Hélène Sicard

In Vivo Immunomanipulation of Vγ9Vδ2 T Cells with a Synthetic Phosphoantigen in a Preclinical Nonhuman Primate Model

Vγ9Vδ2+ cells represent the major population of γδ T cells in primate blood and react in an MHC-unrestricted fashion to a set of low m.w. nonpeptide phosphoantigens. Two types of structurally related agonists have been discovered so far: the natural phosphoantigens (hydroxydimethyl allyl-pyrophosphate or isopentenyl-pyrophosphate (IPP)) acting directly on Vγ9Vδ2+ TCR and aminobisphosphonates, which block the mevalonate pathway in target cells, leading to accumulation of natural phosphoantigens that in turn activate Vγ9Vδ2+ cells. We demonstrate in the cynomolgus monkey that Vγ9Vδ2 can be manipulated in vivo with bromohydrin pyrophosphate (BrHPP)/Phosphostim, a potent synthetic agonist for which the mechanism of action is similar to natural phosphoantigens. Although of very short half-life, injection of BrHPP leads to strong activation of Vγ9Vδ2, inducing production of a high level of Th1 cytokines. Combination of BrHPP with low-dose rhIL-2 induces specific amplification of effector-memory peripheral Vγ9Vδ2 in blood in a dose-dependant manner. This transient response returns to baseline within 10–15 days. Successive infusions of BrHPP and rhIL-2 induce less vigorous expansions, suggesting a progressive exhaustion of the response. As no toxicity is detected with or without IL-2, this scheme represents a promising immunotherapeutic strategy for induction of systemic Th1 cytokines and massive expansion of γδ T cell subset with antitumor and anti-infectious properties.

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.

What lessons can be learned from γδ T cell-based cancer immunotherapy trials?

During the last several years, research has produced a significant amount of knowledge concerning the characteristics of human γδ T lymphocytes. Findings regarding the immune functions of these cells, particularly their natural killer cell-like lytic activity against tumor cells, have raised expectations for the therapeutic applications of these cells for cancer. Pharmaceutical companies have produced selective agonists for these lymphocytes, and several teams have launched clinical trials of γδ T cell-based cancer therapies. The findings from these studies include hematological malignancies (follicular lymphoma, multiple myeloma, acute and chronic myeloid leukemia), as well as solid tumors (renal cell, breast and prostate carcinomas), consisting of samples from more than 250 patients from Europe, Japan and the United States. The results of these pioneering studies are now available, and this short review summarizes the lessons learned and the role of γδ T cell-based strategies in the current landscape of cancer immunotherapies.

A chemical basis for selective recognition of nonpeptide antigens by human δ T cells 1

Whereas αβ T cell receptors (TCR) recognize processed antigenic peptides or glycolipids bound respectively to major histocompatibility complex or CD1 molecules, γδ TCR react differently to a broad set of native antigens. A major human γδ T cell subset is activated through a mechanism involving Vγ 9Vδ 2 TCR and structurally unrelated phosphorylated nonpeptide antigens (referred to as phosphoantigens). Here, the structure‐function relationship of the strongest natural and synthetic phosphoantigens stimulating γδ cells was analyzed to elucidate the molecular basis of this unconventional recognition. Besides conformational determinants, we found that chemical reactivity of antigens is critical to their bioactivity. For Vγ 9Vδ 2 T cell activation, both organic and phosphorylated moieties of strong ligands undergo rapid and degradative structural changes. Conversely, analogs that are resistant to degradation specifically antagonize phosphoantigen‐mediated γδ T cell activation. These data suggest a novel mode of antigen perception involving both topological recognition and ligand consumption, which confers highly specific γδ T cell activation by structurally diverse ligands.

Synthesis and Biological Activity of Phosphonate Analogues and Geometric Isomers of the Highly Potent Phosphoantigen (E)-1-Hydroxy-2-methylbut-2-enyl 4-Diphosphate

Gammadelta-T-lymphocytes contribute to innate immunity and are selectively activated by nonpeptide phosphorylated molecules (so-called phosphoantigens) produced by organisms responsible for causing a broad range of infectious diseases. gammadelta-T-cells are also activated by synthetic phosphoantigens and are cytotoxic to tumor cells. Here we report the synthesis, NMR characterization, and comparative biological evaluation of new pyrophosphate, phosphonate, and pyrophosphonate monoesters whose structures correspond to isosteric analogues and stereoisomers of the highly potent isoprenoid metabolite ( E)-1-hydroxy-2-methylbut-2-enyl 4-diphosphate called HDMAPP (hydroxy-dimethyl-allyl pyrophosphate). Both pyrophosphate and pyrophosphonate series elicit promising gammadelta-T-cell stimulatory responses in vitro, the pyrophosphonate ester (C-HDMAPP) being by far more stable than its parent pyrophosphate ester (HDMAPP) with improved ADMET properties and a similar pharmacodynamic profile based on in vivo studies in nonhuman primate. In both series, we found that E-stereoisomers are the most active derivatives and that Z-stereoisomers show very marginal bioactivity levels. These results indicate that the use of bioisosteric analogues of HDMAPP may represent promising new leads for immunotherapy.

