CRISPR/Cas9 mediated deletion of the adenosine A2A receptor enhances CAR T cell efficacy.

Adenosine is an immunosuppressive factor that limits anti-tumor immunity through the suppression of multiple immune subsets including T cells via activation of the adenosine A2A receptor (A2AR). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A2AR with a clinically relevant CRISPR/Cas9 strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Effects evoked by CRISPR/Cas9 mediated gene deletion of A2AR are superior to shRNA mediated knockdown or pharmacological blockade of A2AR. Mechanistically, human A2AR-edited CAR T cells are significantly resistant to adenosine-mediated transcriptional changes, resulting in enhanced production of cytokines including IFNγ and TNF, and increased expression of JAK-STAT signaling pathway associated genes. A2AR deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A2AR for the improvement of CAR T cell function in the clinic.

IL-15 Preconditioning Augments CAR T Cell Responses to Checkpoint Blockade for Improved Treatment of Solid Tumors.

Chimeric antigen receptor (CAR) T cell therapy has been highly successful in hematological malignancies leading to their US Food and Drug Administration (FDA) approval. However, the efficacy of CAR T cells in solid tumors is limited by tumor-induced immunosuppression, leading to the development of combination approaches, such as adjuvant programmed cell death 1 (PD-1) blockade. Current FDA-approved methods for generating CAR T cells utilize either anti-CD3 and interleukin (IL)-2 or anti-CD3/CD28 beads, which can generate a T cell product with an effector/exhausted phenotype. Whereas different cytokine preconditioning milieu, such as IL-7/IL-15, have been shown to promote T cell engraftment, the impact of this approach on CAR T cell responses to adjuvant immune-checkpoint blockade has not been assessed. In the current study, we reveal that the preconditioning of CAR T cells with IL-7/IL-15 increased CAR T cell responses to anti-PD-1 adjuvant therapy. This was associated with the emergence of an intratumoral CD8+CD62L+TCF7+IRF4- population that was highly responsive to anti-PD-1 therapy and mediated the vast majority of transcriptional and epigenetic changes in vivo following PD-1 blockade. Our data indicate that preservation of CAR T cells in a TCF7+ phenotype is crucial for their responsiveness to adjuvant immunotherapy approaches and should be a key consideration when designing clinical protocols.

Adoptive cellular therapy with T cells expressing the dendritic cell growth factor Flt3L drives epitope spreading and antitumor immunity.

Adoptive cell therapies using genetically engineered T cell receptor or chimeric antigen receptor T cells are emerging forms of immunotherapy that redirect T cells to specifically target cancer. However, tumor antigen heterogeneity remains a key challenge limiting their efficacy against solid cancers. Here, we engineered T cells to secrete the dendritic cell (DC) growth factor Fms-like tyrosine kinase 3 ligand (Flt3L). Flt3L-secreting T cells expanded intratumoral conventional type 1 DCs and substantially increased host DC and T cell activation when combined with immune agonists poly (I:C) and anti-4-1BB. Importantly, combination therapy led to enhanced inhibition of tumor growth and the induction of epitope spreading towards antigens beyond those recognized by adoptively transferred T cells in solid tumor models of T cell receptor and chimeric antigen receptor T cell therapy. Our data suggest that augmenting endogenous DCs is a promising strategy to overcome the clinical problem of antigen-negative tumor escape following adoptive cell therapy.

CAR T Cells for Acute Myeloid Leukemia: State of the Art and Future Directions.

