14The success of immunotherapy treatment in oncology ushered a new modality for 15 treating a wide variety of cancers. However, lack of effect in some patients made it 16 imperative to identify other pathways that are exploited by cancer cells to 17 circumvent immune surveillance, and possibly synergize immune checkpoint 18 treatment in those cases. It has been recently recognized that adenosine levels 19 increased significantly in the tumor microenvironment and that 2 20 adenosine/adenosine receptors play a powerful role as immunosuppressant and 21 attenuating several effector T cell functions. The two main enzymes responsible for 22 generating adenosine in the microenvironment are the ectonucleotidases CD39 and 23 CD73, the former utilizes both ATP and ADP and producing AMP while the latter 24 utilizes AMP and generates adenosine. Thus, these two enzymes combined are the 25 major source for the bulk of adenosine produced in the microenvironment. They 26 were shown to be validated targets in oncology leading to several clinical trials that 27 include small molecules as well as antibodies, showing positive and encouraging 28 results in the preclinical arena. Towards the development of novel drugs to target 29 these enzymes, we have developed a platform that can be utilized to monitor the 30 activities of both enzymes in vitro (biochemical) as well as in cells (cell based) 31 assays. We have developed very sensitive and homogenous assays that enabled us 32 to monitor the activity of both enzymes and demonstrate selectivity of known 33 inhibitors as well as monoclonal antibodies. This should speed up screening for 34 novel inhibitors leading to more effective cancer therapy 35 Introduction: 36 Although immune-checkpoint blockers drugs showed tremendous success in 37 the clinic, many patients failed to respond to such treatments. Thus, identifying 38 other pathways exploited by cancer cells to circumvent immune surveillance, novel 39 pharmacological agents that will be synergistic with immune checkpoint inhibitors 3 40 are needed (1). The insightful observation that a significant increase in adenosine 41 levels was seen in the cancer microenvironment triggered interest in the role 42 played by this nucleoside in tumor onset and progression (2). Elevated adenosine 43 concentrations within neoplastic milieu, and activation of adenosine receptors were 44 shown to have powerful immunosuppressant activity and attenuation of several 45 effector T cell functions. Adenosine accumulation in solid tumors at high 46 concentrations stimulated tumor growth and angiogenesis, inhibited cytokine 47 synthesis, functions of T cells, macrophages, and natural killer cells (3).
48Overexpression of an ecto-5'-nucleotidase (CD73), the enzyme that 49 dephosphorylates extracellular adenosine monophosphate (AMP) to adenosine, 50 within the cancer microenvironment, was recognized as the leading candidate to 51 the generation of a strong immunosuppressive and pro-angiogenic adenosine halo, 52 that facilitates cancer onset and pr...