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Abstract As a strategy to improve the therapeutic success of chimeric antigen receptor T cells (CART) directed against solid tumors, we here test the combinatorial use of CART and IMSA101, a newly developed stimulator of interferon genes (STING) agonist. In two syngeneic tumor models, improved overall survival is observed when mice are treated with intratumorally administered IMSA101 in addition to intravenous CART infusion. Transcriptomic analyses of CART isolated from tumors show elevated T cell activation, as well as upregulated cytokine pathway signatures, in particular IL-18, in the combination treatment group. Also, higher levels of IL-18 in serum and tumor are detected with IMSA101 treatment. Consistent with this, the use of IL-18 receptor negative CART impair anti-tumor responses in mice receiving combination treatment. In summary, we find that IMSA101 enhances CART function which is facilitated through STING agonist-induced IL-18 secretion.

Abstract Human natural killer (NK) cell-based therapies are under assessment for treating various cancers, but cryopreservation reduces both the recovery and function of NK cells, thereby limiting their therapeutic feasibility. Using cryopreservation protocols optimized for T cells, here we find that ~75% of NK cells die within 24 h post-thaw, with the remaining cells displaying reduced cytotoxicity. Using CRISPR-Cas9 gene editing and confocal microscopy, we find that cryopreserved NK cells largely die via apoptosis initiated by leakage of granzyme B from cytotoxic vesicles. Pretreatment of NK cells with a combination of Interleukins-15 (IL-15) and IL-18 prior to cryopreservation improves NK cell recovery to ~90-100% and enables equal tumour control in a xenograft model of disseminated Raji cell lymphoma compared to non-cryopreserved NK cells. The mechanism of IL-15 and IL-18-induced protection incorporates two mechanisms: a transient reduction in intracellular granzyme B levels via degranulation, and the induction of antiapoptotic genes.

Abstract Loss of inflammatory effector function, such as cytokine production and proliferation, is a fundamental driver of failure in T cell therapies against solid tumors. Here, we used CRISPR/Cas9 to genetically disrupt ZFP36, an RNA binding protein that regulates the stability of mRNAs involved in T cell inflammatory function, such as the cytokines IL2 and IFNγ, in human T cells engineered with a clinical-stage mesothelin-targeting CAR to determine whether its disruption could enhance antitumor responses. ZFP36 disruption slightly increased antigen-independent activation and cytokine responses but did not enhance overall performance in vitro or in vivo in a xenograft tumor model with NSG mice. While ZFP36 disruption does not reduce the function of CAR-T cells, these results suggest that singular disruption of ZFP36 is not sufficient to improve their function and may benefit from a multiplexed approach.

The use of lentiviral vectors in cell and gene therapy is steadily increasing, both in commercial and investigational therapies. Although existing data increasingly support the usefulness and safety of clinical-grade lentiviral vectors used in cell manufacturing, comprehensive studies specifically addressing their long-term stability are currently lacking. This is significant considering the high cost of producing and testing GMP-grade vectors, the limited number of production facilities, and lengthy queue for production slots. Therefore, an extended shelf life is a critical attribute to justify the investment in large vector lots for investigational cell therapies. This study offers a thorough examination of essential stability attributes, including vector titer, transduction efficiency, and potency for a series of clinical-grade vector lots, each assessed at a minimum of 36 months following their date of manufacture. The 13 vector lots included in this study were used for cell product manufacturing in 16 different clinical trials, and at the time of the analysis had a maximum storage time at −80°C of up to 8 years. The results emphasize the long-term durability and efficacy of GMP-grade lentiviral vectors for use in ex vivo cell therapy manufacturing.

