Eager: Biomanufacturing: Liposome mediated targeted expansion and stimulation of CD8+ cytotoxic T-cells

尽管我们体内存在t细胞介导的抗肿瘤免疫机制，但异常细胞偶尔会逃避免疫监视，导致癌症的发生。最近开发的一种抗肿瘤方法，过继细胞转移疗法（ACT），利用t细胞作为“活的药物”，通过扩大其体外（体外）的数量，并通过增强其特异性识别和破坏肿瘤细胞的能力，重新注射到体内。然而，涉及基因修饰和体外增殖的相关技术具有挑战性，并且不适合大规模实施。此外，工程化的t细胞往往不能识别大多数癌症中常见的异质突变肿瘤细胞群。为了解决这些挑战，该项目将使用一种新颖的方法，绕过体外干预的需要，诱导细胞毒性t细胞群的扩增，增强归巢能力和与肿瘤细胞呈现的多种抗原相互作用的能力。这将通过合成专门的包来完成，这些包将允许靶向扩增和刺激对肿瘤细胞具有细胞毒性的免疫细胞亚群。该策略将大大简化体内t细胞的产生，同时抑制与当前免疫疗法相关的毒性和副作用。这种方法有可能利用个体自身的免疫系统来治疗多种类型的肿瘤，这将产生重大的变革性影响。除了医学上的进步，该项目还将成为促进科学教育和青年科学家发展的基础平台。该项目的多学科性质将为微纳米技术、生物工程、生物化学和癌症生物学领域的研究生和本科生提供教育机会。该项目的研究结果将通过在科学和社会媒体上实施课程作业和出版物的结果在学生和一般人群中传播。目前的过继细胞转移疗法（ACTs）筛选肿瘤细胞和肿瘤浸润淋巴细胞（TIL）的特定标记物，并对自身t细胞进行基因修饰以表达相应的受体。虽然这些生物制造的嵌合抗原受体呈递T细胞（CAR - T细胞）对单克隆抗原呈递肿瘤细胞（APCs）表现出高效率，但引入单一类型的抗原特异性受体限制了细胞毒性T细胞（ctl）与许多肿瘤中常见的不同突变表型相互作用的能力。另一个重大挑战是ctl到肿瘤部位的低效率运输。尖刺的外周CAR - t细胞依赖于肿瘤病变的成功浸润，然后才能在与apc相互作用时被激活，从而降低了它们对抗实体肿瘤的有效性。最终，免疫细胞的体外扩增需要大量的资源和专门的设施，这使得现有的ACT技术无法大规模实施。该项目通过利用脂质体选择性地靶向自体ctl的生长和刺激，以有效识别和细胞溶解肿瘤细胞，解决了现有的局限性。白细胞介素-2 （IL-2）在体内平衡和淋巴细胞介导的免疫反应中起着核心作用，并刺激t细胞的增殖和功能活性。IL-2还上调肿瘤相关化学引诱剂（如I-TAC）的独特受体CXCR3的表达，并通过增强内皮粘附和跨细胞因子（tnf - α, ifn - γ）激活的内皮细胞的跨内皮迁移（TEM）介导淋巴细胞募集到炎症部位（归巢）。所提出的技术将利用IL-2的免疫原性潜力，提供多种优势，包括：1)将IL-2包封在脂质体内可以防止细胞因子降解，显著提高其对t细胞的功能活性；2)将抗CD8脂质体偶联可以主要靶向细胞毒性t细胞群（CD8+）。因此，调节性t细胞、CD4+细胞和内皮细胞的非期望共刺激将被抑制，3)CXCR3表达的增强将导致CTL对肿瘤组织的浸润改善，4)体内刺激具有增强浸润肿瘤组织能力的大量效应CTL群体将减少对特定抗原的偏向性监视，并可能更有效地破坏通常存在的异质肿瘤表型群体。脂质体和IL-2的利用是FDA批准的，并且提出的t细胞生物制造方法将对肿瘤治疗产生相当大的变革性影响。由于靶向CD8+ t细胞扩增可以在体内诱导，因此该技术有可能成为一种更普遍的治疗各种肿瘤和感染的方法，对社会产生重大影响。该多学科项目整合了生物工程、生物化学、细胞生物学和微加工，将为乔治梅森大学新的生物工程博士项目提供支持，为博士和本科生提供STEM教育培训的绝佳机会。将特别强调招募代表性不足的群体，包括女性和少数民族学生。该项目的研究结果也将应用于课程中，以激发学生对当前科学发展的认识。此外，项目成果将通过期刊出版物和YouTube视频等社交媒体平台以及实验室小组网页在科学界和一般人群中传播。

1645195 - AgrawalDespite the presence of T-cell mediated antitumor immune mechanism in our body, abnormal cells occasionally elude the immune surveillance, resulting in the onset of cancer. A recently developed antitumor approach, Adoptive Cell Transfer therapy (ACT), utilizes T-cells as "living drugs" by expanding their population outside the body (in vitro) and by enhancing their ability to specifically recognize and destroy tumor cells when reinjected into the body. However, the associated technologies involving genetic modifications and ex vivo proliferation are challenging and impractical for large scale implementation. Furthermore, engineered T-cells often fail to recognize the heterogeneous population of mutated tumor cells commonly found in most cancers. To address these challenges, this project will use a novel approach that bypasses the need for in vitro intervention and induces expansion of cytotoxic T-cell population with enhanced homing capacity and the ability to interact with multiple variants of antigens presented by tumor cells. This will be accomplished by synthesizing specialized packets that will allow targeted expansion and stimulation of the immune cell subpopulation that is cytotoxic to tumor cells. The strategy will significantly simplify the production of T-cells within the body while suppressing toxicity and side effects associated with current immunotherapies. The approach has the potential to utilize the individual's own immune system to treat multiple types of tumors, which will have significant transformative impact. In addition to the medical advancements, the project will serve as a fundamental platform to promote scientific education and development of young scientists. The multidisciplinary nature of the project will provide educational opportunities to graduate and undergraduate students in the areas of micro- and nanotechnology, bioengineering, biochemistry, and cancer biology. Findings from the project will be disseminated among students as well as general population through implementation of results in the coursework and publications in scientific