Development & Characterization of Tyrosinase Epitope Specific TCR Transgenic Mice

发展

• 批准号: 7589415
• 负责人: Shikhar Mehrotra
• 金额: $ 19.91万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-18 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7589415
• 关键词: Address; Affinity; Antigens; Autoantigens; Autologous; Avidity; Belief; Breeding; CD4 Positive T Lymphocytes; CD8B1 gene; Cell Therapy; Cells; Cellular biology; Clinical; Cytotoxic T-Lymphocytes; Data; Development; Enzymes; Epitopes; Exhibits; Frequencies; Future; Goals; Growth; HLA-A2 Antigen; Human; Immunotherapy; In Vitro; MHC Class I Genes; Major Histocompatibility Complex; Malignant Neoplasms; Mature T-Lymphocyte; Melanoma Cell; Metastatic Melanoma; Monophenol Monooxygenase; Mus; Mutate; Names; Patients; Peptides; Peripheral; Physiologic pulse; Pigmentation physiologic function; Proteins; Protocols documentation; Reporting; Role; Self Tolerance; Shapes; Signal Transduction; Source; T-Cell Development; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Subsets; TCR Activation; Testing; Transgenic Mice; Transgenic Organisms; Tumor Antigens; Tumor-Infiltrating Lymphocytes; Vaccination; Vaccines; base; cellular engineering; clinically relevant; design; flexibility; human tissue; improved; in vivo; melanocyte; melanoma; mouse model; public health relevance; receptor; receptor expression; receptor-mediated signaling; response; tumor

DESCRIPTION (provided by applicant): Peptide epitopes derived from non-mutated differentiation proteins that are widely expressed by cells of the melanocyte lineage and naturally presented by class I major histocompatibility complex molecules provide targets for vaccination-induced, cytotoxic T lymphocyte (CTL)-based immunotherapy of malignant melanoma. One of the key enzymes involved in pigmentation is the tyrosinase (Tyr) molecule that is expressed not only by malignant melanomas and normal melanocytes, but also by a broad range of human tissues as well. Available data for anti-self human Tyr (hTyr)-based clinical vaccination trials however shows that limited responses were observed in melanoma patients probably due Tyr-specific self-tolerance on the T cell repertoire limited the frequency and avidity of Tyr- reactive CTL. Since T cell receptor (TCR) transgenic mice have contributed to many aspects in understanding T cell ontogeny and selection of the peripheral repertoire, we propose to develop a TCR transgenic mice using a tyrosinase TCR that was previously isolated from a MHC class I- restricted CD4+ T cell isolated from the tumor infiltrating lymphocytes (TILs) of a HLA-A2+ patient with metastatic melanoma. This particular TCR (referred as TIL1383i) has been well characterized and is a co-receptor independent high affinity TCR. The avidity of TIL 1383i for peptide pulsed targets is 10-100-fold lower than most melanoma-reactive CD8+ T cell clones. In our first submission we hypothesized that owing to its origin and high affinity, this TIL1383i TCR may get selected again on a CD4 cell. Indeed we were able to successfully generate the TIL1383i TCR transgenic and our hypothesis proved to be partially correct when we got TIL1383i TCR expression on both CD4 and CD8 T cells. Surprisingly however the T cells passed thymic selection in mice with C57BL/6J background. Now we propose to carry out comprehensive analysis of this new transgenic mouse in context of tumor immunotherapy with the following aims: 1). To characterize antigen recognition and the functional avidity of CD4+ and CD8+ T cells from TIL1383i TCR mice. 2). To determine if CD4+ and CD8+ T cells in TIL1383i TCR mice can control the growth of B16/A2/Kb tumors. We believe that the T cells obtained from the proposed TCR transgenic mice would closely mimic the human T cells and thus the results obtained from the proposed study would help improve us in designing future immunotherapy trials using this unique TCR. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop and characterize a transgenic mice bearing high affinity T cell receptor (TCR) reactive to tumor associated epitope tyrosinase. The high affinity TCR that was used to develop this TCR transgenic mice was isolated from a unique class-I restricted CD4 cell expanded from tumor infiltrating lymphocytes (TILs) of an HLA-A2+ patient with metastatic melanoma (thus referred as TIL1383i TCR). The unusual origin of the TCR and the unique founder obtained on C57BL/6J mice exhibiting TIL1383i TCR expression on both CD4 and CD8 T cell subsets makes this proposal unique since we now propose to understand the role of TCR affinity in tumor response by altering the signal strength of the TIL1383i TCR. This objective will be accomplished by breeding our TIL1383i/Tg mice with commercially available HLA-A2 and HLA-A/H2-Db mice. Successful completion of this proposal would provide us information about the feasibility of using this TIL1383i TCR in T cell adoptive immunotherapy protocols.

