Hepatitis C virus (HCV) is a global health concern; recent estimates suggest that 2.2 to 3% of the world's population, equivalent to 130 to 170 million individuals, are chronically infected with the virus (13, 31). These patients are at risk of developing debilitating liver diseases such as cirrhosis and hepatocellular carcinoma (1). Furthermore, current models suggest that the burden of HCV-associated disease is set to rise for the next 20 years (6). There is no HCV vaccine; the current standard of care (SOC) involves lengthy treatments with ribavirin and injected pegylated interferon, which exhibit variable efficacies and are associated with severe, and sometimes lifethreatening, side effects. Encouragingly, many direct-acting antiviral (DAA) molecules are in clinical development, and the most advanced (telaprevir and boceprevir) will probably be used to treat HCV-infected patients in 2011 (19,29,42,43,61). However, caution should be employed against overoptimism; attrition rates are high during drug development, and the first drugs will be given in combination with, not instead of, the current SOC. Therefore, the continued development of additional treatments is needed, especially since it is widely acknowledged that to limit the emergence of drug-resistant viral variants, effective therapeutic strategies for HCV will consist of multiple DAAs (50).A multitude of screening campaigns has revealed many diverse and interesting chemical compounds capable of specifically inhibiting HCV RNA replication. Many of these compounds target the HCV-encoded nonstructural (NS) proteins (NS3, NS4A, NS4B, NS5A, and NS5B), which are required for HCV genome synthesis (3, 37). To instigate HCV genome replication, the NS proteins interact with viral genomes and certain host-encoded factors to form multiprotein assemblies termed "replication complexes" (RCs), which are sites of viral RNA synthesis derived from the endoplasmic reticulum (ER) (8,14,45,53). In HCV-infected cells, RCs are juxtaposed to intracellular lipid storage organelles termed lipid droplets (LDs), which are coated with the HCV capsid protein (core) and probably serve as platforms to accept replicated genomes from RCs to initiate virion assembly (26,44,53). Of considerable interest are inhibitors that target the HCV-encoded NS5A protein. These inhibitors were originally discovered from the screening of cells containing HCV subgenomic replicons against libraries of small molecules and were identified as NS5A inhibitors by utilizing a strategy termed "chemical genetics" (12, 32). NS5A-targeting inhibitors are notable for their unprecedented potency in cell-based HCV replication assays: 50% inhibitory concentrations (IC 50 s) in the low-picomolar
BackgroundTreatments that generate T cell-mediated immunity to a patient’s unique neoantigens are the current holy grail of cancer immunotherapy. In particular, treatments that do not require cumbersome and individualized ex vivo processing or manufacturing processes are especially sought after. Here we report that AGI-134, a glycolipid-like small molecule, can be used for coating tumor cells with the xenoantigen Galα1-3Galβ1-4GlcNAc (α-Gal) in situ leading to opsonization with pre-existing natural anti-α-Gal antibodies (in short anti-Gal), which triggers immune cascades resulting in T cell mediated anti-tumor immunity.MethodsVarious immunological effects of coating tumor cells with α-Gal via AGI-134 in vitro were measured by flow cytometry: (1) opsonization with anti-Gal and complement, (2) antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells, and (3) phagocytosis and antigen cross-presentation by antigen presenting cells (APCs). A viability kit was used to test AGI-134 mediated complement dependent cytotoxicity (CDC) in cancer cells. The anti-tumoral activity of AGI-134 alone or in combination with an anti-programmed death-1 (anti-PD-1) antibody was tested in melanoma models in anti-Gal expressing galactosyltransferase knockout (α1,3GT−/−) mice. CDC and phagocytosis data were analyzed by one-way ANOVA, ADCC results by paired t-test, distal tumor growth by Mantel–Cox test, C5a data by Mann–Whitney test, and single tumor regression by repeated measures analysis.ResultsIn vitro, α-Gal labelling of tumor cells via AGI-134 incorporation into the cell membrane leads to anti-Gal binding and complement activation. Through the effects of complement and ADCC, tumor cells are lysed and tumor antigen uptake by APCs increased. Antigen associated with lysed cells is cross-presented by CD8α+ dendritic cells leading to activation of antigen-specific CD8+ T cells. In B16-F10 or JB/RH melanoma models in α1,3GT−/− mice, intratumoral AGI-134 administration leads to primary tumor regression and has a robust abscopal effect, i.e., it protects from the development of distal, uninjected lesions. Combinations of AGI-134 and anti-PD-1 antibody shows a synergistic benefit in protection from secondary tumor growth.ConclusionsWe have identified AGI-134 as an immunotherapeutic drug candidate, which could be an excellent combination partner for anti-PD-1 therapy, by facilitating tumor antigen processing and increasing the repertoire of tumor-specific T cells prior to anti-PD-1 treatment.
