinical settings. Spare respiratory capacity (SRC) is defined as the extra mitochondrial capacity available in a cell to produce energy under conditions of increased work or stress. Memory T cells capable of responding to infection has been shown to possess extra SRC (Windt et al., Immunity 2012). We therefore investigated whether subsets of CD19-specific CAR+ T cells after electro-transfer of Sleeping Beauty (SB) plasmids and propagation on activating and propagating cells (AaPC) could be identified based on SRC. Transmission electron microscopy revealed that genetically modified T cells revert to a condensed state of mitochondria after 2 weeks of activation through a second-generation CD19-specific CAR. However, mock-electroporated T cells activated by cross-linking CD3 (using AaPC loaded with OKT3) retain a classic mitochondrial structure. Moreover, antigen-driven numeric expansion in presence of membrane bound IL-15 led to an increase in mitochondrial biomass in CAR+ T cells. We extended these observations to various CAR+ T cells with unique specificity for tumor antigens and found similar changes in mitochondrial structure and distribution. Next, we examined if an increase in mitochondrial biomass influences functionality of genetically modified T cells. By SB mediated transposition CARs were co-expressed along with a fluorescence reporter protein (EYFP-GRX2) constituting yellow fluorescent protein fused to the mitochondrial localization sequence of GRX2 to track mitochondrial distribution in live cells. The genetically modified T cells were selectively propagated by stimulating the CARs using a proprietary monoclonal antibody that binds to a common extracellular stalk motif in CAR construct. CAR+ T cells that signaled through chimeric CD137z exhibited a high mitochondrial mass (EYFPhigh) and had superior rates of expansion ex vivo. In contrast, CAR+ T cells that signaled through chimeric CD28z had a low mitochondrial mass (EYFPdim), elevated levels of apoptosis, and inferior rates of numeric expansion. Confocal microscopy showed EYFP counts were higher for CAR+ T cells that signaled through CD137 signaling domain. We hypothesize that increased survival of CD137z-CAR T cells in a challenging cell culture environment could be due to reserve bio-energetic potential concomitant with the ability to meet metabolic demand of activated T cells. Further, SRC could be quantified using a fluorescent probe for mitochondrial mass pre-infusion which may be a defining criterion attesting to the fitness of CAR+ T cells for human applications.

Disclosures
Jena: Intrexon: Equity Ownership, Patents & Royalties: Potential royalties (Patent submitted); Ziopharm Oncology: Equity Ownership, Patents & Royalties: Potential roylaties (Patent submitted). Rushworth:Intrexon: Other: Potential Equity ownership; Ziopharam Oncology: Other: Potential Equity Ownership. McNamara:Ziopharm Oncology: Equity Ownership, Patents & Royalties: Potential royalties, Research Funding; Intrexon: Equity Ownership, Patents & Royalties: Potential royalties, Research Funding. Cooper:Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties, Research Funding; Intrexon: Equity Ownership, Patents & Royalties.