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Prior to adoptive transfer, CAR T cells are activated, lentivirally infected with CAR transgenes, and expanded over 9 to 11 days. An unintended consequence of this process is the progressive differentiation of CAR T cells over time in culture. Differentiated T cells engraft poorly, which limits their ability to persist and provide sustained tumor control in hematologic as well as solid tumors. Solid tumors include other barriers to CAR T cell therapies, including immune and metabolic checkpoints that suppress effector function and durability. Sialic acids are ubiquitous surface molecules with known immune checkpoint functions. The enzyme C. perfringens neuraminidase (CpNA) removes sialic acid residues from target cells, with good activity at physiologic conditions. In combination with galactose oxidase (GO), NA has been found to stimulate T cell mitogenesis and cytotoxicity in vitro. Here we determine whether CpNA alone and in combination with GO promotes CAR T cell antitumor efficacy. We show that CpNA restrains CAR T cell differentiation during ex vivo culture, giving rise to progeny with enhanced therapeutic potential. CAR T cells expressing CpNA have superior effector function and cytotoxicity in vitro. In a Nalm-6 xenograft model of leukemia, CAR T cells expressing CpNA show enhanced antitumor efficacy. Arming CAR T cells with CpNA also enhanced tumor control in xenograft models of glioblastoma as well as a syngeneic model of melanoma. Given our findings, we hypothesize that charge repulsion via surface glycans is a regulatory parameter influencing differentiation. As T cells engage target cells within tumors and undergo constitutive activation through their CARs, critical thresholds of negative charge may impede cell-cell interactions underlying synapse formation and cytolysis. Removing the dense pool of negative cell-surface charge with CpNA is an effective approach to limit CAR T cell differentiation and enhance overall persistence and efficacy.

Chimeric antigen receptor (CAR) T cell therapy has shown unprecedented success in treating advanced hematological malignancies. Its effectiveness in solid tumors has been limited due to heterogeneous antigen expression, a suppressive tumor microenvironment, suboptimal trafficking to the tumor site and poor CAR T cell persistence. Several approaches have been developed to overcome these obstacles through various strategies including the genetic engineering of CAR T cells to blunt the signaling of immune inhibitory receptors as well as to modulate signaling of cytokine/chemokine molecules and their receptors. In this review we offer our perspective on how genetically modifying cytokine/chemokine molecules and their receptors can improve CAR T cell qualities such as functionality, persistence (e.g. resistance to pro-apoptotic signals) and infiltration into tumor sites. Understanding how such modifications can overcome barriers to CAR T cell effectiveness will undoubtedly enhance the potential of CAR T cells against solid tumors.

The metabolic milieu of solid tumors provides a barrier to chimeric antigen receptor (CAR) T-cell therapies. Excessive lactate or hypoxia suppresses T-cell growth, through mechanisms including NADH buildup and the depletion of oxidized metabolites. NADH is converted into NAD+ by the enzyme Lactobacillus brevis NADH Oxidase (LbNOX), which mimics the oxidative function of the electron transport chain without generating ATP. Here we determine if LbNOX promotes human CAR T-cell metabolic activity and antitumor efficacy. CAR T-cells expressing LbNOX have enhanced oxygen as well as lactate consumption and increased pyruvate production. LbNOX renders CAR T-cells resilient to lactate dehydrogenase inhibition. But in vivo in a model of mesothelioma, CAR T-cell’s expressing LbNOX showed no increased antitumor efficacy over control CAR T-cells. We hypothesize that T cells in hostile environments face dual metabolic stressors of excessive NADH and insufficient ATP production. Accordingly, futile T-cell NADH oxidation by LbNOX is insufficient to promote tumor clearance.

Chimeric antigen receptor (CAR) T cell therapy has led to a paradigm shift in cancer immunotherapy, but still several obstacles limit CAR T cell efficacy in cancers. Advances in high-throughput technologies revealed new insights into the role that epigenetic reprogramming plays in T cells. Mechanistic studies as well as comprehensive epigenome maps revealed an important role for epigenetic remodeling in T cell differentiation. These modifications shape the overall immune response through alterations in T cell phenotype and function. Here, we outline how epigenetic modifications in CAR T cells can overcome barriers limiting CAR T cell effectiveness, particularly in immunosuppressive tumor microenvironments. We also offer our perspective on how selected epigenetic modifications can boost CAR T cells to ultimately improve the efficacy of CAR T cell therapy.

