Abstract CD39 (ENTPD1) is a cell membrane ectonucleotidase that hydrolyzes extracellular immunoactivating ATP and ADP into AMP, which can be further hydrolyzed by ectonucleotidase CD73 into immunosuppressive adenosine. Within the tumor microenvironment, adenosine accumulation causes immune suppression and dysregulation of immune cell infiltrates resulting in tumor spreading. The role of CD39 expression on both Tregs and on tumor cells in promoting immunosuppression has been demonstrated in several reports. Blockade of CD39 may promote anti-tumor immunity by directly accumulating immunostimulating ATP and indirectly by reducing adenosine accumulation. Here, we describe the discovery and preclinical development of an anti-huCD39 blocking antibody for cancer immunotherapy. Parental anti-huCD39 mouse monoclonal antibody was humanized. The humanized mAb specifically binds huCD39 protein, but not CD39-like proteins. Nanomolar affinities for human CD39 were measured in SPR studies on recombinant CD39 protein and in flow cytometry titration studies on CD39 expressing transfectants and tumor cell lines. The humanized mAb blocked human CD39 ATPase activity in vitro in the nanomolar range, as demonstrated using transfected cells, CD39-expressing tumor cell lines, as well as human PBMC and ex-vivo isolated fresh tumor samples. The humanized mAb cross-reacted on cynomolgus CD39 and blocked ATPase activity on cynomolgus PBMC with similar efficacy as on human PBMC. Finally, treatment with blocking anti-CD39 mAb inhibited tumor growth in vivo in mouse tumor models. Taken together, these data support the clinical development of anti-CD39 neutralizing mAb for cancer immunotherapy. Citation Format: Severine Augier, Ivan Perrot, Cecile Dejou, Rachel Joly, Stephane Delahaye, Helene Rispaud Blanc, Caroline Denis, Laurent Gauthier, Armand Bensussan, Jean-francois Eliaou, Yannis Morel, Nathalie Bonnefoy, Jeremy Bastid, Carine Paturel. Preclinical development of a humanized blocking antibody targeting the CD39 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 3222.
Abstract Progressing tumors in humans and mice are frequently infiltrated by a highly heterogeneous population of inflammatory myeloid cells that contribute to tumor growth. Among these cells, inflammatory Gr-1+ monocytes display a high developmental plasticity in response to specific microenvironmental signals, leading to diverse immune functions. These observations raise the question of the immune mechanisms by which inflammatory monocytes may contribute to tumor development. In this study, we found that adoptive transfer of normal inflammatory Gr-1+ monocytes in tumor-bearing mice promotes tumor growth. In this tumoral environment, these monocytes can differentiate into tolerogenic dendritic cells (DCs) that produce IL-10 and potently induce regulatory T cell responses in vivo. Moreover, diverting the differentiation of Gr-1+ monocytes into tolerogenic DCs by forced expression of IL-10 soluble receptor and IL-3 in tumor cells improves host immunosurveillance by reducing the regulatory T cell frequency and by inducing immunogenic DCs in the tumor. As a consequence, tumor growth is strongly reduced. Our findings indicate that Gr-1+ monocytes represent a valuable target for innovative immunotherapeutic strategies against cancer.
Abstract 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.
Epidermal wound repair is a complex process involving the fine orchestrated regulation of crucial cell functions, such as proliferation, adhesion and migration. Using an in vitro model that recapitulates central aspects of epidermal wound healing, we demonstrate that the transcription factor HIF1 is strongly stimulated in keratinocyte cultures submitted to mechanical injury. Signals generated by scratch wounding stabilise the HIF1α protein, which requires activation of the PI3K pathway independently of oxygen availability. We further show that upregulation of HIF1α plays an essential role in keratinocyte migration during the in vitro healing process, because HIF1α inhibition dramatically delays the wound closure. In this context, we demonstrate that HIF1 controls the expression of laminin-332, one of the major epithelial cell adhesion ligands involved in cell migration and invasion. Indeed, silencing of HIF1α abrogates injury-induced laminin-332 expression, and we provide evidence that HIF1 directly regulates the promoter activity of the laminin α3 chain. Our results suggest that HIF1 contributes to keratinocyte migration and thus to the re-epithelialisation process by regulating laminin-332.
