Granzyme B-activated IL18 potentiates αß and γδ CAR T cell immunotherapy in a tumor-dependent manner.

Interleukin (IL)18 is a potent pro-inflammatory cytokine that is activated upon caspase 1 cleavage of the latent precursor, pro-IL18. Therapeutic T cell armoring with IL18 promotes autocrine stimulation and positive modulation of the tumor microenvironment (TME). However, existing strategies are imperfect since they involve constitutive/poorly regulated activity or fail to modify the TME. Here, we have substituted the caspase 1 cleavage site within pro-IL18 with that preferred by granzyme B, yielding GzB-IL18. We demonstrate that GzB-IL18 is constitutively released but remains functionally latent unless chimeric antigen receptor (CAR) T cells are activated, owing to concomitant granzyme B release. Armoring with GzB-IL18 enhances cytolytic activity, proliferation, interferon (IFN)-γ release, and anti-tumor efficacy by a similar magnitude to constitutively active IL18. We also demonstrate that GzB-IL18 provides a highly effective armoring strategy for γδ CAR T cells, leading to enhanced metabolic fitness and significant potentiation of therapeutic activity. Finally, we show that constitutively active IL18 can unmask CAR T cell-mediated cytokine release syndrome in immunocompetent mice. By contrast, GzB-IL18 promotes anti-tumor activity and myeloid cell re-programming without inducing such toxicity. Using this stringent system, we have tightly coupled the biological activity of IL18 to the activation state of the host CAR T cell, favoring safer clinical implementation of this technology.

Augmented Expansion of Treg Cells From Healthy and Autoimmune Subjects via Adult Progenitor Cell Co-Culture.

Recent clinical experience has demonstrated that adoptive regulatory T (Treg) cell therapy is a safe and feasible strategy to suppress immunopathology via induction of host tolerance to allo- and autoantigens. However, clinical trials continue to be compromised due to an inability to manufacture a sufficient Treg cell dose. Multipotent adult progenitor cells (MAPCⓇ) promote Treg cell differentiation in vitro, suggesting they may be repurposed to enhance ex vivo expansion of Tregs for adoptive cellular therapy. Here, we use a Good Manufacturing Practice (GMP) compatible Treg expansion platform to demonstrate that MAPC cell-co-cultured Tregs (MulTreg) exhibit a log-fold increase in yield across two independent cohorts, reducing time to target dose by an average of 30%. Enhanced expansion is coupled to a distinct Treg cell-intrinsic transcriptional program characterized by elevated expression of replication-related genes (CDK1, PLK1, CDC20), downregulation of progenitor and lymph node-homing molecules (LEF1 CCR7, SELL) and induction of intestinal and inflammatory tissue migratory markers (ITGA4, CXCR1) consistent with expression of a gut homing (CCR7lo ß7hi) phenotype. Importantly, we find that MulTreg are more readily expanded from patients with autoimmune disease compared to matched Treg lines, suggesting clinical utility in gut and/or T helper type1 (Th1)-driven pathology associated with autoimmunity or transplantation. Relative to expanded Tregs, MulTreg retain equivalent and robust purity, FoxP3 Treg-Specific Demethylated Region (TSDR) demethylation, nominal effector cytokine production and potent suppression of Th1-driven antigen specific and polyclonal responses in vitro and xeno Graft vs Host Disease (xGvHD) in vivo. These data support the use of MAPC cell co-culture in adoptive Treg therapy platforms as a means to rescue expansion failure and reduce the time required to manufacture a stable, potently suppressive product.

Approaches for refining and furthering the development of CAR-based T cell therapies for solid malignancies.

Introduction: Chimeric antigen receptor-engineered T-cells typically use the binding domains of antibodies to target cytotoxicity toward tumors. This approach has produced great efficacy against selected hematological cancers, but benefit in solid tumors has been limited. Characteristically, the microenvironment in solid tumors restricts CAR T cell function, thereby limiting success. Enhancing efficacy will depend on novel target discovery to refine specificity and reduce toxicity. Additionally, overcoming immunosuppressive mechanisms may be achieved by altering the structure of the CAR itself, together with ancillary gene expression or additional therapeutic interventions.Areas covered: Herein, the authors discuss approaches for refining and further developing CAR T cell therapies specifically for use with solid malignancies. The authors survey the existing literature and provide perspectives for the future.Expert opinion: Pronounced efficacy in solid tumors will likely require combination therapies, targeting both the tumor itself and associated immunosuppressive mechanisms. Future exploration of CAR T cell therapies for solid tumors is likely to incorporate next-generation designs that couple more precise targeting of cancer-associated targets with enhanced potency and resistance to exhaustion.

Synergistic T cell signaling by 41BB and CD28 is optimally achieved by membrane proximal positioning within parallel chimeric antigen receptors.