Y2K+1 state-of-the-art on non-peptide phosphoantigens, a novel category of immunostimulatory molecules.

Some human T cells are activated in vivo and in vitro by small non-peptide antigens, so-called phosphoantigens. Since their discovery in 1994, several reports have continuously documented novel members of this category of immunostimulatory molecules. This article reviews the current knowledge on their biochemical properties.

A tuberculosis vaccine based on phosphoantigens and fusion proteins induces distinct γδ and αβ T cell responses in primates

Phosphoantigens are mycobacterial non‐peptide antigens that might enhance the immunogenicity of current subunit candidate vaccines for tuberculosis. However, their testing requires monkeys, the only animal models suitable for γδ T cell responses to mycobacteria. Thus here, the immunogenicity of 6‐kDa early secretory antigenic target‐mycolyl transferase complex antigen 85B (ESAT‐6‐Ag85B) (H‐1 hybrid) fusion protein associated or not to a synthetic phosphoantigen was compared by a prime‐boost regimen of two groups of eight cynomolgus. Although phosphoantigen activated immediately a strong release of systemic Th1 cytokines (IL‐2, IL‐6, IFN‐γ, TNF‐α), it further anergized blood γδ T lymphocytes selectively. By contrast, the hybrid H‐1 induced only memory αβ T cell responses, regardless of phosphoantigen. These latter essentially comprised cytotoxic T lymphocytes specific for Ag85B (on average + 430 cells/million PBMC) and few IFN‐γ‐secreting cells (+ 40 cells/million PBMC, equally specific for ESAT‐6 and for Ag85B). Hence, in macaques, a prime‐boost with the H‐1/phosphoantigen subunit combination induces two waves of immune responses, successively by γδ T and αβ T lymphocytes.

Metabolic Routes as Targets for Immunological Discrimination of Host and Parasite

In humans, T lymphocytes bearing a Vγ9Vδ2 antigen receptor (TCR) exhibit strong cytotoxic activity against cells infected by a wide variety of intracellular pathogens, from bacteria (4, 5, 13, 15, 17, 19, 25, 28) to complex eukaryotic parasites (1, 12). It is now well established that the involvement of human γδ T cells in antiinfectious immunity depends on their TCR-dependent activation by small, protease-resistant ligands containing critical phosphate residues (phosphoantigens). Peripheral Vγ9Vδ2 T cells are subjected to an intense postnatal amplification, most probably due to recurrent encounter with these widespread molecules. Such antigens have been isolated from the mycobacteria Plasmodium falciparum and Francisella tularensis (2, 7, 25, 33), and it is suspected that they exist in several other species (15, 18). Thus, it is clear that the phosphoantigens responsible for γδ T-cell activation are broadly distributed in living organisms. It has been shown that the γδ T-cell response is directed towards cells that contain live bacteria (14) as well as towards live parasites (34), which means that the presence of the recognized ligand depends on an active parasitical metabolism rather than on degradation by-products within the host cell. Finally, the absence of a requirement for classical major histocompatibility complex molecules in the activation of Vγ9Vδ2 T cells reveals a mode of antigen recognition totally different from that of αβ T cells, which enables a particularly rapid response.

A chemical basis for recognition of nonpeptide antigens by human gd T cells

Whereas αβ T cell receptors (TCR) recognize processed antigenic peptides or glycolipids bound respectively to major histocompatibility complex or CD1 molecules, γδ TCR react differently to a broad set of native antigens. A major human γδ T cell subset is activated through a mechanism involving Vγ 9Vδ 2 TCR and structurally unrelated phosphorylated nonpeptide antigens (referred to as phosphoantigens). Here, the structure‐function relationship of the strongest natural and synthetic phosphoantigens stimulating γδ cells was analyzed to elucidate the molecular basis of this unconventional recognition. Besides conformational determinants, we found that chemical reactivity of antigens is critical to their bioactivity. For Vγ 9Vδ 2 T cell activation, both organic and phosphorylated moieties of strong ligands undergo rapid and degradative structural changes. Conversely, analogs that are resistant to degradation specifically antagonize phosphoantigen‐mediated γδ T cell activation. These data suggest a novel mode of antigen perception involving both topological recognition and ligand consumption, which confers highly specific γδ T cell activation by structurally diverse ligands.

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.