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Background & Aim The recent success of immunotherapy using chimeric antigen receptor modified T cells (CAR T) in B-cell malignancies highlights the potential of these cytotoxic “drugs” for cancer therapy. CAR T therapies generally relies upon viral transduction of T cell-receptor (TCR) activated T cell followed by ex vivo expansion of patient-derived T cells over days to weeks prior to infusion. In addition to the required time and labor, we previously reported that TCR/CD3 activation and ex vivo expansion leads to progressive differentiation of the CAR T cells with associated loss of CAR T cell potency. Since cell division is not a prerequisite for lentiviral vector-mediated gene delivery, we hypothesized that elimination of the CD3/CD28 activation step would yield a T cell product with high functional potency. Methods, Results & Conclusion Here, we show that functional CD19-specific CAR T cells (CART19) can be generated in as little as 24 hours using lentiviral vectors without the need for prior T cell activation. We further demonstrate that some of the restriction factors that limit the infection of quiescent T cells can be overcome by changes to the medium formulation. Most importantly, we show that non-activated T cells exhibit potent antitumor function with deep and durable control of leukemia achieved in vivo with greater than 1-log fewer CD19-specific CAR T cells compared with a 9-day process currently used by tisagenlecleucel (Fig. 1). These findings confirmed that as few as an estimated 12,000-32,000 non-activated CAR T cells produced within 24 hours from mononuclear cell collection could eradicate leukemia. These results demonstrate the potential for vastly reducing the time, materials and labor required to generate CAR T cells, which could be especially beneficial in patients with rapidly progressive disease and in resource-poor health care environments.

Background & Aim Effective 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. In this regard, the outcome of the ex-vivo process is largely dependent on the properties of the medium. Media formulations used in CAR T-cell manufacturing have not been optimized either for signaling studies, gene delivery or cell expansion. 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 if a concentrated growth factor extract, purified from human transfusion grade whole blood fractions, and marketed as PhysiologixTM XF hGFC (Phx), supports CAR T-cell expansion and function. Methods, Results & Conclusion We show that Phx supports T cell proliferation in clinical as well as research-grade media. As cytokine stimulation regulates the exo and endocytotic machinery, we hypothesized that factors unique to the growth factors within blood cell fractions would enhance the dynamic process of viral entry. We 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 CAR T-cells expanded in Phx have superior engraftment and potency in vivo. Metabolomic profiling revealed several factors enriched in a blood cell-derived growth factor concentrate that may enhance lentiviral-mediated gene delivery and CAR T-cell differentiation for preclinical discovery, process development, as well as manufacturing. These findings are also informative regarding potential derivatives to include in medium customized for gene delivery, T cell activation, and expansion for adoptive immunotherapies.

The success of chimeric antigen receptor (CAR)–mediated immunotherapy in acute lymphoblastic leukemia (ALL) highlights the potential of T-cell therapies with directed cytotoxicity against specific tumor antigens. The efficacy of CAR T-cell therapy depends on the engraftment and persistence of T cells following adoptive transfer. Most protocols for T-cell engineering routinely expand T cells ex vivo for 9 to 14 days. Because the potential for engraftment and persistence is related to the state of T-cell differentiation, we hypothesized that reducing the duration of ex vivo culture would limit differentiation and enhance the efficacy of CAR T-cell therapy. We demonstrated that T cells with a CAR-targeting CD19 (CART19) exhibited less differentiation and enhanced effector function in vitro when harvested from cultures at earlier (day 3 or 5) compared with later (day 9) timepoints. We then compared the therapeutic potential of early versus late harvested CART19 in a murine xenograft model of ALL and showed that the antileukemic activity inversely correlated with ex vivo culture time: day 3 harvested cells showed robust tumor control despite using a 6-fold lower dose of CART19, whereas day 9 cells failed to control leukemia at limited cell doses. We also demonstrated the feasibility of an abbreviated culture in a large-scale current good manufacturing practice–compliant process. Limiting the interval between T-cell isolation and CAR treatment is critical for patients with rapidly progressing disease. Generating CAR T cells in less time also improves potency, which is central to the effectiveness of these therapies. Cancer Immunol Res; 6(9); 1100–9. ©2018 AACR.