Relapse after conventional chemotherapy remains a major problem in patients with myeloid malignancies such as acute myeloid leukemia (AML), and the major cause of death after diagnosis of AML is from relapsed disease. The only potentially curative treatment option currently available is allogeneic hematopoietic stem cell transplantation (allo-HSCT), which through its graft-vs.-leukemia effects has the ability to eliminate residual leukemia cells. Despite its long history of success however, relapse following allo-HSCT is still a major challenge and is associated with poor prognosis. In the field of adoptive therapy, CD19-targeted chimeric antigen receptor (CAR) T cells have yielded remarkable clinical success in certain types of B-cell malignancies, and substantial efforts aimed at translating this success to myeloid malignancies are currently underway. While complete ablation of CD19-expressing B cells, both cancerous and healthy, is clinically tolerated, the primary challenge limiting the use of CAR T cells in myeloid malignancies is the absence of a dispensable antigen, as myeloid antigens are often co-expressed on normal hematopoietic stem/progenitor cells (HSPCs), depletion of which would lead to intolerable myeloablation. This review provides a discussion on the current state of CAR T cell therapy in myeloid malignancies, limitations for clinical translation, as well as the most recent approaches to overcome these barriers, through various genetic modification and combinatorial strategies in an attempt to make CAR T cell therapy a safe and viable option for patients with myeloid malignancies.

Macrophage-Derived CXCL9 and CXCL10 Are Required for Antitumor Immune Responses Following Immune Checkpoint Blockade.

PURPOSE: Response rates to immune checkpoint blockade (ICB; anti-PD-1/anti-CTLA-4) correlate with the extent of tumor immune infiltrate, but the mechanisms underlying the recruitment of T cells following therapy are poorly characterized. A greater understanding of these processes may see the development of therapeutic interventions that enhance T-cell recruitment and, consequently, improved patient outcomes. We therefore investigated the chemokines essential for immune cell recruitment and subsequent therapeutic efficacy of these immunotherapies. EXPERIMENTAL DESIGN: The chemokines upregulated by dual PD-1/CTLA-4 blockade were assessed using NanoString-based analysis with results confirmed at the protein level by flow cytometry and cytometric bead array. Blocking/neutralizing antibodies confirmed the requirement for key chemokines/cytokines and immune effector cells. Results were confirmed in patients treated with immune checkpoint inhibitors using single-cell RNA-sequencing (RNA-seq) and paired survival analyses. RESULTS: The CXCR3 ligands, CXCL9 and CXCL10, were significantly upregulated following dual PD-1/CTLA-4 blockade and both CD8+ T-cell infiltration and therapeutic efficacy were CXCR3 dependent. In both murine models and patients undergoing immunotherapy, macrophages were the predominant source of CXCL9 and their depletion abrogated CD8+ T-cell infiltration and the therapeutic efficacy of dual ICB. Single-cell RNA-seq analysis of patient tumor-infiltrating lymphocytes (TIL) revealed that CXCL9/10/11 was predominantly expressed by macrophages following ICB and we identified a distinct macrophage signature that was associated with positive responses to ICB. CONCLUSIONS: These data underline the fundamental importance of macrophage-derived CXCR3 ligands for the therapeutic efficacy of ICB and highlight the potential of manipulating this axis to enhance patient responses.

Tissue-specific tumor microenvironments influence responses to immunotherapies.

OBJECTIVES: Investigation of variable response rates to cancer immunotherapies has exposed the immunosuppressive tumor microenvironment (TME) as a limiting factor of therapeutic efficacy. A determinant of TME composition is the tumor location, and clinical data have revealed associations between certain metastatic sites and reduced responses. Preclinical models to study tissue-specific TMEs have eliminated genetic heterogeneity, but have investigated models with limited clinical relevance. METHODS: We investigated the TMEs of tumors at clinically relevant sites of metastasis (liver and lungs) and their impact on αPD-1/αCTLA4 and trimAb (αDR5, α4-1BB, αCD40) therapy responses in the 67NR mouse breast cancer and Renca mouse kidney cancer models. RESULTS: Tumors grown in the lungs were resistant to both therapies whereas the same tumor lines growing in the mammary fat pad (MFP), liver or subcutaneously could be completely eradicated, despite greater tumor burden. Assessment of tumor cells and drug delivery in 67NR lung or MFP tumors revealed no differences and prompted investigation into the immune TME. Lung tumors had a more immunosuppressive TME with increased myeloid-derived suppressor cell infiltration, decreased T cell infiltration and activation, and decreased NK cell activation. Depletion of various immune cell subsets indicated an equivalent role for NK cells and CD8+ T cells in lung tumour control. Thus, targeting T cells with αPD-1/αCTLA4 or trimAb was not sufficient to elicit a robust antitumor response in lung tumors. CONCLUSION: Taken together, these data demonstrate that tissue-specific TMEs influence immunotherapy responses and highlight the importance in defining tissue-specific response patterns in patients.

