Cécile Bonnafous

Anti-KIR Antibody Enhancement Of Anti-Lymphoma Activity Of Natural Killer Cells As Monotherapy and In Combination With Anti-CD20 Antibodies

Natural killer (NK) cells mediate antilymphoma activity by spontaneous cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) when triggered by rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B-cell lymphomas. The balance of inhibitory and activating signals determines the magnitude of the efficacy of NK cells by spontaneous cytotoxicity. Here, using a killer-cell immunoglobulin-like receptor (KIR) transgenic murine model, we show that blockade of the interface of inhibitory KIRs with major histocompatibility complex (MHC) class I antigens on lymphoma cells by anti-KIR antibodies prevents a tolerogenic interaction and augments NK-cell spontaneous cytotoxicity. In combination with anti-CD20 mAbs, anti-KIR treatment induces enhanced NK-cell-mediated, rituximab-dependent cytotoxicity against lymphoma in vitro and in vivo in KIR transgenic and syngeneic murine lymphoma models. These results support a therapeutic strategy of combination rituximab and KIR blockade through lirilumab, illustrating the potential efficacy of combining a tumor-targeting therapy with an NK-cell agonist, thus stimulating the postrituximab antilymphoma immune response.

IPH2101, a novel anti-inhibitory KIR antibody, and lenalidomide combine to enhance the natural killer cell versus multiple myeloma effect.

Multiple myeloma (MM) patients who receive killer cell Ig-like receptor (KIR) ligand-mismatched, T cell-depleted, allogeneic transplantation may have a reduced risk of relapse compared with patients who receive KIR ligand-matched grafts, suggesting the importance of this signaling axis in the natural killer (NK) cell-versus-MM effect. Expanding on this concept, IPH2101 (1-7F9), an anti-inhibitory KIR mAb, enhances NK-cell function against autologous MM cells by blocking the engagement of inhibitory KIR with cognate ligands, promoting immune complex formation and NK-cell cytotoxicity specifically against MM cell targets but not normal cells. IPH2101 prevents negative regulatory signals by inhibitory KIR, whereas lenalidomide augments NK-cell function and also appears to up-regulate ligands for activating NK-cell receptors on MM cells. Lenalidomide and a murine anti-inhibitory NK-cell receptor Ab mediate in vivo rejection of a lenalidomide-resistant tumor. These mechanistic, preclinical data support the use of a combination of IPH2101 and lenalidomide in a phase 2 trial for MM.

Bromohydrin pyrophosphate enhances antibody-dependent cell-mediated cytotoxicity induced by therapeutic antibodies

In human blood, 1% to 5% of lymphocytes are gammadelta T cells; they mostly express the gammadelta T-cell receptor (TCR)Vgamma9, recognize nonpeptide phosphoantigens (PAgs) produced by microbes and tumor cells, and mediate different modes of lytic activities directed against tumor target cells. Antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by cytolytic lymphoid cells is essential for the clinical activity of anticancer monoclonal antibodies (mAbs), but whether PAgs affect ADCC by gammadelta T cells is unknown. Here we report that, in association with the CD20(+)-specific mAb rituximab (RTX), the synthetic PAg bromohydrin pyrophosphate (BrHPP) increased TCRVgamma9(+) cell binding to CD20(+) lymphoma cells in vitro. This combination activated phospho-ZAP70 and phospho-ERK1/2 signaling in TCRVgamma9(+) cells and strongly enhanced their ADCC activity. We obtained similar results with BrHPP in the context of the mAbs alemtuzumab and trastuzumab. Furthermore, BrHPP enhanced RTX-mediated depletion of CD20(+) cells in vitro from peripheral blood mononuclear cells of healthy subjects and enhanced ADCC by gammadelta T cells from patients with chronic lymphocytic leukemia. In cynomolgus macaques, a regimen combining RTX, BrHPP, and IL2 activated TCRVgamma9(+) lymphocytes and enhanced B-cell depletion from blood and lymph nodes. Thus, the combination with BrHPP PAg is able to improve the efficacy of cancer immunotherapy by therapeutic mAbs.