The recent success of immunotherapy using chimeric antigen receptor modified T cells (CAR T) in B-cell malignancies highlights the potential of these cytotoxic "drugs" for cancer therapy. CAR T therapies generally rely upon manufacturing approaches that include prior T cell activation through engagement of the TCR and costimulatory receptors followed by ex vivo expansion of patient-derived T cells over days to weeks. We previously reported that CD3/CD28 stimulation prior to transduction promotes progressive T cell effector differentiation over time in culture with loss of CAR T cell potency (Ghassemi et al. 2018 PMID: 30030295). Since cell division is not a prerequisite for lentiviral vector-mediated gene delivery, we hypothesized that lentiviral transduction of quiescent T cells without prior activation will enhance engraftment and persistence of CART cells that is associated with long-term leukemia control. Here, we show that functional CD19-specific CAR T cells (CART19) can be generated in as little as 24 hours using lentiviral vectors without the need for prior T cell activation. We showed using a non-optimized process that a mean of 6.5% (range 2%-10%) of freshly isolated quiescent T cells can be transduced using an infrared red fluorescent protein (iRFP)-expressing lentiviral vector with slower kinetics of expression compared with activated T cell transduction (peak at 96 hrs vs. 48 hrs for quiescent and activated T cells, respectively). Although substantially less efficient compared to activated T cells, transduction was detected across all T cells subsets with central memory T cells showing the greatest transduction efficiency with a mean of 4-fold greater transduction compared with naïve T cells. Somewhat unexpectedly, CART19 cells generated from quiescent T cells using a CD19-specific CAR vector showed a 3-5 fold greater transgene expression compared with iRFP vectors transduced at similar MOI. However, we show that CAR expression can occur in quiescent T cells even without reverse transcription or integrase function, so called "pseudotransduction". Importantly, we show that this CAR expression produces T cells with cytolytic activity and effector cytokine production in response to antigen that is similar to activated and transduced CAR T cells. Using the well-characterized Nalm6 model of acute lymphoblastic leukemia, we show that CART19 cells generated by transduction of quiescent T cells for 16 hours followed by washing to remove vector exhibit dose-dependent anti-leukemic activity that is durable with injection of as little as 2x105 total T cells. We estimate the latter to contain ~2x104 T cells with integrated lentiviral vector based upon transduction efficiency determined in studies using non-tumor bearing mice. (Fig 1). In summary, our results support the need for further investigation of CAR T cells that are generated using engineering of quiescent T cells. Taking advantage of the ability of lentiviral vectors to transfer genes to quiescent T cells, the highly abbreviated and simplified manufacturing approach described here has the potential to enhance therapeutic potency while also substantially reducing the materials and labor costs associated with current manufacturing approaches that use activated and expanded T cells. In addition, the rapid nature of this manufacturing has the potential to extend the population of patients that may be treated with these therapies by shortening the interval between aphaeresis collection and re-infusion of CAR T cells, which prevents the treatment of some patients with rapidly progressive disease. Disclosures Ghassemi: Novartis: Patents & Royalties. Milone:Novartis: Patents & Royalties, Research Funding.

Recent advances in genetic engineering have resulted in exponential growth in cell therapy technologies. As an increasing number of CAR-T therapies are entering clinical trials, there is a scarcity of resources put into optimizing production strategies. This is largely due to the highly competitive timelines for these companies to advance their drug candidate. Yet, due to this pressure, there is a real need for advances in the cell culture media used to recover, sustain and expand these importanT-cells. Here we show that a novel xeno-free media supplement, PhysiologixTM XF serum replacement, results in improved human primary T-cell growth, preservation of naïve and central memory phenotype, and enhanced transduction efficiency when used in place of traditional media supplements. In the presence of this supplement, T-cell proliferation was nearly doubled in comparison to industry-leading serum free media. In a separate study, we compared PhysiologixTM XF to a control media containing the traditional 10% fetal bovine serum for bulk T-cell culture. We observed that T-cells in both control and test media had similar cell size and population doublings following 48 hours of CD3/CD28 activation. However, FACS analysis revealed thaT-cells grown in PhysiologixTM XF showed a 33% increase in beneficial naïve-like and central memory T-cell phenotypes after nine days in culture when compared to control media. Additionally, lentiviral transduction efficiency was increased from 74% to 94% in PhysiologixTM XF supplemented cells compared to control cells. Taken together, these data demonstrate that T-cell culture with PhysiologixTM XF results in improved growth characteristics, desirable cell phenotypes for patient infusion, and improved ease of transduction, all of which may prove beneficial in the CAR-T race from bench to bedside. Citation Format: Alyssa Master, Roddy S. O'Connor, Saba Ghassemi, Dina A. Schneider, Lisa Karimi-Naser. A novel media supplementation strategy for improved T-cell culture and preservation of naivety [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B029.

