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Chimeric antigen receptor (CAR) T cell immunotherapy is promising for treatment of blood cancers; however, clinical benefits remain unpredictable, necessitating development of optimal CAR T cell products. Unfortunately, current preclinical evaluation platforms are inadequate due to their limited physiological relevance to humans. We herein engineered an organotypic immunocompetent chip that recapitulates microarchitectural and pathophysiological characteristics of human leukemia bone marrow stromal and immune niches for CAR T cell therapy modeling. This leukemia chip empowered real-time spatiotemporal monitoring of CAR T cell functionality, including T cell extravasation, recognition of leukemia, immune activation, cytotoxicity, and killing. We next on-chip modelled and mapped different responses post CAR T cell therapy, i.e., remission, resistance, and relapse as observed clinically and identify factors that potentially drive therapeutic failure. Finally, we developed a matrix-based analytical and integrative index to demarcate functional performance of CAR T cells with different CAR designs and generations produced from healthy donors and patients. Together, our chip introduces an enabling ‘(pre-)clinical-trial-on-chip’ tool for CAR T cell development, which may translate to personalized therapies and improved clinical decision-making.

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.

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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.

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., Graphical Abstract, Media formulations used in CAR-T cell therapies have not been optimized for gene delivery, cell expansion, and overall potency. Ghassemi et al. report that anti-GD2 CAR-T cells expanded in medium conditioned with a concentrated growth factor extract have superior engraftment and potency in a human xenograft model of neuroblastoma.

Longevity, functionality, and metabolic fitness are key determinants of chimeric antigen receptor (CAR) T cell efficacy. Activated T cells follow an ordered differentiation program which is facilitated by metabolic adaptations. In response to antigen, T cells undergo a highly-regulated shift to glycolysis. Committing to, and engaging in, glycolysis supports T cell expansion and effector function. Inside tumors, heightened tumor cell metabolism and dysregulated perfusion create a competition for nutrients. As local metabolism supports the differentiation of T cells into functionally-competent progeny, nutrient depletion coupled with persisting antigen can trigger T cell exhaustion. Emerging insights into the barriers impeding CAR T cell function in hostile tumor microenvironments (TME) reveal that metabolic intermediates shape the immune response by influencing epigenetic programs and the control of gene expression. In this review, we discuss recent progress connecting cellular metabolism with epigenetic states in CAR T cells. Given that CAR T cell metabolism can be dynamically regulated, we introduce the concepts of "metabolic-based epigenetic altering" and "epigenetic-based metabolism altering" to restore functional competence in CARTs traversing solid TMEs.

BackgroundAdaptive CD19-targeted chimeric antigen receptor (CAR) T-cell transfer has become a promising treatment for leukemia. Although patient responses vary across different clinical trials, reliable methods to dissect and predict patient responses to novel therapies are currently lacking. Recently, the depiction of patient responses has been achieved using in silico computational models, with prediction application being limited.MethodsWe established a computational model of CAR T-cell therapy to recapitulate key cellular mechanisms and dynamics during treatment with responses of continuous remission (CR), non-response (NR), and CD19-positive (CD19+) and CD19-negative (CD19−) relapse. Real-time CAR T-cell and tumor burden data of 209 patients were collected from clinical studies and standardized with unified units in bone marrow. Parameter estimation was conducted using the stochastic approximation expectation maximization algorithm for nonlinear mixed-effect modeling.ResultsWe revealed critical determinants related to patient responses at remission, resistance, and relapse. For CR, NR, and CD19+relapse, the overall functionality of CAR T-cell led to various outcomes, whereas loss of the CD19+antigen and the bystander killing effect of CAR T-cells may partly explain the progression of CD19−relapse. Furthermore, we predicted patient responses by combining the peak and accumulated values of CAR T-cells or by inputting early-stage CAR T-cell dynamics. A clinical trial simulation using virtual patient cohorts generated based on real clinical patient datasets was conducted to further validate the prediction.ConclusionsOur model dissected the mechanism behind distinct responses of leukemia to CAR T-cell therapy. This patient-based computational immuno-oncology model can predict late responses and may be informative in clinical treatment and management.

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.