Background: AGI-134 is a fully synthetic α-Gal (Galα1-3Galβ1-4GlcNAc-R) glycolipid that is being developed for the treatment of solid tumors. The α-Gal epitope is not expressed in humans, who, as a result of constant antigenic stimulation by α-Gal-bearing commensal bacteria, develop high titer natural antibodies to α-Gal. We have previously demonstrated that AGI-134 recruits anti-Gal antibodies to tumor cells in vitro, activating complement and driving phagocytosis by antigen presenting cells. AGI-134 also confers systemic protection from distal lesion development in a mouse model of melanoma and synergizes with anti-PD-11. Here we present further data characterizing the multiple pathways activated by the anti-Gal subclasses to drive AGI-134-mediated anti-tumor immunity. Results: Using quantitative methods, we demonstrate that human anti-Gal is composed of a diverse repertoire of effector antibodies in a panel of serum samples, with IgM, IgG1 and IgG2 being the major subclasses. Polyclonal anti-Gal IgG purified from human IVIG was, like human serum, comprised mainly of IgG1 and IgG2. When AGI-134 treated cells were incubated with human serum, binding of all anti-Gal subclasses was detected by flow cytometry, demonstrating that all anti-Gal subclasses can interact with AGI-134 treated cells and are available to activate downstream effector functions. When human serum was depleted of IgG, AGI-134 stimulated complement dependent cytotoxicity (CDC) was still effective, indicating that anti-Gal IgM is primarily responsible for deposition of complement on AGI-134 treated cells. AGI-134 treated cells incubated with purified polyclonal anti-Gal IgG activated FcγRIIIA on a reporter cell line and promoted natural killer cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC). Opsonization of AGI-134 treated human cancer cells with human anti-Gal and complement stimulated their phagocytosis by human monocyte-derived macrophages. Ovalbumin-expressing cells treated with AGI-134 and then incubated with human serum to initiate CDC were specifically phagocytosed by murine CD8α+ dendritic cells and the immunodominant antigen of ovalbumin, SIINFEKL, was cross-presented to CD8+ T cells. In conclusion, AGI-134 stimulates adaptive anti-tumor immunity through immune activation and antigen cross-presentation, which is driven by the diverse repertoire of anti-Gal antibodies. 1. Shaw, S. et al. Abstract 4862: AGI-134: a fully synthetic alpha-Gal glycolipid that prevents the development of distal lesions and is synergistic with an anti-PD-1 antibody in a mouse melanoma model. [abstract]. AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4862. Citation Format: Jenny L. Middleton, Oliver Schulz, Amber Charlemagne, Sascha A. Kristian, Stephen Michael Shaw. The novel α-Gal-based immunotherapy AGI-134 invokes CD8+ T cell-mediated immunity by driving tumor cell destruction, phagocytosis and tumor-associated antigen cross-presentation via multiple antibody-mediated effector functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 616. doi:10.1158/1538-7445.AM2017-616
Background: AGI-134 is a fully synthetic glycolipid, composed of an alpha-Gal (Galá1-3Galâ1-4GlcNAc-R) sugar epitope attached via a linker to a lipid tail. Natural antibodies to the alpha-Gal epitope are responsible for the hyperacute rejection of xenografts in humans. It is proposed that intratumorally administered AGI-134 will incorporate into the cell membranes of the tumor cells, presenting the alpha-Gal epitope for binding of anti-Gal antibodies to the tumor cells. This will initiate an immune response that attacks the injected tumor and, through uptake of immune-complexed tumor antigens by antigen presenting cells, will create a patient-specific, systemic anti-tumor response against distant metastases. Results: We demonstrate that AGI-134 incorporates into tumor cell membranes in vitro and that the exposed alpha-Gal epitope binds anti-Gal IgG and IgM antibodies from human serum to the tumor cell surface. Using flow cytometry and a complement-dependent cytotoxicity assay we show that tumor cell opsonization with anti-Gal antibodies leads to deposition of complement proteins C3b and C5b-9, which ultimately leads to tumor cell lysis. Furthermore, we demonstrate that AGI-134-labeled tumor cells opsonized with human serum proteins are phagocytosed by professional APCs. Using the B16-F10 melanoma model in anti-Gal producing á1,3-galactosyltransferase knockout (GT KO) mice we present data to demonstrate that AGI-134 injection into a primary tumor provides significant dose-dependent protection from the development of established distant lesions. Using GT KO mouse serum we demonstrate in vitro that deposition of complement on AGI-134-labeled mouse tumor cells is both alpha-Gal and anti-Gal dependent. In vivo, we demonstrate that the effect of AGI-134 is due to the alpha-Gal moiety by replacing it with human blood group antigens. The protection from secondary lesions conferred by AGI-134 is long lasting in the GT KO mouse melanoma model (monitored up to 90 days). Importantly, when sub-optimal concentrations of AGI-134 were tested in vivo in combination with an anti-PD-1 antibody (RMP1-14), a significant enhancement in efficacy over either of the agents administered alone was observed. Citation Format: Stephen Shaw, Sascha Kristian, Kim Wigglesworth, Jenny Middleton, Mel Glossop, Giles Whalen, Robert Old, Mike Westby, Chris Pickford. AGI-134: a fully synthetic alpha-Gal glycolipid that prevents the development of distal lesions and is synergistic with an anti-PD-1 antibody in a mouse melanoma model. [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 4862.
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