Adenosine receptor 2A blockade increases the efficacy of anti-PD-1 through enhanced anti-tumor T cell responses

Immunotherapy is rapidly emerging as a cancer treatment with high potential. Recent clinical trials with anti-CTLA-4 and anti-PD-1/PDL-1 antibodies (mAbs) suggest that targeting multiple immunosuppressive pathways may significantly improve patient survival. The generation of adenosine by CD73 also suppresses anti-tumor immune responses through the activation of A 2A receptors on T cells and NK cells. We sought to determine whether blockade of A 2A 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 A 2A adenosine receptor antagonist. The blockade of PD-1 enhanced A 2A receptor expression on tumor-infiltrating CD8 + T cells, making them more susceptible to A 2A mediated suppression. Thus, dual blockade of PD-1 and A 2A 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. Our study indicates that CD73 expression may constitute a potential biomarker for the efficacy of anti-PD-1 mAb in cancer patients and that the efficacy of anti-PD-1 mAb can be significantly enhanced by A 2A antagonists. We have therefore revealed a potentially novel biomarker for the efficacy of anti-PD-1 that warrants further investigation in patients. Our studies utilized SYN-115, a drug that has already undergone phase IIb testing in Parkinson’s Disease and so our findings therefore have immediate translational relevance for cancer patients.


Abstract
Immunotherapy is rapidly emerging as a cancer treatment with high potential. Recent can be significantly enhanced by A 2A antagonists. We have therefore revealed a potentially novel biomarker for the efficacy of anti-PD-1 that warrants further investigation in patients. Our studies utilized SYN-115, a drug that has already undergone phase IIb testing in Parkinson's Disease and so our findings therefore have immediate translational relevance for cancer patients.

Introduction
The importance of the immune system in the control of cancers is increasingly being recognised. Tumors utilize multiple inhibitory pathways in order to suppress the immune response and thus lead to 'Immune Escape' (1). The clinical success of therapies that block these inhibitory pathways such as anti-PD-1 (2) and anti-CTLA-4 (3) mAbs underlines the potential of immunotherapy to treat cancer in a more specific fashion with fewer adverse events than with conventional cancer therapies.
One mechanism of tumor-induced immune suppression is the generation of adenosine by the ectoenzyme CD73 (4). In terms of the generation of adenosine, CD73 sits downstream of the ectoenzyme CD39 which catalyzes the degradation of ATP into AMP via the intermediate ADP. AMP then acts as a substrate for CD73, resulting in the production of immunosuppressive adenosine (4). A major enzyme for the degradation of adenosine is adenosine deaminase (ADA). ADA can be expressed on the cell surface when bound to CD26, however CD26 expression is reduced on tumor cells (5). Thus, the tumor microenvironment contains concentrations of adenosine sufficient to inhibit anti-tumor T cell responses (6).
CD73 is endogenously expressed on epithelial cells, endothelial cells and some immune subsets and its expression has also been observed in several cancer types including colon cancer, melanoma, breast cancer, ovarian cancer and leukaemia (4).
Notably, the expression of CD73 by tumor cells in triple negative breast cancer patients correlates with an increased risk of metastasis and consequently poor patient outcomes (7,8). The drivers of CD73 expression within the tumor microenvironment include hypoxia, type I IFNs, TNFα, IL-1β , prostaglandin (PG)E 2 , and TGFβ (4).
Moreover it has been shown that certain chemotherapies including anthracyclines can drive CD73 expression, contributing to chemoresistance of tumor cells (8). The protumoral effect of CD73 has been validated in preclinical models showing that targeting CD73 with a monoclonal antibody can reduce primary tumor growth and metastasis (9). Moreover, anti-CD73 has been shown to enhance the efficacy of chemotherapy and other immunotherapies (8,10).
Although targeting CD73 directly has shown great therapeutic promise, there is as yet no clinical development of anti-CD73 mAbs. However, since the immunosuppressive effects of CD73 are mediated by adenosine, targeting the downstream adenosine receptors has high translational potential. Activation of A 2A and A 2B receptors is known to suppress multiple immune cells including the proliferation, cytokine production and cytotoxicity of T cells (4,11). Notably in the context of cancer, activation of either A 2A or A 2B receptors has been shown to reduce anti-tumor immunity (6,12,13) and enhance the metastasis of CD73 + tumors (14). Moreover, antagonists targeting the A 2A and A 2B receptors have undergone clinical trials for other indications and have shown a good safety profile (15,16).
In the current study we investigated whether blockade of A 2A receptors could enhance the efficacy of anti-PD-1. Since PD-1 blockade is known to enhance T-lymphocyte function, and that A 2A expression is increased on T-lymphocytes following activation (17), we hypothesized that A 2A blockade would enhance the efficacy of anti-PD-1 by augmenting anti-tumor T-lymphocyte responses.