as well as social media.Current adoptive cell transfer therapies (ACTs) screen tumor cells and tumor infiltrating lymphocytes (TIL) for specific markers and genetically modify autologous T-cells to express the corresponding receptors. While these biomanufactured chimeric antigen receptor presenting T-cells (CAR T-cells) demonstrate high efficacy against a monoclonal population of the antigen presenting tumor cells (APCs), introduction of a single type of antigen specific receptor limits the ability of cytotoxic T cells (CTLs) to interact with different mutated phenotypes commonly found in many tumors. Another significant challenge is the inefficient trafficking of CTLs to the tumor sites. The spiked peripheral CAR T-cells rely on successful infiltration of the tumor lesion before they can be activated upon interaction with APCs, diminishing their effectiveness against solid tumors. Ultimately, the ex vivo expansion of immune cells requires substantial resources and specialized facilities making the existing ACT technologies impractical to implement on a large scale. The project addresses the existing limitations by utilizing liposomes to selectively target the growth and stimulation of autologous CTLs for efficient recognition and cytolysis of tumor cells. Interleukin-2 (IL-2) plays a central role during homeostasis and lymphocyte mediated immune responses and stimulates proliferation as well as functional activity of T-cells. IL-2 also upregulates expression of CXCR3, a unique receptor for tumor associates chemoattractants (e.g. I-TAC), and mediates recruitment of lymphocytes to the site of inflammation (homing) via enhanced endothelial adhesion and transendothelial migration (TEM) across cytokine (TNF-alpha, IFN-gamma) activated endothelial cells. The proposed technique will harness the immunogenic potential of IL-2 providing multiple advantages including: 1) Encapsulation of IL-2 within liposomes will keep the cytokine from degradation, significantly improving its functional activity on T-cells, 2) Conjugation of liposomes with anti-CD8 will allow targeting primarily the cytotoxic T-cell population (CD8+). Thus, the undesired co-stimulation of regulatory T-cells, CD4+ cells and endothelial cells will be suppressed, 3) Enhanced CXCR3 expression will lead to improved infiltration of the tumor tissue by CTLs, 4) Stimulation of a large effector CTL population in vivo with enhanced capability to infiltrate the tumor tissue will reduce the biased surveillance of specific antigens and will likely be more effective in destroying the heterogeneous population of tumor phenotypes that are usually present. The utilization of both liposomes as well as IL-2 is FDA approved and presented T-cell biomanufacturing approach will have a considerable transformative impact on tumor therapies. Since the targeted CD8+ T-cell amplification can be induced in vivo, the technique has the potential for a more universal approach against various tumors and infections, greatly impacting the society. The multidisciplinary project, integrating bioengineering, biochemistry, cell biology, and microfabrication, will support the new Bioengineering PhD program at George Mason University offering an excellent opportunity for training in STEM education by involving PhD and undergraduate students. Particular emphasis will be given on the recruitment of underrepresented groups including females and minority students. Findings from the project will also be implemented in the coursework to stimulate student awareness on current scientific developments. Additionally, project outcomes will be disseminated within the scientific community and general population through journal publications and postings in the social media platforms such as YouTube videos as well as laboratory group webpage.