SummaryShape is an indicator of cell health. But how is the information in shape decoded? We hypothesize that decoding occurs by modulation of signaling through changes in plasma membrane curvature. Using analytical approaches and numerical simulations, we studied how elongation of cell shape affects plasma membrane signaling. Mathematical analyses reveal transient accumulation of activated receptors at regions of higher curvature with increasing cell eccentricity. This distribution of activated receptors is periodic, following the Mathieu function, and it arises from local imbalance between reaction and diffusion of soluble ligands and receptors in the plane of the membrane. Numerical simulations show that transient microdomains of activated receptors amplify signals to downstream protein kinases. For growth factor receptor pathways, increasing cell eccentricity elevates the levels of activated cytoplasmic Src and nuclear MAPK1,2. These predictions were experimentally validated by changing cellular eccentricity, showing that shape is a locus of retrievable information storage in cells.

During cell migration, the cell edge undergoes periodic protrusion–retraction cycles. Quantitative analyses of the forces at the cell edge that drive these cycles are provided. We show that α-actinin links local contractile units and the global actin flow forces at the cell edge and present a novel model based on these results., During spreading and migration, the leading edges of cells undergo periodic protrusion–retraction cycles. The functional purpose of these cycles is unclear. Here, using submicrometer polydimethylsiloxane pillars as substrates for cell spreading, we show that periodic edge retractions coincide with peak forces produced by local contractile units (CUs) that assemble and disassemble along the cell edge to test matrix rigidity. We find that, whereas actin rearward flow produces a relatively constant force inward, the peak of local contractile forces by CUs scales with rigidity. The cytoskeletal protein α-actinin is shared between these two force-producing systems. It initially localizes to the CUs and subsequently moves inward with the actin flow. Knockdown of α-actinin causes aberrant rigidity sensing, loss of CUs, loss of protrusion–retraction cycles, and, surprisingly, enables the cells to proliferate on soft matrices. We present a model based on these results in which local CUs drive rigidity sensing and adhesion formation.

Extracellular matrices in vivo are heterogeneous structures containing gaps that cells bridge with an actomyosin network. To understand the basis of bridging, we plated cells on surfaces patterned with fibronectin (FN)-coated stripes separated by non-adhesive regions. Bridges developed large tensions where concave cell edges were anchored to FN by adhesion sites. Actomyosin complexes assembled near those sites (both actin and myosin filaments) and moved towards the centre of the non-adhesive regions in a treadmilling network. Inhibition of myosin-II (MII) or Rho-kinase collapsed bridges, whereas extension continued over adhesive areas. Inhibition of actin polymerization (latrunculin-A, jasplakinolide) also collapsed the actomyosin network. We suggest that MII has distinct functions at different bridge regions: (1) at the concave edges of bridges, MIIA force stimulates actin filament assembly at adhesions and (2) in the body of bridges, myosin cross-links actin filaments and stimulates actomyosin network healing when breaks occur. Both activities ensure turnover of actin networks needed to maintain stable bridges from one adhesive region to another.