Chimaeric antigen receptor (CAR) T cells can generate durable clinical responses in B-cell haematologic malignancies. The manufacturing of these T cells typically involves their activation, followed by viral transduction and expansion ex vivo for at least 6 days. However, the activation and expansion of CAR T cells leads to their progressive differentiation and the associated loss of anti-leukaemic activity. Here we show that functional CAR T cells can be generated within 24 hours from T cells derived from peripheral blood without the need for T-cell activation or ex vivo expansion, and that the efficiency of viral transduction in this process is substantially influenced by the formulation of the medium and the surface area-to-volume ratio of the culture vessel. In mouse xenograft models of human leukaemias, the rapidly generated non-activated CAR T cells exhibited higher anti-leukaemic in vivo activity per cell than the corresponding activated CAR T cells produced using the standard protocol. The rapid manufacturing of CAR T cells may reduce production costs and broaden their applicability.

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.

Adoptive T cell transfer (ACT) is a new area of transfusion medicine involving the infusion of lymphocytes to mediate antitumor, antiviral, or anti-inflammatory effects. The field has rapidly advanced from a promising form of immuno-oncology in preclinical models to the recent commercial approvals of chimeric antigen receptor (CAR) T cells to treat leukemia and lymphoma. This Review describes opportunities and challenges for entering mainstream oncology that presently face the CAR T field, with a focus on the challenges that have emerged over the past several years.

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.

Etomoxir (ETO) is a widely used small-molecule inhibitor of fatty acid oxidation (FAO) through its irreversible inhibitory effects on the carnitine palmitoyl-transferase 1a (CPT1a). We used this compound to evaluate the role of fatty acid oxidation in rapidly proliferating T cells following costimulation through the CD28 receptor. We show that ETO has a moderate effect on T cell proliferation with no observable effect on memory differentiation, but a marked effect on oxidative metabolism. We show that this oxidative metabolism is primarily dependent upon glutamine rather than FAO. Using an shRNA approach to reduce CPT1a in T cells, we further demonstrate that the inhibition of oxidative metabolism in T cells by ETO is independent of its effects on FAO at concentrations exceeding 5 μM. Concentrations of ETO above 5 μM induce acute production of ROS with associated evidence of severe oxidative stress in proliferating T cells. In aggregate, these data indicate that ETO lacks specificity for CTP1a above 5 μM, and caution should be used when employing this compound for studies in cells due to its non-specific effects on oxidative metabolism and cellular redox.

A microneedle patch containing melanin promotes systemic antitumor response upon photothermal irradiation. See the related research article by Ye et al .

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.

The field of cell therapy is shifting away from animal origin components used in the culture or preservation of cells and replacing them with human or chemically defined components. However, this shift often leads to diminished cell proliferation and functionality due to the lack of physiologically relevant proteins. Therefore, there is an important need for generation of media supplements to optimize cell therapy. PhysiologixTM XF Human Growth Factor Concentrate (hGFC) is a cGMP, xeno-free media supplement that can be used as a replacement to supplements such as human serum. In this study, T cells were isolated from blood and cultured for 14 days in OpTmizer or X-VIVO 15 supplemented with 5% human serum or 2% PhysiologixTM XF. Population doublings and cell size were defined, as well as CD4/CD8 and memory markers (CCR7 and CD45RO) to assess phenotype. We detected an increase in naïve and central memory populations in 2% PhysiologixTM XF compared to human serum across all tested basal media. These populations have been described as highly relevant for the outcome of patients undergoing CAR-T cell therapy. We compared the transduction efficiency of a lentiviral-GFP in OpTmizer, X-VIVO 15 or RPMI, supplemented with 2% PhysiologixTM XF or 5% human serum at different multiplicities of infection (MOIs). Higher transduction efficiency was observed when using 2% PhysiologixTM XF at a high MOI of 4 and was conserved across all dilutions including a MOI of

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.

Cells test the rigidity of the extracellular matrix by applying forces to it through integrin adhesions. Recent measurements show that these forces are applied by local micrometre-scale contractions, but how contraction force is regulated by rigidity is unknown. Here we performed high temporal- and spatial-resolution tracking of contractile forces by plating cells on sub-micrometre elastomeric pillars. We found that actomyosin-based sarcomere-like contractile units (CUs) simultaneously moved opposing pillars in net steps of ∼2.5 nm, independent of rigidity. What correlated with rigidity was the number of steps taken to reach a force level that activated recruitment of α-actinin to the CUs. When we removed actomyosin restriction by depleting tropomyosin 2.1, we observed larger steps and higher forces that resulted in aberrant rigidity sensing and growth of non-transformed cells on soft matrices. Thus, we conclude that tropomyosin 2.1 acts as a suppressor of growth on soft matrices by supporting proper rigidity sensing.