Supercharging adoptive T cell therapy to overcome solid tumor-induced immunosuppression.

The development of new cancer immunotherapies including checkpoint blockade and chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer treatment. CAR T cells have shown tremendous success in certain B cell malignancies, resulting in U.S. Food and Drug Administration (FDA) approval of this approach for certain types of leukemia and lymphoma. However, response rates against solid cancer have been less successful to date. Approaches to modulate the immunosuppressive tumor microenvironment including targeting checkpoint pathways, modulating metabolic pathways, and generating cytokine-producing T cells have led to considerable enhancement of adoptive T cell immunotherapy, first in preclinical models and now in patients. This review provides a discussion of the most recent strategies to enhance the efficacy of CAR T cell antitumor responses in solid cancers.

Switching on the green light for chimeric antigen receptor T-cell therapy.

Adoptive cellular therapy involving genetic modification of T cells with chimeric antigen receptor (CAR) transgene offers a promising strategy to broaden the efficacy of this approach for the effective treatment of cancer. Although remarkable antitumor responses have been observed following CAR T-cell therapy in a subset of B-cell malignancies, this has yet to be extended in the context of solid cancers. A number of promising strategies involving reprogramming the tumor microenvironment, increasing the specificity and safety of gene-modified T cells and harnessing the endogenous immune response have been tested in preclinical models that may have a significant impact in patients with solid cancers. This review will discuss these exciting new developments and the challenges that must be overcome to deliver a more sustained and potent therapeutic response.

Dual PD-1 and CTLA-4 Checkpoint Blockade Promotes Antitumor Immune Responses through CD4+Foxp3- Cell-Mediated Modulation of CD103+ Dendritic Cells.

Immunotherapy is widely accepted as a powerful new treatment modality for the treatment of cancer. The most successful form of immunotherapy to date has been the blockade of the immune checkpoints PD-1 and CTLA-4. Combining inhibitors of both PD-1 and CTLA-4 increases the proportion of patients who respond to immunotherapy. However, most patients still do not respond to checkpoint inhibitors, and prognostic biomarkers are currently lacking. Therefore, a better understanding of the mechanism by which these checkpoint inhibitors enhance antitumor immune responses is required to more accurately predict which patients are likely to respond and further enhance this treatment modality. Our current study of two mouse tumor models revealed that CD4+Foxp3- cells activated by dual PD-1/CTLA-4 blockade modulated the myeloid compartment, including activation of conventional CD103+ dendritic cells (DC) and expansion of a myeloid subset that produces TNFα and iNOS (TIP-DCs). CD4+Foxp3- T cell-mediated activation of CD103+ DCs resulted in enhanced IL12 production by these cells and IL12 enhanced the therapeutic effect of dual PD-1/CTLA-4 blockade. Given the importance of these myeloid subsets in the antitumor immune response, our data point to a previously underappreciated role of CD4+Foxp3- cells in modulating this arm of the antitumor immune response. Cancer Immunol Res; 6(9); 1069-81. ©2018 AACR.

Targeting the adenosine 2A receptor enhances chimeric antigen receptor T cell efficacy.