Abstract Background: Nectin-4 is a cell membrane adhesion protein overexpressed in several solid tumors, including urothelial, breast, lung, ovarian, and pancreatic cancers, with limited expression in normal tissues, making it an ideal target for antibody-drug conjugates (ADC). Enfortumab Vedotin (EV, PADCEV®) is an ADC targeting Nectin-4 with a monomethyl auristatin E (MMAE) payload approved for the treatment of patients with urothelial carcinoma (UC), but with limited clinical activity reported beyond UC. In addition, EV causes peripheral neuropathy and skin toxicity, leading to treatment discontinuation. To overcome these limitations and the mechanism of resistance to EV, IPH45, a novel topoisomerase I inhibitor ADC targeting Nectin-4 with an improved therapeutic index, was developed. Methods: In vitro studies were performed to investigate internalization, tumor cell killing and bystander effect, antibody-dependent cellular cytotoxicity (ADCC) and complement activation. In vivo efficacy studies were performed on cell line-derived xenografts (CDX) of triple negative breast cancer, patient-derived xenografts (PDX) of urothelial carcinoma and syngeneic tumor models expressing human Nectin-4. IPH45 efficacy was compared to EV. Toxicology studies to determine the dose range were conducted in rat and non-human primates (NHP). Results: IPH45 consists of a humanized IgG1 targeting a unique epitope in human Nectin-4, non-overlapping with EV, a cleavable linker, and a topoisomerase I inhibitor with a high drug-to-antibody ratio (DAR). In vitro, IPH45 is internalized upon binding to its target and induces direct killing of Nectin-4 expressing tumor cells as well as bystander killing of Nectin-4 non-expressing tumor cells in mixed culture. IPH45 mediates ADCC and complement-mediated killing. IPH45 demonstrates activity in CDX and PDX models with similar effective dose as EV. Moreover, IPH45 shows anti-tumor efficacy in vitro and in vivo in primary and secondary EV resistant models. In dose-range finding toxicology studies in rats and NHP, IPH45, unlike EV, does not elicit skin toxicity. IPH45 remains well tolerated up to the highest dose tested, suggesting a potentially larger therapeutic index than MMAE-based ADCs. Pharmacokinetic analyses in mice, rats, and NHP show minimal release of free toxin in the serum and half-life compatible with Q2W or less frequent dosing schedules in clinic. Conclusion: IPH45 is a topoisomerase I ADC targeting Nectin-4 with the potential for larger therapeutic index than EV, improved safety and dosing regimen, and ability to overcome primary and secondary resistance to EV or other Nectin-4 ADC in development. IPH45 is progressing to the clinic where the translatability of this unique profile into improved patient outcomes will be assessed across indications. Citation Format: Romain Remark, Cécile Bonnafous, Laura Chiossone, Caroline Soulas, Cyril Perrier, Guillaume Habif, Sivan Bokobza, Aurélie Maguer, Rachel Courtois, Julie Lopez, Grégory Fenaux, Olivier Benac, Barbara Carrette, Robin Letay-Drouet, Raja Bonifay, Séverine Augier, Léa Simon, Benjamin Rossi, Ariane Morel, Agnès Represa, Nicola Beltraminelli, Yannis Morel, Carine Paturel, Eric Vivier. Preclinical characterization of IPH45, a novel topoisomerase I inhibitor ADC targeting Nectin-4 for the treatment of Nectin-4 expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6582.
Gene therapy offers exciting opportunities for the treatment of innate or acquired genetic diseases. However, there is still a need for a safe and efficient strategy to deliver nucleic acids into cells while overcoming the current limitations faced with standard viral vectors. Intensive researches have been carried out over the past decade, focusing both on viral and non-viral (i.e. physical or chemical) strategies. Of these numerous attempts, magnetofection, defined as the combination of nucleic acid vectors with magnetic nanoparticles, holds the promise to achieve high transfection efficiency with reduced toxicity by magnetically focusing the genetic material to be delivered on its cellular target. In vitro as well as in vivo results already demonstrated that this strategy may become a valuable tool towards practical gene therapy.
