An NK-like CAR T cell transition in CAR T cell dysfunction.

Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains ineffective in solid tumors, due in part to CAR T cell exhaustion in the solid tumor microenvironment. To study dysfunction of mesothelin-redirected CAR T cells in pancreatic cancer, we establish a robust model of continuous antigen exposure that recapitulates hallmark features of T cell exhaustion and discover, both in vitro and in CAR T cell patients, that CAR dysregulation is associated with a CD8+ T-to-NK-like T cell transition. Furthermore, we identify a gene signature defining CAR and TCR dysregulation and transcription factors, including SOX4 and ID3 as key regulators of CAR T cell exhaustion. Our findings shed light on the plasticity of human CAR T cells and demonstrate that genetic downmodulation of ID3 and SOX4 expression can improve the efficacy of CAR T cell therapy in solid tumors by preventing or delaying CAR T cell dysfunction.

Synthetic manipulation of the cancer-immunity cycle: CAR-T cell therapy.

Synthetic immunity to cancer has been pioneered by the application of chimeric antigen receptor (CAR) engineering into autologous T cells. CAR T cell therapy is highly amenable to molecular engineering to bypass barriers of the cancer immunity cycle, such as endogenous antigen presentation, immune priming, and natural checkpoints that constrain immune responses. Here, we review CAR T cell design and the mechanisms that drive sustained CAR T cell effector activity and anti-tumor function. We discuss engineering approaches aimed at improving anti-tumor function through a variety of mechanistic interventions for both hematologic and solid tumors. The ability to engineer T cells in such a variety of ways, including by modifying their trafficking, antigen recognition, costimulation, and addition of synthetic genes, circuits, knockouts and base edits to finely tune complex functions, is arguably the most powerful way to manipulate the cancer immunity cycle in patients.

Costimulatory domains direct distinct fates of CAR-driven T-cell dysfunction.

T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have demonstrated impressive activity against relapsed or refractory B-cell cancers yet fail to induce durable remissions for nearly half of all patients treated. Enhancing the efficacy of this therapy requires detailed understanding of the molecular circuitry that restrains CAR-driven antitumor T-cell function. We developed and validated an in vitro model that drives T-cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains, central components of CAR structure and function, contribute to T-cell failure. We found that chronic activation of CD28-based CARs results in activation of classical T-cell exhaustion programs and development of dysfunctional cells that bear the hallmarks of exhaustion. In contrast, 41BB-based CARs activate a divergent molecular program and direct differentiation of T cells into a novel cell state. Interrogation using CAR T cells from a patient with progressive lymphoma confirmed the activation of this novel program in a failing clinical product. Furthermore, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is directly responsible for impairing CAR T-cell function. These findings identify that costimulatory domains are critical regulators of CAR-driven T-cell failure and that targeted interventions are required to overcome costimulation-dependent dysfunctional programs.

Perspectives in Immunotherapy: meeting report from Immunotherapy Bridge (Naples, November 30th-December 1st, 2022).

The discovery and development of novel treatments that harness the patient's immune system and prevent immune escape has dramatically improved outcomes for patients across cancer types. However, not all patients respond to immunotherapy, acquired resistance remains a challenge, and responses are poor in certain tumors which are considered to be immunologically cold. This has led to the need for new immunotherapy-based approaches, including adoptive cell transfer (ACT), therapeutic vaccines, and novel immune checkpoint inhibitors. These new approaches are focused on patients with an inadequate response to current treatments, with emerging evidence of improved responses in various cancers with new immunotherapy agents, often in combinations with existing agents. The use of cell therapies, drivers of immune response, and trends in immunotherapy were the focus of the Immunotherapy Bridge (November 30th-December 1st, 2022), organized by the Fondazione Melanoma Onlus, Naples, Italy, in collaboration with the Society for Immunotherapy of Cancer.

Mechanisms of resistance to CAR T cell therapies.

