Hélène Haegel

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.

Bone‐ and Cartilage‐Protective Effects of a Monoclonal Antibody Against Colony‐Stimulating Factor 1 Receptor in Experimental Arthritis

Colony‐stimulating factor 1 receptor (CSF‐1R) essentially modulates monocyte proliferation, migration, and activation, which are considered important for the pathogenesis of rheumatoid arthritis (RA). We undertook this study to determine CSF‐1R expression in human RA as well as the efficacy of a specific anti–CSF‐1R monoclonal antibody (AFS98) in 2 different animal models of RA.

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.

TLR2 ligation protects effector T cells from regulatory T-cell mediated suppression and repolarizes T helper responses following MVA-based cancer immunotherapy

Cancer immunotherapy is hampered by the immunosuppression maintained by regulatory T cells (Tregs) in tumor-bearing hosts. Stimulation of the Toll-like receptor 2 (TLR2) by Pam3Cys is known to affect Treg-mediated suppression. We found that Pam3Cys increases the proliferation of both CD4+ effector T cells (Teffs) and Tregs co-cultured in vitro, but did not induce the proliferation of Tregs alone upon CD3 and CD28 stimulation. In a mouse model of RMA-MUC1 tumors, Pam3Cys was administered either alone or in combination with a modified vaccinia ankara (MVA)-based mucin 1 (MUC1) therapeutic vaccine. The combination of Pam3Cys with MVA-MUC1 (1) diminished splenic Treg/CD4+ T-cell ratios to those found in tumor-free mice, (2) stimulated a specific anti-MUC1 interferon γ (IFNγ) response and (3) had a significant therapeutic effect on tumor growth and mouse survival. When CD4+ Teffs and Tregs were isolated from Pam3Cys-treated mice, Teffs had become resistant to Treg-mediated suppression while upregulating the expression of BclL-xL. Tregs from Pam3Cys-treated mice were fully suppressive for Teffs from naïve mice. Bcl-xL was induced by Pam3Cys with different kinetics in Tregs and Teffs. Teff from Pam3Cys-treated mice produced increased levels of Th1 and Th2-type cytokines and an interleukin (IL)-6-dependent secretion of IL-17 was observed in Teff:Treg co-cultures, suggesting that TLR2 stimulation had skewed the immune response toward a Th17 profile. Our results show for the first time that in a tumor-bearing host, TLR2 stimulation with Pam3Cys affects both Tregs and Teffs, protects Teff from Treg-mediated suppression and has strong therapeutic effects when combined with an MVA-based antitumor vaccine.

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.

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