Thierry Menguy

TG1050, an immunotherapeutic to treat chronic hepatitis B, induces robust T cells and exerts an antiviral effect in HBV-persistent mice

Objective To assess a new adenovirus-based immunotherapy as a novel treatment approach to chronic hepatitis B (CHB). Methods TG1050 is a non-replicative adenovirus serotype 5 encoding a unique large fusion protein composed of a truncated HBV Core, a modified HBV Polymerase and two HBV Envelope domains. We used a recently described HBV-persistent mouse model based on a recombinant adenovirus-associated virus encoding an over length genome of HBV that induces the chronic production of HBsAg, HBeAg and infectious HBV particles to assess the ability of TG1050 to induce functional T cells in face of a chronic status. Results In in vitro studies, TG1050 was shown to express the expected large polyprotein together with a dominant, smaller by-product. Following a single administration in mice, TG1050 induced robust, multispecific and long-lasting HBV-specific T cells detectable up to 1 year post-injection. These cells target all three encoded immunogens and display bifunctionality (ie, capacity to produce both interferon γ and tumour necrosis factor α as well as cytolytic functions). In addition, control of circulating levels of HBV DNA and HBsAg was observed while alanine aminotransferase levels remain in the normal range. Conclusions Injection of TG1050 induced both splenic and intrahepatic functional T cells producing cytokines and displaying cytolytic activity in HBV-naïve and HBV-persistent mouse models together with significant reduction of circulating viral parameters. These results warrant clinical evaluation of TG1050 in the treatment of CHB.

Therapeutic Effects of Anti-CD115 Monoclonal Antibody in Mouse Cancer Models through Dual Inhibition of Tumor-Associated Macrophages and Osteoclasts

Tumor progression is promoted by Tumor-Associated Macrophages (TAMs) and metastasis-induced bone destruction by osteoclasts. Both myeloid cell types depend on the CD115-CSF-1 pathway for their differentiation and function. We used 3 different mouse cancer models to study the effects of targeting cancer host myeloid cells with a monoclonal antibody (mAb) capable of blocking CSF-1 binding to murine CD115. In mice bearing sub-cutaneous EL4 tumors, which are CD115-negative, the anti-CD115 mAb depleted F4/80+ CD163+ M2-type TAMs and reduced tumor growth, resulting in prolonged survival. In the MMTV-PyMT mouse model, the spontaneous appearance of palpable mammary tumors was delayed when the anti-CD115 mAb was administered before malignant transition and tumors became palpable only after termination of the immunotherapy. When administered to mice already bearing established PyMT tumors, anti-CD115 treatment prolonged their survival and potentiated the effect of chemotherapy with Paclitaxel. As shown by immunohistochemistry, this therapeutic effect correlated with the depletion of F4/80+CD163+ M2-polarized TAMs. In a breast cancer model of bone metastasis, the anti-CD115 mAb potently blocked the differentiation of osteoclasts and their bone destruction activity. This resulted in the inhibition of cancer-induced weight loss. CD115 thus represents a promising target for cancer immunotherapy, since a specific blocking antibody may not only inhibit the growth of a primary tumor through TAM depletion, but also metastasis-induced bone destruction through osteoclast inhibition.

A unique anti-CD115 monoclonal antibody which inhibits osteolysis and skews human monocyte differentiation from M2-polarized macrophages toward dendritic cells

Cancer progression has been associated with the presence of tumor-associated M2-macrophages (M2-TAMs) able to inhibit anti-tumor immune responses. It is also often associated with metastasis-induced bone destruction mediated by osteoclasts. Both cell types are controlled by the CD115 (CSF-1R)/colony-stimulating factor-1 (CSF-1, M-CSF) pathway, making CD115 a promising target for cancer therapy. Anti-human CD115 monoclonal antibodies (mAbs) that inhibit the receptor function have been generated in a number of laboratories. These mAbs compete with CSF-1 binding to CD115, dramatically affecting monocyte survival and preventing osteoclast and macrophage differentiation, but they also block CD115/CSF-1 internalization and degradation, which could lead to potent rebound CSF-1 effects in patients after mAb treatment has ended. We thus generated and selected a non-ligand competitive anti-CD115 mAb that exerts only partial inhibitory effects on CD115 signaling without blocking the internalization or the degradation of the CD115/CSF-1 complex. This mAb, H27K15, affects monocyte survival only minimally, but downregulates osteoclast differentiation and activity. Importantly, it inhibits monocyte differentiation to CD163+CD64+ M2-polarized suppressor macrophages, skewing their differentiation toward CD14-CD1a+ dendritic cells (DCs). In line with this observation, H27K15 also drastically inhibits monocyte chemotactic protein-1 secretion and reduces interleukin-6 production; these two molecules are known to be involved in M2-macrophage recruitment. Thus, the non-depleting mAb H27K15 is a promising anti-tumor candidate, able to inhibit osteoclast differentiation, likely decreasing metastasis-induced osteolysis, and able to prevent M2 polarization of TAMs while inducing DCs, hence contributing to the creation of more efficient anti-tumor immune responses.