Chimeric antigen receptor (CAR)-engineered T cells have demonstrated remarkable success in the treatment of B cell malignancies. FDA approval of these therapies represents a watershed moment in the development of therapies for cancer. Despite the successes of the last decade, many patients will unfortunately not experience durable responses to CAR therapy. Emerging research has shed light on the biology responsible for these failures, and further highlighted the hurdles to broader success. Here, we review the recent research identifying how interactions between cancer cells and engineered immune cells result in resistance to CAR therapies.

Ionizable Lipid Nanoparticle-Mediated mRNA Delivery for Human CAR T Cell Engineering.

Chimeric antigen receptor (CAR) T cell therapy relies on the ex vivo manipulation of patient T cells to create potent, cancer-targeting therapies, shown to be capable of inducing remission in patients with acute lymphoblastic leukemia and large B cell lymphoma. However, current CAR T cell engineering methods use viral delivery vectors, which induce permanent CAR expression and could lead to severe adverse effects. Messenger RNA (mRNA) has been explored as a promising strategy for inducing transient CAR expression in T cells to mitigate the adverse effects associated with viral vectors, but it most commonly requires electroporation for T cell mRNA delivery, which can be cytotoxic. Here, ionizable lipid nanoparticles (LNPs) were designed for ex vivo mRNA delivery to human T cells. A library of 24 ionizable lipids was synthesized, formulated into LNPs, and screened for luciferase mRNA delivery to Jurkat cells, revealing seven formulations capable of enhanced mRNA delivery over lipofectamine. The top-performing LNP formulation, C14-4, was selected for CAR mRNA delivery to primary human T cells. This platform induced CAR expression at levels equivalent to electroporation, with substantially reduced cytotoxicity. CAR T cells engineered via C14-4 LNP treatment were then compared to electroporated CAR T cells in a coculture assay with Nalm-6 acute lymphoblastic leukemia cells, and both CAR T cell engineering methods elicited potent cancer-killing activity. These results demonstrate the ability of LNPs to deliver mRNA to primary human T cells to induce functional protein expression, and indicate the potential of LNPs to enhance mRNA-based CAR T cell engineering methods.

Erythropoietic protoporphyria in an adult with sequential liver and hematopoietic stem cell transplantation: A case report.

Erythropoietic protoporphyria (EPP) is a rare inherited disorder of the heme biosynthesis pathway resulting in the accumulation of protoporphyrins in the blood, erythrocytes, and other tissues. Because of a gene mutation in the FECH gene, ferrochelatase, the enzyme involved in the final step of heme synthesis, is deficient in these patients. Although the major symptom of this disorder is photosensitivity, rarely, it can cause progressive liver disease requiring liver transplantation (LT). However, LT is not curative and only bone marrow transplantation (BMT) can correct the underlying enzymatic defect. Because liver disease results from accumulation of protoporphyrin in the liver, LT without hematopoietic stem cell transplantation leaves the new liver at risk for similar EPP-related damage. A handful of pediatric patients undergoing sequential LT and stem cell transplantation have been described in the literature; however, to date none has been described in detail in adults. We report a case of an adult male with EPP and liver failure who successfully underwent a sequential liver and hematopoietic stem cell transplantation (HSCT).

Monocyte lineage-derived IL-6 does not affect chimeric antigen receptor T-cell function.

BACKGROUND AIMS: Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 has demonstrated remarkable success in targeting B-cell malignancies but is often complicated by serious systemic toxicity in the form of cytokine release syndrome (CRS). CRS symptoms are primarily mediated by interleukin 6 (IL-6), and clinical management has focused on inhibition of IL-6 signaling. The cellular source and function of IL-6 in CRS remain unknown. METHODS: Using co-culture assays and data from patients on our clinical CAR T-cell trials, we investigated the cellular source of IL-6, as well as other CRS-associated cytokines, during CAR T-cell activation. We also explored the effect that IL-6 has on T-cell function. RESULTS: We demonstrated that IL-6 is secreted by monocyte-lineage cells in response to CAR T-cell activation in a contact-independent mechanism upon T-cell engagement of target leukemia. We observed that the presence of antigen-presenting cell-derived IL-6 has no impact on CAR T-cell transcriptional profiles or cytotoxicity. Finally, we confirm that CAR T cells do not secrete IL-6 in vivo during clinical CRS. DISCUSSION: These findings suggest that IL-6 blockade will not affect CD19 CAR T-cell-driven anti-leukemic cytotoxicity, permitting enhanced control of CRS while maintaining CAR T-cell efficacy.

