CD4 CAR-T cells targeting CD19 play a key role in exacerbating cytokine release syndrome, while maintaining long-term responses.

BACKGROUND: To date, T cells redirected with CD19-specific chimeric antigen receptors (CAR) have gained impressive success in B-cell malignancies. However, treatment failures are common and the occurrence of severe toxicities, such as cytokine release syndrome (CRS), still limits the full exploitation of this approach. Therefore, the development of cell products with improved therapeutic indexes is highly demanded. METHODS: In this project, we investigated how CD4 and CD8 populations cooperate during CD19 CAR-T cell responses and what is their specific role in CRS development. To this aim, we took advantage of immunodeficient mice reconstituted with a human immune system (HuSGM3) and engrafted with the B-cell acute lymphoblastic leukemia cell line NALM-6, a model that allows to thoroughly study efficacy and toxicity profiles of CD19 CAR-T cell products. RESULTS: CD4 CAR-T cells showed superior proliferation and activation potential, which translated into stronger stimulation of myeloid cells, the main triggers of adverse events. Accordingly, toxicity assessment in HuSGM3 mice identified CD4 CAR-T cells as key contributors to CRS development, revealing a safer profile when they harbor CARs embedded with 4-1BB, rather than CD28. By comparing differentially co-stimulated CD4:CD8 1:1 CAR-T cell formulations, we observed that CD4 cells shape the overall expansion kinetics of the infused product and are crucial for maintaining long-term responses. Interestingly, the combination of CD4.BBz with CD8.28z CAR-T cells resulted in the lowest toxicity, without impacting antitumor efficacy. CONCLUSIONS: Taken together, these data point out that the rational design of improved adoptive T-cell therapies should consider the biological features of CD4 CAR-T cells, which emerged as crucial for maintaining long-term responses but also endowed by a higher toxic potential.

Time to evolve: predicting engineered T cell-associated toxicity with next-generation models.

Despite promising clinical results in a small subset of malignancies, therapies based on engineered chimeric antigen receptor and T-cell receptor T cells are associated with serious adverse events, including cytokine release syndrome and neurotoxicity. These toxicities are sometimes so severe that they significantly hinder the implementation of this therapeutic strategy. For a long time, existing preclinical models failed to predict severe toxicities seen in human clinical trials after engineered T-cell infusion. However, in recent years, there has been a concerted effort to develop models, including humanized mouse models, which can better recapitulate toxicities observed in patients. The Accelerating Development and Improving Access to CAR and TCR-engineered T cell therapy (T2EVOLVE) consortium is a public-private partnership directed at accelerating the preclinical development and increasing access to engineered T-cell therapy for patients with cancer. A key ambition in T2EVOLVE is to design new models and tools with higher predictive value for clinical safety and efficacy, in order to improve and accelerate the selection of lead T-cell products for clinical translation. Herein, we review existing preclinical models that are used to test the safety of engineered T cells. We will also highlight limitations of these models and propose potential measures to improve them.

CAR T cell manufacturing from naive/stem memory T lymphocytes enhances antitumor responses while curtailing cytokine release syndrome.

Chimeric antigen receptor (CAR) T cell expansion and persistence represent key factors to achieve complete responses and prevent relapses. These features are typical of early memory T cells, which can be highly enriched through optimized manufacturing protocols. Here, we investigated the efficacy and safety profiles of CAR T cell products generated from preselected naive/stem memory T cells (TN/SCM), as compared with unselected T cells (TBULK). Notwithstanding their reduced effector signature in vitro, limiting CAR TN/SCM doses showed superior antitumor activity and the unique ability to counteract leukemia rechallenge in hematopoietic stem/precursor cell-humanized mice, featuring increased expansion rates and persistence together with an ameliorated exhaustion and memory phenotype. Most relevantly, CAR TN/SCM proved to be intrinsically less prone to inducing severe cytokine release syndrome, independently of the costimulatory endodomain employed. This safer profile was associated with milder T cell activation, which translated into reduced monocyte activation and cytokine release. These data suggest that CAR TN/SCM are endowed with a wider therapeutic index compared with CAR TBULK.

Lenalidomide enhances CD23.CAR T cell therapy in chronic lymphocytic leukemia.

