A high-density microfluidic bioreactor for the automated manufacturing of CAR T cells.

The manufacturing of autologous chimaeric antigen receptor (CAR) T cells largely relies either on fed-batch and manual processes that often lack environmental monitoring and control or on bioreactors that cannot be easily scaled out to meet patient demands. Here we show that human primary T cells can be activated, transduced and expanded to high densities in a 2 ml automated closed-system microfluidic bioreactor to produce viable anti-CD19 CAR T cells (specifically, more than 60 million CAR T cells from donor cells derived from patients with lymphoma and more than 200 million CAR T cells from healthy donors). The in vitro secretion of cytokines, the short-term cytotoxic activity and the long-term persistence and proliferation of the cell products, as well as their in vivo anti-leukaemic activity, were comparable to those of T cells produced in a gas-permeable well. The manufacturing-process intensification enabled by the miniaturized perfusable bioreactor may facilitate the analysis of the growth and metabolic states of CAR T cells during ex vivo culture, the high-throughput optimization of cell-manufacturing processes and the scale out of cell-therapy manufacturing.

Current perspectives on resistance to chimeric antigen receptor T-cell therapy and strategies to improve efficacy in B-cell lymphoma.

Although chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable efficacy in patients with chemo-refractory B-cell lymphoma, a significant portion is refractory or relapse. Resistance is a major barrier to improving treatment efficacy and long-term survival in CAR T-cell therapy, and clinicians have very limited tools to discriminate a priori patients who will or will not respond to treatment. While CD19-negative relapses due to loss of target antigen is well described, it accounts for only about 30% of cases with treatment failure. Recent efforts have shed light on mechanisms of CD19-positive relapse due to tumor intrinsic resistance, T-cell quality/manufacturing, or CAR T-cell exhaustion mediated by hostile tumor microenvironment. Here, we review the latest updates of preclinical and clinical trials to investigate the mechanisms of resistance and relapse post CAR T-cell therapy in B cell lymphoma and discuss novel treatment strategies to overcome resistance as well as advances that are useful for a CAR T therapist to optimize and personalize CAR T-cell therapy.

Conjunctival T Cell Profile in Allogeneic Hematopoietic Stem Cell Transplant Patients after Instilling Topical Cyclosporine-A 0.1% Cationic Emulsion.

INTRODUCTION: To profile conjunctival T cell populations in allogeneic hematopoietic stem cell transplant (HSCT) patients after instillation of daily topical cyclosporine-A (CsA) 0.1% cationic emulsion (Ikervis), and to evaluate patients' tolerance to these eye drops. METHODS: Nineteen participants were prescribed Ikervis prophylaxis once daily to both eyes from 3-5 weeks pre-HSCT to 12 months post-HSCT. The outcome measure was conjunctival T cell proportions from flow cytometry after impression cytology. Covariates included visual acuity, intraocular pressure, slit lamp and fundal examination, dry eye (SPEED) and quality of life questionnaires, non-invasive keratograph tear break-up time (NIKBUT), conjunctival redness, meibography, lipid thickness, Schirmer test, tear cytokines, fluorescein staining, tear osmolarity, and meibomian gland expressibility. RESULTS: The conjunctival T cell analysis showed either stable or decreased proportions of conjunctival CD4 T cells at the last visit from baseline in compliant patients. CD4 proportions were increased in non-compliant patients and in the single patient who developed ocular graft-versus-host disease (GVHD). All patients were tolerant to Ikervis but 6/19 were not compliant. In the majority of patients, vision did not affect activities of daily living. Pre- and post-HSCT up to the last study visit, there was no statistically significant change in clinical covariates. Only one participant developed ocular GVHD at 9 months post-HSCT. CONCLUSION: Superficial conjunctival T cell profile reflects compliance to daily topical Ikervis eye drops and clinical ocular surface parameters in allogenic HSCT patients. Tolerance is comparable to other formulations of topical CsA in the first 12 months. GOV IDENTIFIER: NCT04636918. URL: https://clinicaltrials.gov/ct2/show/NCT04636918?cond=ocular+Graft+Versus+Host+Disease&cntry=SG&draw=2&rank=2 .

Activated B cells suppress T-cell function through metabolic competition.

BACKGROUND: B cells play a pivotal role in regulating the immune response. The induction of B cell-mediated immunosuppressive function requires B cell activating signals. However, the mechanisms by which activated B cells mediate T-cell suppression are not fully understood. METHODS: We investigated the potential contribution of metabolic activity of activated B cells to T-cell suppression by performing in vitro experiments and by analyzing clinical samples using mass cytometry and single-cell RNA sequencing. RESULTS: Here we show that following activation, B cells acquire an immunoregulatory phenotype and promote T-cell suppression by metabolic competition. Activated B cells induced hypoxia in T cells in a cell-cell contact dependent manner by consuming more oxygen via an increase in their oxidative phosphorylation (OXPHOS). Moreover, activated B cells deprived T cells of glucose and produced lactic acid through their high glycolytic activity. Activated B cells thus inhibited the mammalian target of rapamycin pathway in T cells, resulting in suppression of T-cell cytokine production and proliferation. Finally, we confirmed the presence of tumor-associated B cells with high glycolytic and OXPHOS activities in patients with melanoma, associated with poor response to immune checkpoint blockade therapy. CONCLUSIONS: We have revealed for the first time the immunomodulatory effects of the metabolic activity of activated B cells and their possible role in suppressing antitumor T-cell responses. These findings add novel insights into immunometabolism and have important implications for cancer immunotherapy.