Mechanical cues may have important roles in tissue morphogenesis; progression through complex functions like differentiation may be associated with changes in cellular force generation and mechanosensing. To explore this concept, we use elastomer pillar arrays to map forces generated by neural stem cells in vitro, and identify two distinct dynamics of force generation. First, cell generated forces decrease as cells transition from a proliferative mode to differentiation, a process covering several days. This change in force generation correlated with a loss of sensitivity to substrate rigidity over a series of polydimethylsiloxane substrates. Second, neural stem cells exhibit a faster pattern of localized contractions at the cell body and outlying processes; each lasts on the order of minutes, and is not synchronized across the cell. This faster process is reminiscent of migratory behavior observed in vivo, and may be involved in controlling the motion of internal structures such as the cell nucleus. These results together provide new clues into the role of forces during development, and may lead to design principles for materials targeted for use in the central nervous system.

Adoptive T cell immunotherapy involves the isolation, ex vivo expansion and reinfusion of patient T cells. The efficacy of adoptive immunotherapy is dependent on the ability of T cells to engraft, expand and persist upon adoptive transfer. In this therapy, T cells are cultured ex vivo using natural or artificial antigen presenting cells that deliver signal 1 (TCR/CD3) and signal 2 (e.g. CD28 co-stimulation) along with exogenously added cytokines. IL-2 is the most commonly used cytokine for ex vivo T cell culture; however, there is renewed interest in IL-7 and IL-15 due to their ability to enhance the survival and proliferation of stem cell memory (Tscm) and central memory (Tcm) T cells. We show that primary human T cells freshly isolated from peripheral blood are heterogeneous with substantial numbers of Tscm and Tcm cells in addition to effector differentiated T cells. During ex vivo culture, these cells progressively differentiate into a population of T cells with a predominantly CD45RO+, CD27-, CCR7- effector differentiated phenotype. Exogenous IL-7 and IL-15 delay this transition in T cell phenotype and preserve a greater proportion of Tscm and Tcm cells in the final ex vivo culture product. We hypothesize that limited ex vivo culture of T cells in the presence of IL-7 and IL-15 rather than IL-2 will enhance engraftment and persistence of T cells in vivo contributing to enhanced efficacy in adoptive transfer. We show that T cells can be harvested and viably frozen from ex vivo cultures as early as day 3 following activation. Early activated T cells expressing a chimeric antigen receptor targeting CD19 (CART-19) show potent yet specific cytotoxicity and cytokine production in vitro. We investigated the therapeutic potential of cells harvested at day 3 versus later time points using a Nalm-6 leukemic cell xenograft mouse model. We demonstrate that day 3 CART-19 cells show potent anti-leukemic activity compared to day 5 or day 9 cells. Comparing CART19 cells cultured in either IL-2 or IL-7/15, we show that mice treated at a 10-fold lower dose with day 3 cells cultured in IL-7/15 exhibit the greatest anti-leukemic efficacy compared with day 9 cells where the latter fail to control leukemia. In summary, we show that limiting T cell culture ex vivo to the minimum required for lentiviral transduction in the presence of IL-7 and IL-15 provides the most efficacious T cells for adoptive T cell immunotherapy.

Extracellular matrix rigidity directs cell behavior and key processes in development and tumorigenesis. During spreading and migration, cells produce periodic lamellipodial protrusion retractions cycles that require a rigid substrate and the development of adhesions1. Moreover, there is evidence that rigidity is detected locally in the region near the cell edge2. The motor protein myosin II powers cell retractions, which are simultaneous with rearward flow of the actin filament network1. However, the pattern and mechanism of forces exerted by cells to test matrix rigidity have not been characterized. using high-resolution force sensing pillars, we show here that the primary forces powering periodic edge retraction come from local contraction units, which exert inward forces near the cell edge, correlated with outward forces 2-3 micrometers rearwards from the edge. Pillar displacement correlates with the local concentrations of active myosin II and the actin crosslinking protein alpha-actinin, but not with local levels of other common actin binding proteins. The contractile units therefore display similarity to striated muscle and stress fiber units, which are ∼1-3 micrometers long, rely on myosin II for contractility, and contain alpha-actinin as a major component of the complex that anchors actin filaments. Thus, our results indicate that local contractile units, with sarcomere-like organization, are responsible for periodic cell edge retractions and rigidity sensing. References: [1] G. Giannone, B. Dubin-Thaler, M. Sheetz et al. (2007) Cell 128, 561-575. [2] S. Ghassemi, G. Meacci, J. Hone et al. (2012) Proc. Natl. Acad. Sci. USA, 109, 5328-5333