3D modeling and characterization of the human CD115 monoclonal antibody H27K15 epitope and design of a chimeric CD115 target

The humanized monoclonal antibody H27K15 specifically targets human CD115, a type III tyrosine kinase receptor involved in multiple cancers and inflammatory diseases. Binding of H27K15 to hCD115 expressing cells inhibits the functional effect of colony-stimulating factor-1 (CSF-1), in a non-competitive manner. Both homology modeling and docking programs were used here to model the human CD115 extracellular domains, the H27K15 variable region and their interaction. The resulting predicted H27K15 epitope includes mainly the D1 domain in the N-terminal extracellular region of CD115 and some residues of the D2 domain. Sequence alignment with the non-binding murine CD115, enzyme-linked immunosorbent assay, nuclear magnetic resonance spectroscopy and affinity measurements by quartz crystal microbalance revealed critical residues of this epitope that are essential for H27K15 binding. A combination of computational simulations and biochemical experiments led to the design of a chimeric CD115 carrying the human epitope of H27K15 in a murine CD115 backbone that is able to bind both H27K15 as well as the murine ligands CSF-1 and IL-34. These results provide new possibilities to minutely study the functional effects of H27K15 in a transgenic mouse that would express this chimeric molecule.

Comparative analysis of immunization schedules using a novel adenovirus-based immunotherapeutic targeting hepatitis B in naïve and tolerant mouse models

Development of active targeted immunotherapeutics is a rapid developing field in the arena of chronic infectious diseases. The question of repeated, closely spaced administration of immunotherapeutics to achieve a rapid impact on the replicating agent is an important one. We analyzed here, using a prototype adenovirus-based immunotherapeutic encoding Core and Polymerase from the hepatitis B virus (Ad-HBV), the influence of closely spaced repeated immunizations on the level and quality of induced HBV-specific and vector-specific immune responses in various mouse models. Ad-HBV, whether injected once or multiple times, was able to induce HBV- and adeno-specific T cells both in HBV-free mice and in a HBV tolerant mouse model. Adenovirus-specific T cell responses and titers of neutralizing anti-Ad5 antibodies increased from time of the 3rd injection. Interestingly, single or multiple Ad-HBV injections resulted in detection of Polymerase-specific functional T cells in HBV tolerant mice. Overall no modulation of the levels of HBV-specific cytokine-producing (IFNγ/TNFα) and cytolytic T cells was observed following repeated administrations (3 or 6 weekly injections) when compared with levels detected after a single injection with the exception of two markers: 1. the proportion of HBV-specific IFNγ-producing cells bearing the CD27+/CD43+ phenotype appeared to be sustained in C57BL/6J mice following 6 weekly injections; 2. the percentage of IFNγ/TNFα Core-specific producing cells observed in spleens of HLA-A2 mice as well as of that specific of Polymerase observed in livers of HBV tolerant mice was maintained. In addition, percentage of HBV-specific T cells expressing PD-1 was not increased by multiple injections. Overall these data show that, under experimental conditions used, rapid, closely spaced administrations of an adenovirus-based HBV immunotherapeutics does not inhibit induced T-cell responses including in a HBV-tolerant environment.

