Carine Paturel

The Development of Murine Plasmacytoid Dendritic Cell Precursors Is Differentially Regulated by FLT3-ligand and Granulocyte/Macrophage Colony-Stimulating Factor

Plasmacytoid predendritic cells or type 1 interferon (IFN)-producing cells (IPCs) have recently been identified in mice. Although culture systems giving rise to different murine dendritic cell subsets have been established, the developmental regulation of murine plasmacytoid IPCs and the culture conditions leading to their generation remain unknown. Here we show that large numbers of over 40% pure CD11c+CD11b−B220+Gr-1+ IPCs can be generated from mouse bone marrow cultures with FLT3-ligand. By contrast GM-CSF or TNF-α, which promote the generation of CD11c+CD11b+B220− myeloid DCs, block completely the development of IPCs. IPCs generated display similar features to human IPCs, such as the plasmacytoid morphology, the ability to produce large amounts of IFN-α in responses to herpes simplex virus, and the capacity to respond to ligands for Toll-like receptor 9 (TLR-9; CpG ODN 1668), but not to ligands for TLR-4 (lipopolysaccharide [LPS]). Unlike human IPCs which produce little IL-12p70, mouse IPCs produce IL-12p70 in response to CpG ODN 1668 and herpes simplex virus. This study demonstrates that the development of murine CD11c+CD11b−B220+Gr-1+ IPCs and CD11c+CD11b+B220− myeloid DCs is differentially regulated by FLT3-ligand and granulocyte/macrophage colony-stimulating factor. Human IPCs and mouse IPCs display different ability to produce IL-12p70. Large numbers of mouse IPCs can now be obtained from total bone marrow culture.

TLR3 and Rig-like receptor on myeloid dendritic cells and Rig-like receptor on human NK cells are both mandatory for production of IFN-gamma in response to double-stranded RNA.

Cross-talk between NK cells and dendritic cells (DCs) is critical for the potent therapeutic response to dsRNA, but the receptors involved remained controversial. We show in this paper that two dsRNAs, polyadenylic-polyuridylic acid and polyinosinic-polycytidylic acid [poly(I:C)], similarly engaged human TLR3, whereas only poly(I:C) triggered human RIG-I and MDA5. Both dsRNA enhanced NK cell activation within PBMCs but only poly(I:C) induced IFN-γ. Although myeloid DCs (mDCs) were required for NK cell activation, induction of cytolytic potential and IFN-γ production did not require contact with mDCs but was dependent on type I IFN and IL-12, respectively. Poly(I:C) but not polyadenylic-polyuridylic acid synergized with mDC-derived IL-12 for IFN-γ production by acting directly on NK cells. Finally, the requirement of both TLR3 and Rig-like receptor (RLR) on mDCs and RLRs but not TLR3 on NK cells for IFN-γ production was demonstrated using TLR3- and Cardif-deficient mice and human RIG-I–specific activator. Thus, we report the requirement of cotriggering TLR3 and RLR on mDCs and RLRs on NK cells for a pathogen product to induce potent innate cell activation.

IPH4102, a humanized KIR3DL2 antibody with potent activity against cutaneous T-cell lymphoma.

Advanced cutaneous T-cell lymphoma (CTCL) remains an unmet medical need, which lacks effective targeted therapies. In this study, we report the development of IPH4102, a humanized monoclonal antibody that targets the immune receptor KIR3DL2, which is widely expressed on CTCL cells but few normal immune cells. Potent antitumor properties of IPH4102 were documented in allogeneic human CTCL cells and a mouse model of KIR3DL2(+) disease. IPH4102 antitumor activity was mediated by antibody-dependent cell cytotoxicity and phagocytosis. IPH4102 improved survival and reduced tumor growth in mice inoculated with KIR3DL2(+) tumors. Ex vivo efficacy was further evaluated in primary Sézary patient cells, sorted natural killer-based autologous assays, and direct spiking into Sézary patient peripheral blood mononuclear cells. In these settings, IPH4102 selectively and efficiently killed primary Sézary cells, including at unfavorable effector-to-target ratios characteristic of unsorted PBMC. Together, our results offer preclinical proof of concept for the clinical development of IPH4102 to treat patients with advanced CTCL.

Targeting MICA with therapeutic antibodies for the treatment of cancer.