Chimeric antigen receptor therapy for cancer.

Improved outcomes for patients with cancer hinge on the development of new targeted therapies with acceptable short-term and long-term toxicity. Progress in basic, preclinical, and clinical arenas spanning cellular immunology, synthetic biology, and cell-processing technologies has paved the way for clinical applications of chimeric antigen receptor-based therapies. This new form of targeted immunotherapy merges the exquisite targeting specificity of monoclonal antibodies with the potent cytotoxicity and long-term persistence provided by cytotoxic T cells. Although this field is still in its infancy, clinical trials have already shown clinically significant antitumor activity in neuroblastoma, chronic lymphocytic leukemia, and B cell lymphoma, and trials targeting a variety of other adult and pediatric malignancies are under way. Ongoing work is focused on identifying optimal tumor targets and on elucidating and manipulating both cell- and host-associated factors to support expansion and persistence of the genetically engineered cells in vivo. The potential to target essentially any tumor-associated cell-surface antigen for which a monoclonal antibody can be made opens up an entirely new arena for targeted therapy of cancer.

CAR T Cell Therapy in Acute Lymphoblastic Leukemia and Potential for Chronic Lymphocytic Leukemia.

OPINION STATEMENT: Adoptive transfer of autologous T cells engineered to express a chimeric antigen receptor (CAR) represents a powerful targeted immunotherapy that has shown great promise in some of the most refractory leukemias. CAR-modified T cells directed against CD19 have led the way, setting a high standard with remission rates as high as 90 % in clinical trials for relapsed/refractory acute lymphoblastic leukemia (ALL). Yet, the first demonstration of efficacy was in another disease, chronic lymphocytic leukemia (CLL), in which CD19-targeted CAR T cells eradicated bulky, highly refractory disease. Despite early encouraging results, clinical trials in CLL have yielded lower response rates, revealing disease-specific differences in response in this form of immunotherapy. Ongoing research focused on identifying and overcoming these limitations, promises to improve response rates. Beyond the induction of remission, the transformative impact of engineered T cell therapy lies in its potential for long-term disease control. With longer follow-up and durable T cell persistence now reported, we are closer to answering the question of whether sustained remissions are possible with CAR T cell monotherapy. As might be expected with a highly effective therapy using a single mechanism of action, escape pathways have emerged. Combinatorial approaches are needed to anticipate and prevent this mode of relapse. Lastly, toxicity management is vital to ensure the safety of this exciting cancer immunotherapy.

Donor-derived CD19 chimeric antigen receptor T cells.

PURPOSE OF REVIEW: As immunotherapy matures into possible front-line therapy, new approaches are necessary to expand the capacity to treat more patients. Although most technologies for chimeric antigen receptor (CAR) therapies require autologous T cells, 'off the shelf' sources are highly desired. RECENT FINDINGS: Sources of T cells for modification with CARs include cord blood and either related or unrelated allogeneic donors. Strategies to manipulate these sources focus on reducing the risk of alloreactivity, while maintaining the potential for high function and long persistence associated with successful CAR T-cell therapies. SUMMARY: Recent research implies that manipulating nonautologous T-cell sources can result in well tolerated and effective products, but work remains to determine if these approaches will reach the efficacy of autologous products.

Early T2 changes predict onset of radiographic knee osteoarthritis: data from the osteoarthritis initiative.