Abstract The desmoplastic stroma in solid tumors presents a formidable challenge to immunotherapies that rely on endogenous or adoptively transferred T cells, however, the mechanisms are poorly understood. To define mechanisms involved, here we treat established desmoplastic pancreatic tumors with CAR T cells directed to fibroblast activation protein (FAP), an enzyme highly overexpressed on a subset of cancer-associated fibroblasts (CAFs). Depletion of FAP+ CAFs results in loss of the structural integrity of desmoplastic matrix. This renders these highly treatment-resistant cancers susceptible to subsequent treatment with a tumor antigen (mesothelin)-targeted CAR T cells and to anti-PD-1 antibody therapy. Mechanisms include overcoming stroma-dependent restriction of T cell extravasation and/or perivascular invasion, reversing immune exclusion, relieving T cell suppression, and altering the immune landscape by reducing myeloid cell accumulation and increasing endogenous CD8+ T cell and NK cell infiltration. These data provide strong rationale for combining tumor stroma- and malignant cell-targeted therapies to be tested in clinical trials.

BACKGROUND COVID-19 remains a global health emergency with limited treatment options, lagging vaccine rates, and inadequate healthcare resources in the face of an ongoing calamity. The disease is characterized by immune dysregulation and cytokine storm. Cyclosporine A (CSA) is a calcineurin inhibitor that modulates cytokine production and may have direct antiviral properties against coronaviruses.METHODS To test whether a short course of CSA was safe in patients with COVID-19, we treated 10 hospitalized, oxygen-requiring, noncritically ill patients with CSA (starting at a dose of 9 mg/kg/d). We evaluated patients for clinical response and adverse events, measured serum cytokines and chemokines associated with COVID-19 hyperinflammation, and conducted gene-expression analyses.RESULTS Five participants experienced adverse events, none of which were serious; transaminitis was most common. No participant required intensive care unit–level care, and all patients were discharged alive. CSA treatment was associated with significant reductions in serum cytokines and chemokines important in COVID-19 hyperinflammation, including CXCL10. Following CSA administration, we also observed a significant reduction in type I IFN gene expression signatures and other transcriptional profiles associated with exacerbated hyperinflammation in the peripheral blood cells of these patients.CONCLUSION Short courses of CSA appear safe and feasible in patients with COVID-19 who require oxygen and may be a useful adjunct in resource-limited health care settings.TRIAL REGISTRATION This trial was registered on ClinicalTrials.gov (Investigational New Drug Application no. 149997; ClinicalTrials.gov NCT04412785).FUNDING This study was internally funded by the Center for Cellular Immunotherapies.

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.

As Nature Communications celebrates a 10-year anniversary, the field has witnessed the transition of cancer immunotherapy from a pipe dream to an established powerful cancer treatment modality. Here we discuss the opportunities and challenges for the future.

Abstract: Tisagenlecleucel, a chimeric antigen receptor (CAR) T-cell product targeting CD19 is approved for relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, the impact of pretreatment variables, such as CD19 expression level, on leukemic blasts, the presence of CD19– subpopulations, and especially prior CD19-targeted therapy, on the response to CAR T-cell therapy has not been determined. We analyzed 166 patients treated with CAR T-cell therapy at our institution. Eleven patients did not achieve a minimal residual disease (MRD)– deep remission, whereas 67 patients had a recurrence after achieving a MRD– deep remission: 28 patients with CD19+ leukemia and 39 patients with CD19– leukemia. Return of CD19+ leukemia was associated with loss of CAR T-cell function, whereas CD19– leukemia was associated with continued CAR T-cell function. There were no significant differences in efficacy of CAR T cells in CD19-dim B-ALL, compared with CD19-normal or -bright B-ALL. Consistent with this, CAR T cells recognized and lysed cells with very low levels of CD19 expression in vitro. The presence of dim CD19 or rare CD19– events by flow cytometry did not predict nonresponse or recurrence after CAR T-cell therapy. However, prior therapy with the CD19-directed, bispecific T-cell engager blinatumomab was associated with a significantly higher rate of failure to achieve MRD– remission or subsequent loss of remission with antigen escape. Finally, immunophenotypic heterogeneity and lineage plasticity were independent of underlying clonotype and cytogenetic abnormalities.