Selective patterning of bio-substances onto solid platforms is of increasing importance in many areas and widely used for various applications ranging from bio-sensing to cell and tissue engineering. In this study, a new fabrication scheme for the construction of highly selective bio-platforms is presented. The method is based on a direct patterning of poly(ethyleneglycol) (PEG) bio-inert layers on a conducting indium tin oxide (ITO) substrate using electron beam lithography and subsequent assembly of modified amine reactive layers onto the exposed areas. The process is found to create very high “surface contrast” between adhesive and repulsive regions onto the substrate. The platforms are shown to be enable efficient for selective adsorption of a variety of bio-substances including protein arrays, latex beads, and single cells. The high resolution of the technique makes it also applicable for the construction and deposition of bio-structures at the sub-micron scale. The reported technique employs standard lithography and surface chemistry processes, which makes it useful and easy to adopt for a variety of applications and other conductive substrates.

The results of this work show that significant changes to the spectral properties of thermal emission may be accomplished by taking advantage of surface phonon polariton (SPP) modes that are very sensitive to small refractive index changes. Surface phonon polaritons are interface restricted wave modes that do not usually participate in thermal radiative energy exchange. In this work, the possibility of varying the spectral properties by using a thin-film surface active material, layered between dielectrics, is explored. Unlike surface gratings, the multilayered structure discussed in this work couples the surface modes into radiative modes using subtle changes in refractive index. The proposed multilayer effect is studied by first developing the SPP mode dispersion relation to identify the spectral location of the surface modes. The relationship between SPP coupling and refractive index change is then developed. The fluctuation-dissipation theorem and an impedance boundary condition is used to theoretically show how thermal radiation can excite SPP modes. Results are then presented that show how film thickness and substrate properties act to support SPP modes that are particularly sensitive to changes in refractive index and therefore offers opportunities for dramatic changes to the spectral properties.

In response to antigen and costimulation, T cells undergo a series of metabolic transitions supporting clonal expansion and differentiation. We recently showed how CD28 and 4-1BB intracellular co-stimulatory domains induce glycolytic vs oxidative metabolic programs, respectively, in the context of a chimeric antigen receptor. Understanding how endogenous co-stimulatory pathways, in particular CD28, supports differentiation and memory cell development despite encountering hostile environments characterized by varying oxygen tension and metabolic checkpoints is of clear importance to cellular immunotherapy. Immune checkpoint molecule (ICM) expression is associated with T cell exhaustion characterized by reduced proliferative capacity and diminished effector function. While studying the role of LCFAO in primary human T cell differentiation, we uncovered that the CPT1a inhibitor, etomoxir (ETO), induces expression of the PD-1, Tim-3, and Lag-3 ICMs. This is mediated by non-specific effects on oxidative metabolism, culminating in reactive oxygen species (ROS) production. The induction of ICM expression by ETO is calcineurin-dependent and reversed by the antioxidant N-acetylcysteine (NAC). We show that Tim-3 is upregulated by T cells in highly hypoxic xenograft tumors independent of TCR signaling. Transition of highly activated T cell cultures from normoxia (21% O2) to hypoxia (1% O2) induces Tim-3, which is reversed by NAC. These results suggest that ICMs, especially Tim-3, are part of a natural regulatory system that responds to oxidative stress and tempers T cell activation and associated metabolism to minimize oxidative damage to T cell clones expanding following antigen receptor activation.

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.

The mechanical properties of a cell's environment can alter behavior such as migration and spreading, and control the differentiation path of stem cells. Here we describe a technique for fabricating substrates whose rigidity can be controlled locally without altering the contact area for cell spreading. The substrates consist of elastomeric pillar arrays in which the top surface is uniform but the pillar height is changed across a sharp step. Preliminary results demonstrate the effects on cell migration and morphology at the step boundary.

Extracellular matrix (ECM) rigidity is a critical regulator of important cellular processes such as survival, proliferation, and differentiation. To measure ECM rigidity, cells form actomyosin-based contractile units at the lamellipodium that pull on the matrix with forces proportional to its stiffness. However, the mechanisms that regulate force production are unknown. Here we show that generation of force involves constant step-wise movements of myosin that are controlled by tropomyosin-1. Nanometer- and millisecond-scale measurements of fibroblasts pulling on elastomeric PDMS pillars show that pillar displacement occurs by discrete steps of ∼1 nm. In contractile pairs, simultaneous steps of opposing pillars give a net movement of ∼2.2 nm, independent of rigidity. Changes in the stepping patterns on different rigidities indicate that the level of force is critical for sensing pillar stiffness. Importantly, knockdown of tropomyosin-1 causes larger steps and increased forces that result in aberrant rigidity sensing. These results indicate that tropomyosin-1 is critical for rigidity sensing by controlling myosin movements on actin.