OBJECTIVE: To evaluate whether T2 relaxation time measurements obtained at 3 T MRI predict the onset of radiographic knee osteoarthritis (OA). MATERIALS AND METHODS: We performed a nested case-control study of incident radiographic knee OA in the Osteoarthritis Initiative cohort. Cases were 50 knees with baseline Kellgren-Lawrence (KL) grade of 0 that developed KL grade of 2 or more over a 4-year period. Controls were 80 knees with KL grade of 0 after 4 years of follow-up. Baseline T2 relaxation time measurements and laminar analysis of T2 in deep and superficial layers were performed in all knee compartments. The association of T2 values with incident OA was assessed with logistic regression and differences in T2 values by case-control status with linear regression, adjusting for age, sex, body mass index (BMI) and other covariates. RESULTS: Baseline T2 values in all compartments except the medial tibia were significantly higher in knees that developed OA compared with controls and were particularly elevated in the superficial cartilage layers in all compartments. There was an increased likelihood of incident knee OA associated with higher baseline T2 values, particularly in the patella, adjusted OR per 1 SD increase in T2 (3.37 (95% CI 1.72 to 6.62)), but also in the medial femur (1.90 (1.07 to 3.39)), lateral femur (2.17 (1.11 to 4.25)) and lateral tibia (2.23 (1.16 to 4.31)). CONCLUSIONS: These findings suggest that T2 values assessed when radiographic changes are not yet apparent may be useful in predicting the development of radiological tibiofemoral OA.

Relation of clinical culture method to T-cell memory status and efficacy in xenograft models of adoptive immunotherapy.

BACKGROUND AIMS: Cytotoxic T lymphocytes modified with chimeric antigen receptors (CARs) for adoptive immunotherapy of hematologic malignancies are effective in pre-clinical models, and this efficacy has translated to success in several clinical trials. Many early trials were disappointing in large part because of the lack of proliferation and subsequent persistence of transferred cells. Recent investigations have pointed to the importance of delivering highly proliferative cells, whether of naive or early memory phenotypes. METHODS: We investigated the influence of two common cell culturing methods used in early trials and their relationship to T-cell phenotype and pre-clinical efficacy. RESULTS: We observed that stimulation with soluble anti-CD3 antibody OKT-3 and high-dose interleukin-2 produces more effector memory-type T cells with shorter average telomeres when compared with cells generated with the use of CD3/CD28 beads. When used in xenograft models of leukemia, bead-stimulated cells proliferated earlier and to a higher degree than those generated with the use of OKT-3/IL2 and resulted in better disease control despite no difference in distribution or migration throughout the mouse. Inclusion of the known successful clinical 4-1BB endodomain in the CAR could not rescue the function of OKT-3/IL-2-cultured cells. T cells isolated from animals that survived long-term (>120 days) retained a central memory-like phenotype and demonstrated a memory response to a large re-challenge of CD19-positive leukemia. CONCLUSIONS: In summary, we confirm that cells with a younger phenotype or higher proliferative capacity perform better in pre-clinical models and that cell culturing influences cell phenotype seemingly independent of the 4-1BB endodomain in the CAR structure.

Regulatory Considerations for Genome-Edited T-cell Therapies.

Methods to engineer the genomes of human cells for therapeutic intervention continue to advance at a remarkable pace. Chimeric antigen receptor-engineered T lymphocytes have pioneered the way for these therapies, initially beginning with insertions of chimeric antigen receptor transgenes into T-cell genomes using classical gene therapy vectors. The broad use of clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies to edit endogenous genes has now opened the door to a new era of precision medicine. To add complexity, many engineered cellular therapies under development integrate gene therapy with genome editing to introduce novel biological functions and enhance therapeutic efficacy. Here, we review the current state of scientific, translational, and regulatory oversight of gene-edited cell products.

Rational protein engineering to enhance MHC-independent T cell receptors.

Chimeric antigen receptor (CAR)-based therapies have pioneered synthetic cellular immunity but remain limited in their long-term efficacy. Emerging data suggest that dysregulated CAR-driven T cell activation causes T cell dysfunction and therapeutic failure. To re-engage the precision of the endogenous T cell response, we designed MHC-independent T cell receptors (miTCRs) by linking antibody variable domains to TCR constant chains. Using predictive modeling, we observed that this standard "cut and paste" approach to synthetic protein design resulted in myriad biochemical conflicts at the hybrid variable-constant domain interface. Through iterative modeling and sequence modifications we developed structure-enhanced miTCRs which significantly improved receptor-driven T cell function across multiple tumor models. We found that 41BB costimulation specifically prolonged miTCR T cell persistence and enabled improved leukemic control in vivo compared to classic CAR T cells. Collectively, we have identified core features of hybrid receptor structure responsible for regulating function.

