Paul A. Beavis

Anti-PD-1 Antibody Therapy Potently Enhances the Eradication of Established Tumors By Gene-Modified T Cells

Purpose: To determine the antitumor efficacy and toxicity of a novel combination approach involving adoptive T-cell immunotherapy using chimeric antigen receptor (CAR) T cells with an immunomodulatory reagent for blocking immunosuppression. Experimental Design: We examined whether administration of a PD-1 blocking antibody could increase the therapeutic activity of CAR T cells against two different Her-2+ tumors. The use of a self-antigen mouse model enabled investigation into the efficacy, mechanism, and toxicity of this combination approach. Results: In this study, we first showed a significant increase in the level of PD-1 expressed on transduced anti-Her-2 CD8+ T cells following antigen-specific stimulation with PD-L1+ tumor cells and that markers of activation and proliferation were increased in anti-Her-2 T cells in the presence of anti-PD-1 antibody. In adoptive transfer studies in Her-2 transgenic recipient mice, we showed a significant improvement in growth inhibition of two different Her-2+ tumors treated with anti-Her-2 T cells in combination with anti-PD-1 antibody. The therapeutic effects observed correlated with increased function of anti-Her-2 T cells following PD-1 blockade. Strikingly, a significant decrease in the percentage of Gr1+ CD11b+ myeloid-derived suppressor cells (MDSC) was observed in the tumor microenvironment of mice treated with the combination therapy. Importantly, increased antitumor effects were not associated with any autoimmune pathology in normal tissue expressing Her-2 antigen. Conclusion: This study shows that specifically blocking PD-1 immunosuppression can potently enhance CAR T-cell therapy that has significant implications for potentially improving therapeutic outcomes of this approach in patients with cancer. Clin Cancer Res; 19(20); 5636–46. ©2013 AACR.

CD73 promotes anthracycline resistance and poor prognosis in triple negative breast cancer

Using gene-expression data from over 6,000 breast cancer patients, we report herein that high CD73 expression is associated with a poor prognosis in triple-negative breast cancers (TNBC). Because anthracycline-based chemotherapy regimens are standard treatment for TNBC, we investigated the relationship between CD73 and anthracycline efficacy. In TNBC patients treated with anthracycline-only preoperative chemotherapy, high CD73 gene expression was significantly associated with a lower rate of pathological complete response or the disappearance of invasive tumor at surgery. Using mouse models of breast cancer, we demonstrated that CD73 overexpression in tumor cells conferred chemoresistance to doxorubicin, a commonly used anthracycline, by suppressing adaptive antitumor immune responses via activation of A2A adenosine receptors. Targeted blockade of CD73 enhanced doxorubicin-mediated antitumor immune responses and significantly prolonged the survival of mice with established metastatic breast cancer. Taken together, our data suggest that CD73 constitutes a therapeutic target in TNBC.

CD73: a potent suppressor of antitumor immune responses

Tumors use several strategies to evade immunosurveillance. One such mechanism is the generation of adenosine within the tumor microenvironment, which potently suppresses antitumor T cell responses. Adenosine within the tumor is generated by CD73, a membrane-bound nucleotidase that is expressed by tumor cells, suppressive immune subsets such as T regulatory cells (Tregs) and myeloid-derived suppressor cells and endothelial cells. Recent evidence suggests that targeted inhibition of CD73 has the potential to reduce tumorigenesis and metastasis, as well as enhancing the potency of T-cell-directed therapies. This review outlines the impact of adenosine on suppressing the antitumor response and the evidence supporting the rationale for CD73 targeting in the treatment of cancer.