MICA and MICB, along with ULPBs, are ligands for the activating receptor NKG2D expressed on NK cells and subsets of T cells in Human. NKG2D ligands are induced by cellular stress and pathogen infections. Their expression is tightly regulated by complex mechanisms both at the mRNA and protein levels. In the case of MICA and MICB, more than 65 and 30 alleles respectively were described with different properties regarding to their cellular location adding to the complexity of this recognition system. Nevertheless, as markers of cellular stress, in particular in tumorigenesis, MICA and the closely related MICB proteins are candidates of choice to be targeted by a cytotoxic therapeutic antibody. We first evaluated MICA/B expression by immunohistochemistry on healthy tissues and tumors to validate these antigens as therapeutic targets. Then, using mouse immunization, we generated a panel of chimeric human IgG1 monoclonal antibodies targeting MICA and MICB. These mAbs have the ability to bind to several structurally different alleles and to cross-react on MIC proteins from cynomolgus macaques. Their capacity to block the MICA/NKG2D interaction was assessed by surface plasmon resonance as well as by using cell-based assays. In vitro efficacy was measured by the capacity to mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cell cytotoxicity (ADCC) towards MICA expressing cells. In vivo efficacy of the anti-MICA mAbs was measured in both a preventive and a curative setting using MICA expressing cell lines. Altogether, we have generated a panel of anti-MICA mAbs with diverse functional properties. Ongoing work aims to choose the best candidate for humanization and further clinical development.

Abstract 2344: Discovery and characterization of new original blocking antibodies targeting the CD73 immune checkpoint for cancer immunotherapy

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 Community9s 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, Celine Rodriguez, Helene Rispaud Blanc, Stephanie Chanteux, Nicolas Gourdin, Laurent Gauthier, Christine Menetrier 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.

Novel therapeutic and diagnostic antibodies against KIR3DL2, a unique tumor antigen overexpressed on subtypes of T cell lymphomas

KIR3DL2 belongs to the killer immunoglobulin (Ig)-like receptors (KIRs) family and is composed of three extracellular Ig-like domains. KIR3DL2 is naturally expressed on some NK cells and minor subpopulations of CD8+ and CD4+ T cells. Physiologically, KIRD3L2 is an inhibitory receptor for human leukocyte antigen (HLA) class I molecules regulating NK cell activation. Remarkably, KIR3DL2 is also aberrantly overexpressed on several subtypes of T lymphomas/leukemias, such as Sezary Syndrome, transformed Mycosis Fungoides and HTLV1+ Adult T Cell Leukemia, making it a unique therapeutic target in cancer. We have generated a series of anti-KIR3DL2 monoclonal antibodies (mAbs) binding selectively to KIR3DL2, spanning epitopes on all three Ig domains. Their efficacy was evaluated in vitro and in vivo against KIR3DL2-expressing tumors and Sezary cell lines as disease model. An autologous assay was set up to evaluate the killing of primary Sezary cells by patients’ NK effectors in the presence of those mAbs. Potent antibody-dependent cell cytotoxicity (ADCC) was found the main mode of action involved in their anti-tumor activity. The most promising candidates were humanized as IgG1 mAbs and the final lead molecule was selected for further development, based on several predefined criteria. In parallel, anti-KIR3DL2 mAbs were also developed as unique and sensitive tools for the detection by immunohistochemistry of KIR3DL2 on tumor biopsies. Owing to the promising efficacy profile of our anti-KIR3DL2 mAb candidate and to the highly restricted expression pattern of the target on some T leukemia/lymphoma cells, an antibody-based therapy targeting KIR3DL2 stands as a potentially unequalled strategy in several orphan diseases with critical unmet medical need.

A phase 1 study of lirilumab (antibody against killer immunoglobulin-like receptor antibody KIR2D; IPH2102) in patients with solid tumors and hematologic malignancies

Purpose Anti-KIR monoclonal antibodies (mAbs) can enhance the antitumor responses of natural killer (NK) cells. We evaluated the safety of the anti-KIR2D mAb lirilumab in patients with various cancers. Experimental design Thirty-seven patients with hematological malignancies (n = 22) or solid tumors (n = 15) were included in the study. Dose escalation (0.015 to 10 mg/kg) was conducted following a 3 + 3 design. Patients were scheduled to receive four cycles of treatment. In a second (extension) phase 17 patients were treated at 0.015 (n = 9) or 3 mg/kg (n = 8). Results No dose-limiting toxicity was recorded. The most frequent lirilumab-related adverse events were pruritus (19%), asthenia (16%), fatigue (14%), infusion-related reaction (14%), and headache (11%), mostly mild or moderate. Pharmacokinetics was dose-dependent and linear, with minimal accumulation resulting from the 4-weekly repeated administrations. Full KIR occupancy (>95%) was achieved with all dosages, and the duration of occupancy was dose-related. No significant changes were observed in the number or distribution of lymphocyte subpopulations, nor was any reduction in the distribution of KIR2D-positive NK cells. Conclusions This phase 1 trial demonstrated the satisfactory safety profile of lirilumab up to doses that enable full and sustained blockade of KIR.

