We explored the mechanism of action of CD39 antibodies that inhibit ectoenzyme CD39 conversion of extracellular ATP (eATP) to AMP and thus potentially augment eATP-P2-mediated proinfl ammatory responses. Using syngeneic and humanized tumor models, we contrast the potency and mechanism of anti-CD39 mAbs with other agents targeting the adenosinergic pathway. We demonstrate the critical importance of an eATP-P2X7-ASC-NALP3infl ammasome-IL18 pathway in the antitumor activity mediated by CD39 enzyme blockade, rather than simply reducing adenosine as mechanism of action. Effi cacy of anti-CD39 activity was underpinned by CD39 and P2X7 coexpression on intratumor myeloid subsets, an early signature of macrophage depletion, and active IL18 release that facilitated the signifi cant expansion of intratumor effector T cells. More importantly, anti-CD39 facilitated infi ltration into T cell-poor tumors and rescued anti-PD-1 resistance. Anti-human CD39 enhanced human T-cell proliferation and Th1 cytokine production and suppressed human B-cell lymphoma in the context of autologous Epstein-Barr virus-specifi c T-cell transfer. SIGNIFICANCE :Overall, these data describe a potent and novel mechanism of action of antibodies that block mouse or human CD39, triggering an eATP-P2X7-infl ammasome-IL18 axis that reduces intratumor macrophage number, enhances intratumor T-cell effector function, overcomes anti-PD-1 resistance, and potentially enhances the effi cacy of adoptive T-cell transfer.

Summary Background Histologically non-responsive celiac disease (NRCD) is a potentially serious condition diagnosed during the follow-up of celiac disease (CD) when patients have persistent villous atrophy despite following a gluten-free diet (GFD). Aim Since current assessments of recovery are limited to invasive and costly serial duodenal biopsies, we sought to identify antibody biomarkers for CD patients that do not respond to traditional therapy. Methods Bacterial display peptide libraries were screened by flow cytometry to identify epitopes specifically recognized by antibodies from patients with NRCD but not by antibodies from responsive CD patients. Deamidated gliadin was confirmed to be the antigen mimicked by library peptides using ELISA with sera from NRCD (n = 15) and responsive CD (n = 45) patients on a strict GFD for at least one year. Results The dominant consensus epitope sequence identified by unbiased library screening QPxx(A/P)FP(E/D) was highly similar to reported deamidated gliadin peptide (dGP) B-cell epitopes. Measurement of anti-dGP IgG titer by ELISA discriminated between NRCD and responsive CD patients with 87% sensitivity and 89% specificity. Importantly, dGP antibody titer correlated with the severity of mucosal damage indicating that IgG dGP titers may be useful to monitor small intestinal mucosal recovery on a GFD. Conclusions The finding of increased levels of anti-dGP IgG antibodies in CD patients on strict GFDs effectively identifies patients with NRCD. Finally, anti-dGP IgG assays may be useful to monitor mucosal damage and histological improvement in CD patients on a strict GFD.

The extracellular ATP/adenosine axis in the tumor microenvironment (TME) has emerged as an important immune-regulatory pathway. Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), otherwise known as CD39, is highly expressed in the TME, both on infiltrating immune cells and tumor cells across a broad set of cancer indications. CD39 processes pro-inflammatory extracellular ATP to ADP and AMP, which is then processed by Ecto-5ʹ-nucleotidase/CD73 to immunosuppressive adenosine. Directly inhibiting the enzymatic function of CD39 via an antibody has the potential to unleash an immune-mediated anti-tumor response via two mechanisms: 1) increasing the availability of immunostimulatory extracellular ATP released by damaged and/or dying cells, and 2) reducing the generation and accumulation of suppressive adenosine within the TME. Tizona Therapeutics has engineered a novel first-in-class fully human anti-CD39 antibody, TTX-030, that directly inhibits CD39 ATPase enzymatic function with sub-nanomolar potency. Further characterization of the mechanism of inhibition by TTX-030 using CD39 + human melanoma cell line SK-MEL-28 revealed an uncompetitive allosteric mechanism (α < 1). The uncompetitive mechanism of action enables TTX-030 to inhibit CD39 at the elevated ATP concentrations reported in the TME. Maximal inhibition of cellular CD39 ATPase velocity was 85%, which compares favorably to results reported for antibody inhibitors to other enzyme targets. The allosteric mechanism of TTX-030 was confirmed via mapping the epitope to a region of CD39 distant from its active site, which suggests possible models for how potent inhibition is achieved. In summary, TTX-030 is a potent allosteric inhibitor of CD39 ATPase activity that is currently being evaluated in clinical trials for cancer therapy.