Chimeric antigen receptors (CAR)-modified T cells are an emerging therapeutic tool for chronic lymphocytic leukemia (CLL). However, in patients with CLL, well-known T-cell defects and the inhibitory properties of the tumor microenvironment (TME) hinder the efficacy of CAR T cells. We explored a novel approach combining CARs with lenalidomide, an immunomodulatory drug that tempers the immunosuppressive activity of the CLL TME. T cells from patients with CLL were engineered to express a CAR specific for CD23, a promising target antigen. Lenalidomide maintained the in vitro effector functions of CD23.CAR+ T cells effector functions in terms of antigen-specific cytotoxicity, cytokine release and proliferation. Overall, lenalidomide preserved functional CAR T-CLL cell immune synapses. In a Rag2-/-γc-/--based xenograft model of CLL, we demonstrated that, when combined with low-dose lenalidomide, CD23.CAR+ T cells efficiently migrated to leukemic sites and delayed disease progression when compared to CD23.CAR+ T cells given with rhIL-2. These observations underline the therapeutic potential of this novel CAR-based combination strategy in CLL.

Disrupting N-glycan expression on tumor cells boosts chimeric antigen receptor T cell efficacy against solid malignancies.

Immunotherapy with chimeric antigen receptor (CAR)­engineered T cells showed exceptional successes in patients with refractory B cell malignancies. However, first-in-human studies in solid tumors revealed unique hurdles contributing to poor demonstration of efficacy. Understanding the determinants of tumor recognition by CAR T cells should translate into the design of strategies that can overcome resistance. Here, we show that multiple carcinomas express extracellular N-glycans, whose abundance negatively correlates with CAR T cell killing. By knocking out mannoside acetyl-glucosaminyltransferase 5 (MGAT5) in pancreatic adenocarcinoma (PAC), we showed that N-glycans protect tumors from CAR T cell killing by interfering with proper immunological synapse formation and reducing transcriptional activation, cytokine production, and cytotoxicity. To overcome this barrier, we exploited the high metabolic demand of tumors to safely inhibit N-glycans synthesis with the glucose/mannose analog 2-deoxy-d-glucose (2DG). Treatment with 2DG disrupts the N-glycan cover on tumor cells and results in enhanced CAR T cell activity in different xenograft mouse models of PAC. Moreover, 2DG treatment interferes with the PD-1­PD-L1 axis and results in a reduced exhaustion profile of tumor-infiltrating CAR T cells in vivo. The combined 2DG and CAR T cell therapy was successful against multiple carcinomas besides PAC, including those arising from the lung, ovary, and bladder, and with different clinically relevant CAR specificities, such as CD44v6 and CEA. Overall, our results indicate that tumor N-glycosylation regulates the quality and magnitude of CAR T cell responses, paving the way for the rational design of improved therapies against solid malignancies.

Overcoming key challenges in cancer immunotherapy with engineered T cells.

PURPOSE OF REVIEW: A number of clinical trials are currently testing chimeric antigen receptor (CAR) and T cell receptor (TCR) engineered T cells for the treatment of haematologic malignancies and selected solid tumours, and CD19-CAR-T cells have produced impressive clinical responses in B-cell malignancies. Here, we summarize the current state of the field, highlighting the key aspects required for the optimal application of CAR and TCR-engineered T cells for cancer immunotherapy. RECENT FINDINGS: Toxicities, treatment failure and disease recurrence have been observed at different rates and kinetics. Several strategies have been designed to overcome these hurdles: the identification and combination of known and new antigens, together with the combination of immunotherapeutic and classical approaches may overcome cancer immune evasion. New protocols for genetic modification and T cell culture may improve the overall fitness of cellular products and their resistance to hostile tumour immunomodulatory signals. Finally, the schedules of T cell administration and toxicity management have been adapted to improve the safety of this transformative therapeutic approach. SUMMARY: In order to develop effective adoptive T cell treatments for cancer, therapeutic optimization of engineered CAR and TCR T cells is crucial, by simultaneously focusing on intrinsic and extrinsic factors. This review focuses on the innovative approaches designed and tested to overcome the hurdles encountered so far in the clinical practice, with new excitement on novel laboratory insights and ongoing clinical investigations.

Next-Generation Manufacturing Protocols Enriching TSCM CAR T Cells Can Overcome Disease-Specific T Cell Defects in Cancer Patients.

Chimeric antigen receptor (CAR) T cell expansion and persistence emerged as key efficacy determinants in cancer patients. These features are typical of early-memory T cells, which can be enriched with specific manufacturing procedures, providing signal one and signal two in the proper steric conformation and in the presence of homeostatic cytokines. In this project, we exploited our expertise with paramagnetic beads and IL-7/IL-15 to develop an optimized protocol for CAR T cell production based on reagents, including a polymeric nanomatrix, which are compatible with automated manufacturing via the CliniMACS Prodigy. We found that both procedures generate similar CAR T cell products, highly enriched of stem cell memory T cells (TSCM) and equally effective in counteracting tumor growth in xenograft mouse models. Most importantly, the optimized protocol was able to expand CAR TSCM from B-cell acute lymphoblastic leukemia (B-ALL) patients, which in origin were highly enriched of late-memory and exhausted T cells. Notably, CAR T cells derived from B-ALL patients proved to be as efficient as healthy donor-derived CAR T cells in mediating profound and prolonged anti-tumor responses in xenograft mouse models. On the contrary, the protocol failed to expand fully functional CAR TSCM from patients with pancreatic ductal adenocarcinoma, suggesting that patient-specific factors may profoundly affect intrinsic T cell quality. Finally, by retrospective analysis of in vivo data, we observed that the proportion of TSCM in the final CAR T cell product positively correlated with in vivo expansion, which in turn proved to be crucial for achieving long-term remissions. Collectively, our data indicate that next-generation manufacturing protocols can overcome initial T cell defects, resulting in TSCM-enriched CAR T cell products qualitatively equivalent to the ones generated from healthy donors. However, this positive effect may be decreased in specific conditions, for which the development of further improved protocols and novel strategies might be highly beneficial.