Cell lines and mice
The C57BL/6 mouse breast carcinoma cell line AT-3, was obtained from Dr. Trina Stewart (Griffith University, Queensland) (18), MC38 cells were obtained from Dr.
Nicole Haynes (Peter Mac, Victoria) and 4T1.2 cells were obtained from Prof. Robin Anderson (Peter Mac, Victoria). Tumor cell lines were transduced to express chicken ovalbumin peptide as previously described (19,20). These cell lines were originally obtained from the ATCC, actively passaged for less than 6 months and were authenticated using short tandem repeat profiling. United Kingdom Co-ordinating

Vectors and tumor cell transduction
Murine CD73 cDNA (Open Biosystems) was cloned into the pMSCV-Cherry retroviral vector. Retroviruses were made by transfection of either pMSCV-Cherry or pMSCV-Cherry-CD73 in combination with the pMD2.G envelope vector into HEK293gagpol cells using lipofectamine 2000 (Invitrogen) as per manufacturer's instructions. AT-3ova dim and MC38ova dim were transduced with pMSCV Cherry-CD73 as previously described (14).

Analysis of adenosine receptor expression by RT-PCR
RNA was isolated from T lymphocytes using the Qiagen RNAEasy Mini Kit as per manufacturer's instructions. To generate cDNA, mRNA was added to 1 μl of oligo dT and incubated at 65ºC for 5 minutes. RNA was then added to a mixture of 4 μl 5X cDNA synthesis buffer (Promega), 2 μl 10 mM dNTP mix (Promega), 1  (Promega) and 1 μl RNAase free H 2 O. cDNA was generated at 50ºC for 50 minutes and 85ºC for 5 minutes before storage at -20ºC until analysis of mRNA expression by qRT-PCR. Adenosine receptor expression was determined using the Taqman Gene Expression Master Mix and primers for A 2A and A 2B as previously described (9).

Analysis of tumor-infiltrating immune subsets
After euthanizing mice, tumors were excised and digested using a mix of 1 mg/ml collagenase type IV (Sigma-Aldrich) and 0.02 mg/ml DNAase (Sigma-Aldrich). After 30 minutes digestion at 37ºC, cells were twice passed through a 70 μm filter. Single cell suspensions were then prepared and analyzed by flow cytometry as previously described (14). IFNγ and Granzyme B expression was determined directly ex vivo using antibody clones XMG1.2 and GB11 respectively.

Determination of cAMP content of human PBMCs
PBMCs from a normal donor were isolated using Ficoll and cultured for 10

Combination treatment with anti-PD-1 and A 2A blockade enhances the IFNγ production of CD8 + T cells in vitro
To explore the link between the A 2A and PD-1 suppressive pathways, we first  Figure 1C & 1D). Taken together, this data indicates that combined blockade of A 2A and PD-1 has the capacity to enhance IFNγ production by CD8 + T lymphocytes in the presence of the adenosine analogue NECA and led us to evaluate this combination therapy in vivo.

