Development & Characterization of Tyrosinase Epitope Specific TCR Transgenic Mice

描述（由申请人提供）：来源于非突变分化蛋白的肽表位，由黑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体分子自然呈现，为疫苗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靶标。酪氨酸酶（Tyr）分子是参与色素沉着的关键酶之一，它不仅在恶性黑色素瘤和正常黑色素细胞中表达，而且在广泛的人体组织中表达。然而，基于抗自身人Tyr （hTyr）的临床疫苗接种试验的现有数据显示，在黑色素瘤患者中观察到有限的应答，这可能是由于T细胞库上的Tyr特异性自身耐受性限制了Tyr反应性CTL的频率和活跃度。由于T细胞受体（TCR）转基因小鼠在了解T细胞的个体发生和外周库的选择方面做出了许多贡献，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先前从转移性黑色素瘤HLA-A2+患者的肿瘤浸润淋巴细胞（TILs）中分离出MHC I类限制性CD4+ T细胞。这种特殊的TCR（被称为TIL1383i）已经被很好地表征，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。在我们的第一篇论文中，我们假设由于它的起源和高亲和力，这个TIL1383i TCR可能在CD4细胞上再次被选中。事实上，我们能够成功地产生TIL1383i TCR转基因，当我们在CD4和CD8 T细胞上获得TIL1383i TCR表达时，我们的假设被部分证明是正确的。然而，令人惊讶的是，在具有C57BL/6J背景的小鼠中，T细胞通过了胸腺选择。现拟在肿瘤免疫治疗的背景下，对这种新型转基因小鼠进行综合分析，目的如下：1)。目的研究TIL1383i TCR小鼠CD4+和CD8+ T细胞的抗原识别和功能亲和力。2）. 检测TIL1383i TCR小鼠CD4+和CD8+ T细胞是否能控制B16/A2/Kb肿瘤的生长。我们相信从所提出的TCR转基因小鼠中获得的T细胞将非常接近人类T细胞，因此从所提出的研究中获得的结果将有助于我们设计未来使用这种独特的TCR的免疫治疗试验。公共卫生相关性：该项目的目标是开发和表征具有高亲和力T细胞受体（TCR）对肿瘤相关表位酪氨酸酶反应的转基因小鼠。用于开发这种TCR转基因小鼠的高亲和力TCR是从转移性黑色素瘤HLA-A2+患者的肿瘤浸润淋巴细胞（til）扩增的一种独特的i类限制性CD4细胞中分离出来的（因此称为TIL1383i TCR）。在CD4和CD8 T细胞亚群上均表达TIL1383i TCR的C57BL/6J小鼠上获得的TCR的不同寻常的起源和独特的创始人使这一提议变得独特，因为我们现在提议通过改变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TIL1383i/Tg小鼠与市售的HLA-A2和HLA-A/H2-Db小鼠杂交来实现。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该TIL1383i TCR的可行性的信息。

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.