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.

中文翻译：

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AGI-134：一种全合成的 α-Gal 糖脂，可将肿瘤转化为原位自体疫苗，诱导抗肿瘤免疫，并与小鼠黑色素瘤模型中的抗 PD-1 抗体产生协同作用。

背景 对患者独特的新抗原产生 T 细胞介导的免疫的治疗是当前癌症免疫治疗的圣杯。特别是，不需要繁琐和个性化的离体处理或制造过程的治疗尤其受到追捧。在这里，我们报告了 AGI-134，一种糖脂样小分子，可用于用异种抗原 Galα1-3Galβ1-4GlcNAc (α-Gal) 原位包被肿瘤细胞，导致与预先存在的天然抗α-Gal 进行调理作用抗体（简称抗Gal），它触发免疫级联反应，导致T细胞介导的抗肿瘤免疫。方法 采用流式细胞仪测定体外经 AGI-134 α-Gal 包被肿瘤细胞的各种免疫学效应：（1）抗-Gal 和补体调理作用，(2) NK 细胞的抗体依赖性细胞介导的细胞毒性 (ADCC)，以及 (3) 抗原呈递细胞 (APC) 的吞噬作用和抗原交叉呈递。活力试剂盒用于测试 AGI-134 介导的癌细胞中的补体依赖性细胞毒性 (CDC)。AGI-134 单独或与抗程序性死亡 1（抗 PD-1）抗体组合的抗肿瘤活性在黑色素瘤模型中在抗 Gal 表达半乳糖基转移酶敲除 (α1,3GT-/-) 小鼠中进行了测试. 通过单向方差分析分析CDC和吞噬作用数据，通过配对t检验分析ADCC结果，通过Mantel-Cox检验分析远端肿瘤生长，通过Mann-Whitney检验分析C5a数据，通过重复测量分析分析单个肿瘤消退。结果 在体外，通过 AGI-134 掺入细胞膜对肿瘤细胞进行 α-Gal 标记导致抗 Gal 结合和补体激活。通过补体和ADCC的作用，肿瘤细胞被裂解，APCs对肿瘤抗原的摄取增加。与裂解细胞相关的抗原由 CD8α+ 树突状细胞交叉呈递，导致抗原特异性 CD8+ T 细胞活化。在 α1,3GT-/- 小鼠的 B16-F10 或 JB/RH 黑色素瘤模型中，瘤内 AGI-134 给药导致原发性肿瘤消退并具有强大的远隔效应，即它可以防止远端未注射病变的发展。AGI-134 和抗 PD-1 抗体的组合在保护继发性肿瘤生长方面显示出协同益处。结