Second generation (2G) chimeric antigen receptors (CARs) contain a CD28 or 41BB co-stimulatory endodomain and elicit remarkable efficacy in hematological malignancies. Third generation (3G) CARs extend this linear blueprint by fusing both co-stimulatory units in series. However, clinical impact has been muted despite compelling evidence that co-signaling by CD28 and 41BB can powerfully amplify natural immune responses. We postulate that effective dual co-stimulation requires juxta-membrane positioning of endodomain components within separate synthetic receptors. Consequently, we designed parallel (p)CARs in which a 2G (CD28+CD3ζ) CAR is co-expressed with a 41BB-containing chimeric co-stimulatory receptor. We demonstrate that the pCAR platform optimally harnesses synergistic and tumor-dependent co-stimulation to resist T cell exhaustion and senescence, sustaining proliferation, cytokine release, cytokine signaling, and metabolic fitness upon repeated stimulation. When engineered using targeting moieties of diverse composition, affinity, and specificity, pCAR T cells consistently elicit superior anti-tumor activity compared with T cells that express traditional linear CARs.

Novel approaches to promote CAR T-cell function in solid tumors.

INTRODUCTION: Following recent breakthrough developments in the application of CAR T-cell treatments for hematologic cancers, the potential of this approach to achieve meaningful impact against solid tumors now warrants careful consideration. AREAS COVERED: Effective deployment of CAR T-cell immunotherapy for solid tumors has proven challenging to date, due to a series of formidable hurdles. The first of these is the paucity of safe targets for this highly potent, expensive and potentially toxic form of treatment. Compounding this, the tumor microenvironment (TME) constitutes a nexus of cellular and molecular elements that conspire to suppress effective immunological function at that site, corrupting the physiological reparative processes that operate during wound healing. These obstacles are considered with a view to addressing how next-generation CAR T-cell approaches may be effectively applied to different cancer types. EXPERT OPINION: A variety of novel synthetic biology and combinatorial strategies are being developed that can improve CAR T-cell specificity and combat immunosuppressive pathways found in the TME. In addition, recent advances in genome editing techniques are paving the way toward the production of universally applicable CAR T cells.

Regulatory T cell dysfunction in type 1 diabetes: what's broken and how can we fix it?

Type 1 diabetes is an autoimmune disease characterised by the destruction of insulin producing beta cells in the pancreas. Whilst it remains unclear what the original triggering factors for this destruction are, observations from the natural history of human type 1 diabetes, including incidence rates in twins, suggest that the disease results from a combination of genetic and environmental factors. Whilst many different immune cells have been implicated, including members of the innate and adaptive immune systems, a view has emerged over the past 10 years that beta cell damage is mediated by the combined actions of CD4+ and CD8+ T cells with specificity for islet autoantigens. In health, these potentially pathogenic T cells are held in check by multiple regulatory mechanisms, known collectively as 'immunological tolerance'. This raises the question as to whether type 1 diabetes develops, at least in part, as a result of a defect in one or more of these control mechanisms. Immunological tolerance includes both central mechanisms (purging of the T cell repertoire of high-affinity autoreactive T cells in the thymus) and peripheral mechanisms, a major component of which is the action of a specialised subpopulation of T cells, known as regulatory T cells (Tregs). In this review, we highlight the evidence suggesting that a reduction in the functional capacity of different Treg populations contributes to disease development in type 1 diabetes. We also address current controversies regarding the putative causes of this defect and discuss strategies to correct it as a means to reduce or prevent islet destruction in a clinical setting.

Generation of human islet-specific regulatory T cells by TCR gene transfer.

Based on the success in animal models of type 1 diabetes (T1D), clinical trials of adoptive regulatory T cell (Treg) therapy are underway using ex vivo expanded polyclonal Tregs. However, pre-clinical data also demonstrate that islet-specific Tregs are more potent than polyclonal Tregs at reversing T1D. Translation of this approach into man will require methods to generate large populations of islet-specific Tregs which, to date, has proved to be a major hurdle. Here we demonstrate the feasibility of lentiviral-mediated T cell receptor (TCR) gene transfer to confer antigen specificity on polyclonal human Tregs. Targeting has been achieved using TCRs isolated from human islet-specific and viral-specific CD4+ T cell clones. Engineered T cells demonstrated expression of ectopically-delivered TCRs, resulting in endowment of cognate antigen-specific responses. This enabled antigen-specific suppression at increased potency compared to polyclonal Tregs. However, cells transduced with islet-specific TCRs were less responsive to cognate antigen than viral-specific TCRs, and in some cases, required additional methods to isolate functional antigen-specific Tregs. This study demonstrates the potential of TCR gene transfer to develop islet-specific Treg therapies for effective treatment of T1D, but also highlights that additional optimisation may be required to achieve its full potential.