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

1篇摘要 2乳腺癌（BC）包括由不同分子亚型组成的多种疾病， 3的特征在于不同的致癌驱动因素和独特的治疗方案。尽管存在这些差异， 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、总体生存率低。免疫疗法提供了治疗患者的机会，无论分子水平如何， 6个亚型。该提案利用瘤内（IT）免疫疗法作为“许可”治疗病变的替代方案 7以产生T细胞与抑制性免疫亚群的生产率，同时扩增免疫检查点轴 8，并最终增加对免疫检查点抑制剂（ICI）的敏感性。使用TNBC和单个 9细胞RNA测序，我们证明了IT质粒IL-12（pIL-12）可以将免疫原性差/低的 图10通过协调上调免疫调节因子，将TIL肿瘤转化为高度发炎的免疫活性病变。 11浸润免疫细胞中的CXCR 3轴，其影响两者的迁移、分化和活化 12先天性和适应性免疫细胞。CXCR 3信号也在接受了化疗的患者中显著增强。 图13示出了IT pIL-12治疗后CD 8 T细胞浸润到治疗的肿瘤中的增加和改善的预后。 14总生存率我们假设靶向CXCR 3轴IT将增强TILs并将患者转化为 所有BC分子亚型中的15例ICI应答者。拟议的工作将利用这些初步数据， 16以下3个目标：1。证明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CXCR 3或运输相关基因签名的诱导2.） 18使用免疫组织化学方法评估运输相关蛋白在增强TNBC中对抗PDL 1的反应性中的作用。 19基于CRISPR的筛选; 3.评估质粒CXCL 9的IT注射，以确定CXCR 3的直接靶向是否 20轴足以增强T细胞浸润。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浸润到肿瘤中，以更好地设计、联合收割机和预测 22对免疫疗法的反应，特别关注BC。这类研究的一个关键障碍是 缺乏癌基因驱动的自发性BC肿瘤模型，这阻碍了 24多项临床前发现。克罗斯比博士独立研究的关键资源是自发的HER 2- 25驱动和p53/BRCA 1/PTEN驱动的TNBC模型，她已经创建，出版，并将独特地拥有 26、做好这些工作，做好其他工作。解决如何增强T细胞浸润的基本问题 27转化为肿瘤对于改变BC患者的治疗模式具有重要意义，特别是 28使用肿瘤内质粒方法，该方法易于改变以跟踪新鉴定的靶点。的 这项K22奖提供的资金将保护克罗斯比博士的研究时间，以开发和出版这些 30项基础研究，将支持随后的R 01资助申请。

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.