Synthetic Transcriptional Activators for Cancer Immunotherapy

用于癌症免疫治疗的合成转录激活剂

基本信息

批准号：
10850109
负责人：
IGOR B RONINSON
金额：
$ 19.92万
依托单位：
UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-02-08 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10850109
关键词：
Adoptive Cell Transfers Antigens Applications Grants CRISPR/Cas technology Cell Line Cells Cervix carcinoma Clinic Clinical DNA DNA Binding Development Disease remission Engineering Future Gene Activation Genes Genetic Transcription Goals HIV HIV-1 Hela Cells Human papillomavirus 18 Immune Immune system Immunotherapy In Vitro Investments Length Malignant Neoplasms Measures Mediating Molecular Vaccines Nucleic Acid Regulatory Sequences Nucleotides Patients Peptides Population Preparation Promoter Regions Property Proteins Reporter Safety T-Cell Activation T-Lymphocyte Technology Therapeutic Time Tissues Transcription Coactivator Translations Tumor Antigens Tumor Escape Vaccines Work anti-tumor immune response cancer cell cancer immunotherapy design experimental study expression vector immune checkpoint blockade immunogenicity in vivo neoplastic cell promoter protein expression response targeted treatment therapeutic protein transcription factor

项目摘要

Cancer immunotherapy is a promising approach to treat cancer by activating a patient’s immune system against tumor cells and even providing long-term remission. These clinical benefits have led to recent FDA approvals for immune-checkpoint blockade (ICB), adoptive cell transfer (ACT), and molecular vaccine technologies, and have also motivated substantial commercial investments for their continued development. Nonetheless, these therapies are also associated with nonselective immunogenicity—which limits efficacy and harms healthy tissue. For these treatments to reach their full promise, new strategies are desperately needed to enhance immunotherapy responses while also minimizing off-target effects. One key challenge with developing immunotherapies is achieving robust anti-tumor immune responses. Strategies to promote responses include administering peptide or DNA-encoded T-cell antigens, designed to expand T-cell populations against tumor cells. These vaccines have demonstrated a potential to activate T cells, but often provide limited efficacy due to immune evasion by tumor cells. Recently, engineered transcription factor (ETF) proteins were shown to induce protein expression of latent HIV-infected cell reservoirs by targeting the HIV-1 promoter. Inspired by this approach, we envisioned developing new ETF proteins that induce expression of tumor antigens and, in turn, facilitate immune recognition of cancer cells. Although CRISPR technologies are also used for gene activation, therapeutic applications remain limited due to insufficient safety and intracellular delivery properties. Our overarching goal in this proposal is to demonstrate using an ETF to induce expression of tumor antigens in vitro. Over time, we will establish the efficacy of ETFs, which will guide designs of future in vivo experiments (not included in this proposal) and future grant applications. We will begin these studies by developing ETF activators that induce expression of antigenic proteins associated with human papilloma virus (HPV)-18, which is associated with cervical carcinoma. We selected HPV-18 because it is an ideal starting point for developing ETFs in our group for several reasons. One reason is that the Upstream Regulatory Region (URR) and reporter cell line (HeLa) are well-characterized. Another reason is that these studies build upon the prior work with HIV. If successful, we plan to develop additional ETF proteins as a facile approach for enhancing tumor cell immunogenicity in the clinic. We will develop ETFs through the design, preparation, and characterization of ZF building blocks, which we will assemble to recognize promoter region genes that are 18 nucleotides in length. We will also incorporate a ‘transcriptional activator’ to induce protein transcription and translation. We will evaluate ETF-mediated activation by encoding these constructs into a mammalian expression vector, followed by measuring expression of the protein antigen in cancer cells. We will also prepare synthetic ETFs as therapeutic proteins to enable DNA-binding studies and to enhance intracellular delivery properties.

癌症免疫疗法是通过激活患者的免疫系统来治疗癌症的一种有前途的方法肿瘤细胞，甚至提供长期缓解，这些临床益处导致了 FDA 最近的批准。用于免疫检查点封锁（ICB）、过继细胞转移（ACT）和分子疫苗技术，以及还激发了大量商业投资以促进其持续发展。疗法还与非选择性免疫原性有关，这限制了疗效并损害健康为了使这些治疗充分发挥作用，迫切需要新的策略来增强。免疫疗法反应，同时最大限度地减少脱靶效应是发展的一个关键挑战。免疫疗法正在实现强大的抗肿瘤免疫反应，促进反应的策略包括。施用肽或 DNA 编码的 T 细胞抗原，旨在扩大抗肿瘤的 T 细胞群这些疫苗已被证明具有激活 T 细胞的潜力，但由于以下原因通常效果有限。最近，工程转录因子（ETF）蛋白被证明可以诱导肿瘤细胞的免疫逃避。受此启发，通过靶向 HIV-1 启动子来表达潜伏的 HIV 感染细胞储库的蛋白质。我们设想开发新的 ETF 蛋白来诱导肿瘤抗原的表达，进而尽管CRISPR技术也可用于基因激活，由于安全性和细胞内递送特性不足，治疗应用仍然受到限制。我们本提案的首要目标是证明使用 ETF 诱导肿瘤抗原的表达随着时间的推移，我们将确定 ETF 的功效，这将指导未来体内实验的设计。（不包括在本提案中）和未来的资助申请，我们将通过开发 ETF 来开始这些研究。诱导与人乳头状瘤病毒 (HPV)-18 相关的抗原蛋白表达的激活剂，我们选择 HPV-18，因为它是发展的理想起点。我们组中的 ETF 有几个原因，其中之一是上游监管区域 (URR) 和报告者。另一个原因是这些研究建立在先前的 HIV 研究基础上。如果成功，我们计划开发更多的 ETF 蛋白作为增强肿瘤细胞的简便方法我们将通过 ZF 的设计、制备和表征来开发 ETF。我们将组装这些构件来识别长度为 18 个核苷酸的启动子区域基因。我们还将加入“转录激活剂”来诱导蛋白质转录和翻译。通过将这些构建体编码到哺乳动物表达载体中来评估 ETF 介导的激活，然后通过测量癌细胞中蛋白质抗原的表达，我们还将制备合成 ETF：蛋白质以实现治疗性 DNA 结合研究并增强细胞内递送特性。