Engineering class I MHC molecules to drive enhanced anti-cancer responses

T细胞在识别和摧毁癌细胞方面的重要性已经通过许多不同的方式得到了证明 方法，最近是通过检查点抑制疗法的临床有效性。在过去的几年里 几十年来，一种完全独立的方法揭示了T细胞的效力，这种效应被称为移植物抗病毒 白血病(GVL)。GVL的临床益处在很大程度上来自人们长期欣赏的现象，即 同种异体MHC产品可诱导非常强烈的T细胞反应。在GVL的情况下，效果涉及 白血病细胞表面I类MHC向供体骨源性同种异体反应性T细胞递呈多肽 骨髓。我们最近发现，可以使用KB分子中的一个单一突变来产生‘同种异体’。 Like‘分子可以在诱导CD8+T细胞时克服耐受性；一旦被激活，T细胞就可以 依次识别肿瘤上表达的野生型KB表达的抗原。这种方法的全部潜力 将通过实验确定的MHC突变实现，以诱导最佳的抗肿瘤T细胞 细胞反应。我们将利用我们在深度突变扫描和I类MHC蛋白质工程方面的专业知识来 开发同种异体MHC分子(‘mut-MHC’)，诱导有效的T细胞对癌症抗原的反应。 我们的假设是，被选为多肽结合和/或TCR亲和力改善的MHC突变体将 增强T细胞对多种癌症抗原的反应。此方法的一个关键优势是，与 为了疫苗的目的而努力识别癌症新抗原，是它不需要鉴定的最多 有效的新抗原，并允许变异的自身抗原作为更有效的抗原。 通过设计KB系统，我们将能够在同基因小鼠肿瘤模型中使用 小鼠胶质母细胞瘤细胞系GL261、SMA-560和CT2A。重要的是，我们在工程方面的经验 人类人类白细胞抗原A2系统将使我们能够将这些发现转化为人类癌症。该项目的具体目标 目的：1.使用KB分子的深度突变扫描来表征增强的功能 变种人。我们最近完成了对酵母表面表达的KB的深度突变扫描，检查 每次α1和α2区域的替换(3,420个突变)，使用抗体和TCR探针。这产生了50 到100kb的突变，具有改善多肽稳定性或TCR结合的候选潜力。这一目标将进一步 评估一下这组突变体。目标2.检查突变的KB分子的克服能力 对癌肽抗原的耐受性。我们将检查排名靠前的Kb突变体的呈现能力 克服同基因小鼠胶质母细胞瘤耐受性的肿瘤抗原，如全肿瘤细胞或DC疫苗 型号(GL261、SMA-560和CT2A型)。研究结果将为合理有效地利用资源提供蓝图。 改造MHC分子以克服T细胞对癌症抗原的耐受性。该方法将提供 是其他T细胞导向疗法的替代或辅助疗法，如检查点抑制剂。

The importance of T cells in recognizing and destroying cancer cells has been shown by many different approaches, most recently through the clinical effectiveness of checkpoint inhibitor therapies. Over the past few decades, a completely independent approach has revealed the potency of T cells in an effect called graft vs leukemia (GVL). The clinical benefit of GVL derives in large part from the long-appreciated phenomenon in which allogeneic MHC products induce very strong T cell responses. In the case of GVL, the effect involves the presentation of peptides by class I MHC on leukemia cells to alloreactive T cells derived from the donor bone marrow. We recently showed that it was possible to use a single mutation in the Kb molecule to generate an ‘allo- like’ molecule that could overcome tolerance in the induction of CD8+ T cells; once activated, the T cells could in turn recognize antigens expressed by the wild type Kb expressed on a tumor. The full potential of this approach will be realized with MHC mutations that can be identified experimentally in order to induce optimal anti-tumor T cell responses. We will use our expertise in deep mutational scanning and protein engineering of class I MHC to develop allo-like MHC molecules (‘mut-MHC’) that induce effective T cell responses against cancer antigens. Our hypothesis is that MHC mutants selected for improvements in peptide binding and/or TCR affinity will enhance T cell responses against multiple cancer antigens. A key advantage of this approach, compared to efforts to identify cancer neoantigens for vaccine purposes, is that it does not require identification of the most effective neoantigens and that it allows aberrant self-antigens to serve as more effective antigens. By engineering the Kb system, we will be able to test the hypothesis in syngeneic mouse tumor models using the mouse glioblastoma lines GL261, SMA-560, and CT2A. Importantly, our experience with engineering the human HLA-A2 system will allow us to translate the findings to human cancers. The specific aims of the project are: Aim 1. To use deep mutational scans of the Kb molecule to characterize enhanced functional mutants. We have recently completed a deep mutational scan of Kb expressed on the yeast surface, examining every substitution of the alpha1 and alpha2 domains (3,420 mutations), using antibody and TCR probes. This yielded 50 to 100 Kb mutations with candidate potential for improved peptide stability or TCR binding. This Aim will further evaluate this collection of mutants. Aim 2. To examine mutated Kb molecules for their ability to overcome tolerance against cancer peptide antigens. We will examine the top Kb mutants for their ability to present cancer antigens, as whole tumor cell or DC vaccines, that overcome tolerance in syngeneic mouse glioblastoma models (GL261, SMA-560, and CT2A). The results will provide a blueprint for the effective use of rationally engineered MHC molecules to overcome T cell tolerance against cancer antigens. The approach would provide an alternative, or adjunct, to other T cell directed therapies such as checkpoint inhibitors.