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Simple Summary Recent findings have revealed the contribution of cancer-associated fibroblasts (CAF) in immune escape in breast cancer. Still, how to specifically target immunosuppressive CAF remains an unmet medical question. Here, we provide a promising therapeutic strategy by highlighting the role of CD73 in immunosuppressive CAF. By studying cohorts of breast cancer patients and performing functional assays, our study uncovers how CD73 contributes to immunosuppression by acting in a specific CAF subpopulation (referred to as CAF-S1) in breast cancer. In addition, we validate that using an anti-CD73 antibody significantly reduces CAF-S1-mediated immunosuppression, thereby highlighting a new interesting therapeutic strategy for breast cancer patients. Abstract Background: Cancer-associated fibroblasts (CAF) are heterogeneous with multiple functions in breast cancer. Recently, we identified a specific CAF subpopulation (referred to as CAF-S1), which promotes immunosuppression and immunotherapy resistance. Methods and Results: Here, by studying a large collection of human samples, we highlight the key function of CD73/NT5E in CAF-S1-mediated immunosuppression in breast cancer. We first reveal that CD73 protein level specifically accumulates in CAF-S1 in breast cancer patients. Interestingly, infiltration of regulatory T lymphocytes (Tregs) is significantly correlated with CD73 expression in stroma but not in epithelium, indicating that CD73 contributes to immunosuppression when expressed in CAF-S1 and not in tumor cells. By performing functional assays based on relevant systems using primary CAF-S1 isolated from patients, we demonstrate that CAF-S1 increase the content in both PD-1+ and CTLA-4+ Tregs. Importantly, the use of a blocking anti-CD73 antibody on CAF-S1 reduces CAF-S1-mediated immunosuppression by preventing expression of these immune checkpoints on Tregs. Conclusions: Our data support the potential clinical benefit of using both anti-CD73 and immune-checkpoint inhibitors in breast cancer patients for inhibiting CAF-S1-mediated immunosuppression and enhancing anti-tumor immune response.

BackgroundThere is a clear need for targeted therapies to treat acute myeloid leukemia (AML), the most common acute leukemia in adults. CD123 (IL-3 receptor alpha chain) is an attractive target for AML treatment.1 However, cytotoxic antibody targeting CD123 proved insufficiently effective in a combination setting in phase II/III clinical trials.2 T-cell engagers targeting CD123 displayed some clinical efficacy but were often associated with cytokine release syndrome and neurotoxicity.3 Interest in the use of NK cells for therapeutic interventions has increased in recent years, as a potential safer alternative to T cells. Several NK-cell activating receptors, such as CD16a, NKG2D, and the natural cytotoxicity receptors NKp30 and NKp46, can be targeted to induce antitumor immunity. We previously reported the development of trifunctional NK-cell engagers (NKCEs) targeting a tumor antigen on cancer cells and co-engaging NKp46 and CD16a on NK cells.4MethodsWe report here the design, characterization and preclinical development of a novel trifunctional NK cell engager (NKCE) targeting CD123 on AML cells and engaging the activating receptors NKp46 and CD16a on NK cells. The CD123 NKCE therapeutic molecule was engineered with humanized antibodies targeting NKp464 and CD123.5 We compared CD123-NKCE and a cytotoxic ADCC-enhanced antibody (Ab) targeting CD123, in terms of antitumor activity in vitro, ex vivo and in vivo. Pharmacokinetic, pharmacodynamic and safety profile of CD123-NKCE were evaluated in non-human primate (NHP) studies.ResultsThe expression of the high affinity Fc gamma receptor CD64 on patient-derived AML cells inhibited the ADCC of the Ab targeting CD123 in vitro and ex vivo, but not the antitumor activity of CD123-NKCE. CD123-NKCE had potent antitumor activity against primary AML blasts and AML cell lines, promoted strong NK-cell activation and induced cytokine secretion only in the presence of AML target cells. Its antitumor activity in mouse model was greater than that of the comparator antibody. Moreover, CD123-NKCE had strong and prolonged pharmacodynamic effects in NHP when used at very low doses, was well-tolerated up to high 3 mg/kg dose and triggered only minor cytokine release.ConclusionsThe data for activity, safety, pharmacokinetics, and pharmacodynamics provided here demonstrate the superiority of CD123-NKCE over comparator cytotoxic antibody, in terms of antitumor activity in vitro, ex vivo, in vivo, and its favorable safety profile, as compared to T-cell therapies. These results constitute proof-of-principle for the efficacy of CD123-NKCE for controlling AML tumors in vivo, and provide consistent support for their clinical development.ReferencesEhninger A, Kramer M, Rollig C, et al. Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia. Blood Cancer J 2014;4:e218.Montesinos P, Gail J Roboz GJ, et al. Safety and efficacy of talacotuzumab plus decitabine or decitabine alone in patients with acute myeloid leukemia not eligible for chemotherapy: results from a multicenter, randomized, phase 2/3 study. Leukemia 2021;35(1):62–74.Uy GL, Aldoss I, Foster MC, et al. Flotetuzumab as salvage immunotherapy for refractory acute myeloid leukemia. Blood 2021;137(6):751–762.Gauthier L, Morel A, Anceriz N, et al. Multifunctional natural killer cell engagers targeting NKp46 trigger protective tumor immunity. Cell 2019;177(7):1701–13.Jin L, Lee EM, Ramshaw HS, et al. Monoclonal antibody-mediated targeting of CD123, IL-3 receptor alpha chain, eliminates human acute myeloid leukemic stem cells. Cell Stem Cell 2009;5:31–42.