Abstract 1491: IPH4301, an antibody targeting MICA and MICB exhibits potent cytotoxic activity and immunomodulatory properties for the treatment of cancer

MICA and MICB, and ULPB1-6, are ligands for NKG2D, an activating receptor expressed on NK cells and subsets of T cells. Expression of MICA and MICB is induced by cellular stress in transformed tumor cells, upon infections or at sites of chronic inflammation. Their expression is tightly regulated by complex mechanisms both at the mRNA and protein levels. As markers of cellular stress and tumorigenesis, MICA and MICB proteins are attractive candidates for targeting by a cytotoxic antibody. Moreover, ionizing radiation and various chemotherapies that cause cellular stress have been shown to induce expression of NKG2D ligands, opening interesting options for combination therapies. MICA and MICB are also compelling targets for immunomodulation. MICA and -B cause internalization of NKG2D, leading to reduced cell surface NKG2D levels and desensitization of cytotoxic effector cells in cancer patients. It was recently reported that blockade of the interactions between NKG2D and its ligands could lead to significant anti-tumor responses in mouse models. Moreover, NKG2D ligand expression was induced on immunosuppressive macrophages in cancer patients and in mouse tumor models, raising the possibility that anti-MICA/B antibodies may be used to counter local immunosuppression by targeting myeloid derived suppressor cells. We have selected the IPH4301 antibody for its ability to bind to all allotypes of MICA and MICB, and for its dual action as an immunomodulatory agent, as well as direct cytotoxicity towards MICA/B-expressing tumor cells. First, IPH4301 induces killing of MICA/B expressing tumor cells through antibody-dependent cell cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP) measured towards MICA expressing cells in vitro. In vivo ADCC/ADCP efficacy was demonstrated in several preventive and curative settings using MICA expressing cell lines or endogenous tumors. Second, IPH4301 blocks the binding of MICA/B to NKG2D. In a tumor context of chronic triggering, NKG2D downmodulation has been described in several studies of cancer patients. This modulation is mainly induced by expression of MICA/B, and less by the ULBPs. By blocking the MICA/NKG2D interaction, IPH4301 effectively restored NKG2D expression and function in vitro on primary NK and T cells. Third, we show that IPH4301 can override immunosuppression induced by suppressive myeloid cells. In vitro differentiated M2 macrophages, but not M1 macrophages, have the capacity to impair cytotoxic functions of autologous NK cells towards MICA expressing tumor cell lines. This suppression could be overcome by IPH4301, which triggered ADCC by these otherwise impaired NK cells. Altogether, IPH4301 is a novel, first-in-class anti-MICA/B mAb with both cytotoxic and immunomodulatory properties. Ongoing work aims to perform regulatory toxicology studies and manufacture a clinical grade product for testing in a clinical trial. Citation Format: Ariane Morel, Nicolas Viaud, Cecile Bonnafous, Sylvia Trichard, Alix Joulin-Giet, Samia Mizari, Gwendoline Grondin, Nadia Anceriz, J. Zhang, J. Jarzen, J. Wu, Gwendoline Grondin, Laetitia Cohen-Tannoudji, Yannis Morel, Benjamin Rossi, Carine Paturel, Renaud Buffet, Laurent Gauthier, Nicolai Wagtmann, Mathieu Blery. IPH4301, an antibody targeting MICA and MICB exhibits potent cytotoxic activity and immunomodulatory properties for the treatment of cancer. [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 1491.

Abstract 3218: Disruption of the CD39 immune checkpoint pathway increases the efficacy of various anticancer therapies in syngeneic mouse models

The CD39-CD73-adenosine pathway is an emerging regulator of the immune antitumor response. CD39 is expressed within tumors and the tumor microenvironment by several cell population including immune and cancer cells. In tumor tissues, the pathway leads to the accumulation of immunosuppressive adenosine together with decreased levels of immunoactivating peritumoral ATP. We reported previously that CD39 blockade increased T cell and NK cell-mediated cytotoxic activity in vitro and disclose, during this meeting, the development of the first human-CD39-blocking humanized antibody (S. Augier et al., Preclinical development of a humanized blocking antibody targeting the CD39 immune checkpoint for cancer immunotherapy). Here we demonstrated that this pathway is involved in tumor-induced resistance to various cancer therapies in syngeneic mouse melanoma, colon cancer and fibrosarcoma models. We used therapy-resistant mouse models or inefficacious treatment regimens in the CD39 knockout mice to assess the capacity of CD39 to affect the response to chemotherapies, tumor associated antigen (TAA)-targeting antibodies and immunomodulators such as anti-PD1 antibodies. We achieved increased response rates, increased response duration and some complete and long lasting tumor regressions in the CD39 deficient context. These preclinical proof-of-concept studies highlight the role of the CD39 immune checkpoint pathway in limiting the efficacy of various anticancer therapies in syngeneic mouse models and thereby support the potential clinical value of the humanized CD39-neutralizing antibody under development. Citation Format: Marion Lapierre, Cecile Dejou, Carine Paturel, Henri-Alexandre Michaud, Laurent Gros, Armand Bensussan, Jean-Francois Eliaou, Jeremy Bastid, Nathalie Bonnefoy. Disruption of the CD39 immune checkpoint pathway increases the efficacy of various anticancer therapies in syngeneic mouse models. [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 3218.

Abstract 3222: Preclinical development of a humanized blocking antibody targeting the CD39 immune checkpoint for cancer immunotherapy

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.