Novel cell therapy approaches for molecularly defined subsets of therapy-resistant melanoma

项目摘要/摘要 黑色素瘤(Mel)是研究肿瘤免疫治疗机制的一种模型恶性肿瘤。抗体 阻断T细胞功能的负面调节，称为免疫检查点阻断(ICB)，已经转化为 梅尔和其他实体癌症的治疗。尽管一些患者有持久的疾病控制，但许多人失败了。 在最初经历肿瘤消退后有反应或进展。治疗耐药性在两个方面有所增强 分子定义的MEL亚型。28%的MEL具有激活突变(Mut) 驱动癌基因NRAS，第二常见的Mut RAS亚型。除MEL外，Mut NRAS出现在其他 常见的恶性肿瘤，包括结直肠癌(CRC)。我们和其他人最近发现，患者 对于Mut、NRAS、MEL和CRC，治疗失败的时间明显缩短。另外，约30%的MEL 获得人类白细胞抗原的基本成分β-2-微球蛋白(B2M)的突变 I类(人类白细胞抗原-I)复合体，随着ICB进展。患有Mut B2M的癌症对CD8+T细胞具有天生的抵抗力 细胞杀戮。因此，限制MEL和其他疾病免疫治疗潜力的两个主要知识空白 常见的癌症包括：(1)Mut NRAS肿瘤表达的免疫原性抗原的鉴定；(2) 克服人类白细胞抗原-I基因缺失的治疗策略。我们假设患有哑巴的癌症患者 NRAS或Mut B2M可以使用基于T细胞受体(TCR)的免疫疗法作为治疗靶点。在……里面 支持我们的假设，我们发现使用质谱仪(MS)屏幕上的三个最常见的 NRAS热点替换产生由流行的人类白细胞抗原呈现的共享(或公共)新抗原(NeoAg) 等位基因。使用来自表达NRAS公共NeoAg的患者的独特的生物标本收集，我们 产生这些表位的特异性T细胞，检索它们的TCR基因序列，并转移到公众 NeoAg对多克隆T细胞的反应性。这些结果证实了筛选鉴定的NRAS具有免疫原性。 公共NeoAgs，并使基于TCR的疗法的开发成为可能。我们进一步发现，一个重要的 部分MEL受到表达人类白细胞抗原II类(HLA-II)限制性TCR的T细胞的直接杀伤。vbl.使用 一次基因组规模的CRISPR筛查，我们发现当B2M和其他人类白细胞抗原-I一起根除癌症时，肿瘤被保存下来 基因被破坏了。基于这些初步数据，我们在目标1中建议开发一种新的治疗方法 利用TCR基因工程和过继细胞表达NRAS公共NeoAg的肿瘤研究 调职。在目标2中，我们将研究NRAS公共NeoAg TCR特异性的物理机制， 包括一些TCR适应多个热点替换的独特能力。在《目标3》中，我们将 确定人类白细胞抗原II类限制性TCR直接杀伤癌细胞的分子基础，并测试它们的组合 增强过继转移的CD4+T细胞的抗肿瘤作用。通过实现这些目标，我们将 为Mut NRAS和人类白细胞抗原-I基因缺陷的癌症开发新的、基于机制的细胞免疫疗法。

PROJECT SUMMARY/ABSTRACT Melanoma (MEL) is a model malignancy for studying the mechanisms of cancer immunotherapy. Antibodies that block negative regulators of T cell function, termed immune checkpoint blockade (ICB), have transformed the treatment of MEL and other solid cancers. Although some patients have durable disease control, many fail to respond or progress after initially experiencing tumor regression. Therapeutic resistance is enriched in two molecularly defined MEL subtypes. Twenty eight percent of MELs possess activating mutations (Mut) in the driver oncogene NRAS, the second most common Mut RAS isoform. Beyond MEL, Mut NRAS occurs in other prevalent malignancies, including colorectal cancer (CRC). We and others recently discovered that patients with Mut NRAS MEL and CRC have a significantly shorter time to treatment failure. Separately, ~30% of MELs acquire mutations in beta-2-microglobulin (B2M), an essential component of the human leukocyte antigen class I (HLA-I) complex, following ICB progression. Cancers with Mut B2M are intrinsically resistant to CD8+ T cell killing. Thus, two major gaps in knowledge that limit the potential of immunotherapy in MEL and other common cancers include: (1) identification of immunogenic antigens expressed by Mut NRAS tumors, and (2) therapeutic strategies to overcome genetic loss of HLA-I presentation. We hypothesize that cancers with Mut NRAS or Mut B2M can be therapeutically targeted using T cell receptor (TCR)-based immunotherapies. In support of our hypothesis, we discovered using a mass spectrometry (MS) screen that the three most common NRAS hotspot substitutions generate shared (or “public”) neoantigens (NeoAgs) presented by a prevalent HLA allele. Using a unique collection of biospecimens from patients who express an NRAS public NeoAg, we generated T cells specific for these epitopes, retrieved their TCR gene sequences, and transferred public NeoAg reactivity to polyclonal T cells. These results confirm the immunogenicity of screen-identified NRAS public NeoAgs and enable the development of TCR-based therapies. We further discovered that a significant proportion of MELs undergo direct killing by T cells that express an HLA class II (HLA-II) restricted TCR. Using a genome-scale CRISPR screen, we found that cancer eradication is preserved when B2M and other HLA-I genes are disrupted. Building on these preliminary data, we propose in Aim 1 to develop a novel therapeutic approach for cancers expressing an NRAS public NeoAg using TCR genetic engineering and adoptive cell transfer. In Aim 2, we will study the physical mechanisms underlying NRAS public NeoAg TCR specificity, including the unique capacity of some TCRs to accommodate multiple hotspot substitutions. In Aim 3, we will define the molecular basis for direct cancer cell killing by HLA class II-restricted TCRs and test combinations to enhance the antitumor efficacy of adoptively transferred CD4+ T cells. By completing these aims, we will develop novel, mechanism-based cellular immunotherapies for Mut NRAS and HLA-I deficient cancers.