描述（由申请人提供）：衍生自非突变分化蛋白的肽表位，由黑色素细胞谱系的细胞广泛表达，并由 I 类主要组织相容性复合物分子天然呈现，为疫苗接种诱导的、基于细胞毒性 T 淋巴细胞 (CTL) 的恶性黑色素瘤免疫治疗提供了靶点。参与色素沉着的关键酶之一是酪氨酸酶 (Tyr) 分子，它不仅由恶性黑色素瘤和正常黑色素细胞表达，而且还由多种人体组织表达。然而，基于抗自身人 Tyr (hTyr) 的临床疫苗接种试验的现有数据表明，在黑色素瘤患者中观察到的反应有限，可能是由于 T 细胞库上的 Tyr 特异性自我耐受限制了 Tyr 反应性 CTL 的频率和亲和力。由于 T 细胞受体 (TCR) 转基因小鼠在理解 T 细胞个体发育和外周库选择的许多方面做出了贡献，因此我们建议使用酪氨酸酶 TCR 开发 TCR 转基因小鼠，该酪氨酸酶 TCR 先前是从 MHC I 类限制性 CD4+ T 细胞中分离出来的，该细胞是从 HLA-A2+ 患者的肿瘤浸润淋巴细胞 (TIL) 中分离出来的。 转移性黑色素瘤。这种特殊的 TCR（称为 TIL1383i）已得到充分表征，并且是一种不依赖于共受体的高亲和力 TCR。 TIL 1383i 对肽脉冲靶标的亲和力比大多数黑色素瘤反应性 CD8+ T 细胞克隆低 10-100 倍。在我们的第一份提交中，我们假设由于其来源和高亲和力，该 TIL1383i TCR 可能会在 CD4 细胞上再次被选择。事实上，我们能够成功地产生 TIL1383i TCR 转基因，并且当我们在 CD4 和 CD8 T 细胞上获得 TIL1383i TCR 表达时，我们的假设被证明是部分正确的。然而令人惊讶的是，T 细胞在具有 C57BL/6J 背景的小鼠中通过了胸腺选择。现在我们建议在肿瘤免疫治疗的背景下对这种新型转基因小鼠进行全面分析，目的如下：1）。表征来自 TIL1383i TCR 小鼠的 CD4+ 和 CD8+ T 细胞的抗原识别和功能亲合力。 2）。确定 TIL1383i TCR 小鼠中的 CD4+ 和 CD8+ T 细胞是否可以控制 B16/A2/Kb 肿瘤的生长。我们相信，从拟议的 TCR 转基因小鼠中获得的 T 细胞将非常模仿人类 T 细胞，因此从拟议的研究中获得的结果将有助于改进我们使用这种独特的 TCR 设计未来的免疫治疗试验。 公共健康相关性：该项目的目标是开发和鉴定具有对肿瘤相关表位酪氨酸酶具有反应性的高亲和力 T 细胞受体 (TCR) 的转基因小鼠。用于开发这种 TCR 转基因小鼠的高亲和力 TCR 是从一种独特的 I 类限制性 CD4 细胞中分离出来的，该细胞是从患有转移性黑色素瘤的 HLA-A2+ 患者的肿瘤浸润淋巴细胞 (TIL) 中扩增出来的（因此称为 TIL1383i TCR）。 TCR 的不寻常起源和在 CD4 和 CD8 T 细胞亚群上均表现出 TIL1383i TCR 表达的 C57BL/6J 小鼠上获得的独特创始人使这一提议独一无二，因为我们现在建议通过改变 TIL1383i TCR 的信号强度来了解 TCR 亲和力在肿瘤反应中的作用。这一目标将通过将我们的 TIL1383i/Tg 小鼠与市售 HLA-A2 和 HLA-A/H2-Db 小鼠进行繁殖来实现。该提案的成功完成将为我们提供有关在 T 细胞过继免疫治疗方案中使用 TIL1383i TCR 的可行性的信息。