Adoptive cell therapy employing T cells equipped with a chimeric antigen receptor (CAR) containing a single chain antibody fragment fused to T cell signaling domains 4-1BB and CD3zeta (CTL019) has shown great potency against various hematopoietic malignancies, e.g. B cell acute lymphoblastic leukemia (ALL). However, it has not shown the same response rate in other malignancies such as chronic lymphocytic leukemia (CLL). We recently demonstrated that the in vivo expansion and persistence of CAR T cells is an important predictor of response to CTL019 in CLL (PMID: 26333935) and ALL (Thudium et al., ASH 2016; Fraietta et al., ASH 2016). Furthermore, it is well known that prolonged culture of T cells negatively impacts the in vivo expansion of the adoptively transferred cells. We therefore hypothesized that minimizing the ex vivo manipulation of T cells would improve the efficacy of CAR T cells. We tested this hypothesis by generating CART19 cells using our standard 9-day manufacturing process plus two abbreviated versions. Cells from normal donors (n=9) and from patients with adult ALL (n=6) were stimulated on day 0 followed by transduction with the CAR19-encoding lentiviral vector on day 1. Cells were harvested on days 3, 5, and 9. Cryopreserved aliquots were evaluated for T cell differentiation using polychromatic flow cytometry, cytokine secretion profile using Luminex, cytolytic ability against a leukemia cell line (NALM6), proliferative ability upon restimulation with CD19-expressing target cells, and in vivo control of our well-established xenogeneic ALL model employing NALM6 as the target. Our data show that all cultures contain a substantial proportion (40%-80%) of na•ve-like CD45RO-CCR7+ T cells that progressively differentiate leading to the accumulation of predominantly (60%-90%) central memory T cells by the end of expansion. Comparative assessment of the CART19 cells at all three time points demonstrated that the cells from the shorter cultures displayed a superior in vitrocytolytic activity, and proliferative response compared to the standard process. In addition,the cells from our standard and shortened cultures all secreted comparable levels of type I cytokines (i.e. IFN-g, IL-2, and TNF-α). Importantly, we investigated the therapeutic potential of cells harvested at day 3 versus later time points. We treated NALM6 xenograftmice with a low dose (0.5 x106 CAR+ T cell I.V.) or standard dose (3 x106 CAR+ T cell I.V.).We demonstrate that day 3 CART19 cells show superior anti-leukemic activity compared to day 5 or day 9 cells. Additionally, we show that mice treated at a low dose with day 3 cells exhibit the greatest anti-leukemic efficacy compared with day 9 cells where the latter fail to control leukemia (Figure 1). Our preclinical findings provide evidence that extended ex vivo manipulation of T cells negatively affects their in vivo potency.In summary, we show that limiting T cell culture ex vivo to the minimum required for lentiviral transduction provides the most efficacious T cells for adoptive T cell immunotherapy. Figure 1 Figure 1. Disclosures Ghassemi: Novartis: Research Funding. Scholler:Novartis: Patents & Royalties; University of Pennsylvania: Patents & Royalties: FAP-CAR US Patent 9,365,641 for targeting tumor microenvironment. Nunez-Cruz:Novartis: Research Funding. Barrett:Novartis: Research Funding. Bedoya:Novartis: Patents & Royalties. Fraietta:Novartis: Patents & Royalties: Novartis, Research Funding. Lacey:Novartis: Research Funding. Levine:GE Healthcare Bio-Sciences: Consultancy; Novartis: Patents & Royalties, Research Funding. Grupp:Novartis: Research Funding. June:Johnson & Johnson: Research Funding; Tmunity: Equity Ownership, Other: Founder, stockholder ; University of Pennsylvania: Patents & Royalties; Pfizer: Honoraria; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; Immune Design: Consultancy, Equity Ownership; Celldex: Consultancy, Equity Ownership. Milone:Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding.

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.