Abstract 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.
Abstract Therapeutic antibodies have revolutionized the way we treat cancer. Enhanced activity of therapeutic IgG can be achieved by the modulation of Fc binding to Fcγ receptors (FcγR), which will consequently modulate the Fc-effector functions triggered upon crosslinking of target and effector cells by these antibodies. Assessment of therapeutic antibodies pharmacodynamic (PD) in preclinical models is challenging, as the FcγR are different from mice to human, as well as their expression pattern. FcRn is involved in the recycling and transport of IgG and is a key determinant of the pharmacokinetics (PK) of IgG. We report here a novel mouse model expressing human FcγR with a human-like expression pattern, in which human FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB are expressed and replace their mouse orthologs. The model was intercrossed with the hSA/hFcRn mouse model (Viuff et al., 2016), which has shown to improve translatability of PK assessment of therapeutics with extended half-life via FcRn recycling and/or HSA binding. Investigation of FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB expression on DC, monocytes, granulocytes, NK, T and B cells shows that expression pattern in hFcγR mice is consistent with their expression on human PBMC, with a few exceptions. These receptors are functional, as a human IgG1 targeting mouse CD20 induces approximately 50% of B-cell depletion in blood and liver, after a single iv administration in hFcγR mice. This depletion is Fc-mediated, as it is abrogated when hFcγR mice are treated with an anti-mouse CD20 human IgG1 with a Fc-null mutation. Assessment of antibody-dependent cell cytotoxicity (ADCC) ex vivo to investigate FcγR functionality in NK cells shows that Rituximab, an anti-human CD20 chimeric IgG1 with regular Fc, induces higher ADCC efficacy in NK cells from hFcγR than in NK cells from WT mice. Obinutuzumab, an anti-human CD20 featuring an optimized Fc portion to enhance binding to FcγRIII, shows higher tumor cell lysis than Rituximab in NK cells from hFcγR mice, suggesting this model enables ranking of antibodies. Moreover, assessment of activity of Fc-engineered anti-influenza IgG antibodies also shows that anti-Flu antibody with enhanced Fc binding outperforms the WT antibody, improves protective activity, and demonstrates effector function in influenza challenge model, following prophylactic treatment. Conversely, hFcγR mice treated with an Fc-null version of the anti-Flu antibody shows reduced protective activity and survival compared with the WT antibody. Therefore, the model reported here is a novel tool for testing efficacy of Fc-engineered therapeutic antibodies. We are currently investigating the PK profile of therapeutic antibodies in the hFcγR/hSA/hFcRn model. This novel tool is being improved to enable tolerability to human IgG1, biodistribution to the central nervous system, and flexibility of test of therapeutics through expression of human immune checkpoints. Citation Format: Fabiane Sonego, Angela Pappalardo, Gaëlle Martin, Rachel Courtois, Séverine Augier, Yacine Cherifi, Ariane Morel, Patricia Isnard-Petit, Kader Thiam. Novel FcγR humanized mouse model enables assessment of PK/PD of therapeutic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5341.
NK cells offer a promising alternative to T cell therapies in cancer. We evaluated IPH6501, a clinical-stage, tetraspecific NK cell engager (NKCE) armed with a non-alpha IL-2 variant (IL-2v), which targets CD20 and was developed for treating B cell non-Hodgkin lymphoma (B-NHL). CD20-NKCE-IL2v boosts NK cell proliferation and cytotoxicity, showing activity against a range of B-NHL cell lines, including those with low CD20 density. Whereas it presented reduced toxicities compared with those commonly associated with T cell therapies, CD20-NKCE-IL2v showed greater killing efficacy over a T cell engager targeting CD20 in in vitro preclinical models. CD20-NKCE-IL2v also increased the cell surface expression of NK cell–activating receptors, leading to activity against CD20-negative tumor cells. In vivo studies in nonhuman primates and tumor mouse models further validated its efficacy and revealed that CD20-NKCE-IL2v induces peripheral NK cell homing at the tumor site. CD20-NKCE-IL2v emerges as a potential alternative in the treatment landscape of B-NHL.
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.