Adoptive T cell therapies for solid tumors is challenging. We generated metabolically enhanced co-activated-T cells by transducing intracellular co-stimulatory (41BB, ICOS or ICOS-27) and CD3ζ T cell receptor signaling domains followed by arming with bispecific antibodies (BiAbs) to produce armed “Headless CAR T cells” (hCART). Various hCART armed with BiAb directed at CD3ϵ and various tumor associated antigens were tested for: 1) specific cytotoxicity against solid tumors targets; 2) repeated and dual sequential cytotoxicity; 3) survival and cytotoxicity under in vitro hypoxic condition; and 4) cytokine secretion. The 41BBζ transduced hCART (hCART41BBζ) armed with HER2 BiAb (HER2 hCART41BBζ) or armed with EGFR BiAb (EGFR hCART41BBζ) killed multiple tumor lines significantly better than control T cells and secreted Th1 cytokines/chemokines upon tumor engagement at effector to target ratio (E:T) of 2:1 or 1:1. HER2 hCART serially killed tumor targets up to 14 days. Sequential targeting of EGFR or HER2 positive tumors with HER2 hCART41BBζ followed by EGFR hCART41BBζ showed significantly increased cytotoxicity compared single antigen targeting and continue to kill under in vitro hypoxic conditions. In summary, metabolically enhanced headless CAR T cells are effective serial killers of tumor targets, secrete cytokines and chemokines, and continue to kill under in vitro hypoxic condition.

Autologous chimeric antigen receptor (CAR) T cells targeted to epidermal growth factor receptor variant III (CAR T-EGFRvIII) have been developed and administered experimentally to treat patients with IDH1 wildtype recurrent glioblastoma (rGBM) (NCT02209376). We report the case of a 59-year-old patient who received a single peripheral infusion of CAR T-EGFRvIII cells and survived 36 months after disease recurrence, exceeding expected survival for recurrent glioblastoma. Post-infusion histopathologic analysis of tissue obtained during a second stage surgical resection revealed immunosuppressive adaptive changes in the tumor tissue as well as reduced EGFRvIII expression. Serial brain imaging demonstrated a significant reduction in relative cerebral blood volume (rCBV), a measure strongly associated with tumor proliferative activity, at early time points following CAR T treatment. Notably, CAR T-EGFRvIII cells persisted in her peripheral circulation during 29 months of follow-up, the longest period of CAR T persistence reported in GBM trials to date. These findings in a long-term survivor show that peripherally administered CAR T-EGFRvIII cells can persist for years in the circulation and suggest that this cell therapy approach could be optimized to achieve broader efficacy in recurrent GBM patients.

Tisagenlecleucel is a chimeric antigen receptor–T cell therapy that facilitates the killing of CD19+ B cells. A model was developed for the kinetics of tisagenlecleucel and the impact of therapies for treating cytokine release syndrome (tocilizumab and corticosteroids) on expansion. Data from two phase II studies in pediatric and young adult relapsed/refractory B cell acute lymphoblastic leukemia were pooled to evaluate this model and evaluate extrinsic and intrinsic factors that may impact the extent of tisagenlecleucel expansion. The doubling time, initial decline half‐life, and terminal half‐life for tisagenlecleucel were 0.78, 4.3, and 220 days, respectively. No impact of tocilizumab or corticosteroids on the expansion rate was observed. This work represents the first mixed‐effect model‐based analysis of chimeric antigen receptor–T cell therapy and may be clinically impactful as future studies examine prophylactic interventions in patients at risk of higher grade cytokine release syndrome and the effects of these interventions on chimeric antigen receptor–T cell expansion.