A general strategy to identify serum antibody specificities associated with a given disease state, and peptide reagents for their detection was developed using bacterial display peptide libraries and multiparameter flow cytometry (MPFC). Using sera from patients with celiac disease (CD) (n = 45) or healthy subjects (n = 40), bacterial display libraries were screened for peptides that react specifically with antibodies from CD patients and not those from healthy patients. The libraries were screened for peptides that simultaneously cross-react with CD patient antibodies present in two separate patient groups labeled with spectrally distinct fluorophores, but do not react with unlabeled non-CD antibodies, thus affording a quantitative separation. A panel of six unique peptide sequences yielded 85% sensitivity and 91% specificity (AUC = 0.91) on a set of 60 samples not used for discovery, using leave-one-out cross-validation (LOOCV). Individual peptides were dissimilar with known CD specific antigens tissue transglutaminase (tTG) and deamidated gliadin, and classifier accuracy was independent of anti-tTG antibody titer. These results demonstrate that bacterial display/MPFC provides a highly effective tool for the unbiased discovery of disease-associated antibody specificities and peptide reagents for their detection that may have broad utility for diagnostic development.

The ATP/Adenosine pathway in the tumor microenvironment (TME) has emerged as an important immune-regulatory pathway. The ATPase CD39 is highly expressed in the TME, both on infiltrating immune cells and tumor cells across a broad set of cancer indications. CD39 processes pro-inflammatory extracellular ATP to ADP and AMP, which is then processed by CD73, to immunosuppressive adenosine. Inhibiting the enzymatic function of CD39 has the potential to shift the immunosuppressive milieu of the TME in a 2-pronged fashion: 1) Enhancement of immunostimulatory extracellular ATP released by damaged and/or dying tumor cells and 2) Inhibition of the generation and accumulation of suppressive adenosine within the TME, thereby unleashing an immune-mediated anti-tumor response. Tizona has generated a novel first-in-class fully human anti-CD39 antibody, TTX-030, that inhibits CD39 ATPase enzymatic function with sub-nanomolar affinity and potency. TTX-030 is specific for CD39 and binds to CD39+ cancer cell lines and primary human leukocytes with high affinity. TTX-030 is capable of inhibiting CD39 at elevated ATP concentrations reported in the TME. Enzymatic inhibition by TTX-030 has demonstrated: preservation of pro-inflammatory extracellular ATP, reduction of adenosine accumulation, and inhibition of phosphate release by a variety of CD39-expressing cells, including tumor cells and immune cells. In vitro functional assays using stimulated PBMCs exposed to exogenous ATP demonstrated increased proliferation of both CD4+ and CD8+ T cells in the presence of TTX-030 and increased secretion of the pro-inflammatory cytokines IFN-γ, TNF-α, and IL-2. Treatment of mice with a mouse-specific CD39 inhibitory antibody in a syngeneic tumor model significantly decreased tumor growth. In summary, TTX-030 is a selective and potent CD39 enzymatic inhibitor, capable of preventing adenosine-mediated immune suppression and increasing T-cell activation. Inhibition of CD39 with TTX-030 represents a unique therapeutic target aimed at modulating the immunosuppressive TME in cancer. Citation Format: Alana G. Lerner, Maria Kovalenko, Megan Welch, Tracy dela Cruz, Jeff Jones, Clifford Wong, Bradley Spatola, Meghan Eberhardt, Andrew Wong, Wanchi Fung, Leanna Lagpacan, Karolina Losenkova, Gennady Yegutkin, Vanessa Soros, John Corbin, Courtney Beers, Achim K. Moesta. Targeting CD39 with a first-in-class inhibitory antibody prevents ATP processing and increases T-cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5012.