Intratumoral immunotherapy to enhance T cell infiltration and augment immune checkpoint blockade responses across molecular subtypes of breast cancer

1摘要 乳腺癌包括由不同分子亚型组成的多种疾病，这些亚型包括 3具有明显的致癌驱动因素和独特的治疗方案。尽管存在这些差异，但在所有 4亚型，晚期、复发或转移性疾病患者的治疗选择仍然有限 5总体生存结局较差。免疫疗法提供了治疗患者的机会，而不受分子水平的影响 6个亚型。这项提议利用瘤内(IT)免疫疗法作为‘许可证’治疗的病变的替代方案 7在扩大免疫检查点轴线的同时，产生T细胞与抑制性免疫亚群的比率 8，最终增加对免疫检查点抑制剂(ICI)的敏感性。使用TNBC和Single的模型 9细胞RNA测序，我们证明了IT质粒IL-12(pIL-12)可以将低免疫原性/低免疫 10个TIL肿瘤通过协调上调TIL的表达，形成高度炎症、免疫活跃的病变 11 CXCR3轴影响免疫细胞的迁移、分化和激活 12个先天和获得性免疫细胞。这种CXCR3签名在患有以下疾病的患者中也显著增强 13 IT PIL-12治疗后CD8 T细胞在治疗肿瘤中的浸润增加和预后改善 14总体存活率。我们假设以CXCR3轴为靶点的IT将增强TIL并将患者转化为 BC所有分子亚型的15个ICI应答者。拟议的工作将利用这些初步数据在 16以下3个目标：1.发现IT PIL-12-EP后肿瘤特异性T细胞的浸润增加 17治疗并验证ICI应答者中CXCR3或贩运相关基因签名的诱导2。) 18评估贩运相关蛋白在增强TNBC对抗PDL1的反应中的作用 19基于CRISPR的屏幕；3)评估IT注射CXCL9以确定是否直接靶向CXCR3 20轴足以增强T细胞的浸润。克罗斯比博士的长期目标是建立一个研究项目， 21有助于理解免疫细胞对肿瘤的渗透，以更好地设计、组合和预测 22对免疫疗法的反应，特别侧重于BC。这类研究的一个关键障碍是 23缺乏癌基因驱动的自发BC肿瘤模型，这阻碍了翻译的适用性 24许多临床前发现。克罗斯比博士独立研究的关键资源是自发的HER2- 25个由她创建、发布并将独有的由P53/BRCA1/PTEN驱动的TNBC模型 26进行这些研究和许多其他研究。解决如何增强T细胞渗透的基本问题 27转化为肿瘤对改变BC患者的治疗模式具有重要意义，特别是 28使用瘤内质粒方法，这种方法很容易改变，以跟踪新发现的靶点。这个 这项K22奖项提供的资金将保护克罗斯比博士的研究时间，使其能够开发和发表这些 30项基础性研究，这些研究将支持后续的R01赠款申请。

1 Abstract 2 Breast cancer (BC) encompasses multiple diseases made up of different molecular subtypes that are 3 characterized by distinct oncogenic drivers and unique treatment regimens. Despite these differences, across all 4 subtypes, individuals with advanced, recurrent, or metastatic disease still have limited treatment options and 5 poor overall survival outcomes. Immunotherapies offer an opportunity to treat patients regardless of molecular 6 subtypes. This proposal leverages intratumoral (IT) immunotherapy as an alternative to ‘license’ treated lesions 7 to yield productive ratios of T cells to suppressive immune subsets while amplifying immune checkpoint axes 8 and ultimately increasing sensitivity to immune checkpoint inhibitors (ICI). Using a model of TNBC and single 9 cell RNA sequencing, we demonstrate that IT plasmid IL-12 (pIL-12) can convert poorly immunogenic/low 10 TIL tumors into highly inflamed, immunologically active lesions through the coordinated upregulation of the 11 CXCR3 axis in infiltrating immune cells that impacts the migration, differentiation, and activation of both 12 innate and adaptive immune cells. This CXCR3 signature was also significantly enhanced in patients that had 13 an increase in CD8 T cell infiltration into treated tumors post IT pIL-12 therapy and prognostic of improved 14 overall survival. We hypothesize that targeting the CXCR3 axis IT will enhance TILs and convert patients into 15 ICI responders across all molecular subtypes of BC. The proposed work will leverage this preliminary data in 16 the following 3 aims: 1.) Demonstrate increased infiltration of tumor-specific T cells following IT pIL-12-EP 17 treatment and validate the induction of a CXCR3 or trafficking-associated gene signature in ICI responders 2.) 18 Evaluate the role of trafficking-associated proteins in enhancing responsiveness to anti-PDL1 in TNBC using a 19 CRISPR based screen; 3.) Assess IT injection of plasmid CXCL9 to determine if direct targeting of the CXCR3 20 axis is sufficient to enhance T cell infiltration. Dr. Crosby’s long-term goal is to build a research program that 21 contributes to an understanding of immune cell infiltration into tumors to better design, combine, and predict 22 responses to immunotherapies, with a specific focus on BC. A critical impediment to these types of studies is 23 the lack of oncogene-driven, spontaneous BC tumor models which hampers the translational applicability of 24 many pre-clinical findings. Key resources for Dr. Crosby’s independent research are the spontaneous HER2- 25 driven and p53/BRCA1/PTEN-driven TNBC models that she has created, published, and will uniquely possess 26 to perform these and many other studies. Addressing the basic question of how to enhance T cell infiltration 27 into tumors has significant implications for changing the paradigm of treatment for BC patients, particularly 28 using an intratumoral plasmid approach that is easily altered to follow up on newly identified targets. The 29 funding provided by this K22 award will protect Dr. Crosby’s research time to develop and publish these 30 foundational, studies which will support subsequent R01 grant submissions.