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Abstract Background Treatments 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. Methods Various 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. Results In 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. Conclusions We 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.

Detailed clonal phenotypic/genotypic analyses explored viral-escape mechanisms during maraviroc-based therapy in highly treatment-experienced participants from the MOTIVATE trials. To allow real-time assessment of samples while maintaining a blind trial, the first 267 enrolled participants were selected for evaluation. At failure, plasma samples from 20/50 participants (16/20 maraviroc-treated) with CXCR4-using virus and all 38 (13 maraviroc-treated) with CCR5-tropic virus were evaluated. Of those maraviroc-treated participants with CXCR4-using virus at failure, genotypic and phenotypic clonal tropism determinations showed >90% correspondence in 14/16 at Day 1 and 14/16 at failure. Phylogenetic analysis of clonal sequences detected pre-treatment progenitor CXCR4-using virus, or on-treatment virus highly divergent from the Day 1 R5 virus, excluding possible co-receptor switch through maraviroc-mediated evolution. Re-analysis of pre-treatment samples using the enhanced-sensitivity Trofile® assay detected CXCR4-using virus pre-treatment in 16/20 participants failing with CXCR4-using virus. Post-maraviroc reversion of CXCR4-use to CCR5-tropic occurred in 7/8 participants with follow-up, suggesting selective maraviroc inhibition of CCR5-tropic variants in a mixed-tropic viral population, not emergence of de novo mutations in CCR5-tropic virus, as the main virologic escape mechanism. Maraviroc-resistant CCR5-tropic virus was observed in plasma from 5 treated participants with virus displaying reduced maximal percent inhibition (MPI) but no evidence of IC50 change. Env clones with reduced MPI showed 1-5 amino acid changes specific to each V3-loop region of env relative to Day 1. However, transferring on-treatment resistance-associated changes using site-directed mutagenesis did not always establish resistance in Day 1 virus, and key 'signature' mutation patterns associated with reduced susceptibility to maraviroc were not identified. Evolutionary divergence of the CXCR4-using viruses is confirmed, emphasizing natural selection not influenced directly by maraviroc; maraviroc simply unmasks pre-existing lineages by inhibiting the R5 virus. For R5-viral failure, resistance development through drug selection pressure was uncommon and manifested through reduced MPI and with virus strain-specific mutational patterns.