A 2A blockade enhances the efficacy of anti-PD-1 mAb against CD73 + tumors
Since we hypothesized that adenosine may limit the efficacy of PD-1 blockade, we Tumors were injected subcutaneously into mice and treated at day 14 once they were established (20-50 mm 2 ). Treatment of AT-3ova dim tumors with anti-PD-1 mAb resulted in a profound and significant reduction in primary tumor growth (Figure 2A) with a mean reduction in tumor size at day 14 post therapy of 81% ( Figure 2B).
However, the efficacy of anti-PD-1 mAb against AT-3ova dim CD73 + tumors was significantly less than against AT-3ova dim control tumors, with a mean reduction of tumor size of only 31% ( Figure 2B). Thus, this data shows that the expression of CD73 reduced the efficacy of anti-PD-1 mAb single agent therapy.
We hypothesized that the increased resistance of AT-3ova dim CD73 + tumors to anti-PD-1 monotherapy was due to A 2A -mediated suppression of anti-tumor immune responses induced by anti-PD-1 mAb. To investigate this, we determined whether treatment of mice with the A 2A antagonist SCH58261 could enhance the therapeutic efficacy of anti-PD-1. Although A 2A blockade alone had no effect on the growth of AT-3ova dim CD73 + tumors as a single therapy ( Figure 3B). We next investigated the ability of anti-PD-1 and SCH58261 to treat established 4T1.2 lung metastases. 4T1.2 tumor cells were injected intravenously and after 3 days, mice were treated with a combination of anti-PD-1 or 2A3 isotype control mAb and SCH58261 or vehicle control. Neither anti-PD1 nor SCH58261 were effective in reducing the number of metastases when given as a single agent ( Figure   3C). When anti-PD-1 mAb and SCH58261 were given in combination there was a trend for a reduction in metastases although this did not reach statistical significance.
It has previously been reported that CD8 + T cells coexpress PD-1 and TIM-3 in the 4T1 breast cancer model and that anti-TIM-3 can significantly enhance the efficacy of PD-1 blockade (21). We therefore hypothesized that the inclusion of anti-TIM-3 in

PD-1 blockade enhances A 2A expression on CD8 + tumor infiltrating lymphocytes
Since A 2A expression is known to be increased following TCR stimulation and PD-1 ligation is known to dampen TCR signalling (17,22), we hypothesized that PD-1 blockade may increase A 2A expression on T lymphocytes and thus make them more blockade also occurred in vivo, CD8 + and CD4 + T cells were isolated from AT-3ova dim CD73 + tumors following treatment of mice with either anti-PD-1 or isotype control mAb. Notably, CD8 + T cells isolated from tumors of mice treated with anti-PD-1 had significantly elevated expression of both IFNγ and the A 2A receptor compared to those isolated from isotype control treated mice ( Figure 4C). Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Combination of PD-1 and A 2A blockade significantly enhances the IFNγ production of tumor-infiltrating CD8 + T lymphocytes
To characterize the mechanism by which A 2A blockade enhanced the efficacy of PD-1 mAb in vivo, the number and phenotype of tumor-infiltrating lymphocytes was investigated in mice bearing AT-3ova dim CD73 + tumors treated with either an isotype control antibody (2A3) or anti-PD-1 mAb and either SCH58261 or vehicle control.
TILs were analyzed at days 1, 2 and 7 post treatment. Although the proportion of TCRβ + CD8 + , TCRβ + CD4 + foxp3and TCRβ + CD4 + foxp3 + TILs were not modulated by either anti-PD-1 alone or in combination with A 2A blockade (Supplementary Figure   6A-C), we observed that the combination therapy significantly enhanced CD8 + T cell effector functions. Analysis of TILs at day 1 showed that treatment with anti-PD-1 significantly increased the expression of IFNγ and Granzyme B by CD8 + TILs which was not further enhanced following combination treatment with SCH58261 ( Figure   5A). However 2 days following treatment, a significantly higher proportion of the CD8 + T cells isolated from mice treated with the combination therapy were IFNγ + and Granzyme B + compared to those isolated from mice treated with anti-PD-1 mAb alone ( Figure 5A-B). The trend for increased IFNγ production by CD8 + TILs in the combination group extended to day 7 post therapy with only the combination therapy giving a significantly enhanced proportion of IFNγ + CD8 + TILs compared to isotype control/vehicle treated mice (Supplementary Figure 7). Thus, although the initial response to anti-PD-1 mAb was not affected by A 2A blockade, the induced CD8 + T We next investigated whether the antigen-specific CD8 + T cell population was modulated by anti-PD-1 mAb/SCH58261 treatment. Although PD-1 blockade enhanced the proportion of CD8 + TILs that were ova tetramer specific, the addition of the A 2A antagonist did not further enhance this effect (Supplementary Figure 8D).
Notably however, a significantly higher proportion of these ova tetramer + TILs indicating that the increased IFNγ production induced by the combination of SCH58261 and anti-PD-1 mAb was critical for its improved efficacy (Figure 6).