Cancer is one of the leading causes of death worldwide. Recent advances in cellular therapy have demonstrated that this platform has the potential to give patients with certain cancers a second chance at life. Unlike chemical compounds and proteins, cells are living, self-replicating drugs that can be engineered to possess exquisite specificity. For example, T cells can be genetically modified to express chimeric antigen receptors (CARs), endowing them with the capacity to recognize and kill tumor cells and form a memory pool that is ready to strike back against persisting malignant cells. Anti-CD19 chimeric antigen receptor T cells (CART19s) have demonstrated a remarkable degree of clinical efficacy for certain malignancies. The process of developing CART19 essentially follows the conventional “one gene, one drug, one disease” paradigm derived from Paul Ehrlich’s “magic bullet” concept. With major players within the pharmaceutical industry joining forces to commercialize this new category of “living drugs,” it is useful to use CART19 as an example to examine the similarities and differences in its development, compared with that of a conventional drug. In this way, we can assimilate existing knowledge and identify the most effective approach for advancing similar strategies. This article reviews the use of biomarker-based assays to guide the optimization of CAR constructs, preclinical studies, and the evaluation of clinical efficacy; adverse effects (AEs); and CART19 cellular kinetics. Advanced technologies and computational tools that enable the discovery of optimal targets, novel CAR binding domains, and biomarkers predicting clinical response and AEs are also discussed. We believe that the success of CART19 will lead to the development of other engineered T cell therapies in the same manner that the discovery of arsphenamine initiated the era of synthetic pharmaceuticals. Keywords: Engineered T cell therapies, Chimeric antigen receptor, Drug development process, Biomarkers, CD19-specific chimeric antigen receptor, Anti-CD19 chimeric antigen receptor T cells

Abstract Genome engineering methods have advanced greatly with the development of programmable nucleases, but methods for quantifying on- and off-target cleavage sites and associated deletions remain nascent. Here, we report an improvement of the GUIDE-seq method, iGUIDE, which allows filtering of mispriming events to clarify the true cleavage signal. Using iGUIDE, we specify the locations of Cas9-guided cleavage for four guide RNAs, characterize associated deletions, and show that naturally occurring background DNA double-strand breaks are associated with open chromatin, gene dense regions, and chromosomal fragile sites. iGUIDE is available from https://github.com/cnobles/iGUIDE.

We generated two humanized interleukin-13 receptor α2 (IL-13Rα2) chimeric antigen receptors (CARs), Hu07BBz and Hu08BBz, that recognized human IL-13Rα2, but not IL-13Rα1. Hu08BBz also recognized canine IL-13Rα2. Both of these CAR T cell constructs demonstrated superior tumor inhibitory effects in a subcutaneous xenograft model of human glioma compared with a humanized EGFRvIII CAR T construct used in a recent phase 1 clinical trial (ClinicalTrials.gov: NCT02209376). The Hu08BBz demonstrated a 75% reduction in orthotopic tumor growth using low-dose CAR T cell infusion. Using combination therapy with immune checkpoint blockade, humanized IL-13Rα2 CAR T cells performed significantly better when combined with CTLA-4 blockade, and humanized EGFRvIII CAR T cells’ efficacy was improved by PD-1 and TIM-3 blockade in the same mouse model, which was correlated with the levels of checkpoint molecule expression in co-cultures with the same tumor in vitro. Humanized IL-13Rα2 CAR T cells also demonstrated benefit from a self-secreted anti-CTLA-4 minibody in the same mouse model. In addition to a canine glioma cell line (J3T), canine osteosarcoma lung cancer and leukemia cell lines also express IL-13Rα2 and were recognized by Hu08BBz. Canine IL-13Rα2 CAR T cell was also generated and tested in vitro by co-culture with canine tumor cells and in vivo in an orthotopic model of canine glioma. Based on these results, we are designing a pre-clinical trial to evaluate the safety of canine IL-13Rα2 CAR T cells in dog with spontaneous IL-13Rα2-positive glioma, which will help to inform a human clinical trial design for glioblastoma using humanized scFv-based IL-13Rα2 targeting CAR T cells. Keywords: glioblastoma, chimeric antigen receptor, CAR, IL-13Rα2, minibody, canine, immune checkpoint blockade, PD-1, CTLA-4, TIM-3

Abstract Chimeric Antigen Receptor (CAR) T cell therapies – adoptive T cell therapies that have been genetically engineered for a new antigen-specificity - have displayed significant success in treating patients with hematologic malignancies, leading to three recent US Food and Drug Administration approvals. Based on the promise generated from these successes, the field is rapidly evolving to include new disease indications and CAR designs, while simultaneously reviewing and optimizing toxicity and management protocols. As such, this review provides expert perspective on the significance and clinical considerations of CAR T cell therapies in order to provide timely information to clinicians about this revolutionary new therapeutic class.