A unique hub-and-spoke model to optimize patient management in lymphoma using novel chimeric antigen receptor-T cell therapy in Southeast and South Asia.

Novel therapeutic options for cancer offer hope for patients and their families, particularly when the cancer has not responded to established treatment regimens. The chimeric antigen receptor (CAR)-T cell therapeutic approach has changed the treatment paradigm for relapsed or refractory lymphoma, extending the capacity of the patient's own T cells to detect and eliminate cancer cells through genetic modification of T-cell surface receptors. The process of establishing treatment centers and developing clinical expertize in this novel treatment strategy is complex. Time, resources, and a commitment to focusing health budgets on a new area are required. Currently, Singapore is the only country in southeast and south Asia with market authorization of the CAR-T product, tisagenlecleucel. Availability of CAR-T treatment across international borders provides patients in neighboring countries with choice in therapeutic options. This paper describes the unique hub-and-spoke cross-border collaboration developed between Singapore and its neighbors to provide access to CAR-T cell therapy for patients with relapsed or refractory lymphoma. To date in 2022, four patients have been included in the CAR-T treatment cross-border collaboration. Their stay in Singapore has been about 2 months' duration, including the pre-treatment evaluation, apheresis, CAR-T cell infusion and post-treatment monitoring. Patient support from referring and treating physicians, critical to the success of the undertaking, is characterized by early communication, patient selection, multi-disciplinary care, post-treatment monitoring, and attention to detail. The patient journey and the development and implementation of this unique collaboration are discussed.

CAR T Cell Therapy in Hematological Malignancies: Implications of the Tumor Microenvironment and Biomarkers on Efficacy and Toxicity.

Chimeric antigen receptor (CAR) T cell therapy has ushered in a new era in cancer treatment. Remarkable outcomes have been demonstrated in patients with previously untreatable relapsed/refractory hematological malignancies. However, optimizing efficacy and reducing the risk of toxicities have posed major challenges, limiting the success of this therapy. The tumor microenvironment (TME) plays an important role in CAR T cell therapy's effectiveness and the risk of toxicities. Increasing research studies have also identified various biomarkers that can predict its effectiveness and risk of toxicities. In this review, we discuss the various aspects of the TME and biomarkers that have been implicated thus far and discuss the role of creating scoring systems that can aid in further refining clinical applications of CAR T cell therapy and establishing a safe and efficacious personalised medicine for individuals.

Cost-effectiveness and budget impact analyses of tisagenlecleucel in adult patients with relapsed or refractory diffuse large B-cell lymphoma from Singapore's private insurance payer's perspective.

BACKGROUND: Patients experiencing relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL) have limited treatment options and poor prognosis. Tisagenlecleucel (TIS) has shown improved clinical outcomes, but at a high upfront cost. Singapore has a multi-payer healthcare system where private insurance is one of the major payers. This study evaluated the cost-effectiveness and budget impact of TIS against salvage chemotherapy regimen (SCR) for treating r/r DLBCL patients who have failed ≥2 lines of systemic therapy from Singapore's private insurance payer's perspective. METHODS: Over a life-time horizon, a partitioned survival model with three health-states was developed to evaluate the cost-effectiveness of TIS vs. SCR with or without hematopoietic stem cell transplantation (HSCT). Efficacy inputs for TIS and SCR were based on 43 months of observation data from pooled JULIET and UPenn trials, and CORAL extension studies respectively. Direct costs for pre-treatment, treatment, adverse events, follow-up, subsequent-HSCT, relapse, and terminal care were included. Incremental cost-effectiveness ratios (ICERs) were calculated as the total incremental costs per quality-adjusted life-year (QALY) gained. Additionally, the financial implication of introducing TIS in Singapore from a private payer's perspective was analyzed, comparing the current treatment pathway (without TIS) with a future scenario (with TIS) over 5 years. RESULTS: Compared with SCR, TIS was the dominant option, with cost savings of S$8,477 alongside an additional gain of 2.78 QALYs in privately insured patients who shifted from private to public hospitals for TIS treatment. Scenario analyses for patients starting in public hospitals show ICERs of S$99,623 (no subsidy) and S$133,261 (50% subsidy for SCR treatment, no subsidy for TIS), supporting the base case. The projected annual budget impact ranges from S$850,000 to S$3.4 million during the first 5 years. CONCLUSIONS: TIS for treating r/r DLBCL patients who have failed ≥2 lines of systemic therapies, is likely to be cost effective with limited budget impact.

Targeting a cytokine checkpoint enhances the fitness of armored cord blood CAR-NK cells.

Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2-containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation "armored" chimeric antigen receptor (CAR) engineering of cord blood-derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell antitumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15-secreting armored CAR-NK cells by promoting their metabolic fitness and antitumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.