Identification of core techniques that enhance genome editing of human T cells expressing synthetic antigen receptors.

Genome editing technologies have seen remarkable progress in recent years, enabling precise regulation of exogenous and endogenous genes. These advances have been extensively applied to the engineering of human T lymphocytes, leading to the development of practice changing therapies for patients with cancer and the promise of synthetic immune cell therapies for a variety of non-malignant diseases. Many distinct conceptual and technical approaches have been used to edit T-cell genomes, however targeted assessments of which techniques are most effective for manufacturing, gene editing and transgene expression are rarely reported. Through extensive comparative evaluation, we identified methods that most effectively enhance engineering of research-scale and pre-clinical T-cell products at critical stages of manufacturing.

Inducing T cell dysfunction by chronic stimulation of CAR-engineered T cells targeting cancer cells in suspension cultures.

Several pre-clinical models reveal that chronic chimeric antigen receptor (CAR) stimulation drives a dysfunctional state that mimics in vivo failure. In this protocol, we describe steps to induce T cell dysfunction by persistent and long-term stimulation of CAR-engineered T cells using antigen-expressing cancer cells in suspension cultures. We first described a validated method for manufacturing of CAR T cells, followed by a detailed method for chronic stimulation of CAR T cells and a strategy to evaluate these cells during the process of chronic stimulation. For complete details on the use and execution of this protocol, please refer to Singh et al. (2020).1.

Intrinsic tumor resistance to CAR T cells is a dynamic transcriptional state that is exploitable with low-dose radiation.

Chimeric antigen receptor (CAR) T-cell therapy represents a major advancement for hematologic malignancies, with some patients achieving long-term remission. However, the majority of treated patients still die of their disease. A consistent predictor of response is tumor quantity, wherein a higher disease burden before CAR T-cell therapy portends a worse prognosis. Focal radiation to bulky sites of the disease can decrease tumor quantity before CAR T-cell therapy, but whether this strategy improves survival is unknown. We find that substantially reducing systemic tumor quantity using high-dose radiation to areas of bulky disease, which is commonly done clinically, is less impactful on overall survival in mice achieved by CAR T cells than targeting all sites of disease with low-dose total tumor irradiation (TTI) before CAR T-cell therapy. This finding highlights another predictor of response, tumor quality, the intrinsic resistance of an individual patient's tumor cells to CAR T-cell killing. Little is known about whether or how an individual tumor's intrinsic resistance may change under different circumstances. We find a transcriptional "death receptor score" that reflects a tumor's intrinsic sensitivity to CAR T cells can be temporarily increased by low-dose TTI, and the timing of this transcriptional change correlates with improved in vivo leukemia control by an otherwise limited number of CAR T cells. This suggests an actionable method for potentially improving outcomes in patients predicted to respond poorly to this promising therapy and highlights that intrinsic tumor attributes may be equally or more important predictors of CAR T-cell response as tumor burden.

Costimulatory domains direct distinct fates of CAR-driven T cell dysfunction.

Chimeric antigen receptor (CAR) engineered T cells often fail to enact effector functions after infusion into patients. Understanding the biological pathways that lead CAR T cells to failure is of critical importance in the design of more effective therapies. We developed and validated an in vitro model that drives T cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains contribute to T cell failure. We found that dysfunctional CD28-based CARs targeting CD19 bear hallmarks of classical T cell exhaustion while dysfunctional 41BB-based CARs are phenotypically, transcriptionally and epigenetically distinct. We confirmed activation of this unique transcriptional program in CAR T cells that failed to control clinical disease. Further, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is a significant contributor to this dysfunction and disruption of FOXO3 improves CAR T cell function. These findings identify that chronic activation of 41BB leads to novel state of T cell dysfunction that can be alleviated by genetic modification of FOXO3. Summary: Chronic stimulation of CARs containing the 41BB costimulatory domain leads to a novel state of T cell dysfunction that is distinct from T cell exhaustion.