Genetic and antibody-mediated reprogramming of natural killer cell missing-self recognition in vivo

Natural killer (NK) cells are lymphocytes of the innate immune system able to recognize and kill tumors lacking self-MHC class I molecules. This “missing-self” recognition is mediated by the lack of engagement of MHC class I-specific inhibitory NK cell receptors that include the killer cell Ig-like receptors (KIR) in humans and Ly49 molecules in mice. A promising immunotherapeutic strategy against MHC class I+ cancer cells is to block NK cell inhibitory receptors using monoclonal antibodies (mAb). However, interactions between MHC class I molecules and their inhibitory receptors are also required for the acquisition of NK cell functional competence, a process referred as to “education.” In addition, inhibitory receptors are involved in self-tolerance on educated NK cells. Here, we developed a preclinical mouse model in which all NK cells are educated by a single transgenic inhibitory receptor, human KIR2DL3, through the engagement with its HLA-Cw3 ligand. This approach revealed that NK cells could be reprogrammed to control the development of mouse syngenic tumors in vivo. Moreover, in vivo anti-KIR mAb treatment induced the killing of HLA+ target cells without breaking self-tolerance. Finally, the long-term infusion of anti-KIR mAb neither abolished NK cell education nor tumor cell recognition. Therefore, these results strongly support the use of inhibitory receptor blockade in cancer patients.

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.

A novel targeted immunotherapy for CTCL is on its way: Anti-KIR3DL2 mAb IPH4102 is potent and safe in non-clinical studies

Cutaneous T-cell lymphomas (CTCLs) represent a group of rarely occurring and clinically and pathologically heterogeneous diseases that are considered incurable at advanced stages. Current treatments provide limited clinical benefit and are thus largely amenable to improvement. An antibody-based CTCL-specific immunotherapy targeting the KIR3DL2 receptor expressed by the tumor cells in CTCL is currently under development and has shown encouraging results in pre-clinical studies.

Interferon-α improves phosphoantigen-induced Vγ9Vδ2 T-cells interferon-γ production during chronic HCV infection.

In chronic HCV infection, treatment failure and defective host immune response highly demand improved therapy strategies. Vγ9Vδ2 T-cells may inhibit HCV replication in vitro through IFN-γ release after Phosphoantigen (PhAg) stimulation. The aim of our work was to analyze Vγ9Vδ2 T-cell functionality during chronic HCV infection, studying the role of IFN-α on their function capability. IFN-γ production by Vγ9Vδ2 T-cells was analyzed in vitro in 24 HCV-infected patients and 35 healthy donors (HD) after PhAg stimulation with or without IFN-α. The effect of in vivo PhAg/IFN-α administration on plasma IFN-γ levels was analyzed in M. fascicularis monkeys. A quantitative analysis of IFN-γ mRNA level and stability in Vγ9Vδ2 T-cells was also evaluated. During chronic HCV infection, Vγ9Vδ2 T-cells showed an effector/activated phenotype and were significantly impaired in IFN-γ production. Interestingly, IFN-α was able to improve their IFN-γ response to PhAg both in vitro in HD and HCV-infected patients, and in vivo in Macaca fascicularis primates. Finally, IFN-α increased IFN-γ-mRNA transcription and stability in PhAg-activated Vγ9Vδ2 T-cells. Altogether our results show a functional impairment of Vγ9Vδ2 T-cells during chronic HCV infection that can be partially restored by using IFN-α. A study aimed to evaluate the antiviral impact of PhAg/IFN-α combination may provide new insight in designing possible combined strategies to improve HCV infection treatment outcome.