The role of human CD4+ T cell subsets in cancer immunotherapy is still unclear. Here, the authors show that CD26 identifies three CD4+ T cell subsets with distinct immunological properties in both healthy individuals and cancer patients.

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.

The effects of transgenically encoded human and mouse IL-18 on T cell proliferation and its application in boosting chimeric antigen receptor (CAR) T cells are presented. Robust enhancement of proliferation of IL-18-secreting human T cells occurred in a xenograft model, and this was dependent on TCR and IL-18R signaling. IL-18 augmented IFN-γ secretion and proliferation of T cells activated by the endogenous TCR. TCR-deficient, human IL-18-expressing CD19 CAR T cells exhibited enhanced proliferation and antitumor activity in the xenograft model. Antigen-propelled activation of cytokine helper ensemble (APACHE) CAR T cells displayed inducible expression of IL-18 and enhanced antitumor immunity. In an intact mouse tumor model, CD19-IL-18 CAR T cells induced deeper B cell aplasia, significantly enhanced CAR T cell proliferation, and effectively augmented antitumor effects in mice with B16F10 melanoma. These findings point to a strategy to develop universal CAR T cells for patients with solid tumors.

ABSTRACT The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell-based adoptive immunotherapy. In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous, with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model. To further improve the OKT3/IL-2-based T cell manufacturing procedure, we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB (OKT3-28BB). The expanded T cells were phenotypically and functionally similar to T cells expanded by OKT3/IL-2. Moreover, co-electroporation of CD86 and 4-1BBL could further change the phenotype and enhance the in vivo anti-tumor activity. Although T cells expanded by the co-electroporation of OKT3-28BB with CD86 and 4-1BBL showed an increased central memory phenotype, the T cells still maintained tumor lytic activities as potent as those of OKT3/IL-2 or OKT3-28BB-stimulated T cells. In different tumor mouse models, T cells expanded by OKT3-28BB RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells. Hence, T cells with both a less differentiated phenotype and potent tumor killing ability can be generated by RNA electroporation, and this T cell manufacturing procedure can be further optimized by simply co-delivering other splices of RNA, thus providing a simple and cost-effective method for generating high-quality T cells for adoptive immunotherapy.

A major obstacle for chimeric antigen receptor (CAR) T cell therapy in solid tumors is the lack of truly tumor-specific target antigens, which translates to the targeting of tumor-associated antigens (TAAs) overexpressed on tumors but shared with normal organs, raising safety concerns. In addition, expression of TAAs in solid tumors is particularly heterogeneous. In this regard, it is critical to deeply understand the sensitivity of CAR T cells, especially against low-density targets and the possible therapeutic window of antigen density targeted by CAR T cells. In this review, we discuss the recent findings of mechanisms of antigen recognition through CAR, including immunological synapse formation, and the impact of target antigen density for induction of distinct T cell functions. We also discuss rational strategies to adjust and expand the therapeutic window for effective and safe targeting of solid tumors by CAR T cell platforms.

With the advent and stunning success of combination antiretroviral therapy (ART) to prolong and improve quality of life for persons with HIV infection, HIV research has been afforded the opportunity to pivot towards studies aimed at finding “a cure.” The mere idea that cure of HIV might be possible has energized researchers and the community towards achieving this goal. Funding agencies, both governmental and private, have targeted HIV cure as a high priority; many in the field have responded to these initiatives and the cure research agenda is robust. In this “salon” two editors of Pathogens and Immunity, Michael Lederman and Daniel Douek ask whether curing HIV is a realistic, scalable objective. We start with an overview perspective and have asked a number of prominent HIV researchers to add to the discussion.