Chimeric antigen receptor (CAR) T cells have been highly successful in treating hematological malignancies, including acute and chronic lymphoblastic leukemia. However, treatment of solid tumors using CAR T cells has been largely unsuccessful to date, partly because of tumor-induced immunosuppressive mechanisms, including adenosine production. Previous studies have shown that adenosine generated by tumor cells potently inhibits endogenous antitumor T cell responses through activation of adenosine 2A receptors (A2ARs). Herein, we have observed that CAR activation resulted in increased A2AR expression and suppression of both murine and human CAR T cells. This was reversible using either A2AR antagonists or genetic targeting of A2AR using shRNA. In 2 syngeneic HER2+ self-antigen tumor models, we found that either genetic or pharmacological targeting of the A2AR profoundly increased CAR T cell efficacy, particularly when combined with PD-1 blockade. Mechanistically, this was associated with increased cytokine production of CD8+ CAR T cells and increased activation of both CD8+ and CD4+ CAR T cells. Given the known clinical relevance of the CD73/adenosine pathway in several solid tumor types, and the initiation of phase I trials for A2AR antagonists in oncology, this approach has high translational potential to enhance CAR T cell efficacy in several cancer types.

A Multifunctional Role for Adjuvant Anti-4-1BB Therapy in Augmenting Antitumor Response by Chimeric Antigen Receptor T Cells.

Adoptive immunotherapy utilizing chimeric antigen receptor (CAR) T cells has demonstrated high success rates in hematologic cancers, but results against solid malignancies have been limited to date, due in part to the immunosuppressive tumor microenvironment. Activation of the 4-1BB (CD137) pathway using an agonistic α-4-1BB antibody is known to provide strong costimulatory signals for augmenting and diversifying T-cell responses. We therefore hypothesized that a combination of α-4-1BB and CAR T-cell therapy would result in improved antitumor responses. Using a human-Her2 self-antigen mouse model, we report here that α-4-1BB significantly enhanced CAR T-cell efficacy directed against the Her2 antigen in two different established solid tumor settings. Treatment also increased the expression of IFNγ and the proliferation marker Ki67 in tumor-infiltrating CAR T cells when combined with α-4-1BB. Strikingly, α-4-1BB significantly reduced host immunosuppressive cells at the tumor site, including regulatory T cells and myeloid-derived suppressor cells, correlating with an increased therapeutic response. We conclude that α-4-1BB has a multifunctional role for enhancing CAR T-cell responses and that this combination therapy has high translational potential, given current phase I/II clinical trials with α-4-1BB against various types of cancer. Cancer Res; 77(6); 1296-309. ©2017 AACR.

Dual-specific Chimeric Antigen Receptor T Cells and an Indirect Vaccine Eradicate a Variety of Large Solid Tumors in an Immunocompetent, Self-antigen Setting.

Purpose: While adoptive transfer of T cells bearing a chimeric antigen receptor (CAR) can eliminate substantial burdens of some leukemias, the ultimate challenge remains the eradication of large solid tumors for most cancers. We aimed to develop an immunotherapy approach effective against large tumors in an immunocompetent, self-antigen preclinical mouse model.Experimental Design: In this study, we generated dual-specific T cells expressing both a CAR specific for Her2 and a TCR specific for the melanocyte protein (gp100). We used a regimen of adoptive cell transfer incorporating vaccination (ACTIV), with recombinant vaccinia virus expressing gp100, to treat a range of tumors including orthotopic breast tumors and large liver tumors.Results: ACTIV therapy induced durable complete remission of a variety of Her2+ tumors, some in excess of 150 mm2, in immunocompetent mice expressing Her2 in normal tissues, including the breast and brain. Vaccinia virus induced extensive proliferation of T cells, leading to massive infiltration of T cells into tumors. Durable tumor responses required the chemokine receptor CXCR3 and exogenous IL2, but were independent of IFNγ. Mice were resistant to tumor rechallenge, indicating immune memory involving epitope spreading. Evidence of limited neurologic toxicity was observed, associated with infiltration of cerebellum by T cells, but was only transient.Conclusions: This study supports a view that it is possible to design a highly effective combination immunotherapy for solid cancers, with acceptable transient toxicity, even when the target antigen is also expressed in vital tissues. Clin Cancer Res; 23(10); 2478-90. ©2016 AACR.