In Vivo Manipulation of γ9+ T Cells in the Common Marmoset (Callithrix Jacchus) with Phosphoantigen and Effect on the Progression of Respiratory Melioidosis

Burkholderia pseudomallei is a dangerous human pathogen. Phosphoantigens specifically the target primate specific γ9+δ2+ T cells subset and some have been developed as potential immunotherapeutics. Previously, we demonstrated that, when stimulated with the phosphoantigen CHDMAPP, γ9+δ2+ T cells aid in the killing of intracellular B. pseudomallei bacteria. Moreover, we found that common marmoset (Callithrix Jacchus) γ9+ T cells increase in frequency and respond to the phosphoantigen CHDMAPP and/or B. pseudomallei, in combination with IL-2, in a similar manner to human γ9+δ2+ T cells. Here we evaluate the efficacy of the phosphoantigen CHDMAPP, in combination with IL-2, as a therapy against B. pseudomallei infection, in vivo. We found that the previous studies predicted the in vivo responsiveness of γ9+ T cells to the CHDMAPP+IL-2 treatment and significant expansion of the numbers of peripheral and splenic γ9+ T cells were observed. This effect was similar to those reported in other primate species treated with phosphoantigen. Furthermore, splenocytes were retrieved 7 days post onset of treatment, restimulated with CHDMAPP or heat-killed B. pseudomallei and the cultured γ9+ T cells demonstrated no reduction in IFN-γ response when CHDMAPP+IL-2 animals were compared to IL-2 only treated animals. Using an established model of B. pseudomallei infection in the marmoset, we assessed the potential for using phosphoantigen as a novel immunotherapy. The CHDMAPP treatment regime had no effect on the progression of respiratory melioidosis and this was despite the presence of elevated numbers of γ9+ T cells in the spleen, liver and lung and an increased proportion of IFN-γ+ cells in response to infection. We therefore report that the common marmoset has proven a good model for studying the effect in vivo of γ9+ T cell stimulation; however, γ9+ T cells have little or no effect on the progression of lethal, respiratory B. pseudomallei infection.

Anti-tumoral efficacy of therapeutic human anti-KIR antibody (Lirilumab/BMS-986015/IPH2102) in a preclinical xenograft tumor model

Natural Killer cells (NK cells) are lymphocytes able to recognize and kill tumors for which the expression of Major Histocompatibility Complex (MHC) class I molecules is altered. This “missing self” recognition is mediated in humans by the lack of engagement of MHC class I i.e. Human Leucocyte Antigens (HLA) molecules with NK cell inhibitory receptors that include Killer Immunoglobulin-like Receptors (KIR). Some tumors escape NK cell immune surveillance by increasing the expression of HLA molecules on their surface. Consequently, blocking interactions between KIR and HLA molecules constitutes an interesting therapeutic strategy. The anti-KIR2DL1/2/3-specific monoclonal antibody, lirilumab/BMS-986015/IPH2102, is a human IgG4 that is being developed for treating both hematologic malignancies and solid tumors. In rodents, the MHC class I inhibitory system regulating NK cell activation is based on lectin-like family Ly49 but the KIR molecules are not expressed. The objective of this study was to develop a preclinical model to assess the efficacy of the drug candidate used in clinical trials, lirilumab. Mice expressing the human NK inhibitory KIR2DL3, on the surface of NK cells were generated on a RAG-1deficient background (KIRtgRAG mice). The human B cell lymphoma cell line, 721.221, transduced with HLA-Cw3 molecule, a ligand of KIR2DL3, was intra-venously engrafted in these mice. The expression of HLA-C by tumor cells was sufficient to allow them to escape control of NK cells, leading to mice death in around 30 days. Lirilumab treatment increased mice survival in a dose dependent manner when injected at the same time as the tumor challenge. This protective effect was NK cell-mediated and directly correlated with the duration of KIR saturation. Interestingly, lirilumab treatment also improved survival in therapeutic conditions i.e. when the antibody was injected 5 days after the tumor, also in a NK cell-dependent manner. In conclusion, this study showed efficacy of lirilumab as single agent in a HLA-Cw3-expressing tumor model and this xenogenic pre-clinical model will be an excellent tool to investigate the therapeutic benefits of combination treatments.

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.