Flt3l gene-modified cDC1 in situ vaccination in NSCLC: mechanisms and therapeutic application

项目摘要/摘要 Salehi-Rad博士是退伍军人管理局大洛杉矶医疗系统(VA GLAHS)的肺病专家 对肺癌有临床和研究兴趣，肺癌是美国退伍军人癌症死亡的主要原因。在……里面 申请退伍军人管理局职业发展奖(CDA-2)，Salehi-Rad博士的目标是建立一个独立的 VA GLAHS的翻译研究计划，重点是提高我们对 肺癌的免疫发病机制，以开发癌症免疫治疗的新方法。他是 由史蒂文·杜比内特医学博士(初级导师)支持，他是退伍军人事务部功勋资助的著名内科科学家和 安东尼·里巴斯是领先的肺癌专家，医学博士，博士(共同导师)，国际公认的肺癌领域权威 癌症免疫学博士和著名数据科学家保罗·布特罗斯博士(共同导师)。确定了导师 基于他们在拟议研究中的互补科学专业知识和他们在 指导学术内科医生-科学家。通过加州大学洛杉矶分校临床和翻译科学研究所(CTSI)，Dr。 Salehi-Rad将有机会参加许多职业发展研讨会，这些研讨会讨论的主题包括 写作、手稿准备和伦理研究。他还将参加研究生课程，以获得进一步的培训 在免疫学和生物信息学方面。Salehi-Rad博士将得到退伍军人管理局和加州大学洛杉矶分校的全面机构支持 来开展他的研究。 Salehi-Rad博士建立了临床相关的NSCLC小鼠模型，突变增加 Burden，并确定了LKB1缺陷非小细胞肺癌免疫治疗抵抗的新的靶向机制。 利用这些小鼠模型，Salehi-Rad博士已经证明，与精英抗原的原位免疫(ISV)交叉- 呈现常规的1型DC，经基因修饰后可分泌FMS样酪氨酸激酶3配体(Flt3L- CDC1)，一种促进DC存活和扩增的细胞因子，使免疫难治性非小细胞肺癌对免疫增敏 检查点抑制(ICI)。在这项提案中，Salehi-Rad博士的目标是研究DC ISV的免疫机制。 AIM 1.1建立在初步的体外数据基础上，该数据表明Flt3L-cDc1的活性比cDc1和 旨在确定导致FTL3L-cDC1存活率增加的分子机制。目标1.2和1.3 利用不同的小鼠模型来确定疫苗和内源性DC的活性、抗原转运和 DC ISV后抗原特异性T细胞的激活。该提案的目标2侧重于阐明免疫 DC ISV作为单一疗法或作为与ICI联合治疗的应答决定因素。目标2.1和2.2 流式细胞仪单细胞免疫表型和单细胞RNA测序(scRNA-seq)联合应用 通过多重免疫荧光(MIF)空间分析发展对局部的全面认识 和DC ISV诱导的系统免疫反应。AIM 2.3利用抗体耗竭研究来评估 DC ISV对T细胞和自然杀伤细胞的依赖性。目标3将决定T细胞谱系的演变 通过TCR-βCDR3DNA测序和肿瘤-新抗原全外显子组测序(WES)来评估 DC ISV是否导致TCR谱系的扩大并促进肿瘤免疫编辑。改进 对非小细胞肺癌免疫抑制性质及DC免疫刺激机制的认识 ISV将对肺癌免疫学领域做出重大贡献，并可能促进临床 翻译Flt3L-cDC1 ISV作为这种破坏性疾病的创新治疗策略 我们的许多退伍军人。

PROJECT SUMMARY/ABSTRACT Dr. Salehi-Rad is a Staff Pulmonologist at the VA Greater Los Angeles Healthcare System (VA GLAHS) with a clinical and research interest in lung cancer, the leading cause of cancer death among U.S. Veterans. In applying for the VA Career Development Award (CDA-2), Dr. Salehi-Rad’s goal is to establish an independent translational research program at the VA GLAHS, focused on improving our understanding of the immunopathogenesis of lung cancer for the development of novel approaches for cancer immunotherapy. He is supported by Steven Dubinett, MD (Primary-Mentor), a renowned VA Merit-funded physician-scientist and a leading expert in lung cancer, Antoni Ribas, MD, PhD (Co-Mentor), an internationally recognized authority in cancer immunology, and Paul Boutros, PhD (Co-mentor), a distinguished data scientist. Mentors were identified based on their complementary scientific expertise for the proposed research and their extensive experience in mentoring academic physician-scientists. Through UCLA Clinical and Translational Science Institute (CTSI), Dr. Salehi-Rad will have access to numerous career development seminars that address such topics as grant writing, manuscript preparation, and ethical research. He will also take graduate courses to obtain further training in immunology and bioinformatics. Dr. Salehi-Rad will have the full institutional support of both the VA and UCLA Health Systems to carry out his research. Dr. Salehi-Rad has established clinically relevant murine models of NSCLC with increased mutational burden and identified a novel targetable mechanism of resistance to immunotherapy in LKB1-deficient NSCLC. Utilizing these murine models, Dr. Salehi-Rad has shown that in situ vaccination (ISV) with elite antigen cross- presenting conventional type 1 DCs that are gene-modified to secrete FMS-like tyrosine kinase 3 ligand (FLT3L- cDC1), a cytokine that promotes DC viability and expansion, sensitize immune refractory NSCLC to immune checkpoint inhibition (ICI). In this proposal, Dr. Salehi-Rad aims to study the immune mechanisms of DC ISV. Aim 1.1 builds on preliminary in vitro data indicating enhanced viability of FLT3L-cDC1 compared to cDC1 and seeks to determine the molecular mechanisms that result in increased survival of FTL3L-cDC1. Aim 1.2 & 1.3 utilize various murine models to determine the vaccine and endogenous DC viability, antigen trafficking and antigen-specific T cell priming following DC ISV. Aim 2 of the proposal focuses on elucidating the immune determinants of response to DC ISV as monotherapy or as a combination therapy with ICI. Aim 2.1 & 2.2 combine single cell immunophenotyping by flow cytometry and single cell RNA-sequencing (scRNA-seq) with spatial analysis by multiplex immunofluorescence (MIF) to develop a comprehensive understanding of the local and systemic immune responses induced by DC ISV. Aim 2.3 utilizes antibody depletion studies to evaluate the dependency of DC ISV on T cells and natural killer cells. Aim 3 will determine the evolution of T cell repertoires by TCR-β CDR3 sequencing and tumor-neoantigen profiles by whole-exome sequencing (WES) to assess whether DC ISV induces the expansion of the TCR repertoire and promotes tumor immunoediting. Improved understanding of the nature of immunosuppression in NSCLC and the immunostimulatory mechanisms of DC ISV will represent a significant contribution to the field of lung cancer immunology and could facilitate the clinical translation of FLT3L-cDC1 ISV as an innovative therapeutic strategy for this devastating disease that affects many of our Veterans.