Adoptive T cell immunotherapy involves the isolation, ex vivo expansion and reinfusion of T cells, and is dependent on T cell engraftment and persistence for efficacy. 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) T cells. Peripheral blood (PB) contains substantial numbers of Tscm, central memory (Tcm) and effector subsets. During ex vivo culture, PB T cells differentiate into cells with a predominantly CD45RO+, CD27−, CCR7− effector phenotype. Exogenous IL-7/15 delays this transition in phenotype and preserves a greater proportion of Tscm and Tcm cells. We hypothesize that limited ex vivo T cell culture in the presence of IL-7/15 rather than IL-2 will enhance engraftment and persistence of T cells in vivo. We show that T cells can be harvested from ex vivo cultures as early as day 3 (d3) following activation. T cells expressing a chimeric antigen receptor targeting CD19 (CART-19) show potent yet specific cytotoxicity in vitro. We investigated the therapeutic potential of cells harvested at d3 versus later time points using a Nalm-6 leukemic cell xenograft mouse model. We demonstrate that d3 CART-19 cells show potent anti-leukemic activity compared to day 5 or day 9 cells. Comparing CART19 cells cultured in IL-2 or IL-7/15 for 3 or 9 days, we show that mice treated with d3 cells cultured in IL-7/15 exhibit the greatest anti-leukemic efficacy at a 10-fold lower dose compared with day 9 cells. In summary, we show that limiting T cell culture ex vivo to the minimum required for lentiviral transduction, in the presence of IL-7/15, provides the most efficacious T cells for adoptive immunotherapy.

Cell growth and differentiation are critically dependent upon matrix rigidity, yet many aspects of the cellular rigidity-sensing mechanism are not understood. Here, we analyze matrix forces after initial cell–matrix contact, when early rigidity-sensing events occur, using a series of elastomeric pillar arrays with dimensions extending to the submicron scale (2, 1, and 0.5 μm in diameter covering a range of stiffnesses). We observe that the cellular response is fundamentally different on micron-scale and submicron pillars. On 2-μm diameter pillars, adhesions form at the pillar periphery, forces are directed toward the center of the cell, and a constant maximum force is applied independent of stiffness. On 0.5-μm diameter pillars, adhesions form on the pillar tops, and local contractions between neighboring pillars are observed with a maximum displacement of ∼60 nm, independent of stiffness. Because mutants in rigidity sensing show no detectable displacement on 0.5-μm diameter pillars, there is a correlation between local contractions to 60 nm and rigidity sensing. Localization of myosin between submicron pillars demonstrates that submicron scale myosin filaments can cause these local contractions. Finally, submicron pillars can capture many details of cellular force generation that are missed on larger pillars and more closely mimic continuous surfaces.

Cell motility is crucial for the immune system and its misregulation is associated with cancer and other diseases. Migration is a complex cellular activity with many proteins coordinating within a subcellular motility machine. The machine's parts conspire to propel the cell forward by exerting forces on the environment with spatial variations down to the nanoscale. In this work we measured and statistically characterized the force distributions with the aim of illuminating underlying mechanisms. We cultured mouse embryonic fibroblast cells expressing GFP-actin on dense arrays of flexible nanoengineered pillars. During cell spreading pillar deflections quantitatively revealed cell traction patterns on the substrate. Simultaneous actin imaging enabled the cellular force map to be visualized within the evolving cell contour. The greatest forces were exerted in a ∼10-20 micron strip behind the leading edge and are directed inward. We analyzed time profiles of forces exerted on single pillars as the cell passed over the pillar location. Forces were relatively short lived and showed more fluctuation for pillars with smaller diameter than for larger ones. When the protruding cell edge reaches a pillar a period of large traction force begins, typically lasting several minutes, with maximum stress on the order of several nN/μm2. Stiffer substrates provoked larger tractions than more compliant substrates. Once the front of the cell has passed by, a drastic reduction in traction occurs. Spatial force distribution and its evolution were characterized by correlation functions relative to the leading edge. By varying the geometric parameters of the pillars, we probed a range of effective substrate rigidities. In summary, we combined high resolution measurements, statistical analysis and quantitative modeling to characterize and interpret signature features of migrating and spreading cell force maps.

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

The authors describe a technique for the fabrication of arrays of elastomeric pillars whose top surfaces are treated with selective chemical functionalization to promote cellular adhesion in cellular force transduction experiments. The technique involves the creation of a rigid mold consisting of arrays of circular holes into which a thin layer of Au is deposited, while the top surface of the mold and the sidewalls of the holes are protected by a sacrificial layer of Cr. When an elastomer is formed in the mold, Au adheres to the tops of the molded pillars. This can then be selectively functionalized with a protein that induces cell adhesion, while the rest of the surface is treated with a repellent substance. An additional benefit is that the tops of the pillars can be fluorescently labeled for improved accuracy in force transduction measurements.