Summary: Immune checkpoint inhibitors have revolutionized cancer treatment. However, many cancers are resistant to ICIs, and the targeting of additional inhibitory signals is crucial for limiting tumor evasion. The production of adenosine via the sequential activity of CD39 and CD73 ectoenzymes participates to the generation of an immunosuppressive tumor microenvironment. In order to disrupt the adenosine pathway, we generated two antibodies, IPH5201 and IPH5301, targeting human membrane-associated and soluble forms of CD39 and CD73, respectively, and efficiently blocking the hydrolysis of immunogenic ATP into immunosuppressive adenosine. These antibodies promoted antitumor immunity by stimulating dendritic cells and macrophages and by restoring the activation of T cells isolated from cancer patients. In a human CD39 knockin mouse preclinical model, IPH5201 increased the anti-tumor activity of the ATP-inducing chemotherapeutic drug oxaliplatin. These results support the use of anti-CD39 and anti-CD73 monoclonal antibodies and their combination with immune checkpoint inhibitors and chemotherapies in cancer. : The production of adenosine via CD39 and CD73 ectoenzymes participates in an immunosuppressive tumor microenvironment. Perrot et al. generated two antibodies, IPH5201 and IPH5301, targeting human CD39 and CD73, respectively. In vitro and in vivo data support the use of anti-CD39 and anti-CD73 mAbs in combination cancer therapies. Keywords: CD39, CD73, cancer immunotherapies, therapeutic antibodies, adenosine pathway, tumor micro-environment, immunosuppression

Within the tumor microenvironment, adenosine causes immune suppression and dysregulation of immune cell infiltrates resulting in tumor spreading. Adenosine accumulation results from the hydrolysis of extracellular immunoactivating ATP and ADP into AMP by the CD39 (ENTPD1) ectonucleotidase. AMP can be further hydrolyzed into immunosuppressive Adenosine (Ado) by CD73 (NT5E), a cell membrane ectonucleotidase of the NTPDase family.The immunosuppressive role of CD39 expressed on both Tregs and tumor cells has been demonstrated in several reports. Conversely, CD73 expression in the tumor environment has been associated with poor disease outcome and/or with a pro-metastatic phenotype. Blockade of CD39 and CD73 may promote anti-tumor immunity directly by accumulating immunostimulating ATP for CD39 and indirectly by reducing adenosine accumulation for both targets.In cancer tissue, using IHC and flow cytometry analyses, we observed that while CD73 is often expressed by tumor cells, CD39 is more frequently up-regulated on tumor infiltrating cells compared to PBMC or adjacent non-tumor tissue. We next describe the discovery and preclinical development of a unique anti-huCD39 blocking antibody and of an anti-human CD73 antibody for cancer immunotherapy. These anti-CD39 and anti-CD73 antibodies specifically bind with high affinity to huCD39 and huCD73 proteins, respectively. They potently inhibit enzyme activity of their respective targets under their soluble and membrane-associated forms, without inducing down-modulation of these enzymes expressed at the cell surface. Innate's antibodies efficiently reverse Ado-mediated T cell suppression in vitro in presence of ATP and both CD39- and CD73-expressing immune cells and additionally exhibit unique features. The anti-CD39 Ab maintains high concentration of ATP in the extracellular compartment that enhances DC activation and subsequent T cell proliferation in vitro. The anti-CD73 blocking Ab exhibits a more potent ability to block soluble and membrane-associated CD73 enzyme activity than benchmark Abs currently in clinical development. Finally, in vivo blockade of ATP/Ado pathway in CD39ko mice resulted in improved anti-tumor efficacy of immunogenic cell death inducer chemotherapy and of immune checkpoint therapies, including PD1 and CTLA4.Taken together, these data support the clinical development of anti-CD39 and anti-CD73 neutralizing Abs for cancer immunotherapy, potentially in combination with chemotherapy or Immune Checkpoint therapy. The humanized anti-huCD39 and anti-huCD73 monoclonal antibodies are currently in preclinical development.The research leading to CD73 results were obtained within the TumAdoR collaborative consortium that received funding from the European Community's Seventh Framework Program (FP7/2007-2013) under grant agreement n°602200. Citation Format: Ivan Perrot, Marc Giraudon Paoli, Séverine Augier, Marilyne Royannez Blemont, Marion Gaudin, Frédéric Bosco, Rachel Courtois, Stephane Delahaye, Diana Jecko, Nicolas Gourdin, Maryline Salin Agu, Cyril Perrier, Paul Ricaut, Aurélie Docquier, Stéphanie Chanteux, Benjamin Rossi, Agnès Représa, Caroline Denis, Romain Remark, Cécile Bonnafous, Laurent Gauthier, Ariane Morel, Nathalie Bonnefoy, Jérémy Bastid, Yannis Morel, Carine Paturel. Preclinical development of humanized CD39 and CD73 blocking antibodies targeting the ATP/adenosine immune checkpoint pathway for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2718.