Yeast Virus-Derived Stimulator of the Innate Immune System Augments the Efficacy of Virus Vector-Based Immunotherapy

ABSTRACT To identify novel stimulators of the innate immune system, we constructed a panel of eight HEK293 cell lines double positive for human Toll-like receptors (TLRs) and an NF-κB-inducible reporter gene. Screening of a large variety of compounds and cellular extracts detected a TLR3-activating compound in a microsomal yeast extract. Fractionation of this extract identified an RNA molecule of 4.6 kb, named nucleic acid band 2 (NAB2), that was sufficient to confer the activation of TLR3. Digests with single- and double-strand-specific RNases showed the double-strand nature of this RNA, and its sequence was found to be identical to that of the genome of the double-stranded RNA (dsRNA) L-BC virus of Saccharomyces cerevisiae. A large-scale process of production and purification of this RNA was established on the basis of chemical cell lysis and dsRNA-specific chromatography. NAB2 complexed with the cationic lipid Lipofectin but neither NAB2 nor Lipofectin alone induced the secretion of interleukin-12(p70) [IL-12(p70)], alpha interferon, gamma interferon-induced protein 10, macrophage inflammatory protein 1β, or IL-6 in human monocyte-derived dendritic cells. While NAB2 activated TLR3, Lipofectin-stabilized NAB2 also signaled via the cytoplasmic sensor for RNA recognition MDA-5. A significant increase of RMA-MUC1 tumor rejection and survival was observed in C57BL/6 mice after prophylactic vaccination with MUC1-encoding modified vaccinia virus Ankara (MVA) and NAB2-Lipofectin. This combination of immunotherapies strongly increased at the injection sites the percentage of infiltrating natural killer (NK) cells and plasmacytoid dendritic cells (pDCs), cell types which can modulate innate and adaptive immune responses. IMPORTANCE Virus-based cancer vaccines offer a good alternative to the treatment of cancer but could be improved. Starting from a screening approach, we have identified and characterized an unexplored biological molecule with immunomodulatory characteristics which augments the efficacy of an MVA-based immunotherapeutic agent. The immune modulator consists of the purified dsRNA genome isolated from a commercially used yeast strain, NAB2, mixed with a cationic lipid, Lipofectin. NAB2-Lipofectin stimulates the immune system via TLR3 and MDA-5. When it was injected at the MVA vaccination site, the immune modulator increased survival in a preclinical tumor model. We could demonstrate that NAB2-Lipofectin augments the MVA-induced infiltration of natural killer and plasmacytoid dendritic cells. We suggest indirect mechanisms of activation of these cell types by the influence of NAB2-Lipofectin on innate and adaptive immunity. Detailed analysis of cell migration at the vaccine injection site and the appropriate choice of an immune modulator should be considered to achieve the rational improvement of virus vector-based vaccination by immune modulators.

CD160 Expression in Retinal Vessels Is Associated With Retinal Neovascular Diseases

Purpose Anti-angiogenic agents stand first in the treatment of neovascular diseases of the retina. CD160 appeared in several experimental studies as a marker of activated endothelial cells, suggesting it could represent a promising target for novel anti-angiogenic therapies. The aim of the present study was to assess the distribution of CD160 in the human eye, and to search for a possible correlation with retinal neovascular diseases. Methods The physiological distribution of CD160 in the normal eye was assessed with immunolabeling in 10 human donor eyes. Then, in a retrospective cohort of 75 surgical retinal specimens, the density of CD160+ microvessels was evaluated, along with immunolabeling on serial sections against ERG (pan-endothelial cell marker), CD105 (activated endothelial cell marker), and α-SMA (pericyte cell marker). The cohort was divided into two groups: 29 patients with neovascular disease (NV+) and 46 control patients (NV-). Results CD160 was physiologically expressed by several cell types: endothelial cells of retinal blood vessels, ganglion cells, macrophages, epithelial cells of the conjunctiva, ciliary body, and retinal pigment epithelium. In the patient cohort, the percentage of CD160+ vessels in the retina was significantly and independently higher in patients suffering from neovascular diseases (P = 0.04). On the contrary, the expression of CD105 was correlated neither with retinal neovascular diseases, nor with CD160 expression. Conclusions CD160 was expressed in some retinal vessels in both normal and pathologic eyes. CD160 expression by endothelial cells of retinal vessels was correlated with ocular neovascular diseases. CD160 could therefore represent an interesting target for novel anti-angiogenic therapies.

