CD73 Inhibition Overcomes Immunosuppression and Triggers Autologous T-Cell Mediated Multiple Myeloma Cell Lysis in the Bone Marrow Milieu
Authors: Ray Arghya , Junttila Melissa R , DU Ting , Sutimantanapi Dena , Chen Xi , Warne Bob , Chang Jae , Blank Brian , Wu Kejia , Moore Jared , Ndubaku Chudi , Zavorotinskaya Tatiana , Nadeem Omar , Friedman Lori , Chauhan Dharminder , Anderson Kenneth C. 
Published: November 2021

Abstract
Introduction: Adenosine is an anti-inflammatory and immunosuppressive metabolite, that signals to diminish activation and proliferation of cytotoxic T-cells, impair activity of natural killer cells and CD4 + effector T-cells, and promote the expansion of immunosuppressive cell types. CD73, a cell surface ecto-5'-nucleotidase, is required to convert AMP to adenosine and is a major catalyst of adenosine generation in the tumor microenvironment. Overexpression of CD73 is observed in many tumors and correlates with unfavorable clinical outcome. Bone marrow (BM) aspirates from multiple myeloma (MM) patients have shown increased adenosine levels correspond with disease progression [Horenstein et al. Mol Med. 2016,22:694-704] In addition to the adenosine rich feature of MM, multiple cell types within the MM BM niche express the enzymes required for adenosine production from both NAD and ATP precursors, including CD38, CD203a, CD39 and CD73. Previously, we demonstrated that dysfunctional plasmacytoid dendritic cells (pDCs) predominantly found in the BM of MM patients contribute to MM cell growth, survival, and suppression of antitumor immunity [Chauhan et al, Cancer Cell 2009, 16:309-323; Ray et al, Leukemia 2015, 29:1441-1444]. We recently discovered that the interaction between pDCs and MM cells increased CD73 transcript and protein levels in both cell types, implicating a role for adenosine signaling via CD73 signaling axis in MM. Together, these MM disease features indicate that reducing the level of adenosine via inhibition of CD73 may represent a unique vulnerability and treatment strategy for MM.
Methods: To understand the functional consequence of CD73 inhibition in MM, autologous ex vivo cell assays using freshly isolated BM aspirates from MM patients were used to detect changes in immune cell function and MM cell viability upon treatment with OP-5558, a potent and selective CD73 small molecule inhibitor which is an analog of the clinical candidate, ORIC-533. The majority of BM samples utilized were from patients with relapsed or refractory MM after at least three lines of therapy including immunomodulatory drugs, proteasome inhibitors, and anti-CD38 monoclonal antibodies, as well as a patient with relapsed MM post BCMA-CAR-T therapy.
Results: In BM aspirates from MM patients with relapsed refractory MM, CD73 inhibition by OP-5558 triggered activation of MM pDCs, evidenced by increased expression of CD40/CD83/CD86 (1.2-1.5-fold, OP-5558-treated versus untreated; p &lt; 0.05; n=3). This inhibition of CD73 reversed immunosuppression in MM BM. Specifically, CD73 inhibitor OP-5558 stimulated T-cell activation, associated with increased CD69 cell surface expression on CD3 + T-cells (CD69 MFI:20% increase, treated versus control; p = 0.0031; n = 3). Moreover, CD8 + T-cells from these co-cultures enhanced cytolytic activity against patient MM cells, significantly decreasing autologous MM cell viability (mean 42% decrease in viability; treated versus control; p=0.014; n=5). Of note, OP-5558 treatment did not directly affect viability of MM cells when treated in isolation, indicating that the observed decreased viability occurs via enhanced cytotoxic T-cell activity. Importantly, we show that OP-5558 triggered significant MM cell lysis even within autologous MM bone marrow mononuclear cell (BMNC) cultures, confirming that CD73 inhibition restores MM-specific cytolytic activity of autologous patient T-cells in the MM BM microenvironment. (mean 37% decrease in viability; treated versus control; p=0.009; n=3).
Conclusions: This study therefore demonstrates that: 1. CD73-mediated adenosine activity suppresses the cytolytic activity of T-cells against tumor cells in the MM BM milieu; and 2. CD73 inhibition can overcome immune suppression and restore lysis of MM cells by autologous T-cells. A clinical trial of potent, selective, orally bioavailable CD73 inhibitor ORIC-533 will examine the utility of CD73 inhibition to improve outcome in patients with relapsed refractory MM.

Disclosures
Junttila: ORIC Pharmaceuticals: Current Employment. Sutimantanapi: ORIC Pharmaceuticals: Current Employment. Chen: ORIC Pharmaceuticals: Current Employment. Warne: ORIC Pharmaceuticals: Current Employment. Chang: ORIC Pharmaceuticals: Current Employment. Blank: ORIC Pharmaceuticals: Current Employment. Wu: ORIC Pharmaceuticals: Current Employment. Moore: ORIC Pharmaceuticals: Current Employment. Ndubaku: ORIC Pharmaceuticals: Current Employment. Zavorotinskaya: ORIC Pharmaceuticals: Current Employment. Nadeem: GSK: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees. Friedman: ORIC Pharmaceuticals: Current Employment. Chauhan: C4 Therapeutics: Current equity holder in publicly-traded company; Stemline Therapeutics, Inc: Consultancy. Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.