CD73 (NT5E) is a cell membrane ectoenzyme of the NTPDase family that plays a major role in the conversion of AMP into Adenosine (Ado). Within the tumor microenvironment, accumulation of Ado causes immune suppression and dysregulation of immune cell infiltrates resulting in tumor spreading. CD73 expression in the tumor environment has been associated with poor disease outcome and/or with a pro-metastatic phenotype. Thus, targeting CD73 may promote anti-tumor immunity by reducing Ado accumulation and may block tumor cell metastasis by inhibiting CD73 on tumor cells. Here, we describe the generation and characterization of novel anti-human CD73 antibodies, intended for the treatment of a wide range of cancers. The research leading to these results has received funding from the European Community's Seventh Framework Program (FP7/2007-2013) under grant agreement n°602200. Antibodies were discovered that inhibited CD73 function by different mechanisms, including the direct blocking of CD73 enzymatic activity or the down-modulation of membrane CD73 expression. Epitope mapping revealed that antibodies acting by these different modes of action bound to distinct sites on CD73. All selected antibodies cross-react with cynomolgus CD73 protein and have strong avidity and affinity for membrane or recombinant CD73, by flow cytometry and Surface Plasmon Resonance, respectively. Antibodies that inhibit CD73 enzymatic activity strongly reduce AMP catabolism by both recombinant and cellular CD73 with IC50 in the nanomolar range. They also efficiently reverse ATP- and AMP-mediated T cell suppression in in vitro assays in presence of both CD39+ and CD73+ cells. The antibodies that induce down-modulation of cellular CD73 expression do not block recombinant CD73 enzyme activity and partially inhibit cellular CD73 activity; they reverse ATP- but not AMP-dependent T cell suppression. The antibodies displaying the most interesting features were humanized. Evaluation of their activity in animal models is ongoing. Citation Format: Marc Giraudon Paoli, Severine Augier, Marilyne Royannez Blemont, Céline Rodriguez, Hélène Rispaud Blanc, Stéphanie Chanteux, Nicolas Gourdin, Laurent Gauthier, Christine Ménétrier Caux, Yannis Morel, Christophe Caux, Carine Paturel, Ivan Perrot. Discovery and characterization of new original blocking antibodies targeting the CD73 immune checkpoint for cancer immunotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2344.

Our group and others have previously reported that Treg infiltrating (Ti-Treg) breast tumors have a negative impact on patients’ outcome. We further reported their selective recruitment in the tumor environment through CCL22 secretion and expansion upon ICOS engagement by plasmacytoïd dendritic cells. Here, we investigated the mechanism of Ti-Treg mediated suppression and observed that Ti-Treg express high CD39 levels. CD39 is an ectonucleotidase that cooperates with CD73 to degrade the danger signal ATP into immunosuppressive Adenosine (Ado). The potency of Ado mediated suppression is illustrated in Adenosine-Deaminase (ADA)-deficient patients unable to degrade Ado and developing severe immunodeficiency. We further show that, in contrast to murine Treg, human CD39+Treg do not express CD73 enabling them only to degrade ATP into AMP. Within T cells, CD73 expression was mainly associated with naïve CD8+ T cells and a subset of memory conventional CD4+ T cells (Tconv) that exhibit Th1/Th17 characteristics (CXCR3+CCR6+CCR4negIFNγhighIL17+). CD39+ Treg isolated from healthy donor blood, in presence of exogenous ATP, potently inhibit purified CD73+ but not CD73neg Tconv, proliferation and cytokine production (IFNγ, TNFα). By using enzymatic inhibitors, we demonstrated the involvement of CD39 and CD73 through Treg/Tconv cooperation for Ado mediated immunosuppression. Of importance, when integrated in [Treg-CD4+CD73+] high density coculture, CD4+CD73neg T cells expressing similar levels of Ado receptors (A2A, A2B) are also inhibited. In conclusion, our findings support the existence of an Ado mediated immunosuppression loop in the tumor through cooperation between CD39highTreg and CD73-expressing Th1/Th17 subsets in the breast tumor environment. The research leading to these results has received funding from the European Community's Seventh Framework Program (FP7/2007-2013) under grant agreement n°602200. Citation Format: Nicolas Gourdin, Céline Rodriguez, Selena Vigano, Isabelle Durand, Julien Faget, Camilla Jandus, Jean Yves Blay, Pedro Romero, Christine Menetrier-Caux, Christophe Caux. CD39+ Treg cooperate with a CD73-expressing Th1/Th17 subset for Adenosine-mediated immunosuppression in human breast tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2338.