Blockade of A2A receptors potently suppresses the metastasis of CD73+ tumors

CD73 inhibits antitumor immunity through the activation of adenosine receptors expressed on multiple immune subsets. CD73 also enhances tumor metastasis, although the nature of the immune subsets and adenosine receptor subtypes involved in this process are largely unknown. In this study, we revealed that A2A/A2B receptor antagonists were effective in reducing the metastasis of tumors expressing CD73 endogenously (4T1.2 breast tumors) and when CD73 was ectopically expressed (B16F10 melanoma). A2A−/− mice were strongly protected against tumor metastasis, indicating that host A2A receptors enhanced tumor metastasis. A2A blockade enhanced natural killer (NK) cell maturation and cytotoxic function in vitro, reduced metastasis in a perforin-dependent manner, and enhanced NK cell expression of granzyme B in vivo, strongly suggesting that the antimetastatic effect of A2A blockade was due to enhanced NK cell function. Interestingly, A2B blockade had no effect on NK cell cytotoxicity, indicating that an NK cell-independent mechanism also contributed to the increased metastasis of CD73+ tumors. Our results thus revealed that CD73 promotes tumor metastasis through multiple mechanisms, including suppression of NK cell function. Furthermore, our data strongly suggest that A2A or A2B antagonists may be useful for the treatment of metastatic disease. Overall, our study has potential therapeutic implications given that A2A/A2B receptor antagonists have already entered clinical trials in other therapeutic settings.

Adenosine Receptor 2A Blockade Increases the Efficacy of Anti–PD-1 through Enhanced Antitumor T-cell Responses

Beavis, Milenkovski, and colleagues reveal that adenosine receptor blockade enhanced anti-PD-1 efficacy against CD73+ tumors in twomouse models via augmentation of tumor-infiltrating CD8+ T-cell effector function by increasing IFNγ and Granzyme B production and suggest CD73 expression as a biomarker for anti-PD-1 efficacy. Immunotherapy is rapidly emerging as a cancer treatment with high potential. Recent clinical trials with anti-CTLA-4 and anti–PD-1/PD-L1 antibodies (mAbs) suggest that targeting multiple immunosuppressive pathways may significantly improve patient survival. The generation of adenosine by CD73 also suppresses antitumor immune responses through the activation of A2A receptors on T cells and natural killer (NK) cells. We sought to determine whether blockade of A2A receptors could enhance the efficacy of anti–PD-1 mAb. The expression of CD73 by tumor cells limited the efficacy of anti–PD-1 mAb in two tumor models, and this was alleviated with concomitant treatment with an A2A adenosine receptor antagonist. The blockade of PD-1 enhanced A2A receptor expression on tumor-infiltrating CD8+ T cells, making them more susceptible to A2A-mediated suppression. Thus, dual blockade of PD-1 and A2A significantly enhanced the expression of IFNγ and Granzyme B by tumor-infiltrating CD8+ T cells and, accordingly, increased growth inhibition of CD73+ tumors and survival of mice. The results of our study indicate that CD73 expression may constitute a potential biomarker for the efficacy of anti–PD-1 mAb in patients with cancer and that the efficacy of anti–PD-1 mAb can be significantly enhanced by A2A antagonists. We have therefore revealed a potentially novel biomarker for the efficacy of anti–PD-1 that warrants further investigation in patients. Because our studies used SYN-115, a drug that has already undergone phase IIb testing in Parkinson disease, our findings have immediate translational relevance for patients with cancer. Cancer Immunol Res; 3(5); 506–17. ©2015 AACR.

RAS/MAPK Activation Is Associated with Reduced Tumor-Infiltrating Lymphocytes in Triple-Negative Breast Cancer: Therapeutic Cooperation Between MEK and PD-1/PD-L1 Immune Checkpoint Inhibitors