Targeting CD33 in Chemoresistant AML Patient-Derived Xenografts by CAR-CIK Cells Modified with an Improved SB Transposon System.

The successful implementation of chimeric antigen receptor (CAR)-T cell therapy in the clinical context of B cell malignancies has paved the way for further development in the more critical setting of acute myeloid leukemia (AML). Among the potentially targetable AML antigens, CD33 is insofar one of the main validated molecules. Here, we describe the feasibility of engineering cytokine-induced killer (CIK) cells with a CD33.CAR by using the latest optimized version of the non-viral Sleeping Beauty (SB) transposon system "SB100X-pT4." This offers the advantage of improving CAR expression on CIK cells, while reducing the amount of DNA transposase as compared to the previously employed "SB11-pT" version. SB-modified CD33.CAR-CIK cells exhibited significant antileukemic activity in vitro and in vivo in patient-derived AML xenograft models, reducing AML development when administered as an "early treatment" and delaying AML progression in mice with established disease. Notably, by exploiting an already optimized xenograft chemotherapy model that mimics human induction therapy in mice, we demonstrated for the first time that CD33.CAR-CIK cells are also effective toward chemotherapy resistant/residual AML cells, further supporting its future clinical development and implementation within the current standard regimens.

Balance of Anti-CD123 Chimeric Antigen Receptor Binding Affinity and Density for the Targeting of Acute Myeloid Leukemia.

Chimeric antigen receptor (CAR)-redirected T lymphocytes are a promising immunotherapeutic approach and object of pre-clinical evaluation for the treatment of acute myeloid leukemia (AML). We developed a CAR against CD123, overexpressed on AML blasts and leukemic stem cells. However, potential recognition of low CD123-positive healthy tissues, through the on-target, off-tumor effect, limits safe clinical employment of CAR-redirected T cells. Therefore, we evaluated the effect of context-dependent variables capable of modulating CAR T cell functional profiles, such as CAR binding affinity, CAR expression, and target antigen density. Computational structural biology tools allowed for the design of rational mutations in the anti-CD123 CAR antigen binding domain that altered CAR expression and CAR binding affinity without affecting the overall CAR design. We defined both lytic and activation antigen thresholds, with early cytotoxic activity unaffected by either CAR expression or CAR affinity tuning but later effector functions impaired by low CAR expression. Moreover, the anti-CD123 CAR safety profile was confirmed by lowering CAR binding affinity, corroborating CD123 is a good therapeutic target antigen. Overall, full dissection of these variables offers suitable anti-CD123 CAR design optimization for the treatment of AML.

Acute Myeloid Leukemia Targeting by Chimeric Antigen Receptor T Cells: Bridging the Gap from Preclinical Modeling to Human Studies.

Acute myeloid leukemia (AML) still represents an unmet clinical need for adult and pediatric high-risk patients, thus demanding advanced and personalized therapies. In this regard, different targeted immunotherapeutic approaches are available, ranging from naked monoclonal antibodies (mAb) to conjugated and multifunctional mAbs (i.e., BiTEs and DARTs). Recently, researchers have focused their attention on novel techniques of genetic manipulation specifically to redirect cytotoxic T cells endowed with chimeric antigen receptors (CARs) toward selected tumor associated antigens. So far, CAR T cells targeting the CD19 antigen expressed by B-cell origin hematological cancers have gained impressive clinical results, leading to the possibility of translating the CAR platform to treat other hematological malignancies such as AML. However, one of the main concerns in the field of AML CAR immunotherapy is the identification of an ideal target cell surface antigen, being highly expressed on tumor cells but minimally present on healthy tissues, together with the design of an anti-AML CAR appropriately balancing efficacy and safety profiles. The current review focuses mainly on AML target antigens and the related immunotherapeutic approaches developed so far, deeply dissecting methods of CAR T cell safety improvements, when designing novel CARs approaching human studies.