Anti-CD160, Alone or in Combination With Bevacizumab, Is a Potent Inhibitor of Ocular Neovascularization in Rabbit and Monkey Models

Purpose To assess the efficacy of the murine first-in-class CL1-R2 monoclonal antibody (mAb) targeting human CD160 (alone or in combination with bevacizumab) by using the rabbit corneal neovascularization (CNV) model, and determine the safety and efficacy of ELB01101, a novel CL1-R2-derived humanized IgG4 mAb, in a monkey model of choroidal neovascularization (ChNV). Methods Comparison of effect of CL1-R2, bevacizumab, or aflibercept or IgG1 (control) injections in early and late treatment schemes on evolution of VEGF- or FGF2-induced rabbit CNV was performed. In the combination setting, bevacizumab was coinjected with different doses of CL1-R2. ELB01101 or vehicle was administered intravitreally in monkeys after laser-induced ChNV. Individual laser-induced lesions were semiquantitatively graduated by using fluorescein angiography to determine leakage. Results In the rabbit model, early and late treatments with CL1-R2 significantly decreased both area and length of CNV neovessels. The effect was as potent as produced with anti-VEGF comparators. When combined with bevacizumab, an additive effect of CL1-R2 was measured at all doses tested. In the ChNV model, on day 29, eyes treated with ELB01101 showed a statistically significant reduction in clinically relevant lesions compared to vehicle-treated eyes (∼50%; χ2 test, P = 0.032001). Conclusions The additive effects of anti-CD160 and bevacizumab in the CNV model suggest that these compounds could act via different pathways, opening new therapeutic pathways for cotargeted or combination therapies. In the ChNV model, ELB01101 was well tolerated and prevented approximately 50% of clinically relevant lesions, validating CD160 targeting as a safe approach for treatment of retinal diseases in the most relevant animal model of wet AMD.

Abstract 288: TG3003, an immunomodulatory anti-CD115 mAb targeting M2-macrophage polarization in the tumor microenvironment

Cancer progression has been associated with the presence of tumor-associated M2-type macrophages (M2-TAMs) able to inhibit anti-tumor immune responses, stimulate neo-angiogenesis and facilitate metastasis. Colony-stimulating factor-1 (CSF-1, M-CSF) is a cytokine required for the survival and differentiation of myeloid cell lineages, and CSF-1 signaling is known to polarize macrophages towards the M2-type. M2-TAMs can represent the most abundant immunosuppressive cell population in the tumor microenvironment, notably recruited by CSF-1 and MCP-1/CCL2. Transgene has developed a monoclonal antibody (mAb), TG3003, directed against the CSF-1 cell-surface receptor, CD115 (CSF-1R, M-CSFR). This mAb does not block the binding of CSF-1 to its receptor, but down-modulates CD115 signaling. In contrast to other anti-CD115 mAbs currently in development, whose modes of action rely on the blockade of ligand binding, TG3003 is not cytotoxic to normal myeloid cells that require CD115-mediated signaling for their survival. In vitro, TG3003 skews monocyte differentiation from M2-type macrophages towards dendritic cells, most potent antigen-presenting cells capable of stimulating efficacious T cell responses. It inhibits the secretion of MCP-1/CCL2 by differentiating macrophages and decreases their IL-6 production. Through this inhibition of M2-TAMs, TG3003 may potentiate immune responses in patients and impact on tumor progression. Moreover, due to its unique non-competitive mode of action, TG3003 does not block the physiological pathway for CSF-1 clearing from the circulation, thus avoiding the issue of toxic or rebound effects in treated patients. To investigate the properties of TG3003 in vivo, we have generated a transgenic mouse strain where the mAb epitope has been inserted into murine CD115 without affecting murine CSF-1 binding nor signaling. We will present the results of preclinical proof-of-concept experiments validating the mechanism of action and the immunomodulatory properties of mAb TG3003. Citation Format: Helene Haegel, Christelle Ziller-Remy, Luc Barraud, Jean-Yves Bonnefoy, Sandrine Cochin, Vanessa Duong, Michel Geist, Benoit Grellier, Remy Hallet, Jean-Baptiste Marchand, Thierry Menguy, Ronald Rooke, Christine Thioudellet, Carine Reymann, Xavier Preville. TG3003, an immunomodulatory anti-CD115 mAb targeting M2-macrophage polarization in the tumor microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 288. doi:10.1158/1538-7445.AM2015-288