Abstract 4645: Identification of conditionally active antibodies that selectively block CD39 activity in the acidic tumor microenvironment
Authors: Bai Aiping , Renahan Elise , Malhotra Kanam , Thisted Thomas , Smith F. Donelson , Pierce Robert H , van der Horst Edward H 
Published: April 2023

Abstract
Background CD39 expression is upregulated in the tumor microenvironment (TME), which is characterized by high levels of extracellular ATP (eATP) and low pH. CD39 catalyzes the rate-limiting degradation step of this immunostimulatory ATP, leading to a rise in immunosuppressive adenosine (ADO). CD39 displays broad expression on endothelial cells and macrophages, which represents a significant peripheral sink for CD39 targeting antibodies. Our strategy aims to circumvent this problem through delivery of pH-selective anti-CD39 blocking antibodies that will achieve a high target occupancy in the tumor, maintaining eATP and inhibiting ADO generation to enhance anti-tumor immunity.
Methods Anti-CD39 antibodies were generated through a yeast-based screening platform and characterized for efficacy of blocking CD39 activity. Cell lines (HEK293 stably overexpressing CD39, parental HEK293, CD39+ SK-MEL28, and CD39high ARH77) or soluble CD39 protein were incubated with candidate antibodies. CD39 enzymatic activity was determined using CellTiter Glo 2.0 assay (Promega) and EnzCheck Phosphate Assay (Thermo Fisher).
Results Monovalent affinity of 83 candidate antibodies was measured; KDs ranged from 1 × 10−9 - 5 × 10−7M under both physiological (pH 7.4) and acidic (pH 6.0) conditions. All 83 antibodies bound CD39-overexpressing HEK293 cells at pH 6.0; 76 bound CD39-overexpressing HEK293 cells at pH 7.4. We identified 30 antibodies (100 nM) that block CD39 activity on HEK293-CD39 cells (threshold: &gt; 5% inhibition) under either pH 7.4 or pH 6.0 conditions. 26 antibodies inhibit CD39 activity at pH 7.4, whereas 22 antibodies block CD39 at pH 6.0. Among these 30 antibodies, ten (at 200 nM) inhibit CD39 activity on SK-MEL28 cells (moderate CD39 expression) at both pH 7.4 and pH 6.0; 5 of these preferentially inhibit CD39 at pH 6.0 (threshold: &gt; 7.5% inhibition). The 30 antibodies selected above were tested on ARH77 cells (high CD39 expression). Among them, 16 antibodies inhibited CD39 enzymatic activity beyond 10% at pH 6.0 (maximal inhibition: 43%) and &gt; 6.5% at pH 7.4 (maximal inhibition: 35%). All 16 suppressed soluble CD39 activity under both pH conditions. A set of 8 antibodies demonstrating higher efficacy of CD39 inhibition on both cell lines at pH 6.0 versus pH 7.4 were prioritized for lead optimization.
Summary and conclusions A panel of 83 antibodies were identified and characterized for inhibition of CD39 enzymatic activity at neutral and acidic pH. The 8 most promising antibodies in terms of pH-selective CD39 binding and inhibition are currently undergoing lead optimization. This pH-dependent TME targeting strategy may alleviate undesirable properties of on-target/off-tumor binding such as target-mediated drug disposition (that results in suboptimal pharmacokinetic properties) and effectively bolster the specific blocking of CD39 in the local tumor environs.
Citation Format: Aiping Bai, Elise Renahan, Kanam Malhotra, Thomas Thisted, F. Donelson Smith, Robert H Pierce, Edward H van der Horst. Identification of conditionally active antibodies that selectively block CD39 activity in the acidic tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4645.