Synthetic Transcriptional Activators for Cancer Immunotherapy

用于癌症免疫治疗的合成转录激活剂

• 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
• 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

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.

癌症免疫疗法是一种很有前途的治疗癌症的方法，它通过激活患者的免疫系统来对抗 肿瘤细胞，甚至提供长期缓解。这些临床益处导致了最近FDA的批准 用于免疫检查点阻断(ICB)、过继细胞转移(ACT)和分子疫苗技术，以及 也激励了大量的商业投资，以促进它们的持续发展。尽管如此，这些 治疗还与非选择性免疫原性有关--这限制了疗效并损害了健康 组织。为了使这些治疗方法充分发挥作用，迫切需要新的策略来加强 免疫治疗反应，同时将非靶点效应降至最低。开发的一个关键挑战 免疫疗法正在取得强大的抗肿瘤免疫反应。促进回应的战略包括 注射多肽或DNA编码的T细胞抗原，旨在扩大抗肿瘤的T细胞群 细胞。这些疫苗已经显示出激活T细胞的潜力，但由于以下原因，其疗效往往有限 肿瘤细胞的免疫逃避。最近，工程转录因子(ETF)蛋白被证明能诱导 针对HIV-1启动子的潜伏HIV感染细胞库的蛋白质表达。受此启发 方法，我们设想开发新的ETF蛋白来诱导肿瘤抗原的表达，进而， 促进对癌细胞的免疫识别。虽然CRISPR技术也被用于基因激活， 由于安全性和细胞内给药特性的不足，治疗应用仍然有限。 我们在这项提案中的首要目标是演示使用ETF来诱导肿瘤抗原的表达 在试管中。随着时间的推移，我们将建立ETF的有效性，这将指导未来的活体实验设计 (不包括在本提案中)和未来的赠款申请。我们将从开发ETF开始这些研究 诱导人乳头瘤病毒(HPV)-18相关抗原蛋白表达的激活剂， 与宫颈癌有关。我们选择HPV-18是因为它是开发HPV-18的理想起点 ETF出现在我们的组中有几个原因。一个原因是上游监管区(URR)和记者 细胞系(HeLa)的特性良好。另一个原因是，这些研究建立在以前关于艾滋病毒的工作的基础上。如果 成功后，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 临床上的免疫原性。我们将通过ZF的设计、准备和表征来开发ETF 我们将组装它们来识别长度为18个核苷酸的启动子区域基因。 我们还将加入一种‘转录激活剂’来诱导蛋白质转录和翻译。我们会 通过将这些构建物编码到哺乳动物表达载体中来评估ETF介导的激活， 通过测量癌细胞中蛋白质抗原的表达。我们还将准备合成ETF作为 治疗性蛋白质，使DNA结合研究成为可能，并增强细胞内的递送特性。