Purpose: Tumor-infiltrating lymphocytes (TIL) in the residual disease (RD) of triple-negative breast cancers (TNBC) after neoadjuvant chemotherapy (NAC) are associated with improved survival, but insight into tumor cell-autonomous molecular pathways affecting these features are lacking. Experimental Design: We analyzed TILs in the RD of clinically and molecularly characterized TNBCs after NAC and explored therapeutic strategies targeting combinations of MEK inhibitors with PD-1/PD-L1–targeted immunotherapy in mouse models of breast cancer. Results: Presence of TILs in the RD was significantly associated with improved prognosis. Genetic or transcriptomic alterations in Ras–MAPK signaling were significantly correlated with lower TILs. MEK inhibition upregulated cell surface MHC expression and PD-L1 in TNBC cells both in vivo and in vitro. Moreover, combined MEK and PD-L1/PD-1 inhibition enhanced antitumor immune responses in mouse models of breast cancer. Conclusions: These data suggest the possibility that Ras–MAPK pathway activation promotes immune-evasion in TNBC, and support clinical trials combining MEK- and PD-L1–targeted therapies. Furthermore, Ras/MAPK activation and MHC expression may be predictive biomarkers of response to immune checkpoint inhibitors. Clin Cancer Res; 22(6); 1499–509. ©2015 AACR.

Relevance of tumor-infiltrating lymphocytes in breast cancer.

While breast cancer has not been considered a cancer amenable to immunotherapeutic approaches, recent studies have demonstrated evidence of significant immune cell infiltration via tumor-infiltrating lymphocytes in a subset of patient tumors. In this review we present the current evidence highlighting the clinical relevance and utility of tumor-infiltrating lymphocytes in breast cancer. Retrospective and prospective studies have shown that the presence of tumor-infiltrating lymphocytes is a prognostic marker for higher responses to neoadjuvant chemotherapy and better survival, particularly in triple negative and HER2-positive early breast cancer. Further work is required to determine the immune subsets important in this response and to discover ways of encouraging immune infiltrate in tumor-infiltrating lymphocytes-negative patients.

Immunosuppressive activities of adenosine in cancer

Multiple immunosuppressive mechanisms impede anti-tumor immunity. Among them, the accumulation of extracellular adenosine is a potent and widespread strategy exploited by tumors to escape immunosurveillance through the activation of purinergic receptors. In the immune system, engagement of A2a and A2b adenosine receptors is a critical regulatory mechanism that protects tissues against excessive immune reactions. In tumors, this pathway is hijacked and hinders anti-tumor immunity, promoting cancer progression. Different groups have highlighted the therapeutic potential of blocking CD73-dependent adenosine-mediated immunosuppression to reinstate anti-tumor immunity. Phase clinical trials evaluating anti-CD73 antibodies and A2a receptor antagonists in cancer patients are currently ongoing. We here review the recent literature on the immunosuppressive effects of extracellular adenosine and discuss the development of adenosine inhibitors.

CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity

Cancer cells exploit the expression of the programmed death-1 (PD-1) ligand 1 (PD-L1) to subvert T-cell-mediated immunosurveillance. The success of therapies that disrupt PD-L1-mediated tumour tolerance has highlighted the need to understand the molecular regulation of PD-L1 expression. Here we identify the uncharacterized protein CMTM6 as a critical regulator of PD-L1 in a broad range of cancer cells, by using a genome-wide CRISPR–Cas9 screen. CMTM6 is a ubiquitously expressed protein that binds PD-L1 and maintains its cell surface expression. CMTM6 is not required for PD-L1 maturation but co-localizes with PD-L1 at the plasma membrane and in recycling endosomes, where it prevents PD-L1 from being targeted for lysosome-mediated degradation. Using a quantitative approach to profile the entire plasma membrane proteome, we find that CMTM6 displays specificity for PD-L1. Notably, CMTM6 depletion decreases PD-L1 without compromising cell surface expression of MHC class I. CMTM6 depletion, via the reduction of PD-L1, significantly alleviates the suppression of tumour-specific T cell activity in vitro and in vivo. These findings provide insights into the biology of PD-L1 regulation, identify a previously unrecognized master regulator of this critical immune checkpoint and highlight a potential therapeutic target to overcome immune evasion by tumour cells.

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.