Cell type-directed Tim-3 targeting in melanoma

项目摘要 免疫检查点阻断在转移性乳腺癌患者中引起了前所未有的临床反应。 黑色素瘤和其他癌症。在癌症治疗试验中正在研究的一个有希望的新检查点是T- 细胞免疫球蛋白和粘蛋白结构域3（Tim-3）。Tim-3阻断逆转T细胞损伤，从而 重新激活抗肿瘤T细胞免疫。然而，我们发现Tim-3抑制剂，包括那些在临床上， 试验中，不仅靶向T细胞Tim-3，而且对树突状细胞（DC）上的Tim-3具有不同的亲和力， 巨噬细胞（MΦs）、NK和黑素瘤细胞。因此，临床获益可能并不完全依赖于 T细胞-Tim-3，而且还抑制这些额外的Tim-3表达细胞类型。为了支持，封锁T- 细胞Tim-3抑制，而黑色素瘤定向Tim-3抑制增强小鼠黑色素瘤中的肿瘤生长 模型，从而抵消Tim-3疗法的期望功效。一致，强制表达Tim-3， 黑色素瘤细胞抑制肿瘤发生、转移形成和增殖途径活性。我们 因此，初步研究将黑色素瘤细胞-内在的DC-、MΦ-和NK-细胞-Tim-3确定为意外变量 和/或治疗结果的潜在混杂因素。他们进一步强调需要定义治疗性 Tim-3抗体（Ab）反应在特定细胞类型水平上的后果。Tim-3蛋白熊 多种N-和O-糖结构，在细胞之间的组成、大小和电荷方面存在显著差异 这可能解释了我们在细胞间发现的Tim-3 Ab克隆反应性的显著变化。 类型例如，临床Tim-3试验候选药物TSR-022与T细胞和黑色素瘤紧密结合，但不 NK-、DC-或MΦ-Tim-3，而其他Tim-3 Ab对T细胞、MΦ、DC和/或NK上的Tim-3显示出高亲和力， 而不是黑素瘤细胞。值得注意的是，聚糖修饰方案使抑制剂结合向所需的T细胞结合转移。 Tim-3识别和黑色素瘤-Tim-3反应性降低。我们的初步数据强调了以下方面的迫切需要： 解剖免疫细胞与黑色素瘤细胞-Tim-3糖表位、Ab亲和力、信号传导和免疫生物学。结果 将通过验证优先靶向免疫细胞Tim-3的方案来帮助优化Tim-3的治疗功效 聚糖，同时避免黑色素瘤细胞Tim-3的不必要的封锁。我们的目标是1）定义细胞类型- 相关的Tim-3聚糖部分、配体、Ab亲和力和信号传导网络，2）检查免疫细胞- vs. 黑色素瘤-现有Tim-3拮抗剂的内在作用及其与解释治疗益处的相关性， 和3）鉴定新的Tim-3靶向策略，其加强免疫细胞Tim-3抑制。我们将使用国家- Tim-3功能的最新获得和丧失以及聚糖修饰策略，具有可变组织- 相关亲和力，以及免疫和黑素瘤模型系统，以定义细胞类型特异性Tim-3功能 和糖分子靶点。我们的倡议还实施了临床肿瘤生物标本， 免疫检查点抑制剂。总之，这些研究将为下一代生物标志物铺平道路， 区分免疫细胞和癌细胞Tim-3的治疗模式，以优化免疫治疗结果。

PROJECT SUMMARY Immune checkpoint blockade has elicited unprecedented clinical responses in patients with metastatic melanoma and other cancers. A promising new checkpoint under investigation in cancer therapeutic trials is T- cell immunoglobulin and mucin domain 3 (Tim-3). Tim-3 blockade reverses T-cell impairment, thereby reinvigorating antitumor T-cell immunity. However, we found that Tim-3 inhibitors, including those in clinical trials, not only target T-cell-Tim-3, but also have varying affinity for Tim-3 on dendritic cells (DCs), macrophages (MΦs), NK and melanoma cells. Clinical benefit might thus not exclusively rely on antagonism of T-cell-Tim-3, but also on inhibition of these additional Tim-3-expressing cell types. In support, blockade of T- cell-Tim-3 suppressed, while melanoma-directed Tim-3 inhibition enhanced tumor growth in murine melanoma models, thereby counteracting desired efficacy of Tim-3 therapy. Consistently, enforced expression of Tim-3 on melanoma cells suppressed tumorigenesis, metastasis formation, and proliferative pathway activity. Our preliminary studies thus identify melanoma cell-intrinsic, DC-, MΦ-, and NK-cell-Tim-3 as unexpected variables and/or potential confounders of treatment outcome. They further highlight the need to define therapeutic consequences of Tim-3 antibody (Ab) responses at the level of specific cell types. The Tim-3 protein bears multiple N- and O-glycostructures that differ dramatically in composition, size, and charge between cell lineages and which might explain the marked variations in Tim-3 Ab clone reactivity we found between cell types. For example, the clinical Tim-3 trial candidate, TSR-022, avidly bound T-cell- and melanoma-, but not NK-, DC-, or MΦ-Tim-3, while other Tim-3 Abs showed high affinity for Tim-3 on T-cells, MΦs, DCs, and/or NK, but not melanoma cells. Notably, glycan-modifying regimens shifted inhibitor binding towards desired T-cell- Tim-3 recognition and reduced melanoma-Tim-3 reactivity. Our preliminary data highlights the critical need for dissecting immune- vs. melanoma cell-Tim-3 glyco-epitopes, Ab affinity, signaling, and immunobiology. Results will help optimize Tim-3 therapeutic efficacy by validating regimens that preferentially target immune cell-Tim-3 glycans, while avoiding unwanted blockade of melanoma cell-Tim-3. Our aims are to 1) define cell type- associated Tim-3 glycan moieties, ligands, Ab affinities, and signaling networks, 2) examine immune cell- vs. melanoma-intrinsic effects of existing Tim-3 antagonists and their relevance to interpreting therapeutic benefit, and 3) identify new Tim-3 targeting strategies that accentuate immune cell-Tim-3 inhibition. We will use state- of-the-art gain and loss of Tim-3 function and glycan-modifying strategies, Tim-3 inhibitors with variable tissue- associated affinities, and immune and melanoma model systems to define cell type-specific Tim-3 functions and glycomolecular targets. Our initiative also implements clinical tumor biospecimens from patients receiving immune checkpoint inhibitors. Together, these studies will pave the way for next generation biomarkers and treatment modalities that discriminate immune- from cancer cell-Tim-3 for optimized immunotherapy outcomes.