Enhancement of anti-PD-1 function with the clinical trial drug SYN115
In order to enhance the translational relevance of our study, we also assessed the potential of SYN115, an A 2A antagonist that has undergone phase IIb clinical trials (16), to enhance anti-PD-1 function. Established AT-3ova dim CD73 + tumors were treated with an isotype control or anti-PD-1 mAb in combination with either SCH58261, SYN115 or vehicle. We observed that SYN115 had no single agent activity, but significantly enhanced the anti-tumor efficacy of anti-PD-1 mAb ( Figure   7A) to a similar extent as SCH58261 ( Figure 7B). SYN115 was also able to significantly inhibit the increased cAMP concentrations mediated by the specific A 2A agonist CGS21680 on activated human PBMCs ( Figure 7C) adenosine-mediated immunosuppression. The mechanism by which PD-1 blockade enhances A 2A expression is not known. However A 2A expression is known to be increased in T lymphocytes following TCR activation (17) and PD-1 has been shown to suppress Ca 2+ and Extracellular signal-related kinase (ERK) responses immediately downstream of the TCR (22). Therefore it seems likely that the mechanism by which PD-1 blockade increases A 2A expression is a direct consequence of enhanced TCR signalling, at least in part. Indeed this hypothesis is supported by the fact that upregulation of A 2A on anti-tumor CD8 + T cells was seen both in vitro and in vivo following PD-1 blockade. Furthermore, since our data indicates that A 2A is more highly expressed on PD-1 + cells, the blockade of PD-1 may lead to the enrichment of the PD-1 + subset and consequently increase the proportion of CD8 + T cells susceptible to A 2A -mediated suppression. In the current study we did not observe that A 2A blockade significantly modulated PD-1 expression on CD8 + or CD4 + T cells.
Although this appears to contrast with the observations of Cekic and Linden (37), this may be because in our study A 2A blockade was conducted in established tumors where memory T cells have already differentiated and/or because the pharmacological antagonist SCH58261 does not mediate full A 2A blockade. Therefore the timing and magnitude of A 2A blockade may be crucial to the therapeutic outcome.
It has been proposed that the additional benefit of combined CTLA-4 and PD-1 blockade is attributable to the fact that PD-1 and CTLA-4 inhibit T cell activation via distinct signalling pathways (38,39). This is also the case for A 2A blockade. Whilst blockade. Although the increase in IFNγ production was most marked in the antigenspecific CD8 + PD-1 hi population, we also observed an increased production of IFNγ in the CD8 + PD-1 low subset following combination therapy. The reason for this is not known but it is possible that the PD-1 low subset is indirectly affected due to proinflammatory cytokines secreted by the PD-1 hi cells, leading to increased activation of the PD-1 low cells themselves, as well as activation of other immune subsets including APCs. We observed no increased expression of IFNγ by CD4 + T cells following PD-1 blockade despite expression of PD-1 by these TILs. This may be explained by the fact that CD4 + T cells are less sensitive to PD-1 mediated inhibition than CD8 + T cells (46). However, it should be noted that one caveat with these experiments is that we did not distinguish between foxp3and foxp3 + cells which may on August 10, 2017. © 2015 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
have skewed our interpretation of these results. Nevertheless, the increased efficacy of PD-1 and A 2A blockade, compared to anti-PD-1 mAb alone, seems largely attributable to an increased IFNγ response from CD8 + PD-1 + tumor-antigen specific TILs. Indeed, the combination therapy of PD-1/A 2A blockade was ineffective in IFNγ -/mice. These In the current study we provide evidence that CD73 expression is a negative prognostic marker with regards to the therapeutic efficacy of PD-1 blockade and that A 2A antagonists may be a viable option to improve the clinical efficacy of anti-PD-1 mAb therapy. Interestingly, it has recently been reported that A 2A blockade can also enhance the function of CTLA-4 mAb in a murine melanoma model (50). This