Genetically modified T lymphocytes for redirection of th

摘要：本项目的目的是通过基因工程使T淋巴细胞靶向表达表皮生长因子受体(EGFR)/ErbB家族表面分子异源二聚体的肿瘤并发挥其效应功能。为了实现这一目标，我们将设计跨膜嵌合分子，使其胞外区域表达EGFR/ErbB配体的受体结合区域、heregulins(HRG)和T细胞受体(TCR)的Zeta链的细胞质区域。正在考虑的工作假设是，这些HRG/Zeta链嵌合体的EGF样结合域将介导表达在肿瘤细胞表面的ErbB受体的识别，从而促进转导的T淋巴细胞在肿瘤部位的募集。HRG是一种简便的简单工程分子，具有潜在的动物和人类应用前景。然而，其他结合分子也可以用于嵌合体的胞外部分。嵌合蛋白的细胞质结构域来自TCR Zeta链，包含TCR的主要信号转导。通过其免疫特异性酪氨酸激活基序(ITAM‘s)，该链有望促进下游信号的激活，并在原位诱导特异性的细胞毒性免疫反应。预计对共刺激分子的需求：为此，表达T细胞共刺激分子CD28的HRG结合域和细胞质结构域的嵌合结构的工程也在进行中。HRG/Zeta和HRG/CD28嵌合分子目前正在设计中，并将通过体外(体外)转导的方式在T细胞系和原代分离的T淋巴细胞表面异位表达。转基因的T细胞将进行生化和功能鉴定。已考虑通过使用不同的HRG异构体、胞外铰链(间隔物)和跨膜结构域来优化嵌合结构的表达和功能。已经建立了一个体外模型来测量嵌合分子激活通常跟随TCR与抗原相互作用的一系列事件的能力。表达ErbB分子的肿瘤将被用作靶点。对ErbB阳性肿瘤的信号转导、激活标志物、淋巴因子的分泌、增殖、细胞毒功能等生化参数进行了体外检测。逆转录病毒载体已经被设计用于体内应用。一种用于临床前试验的动物模型正在开发中，用于微调反应特性，例如表达不同ITAM数量和/或类型的工程细胞内Zeta链。

Summary: Aim of this project is to genetically engineer T lymphocytes to be directed at and exert effector function against tumors expressing heterodimers of the epidermal growth factor receptor (EGFR)/ErbB family of surface molecules. To achieve this goal, we will engineer transmembrane chimeric molecules with the extracellular region expressing the receptor-binding domains of the EGFR/ErbB ligands, heregulins (HRG), and the cytoplasmic domains of the zeta chain of the T cell receptor (TCR). The working hypothesis under consideration is that the EGF-like binding domain of these HRG/zeta chain chimeras will mediate the recognition of the ErbB receptors expressed on the surface of tumor cells, thus promoting recruitment of the transduced T lymphocytes at the tumor site. HRG is a convenient molecule of simple engineering with potential application for use in animals and humans. Other binding molecules, however, could be used for the extracellular component of the chimera. The cytoplasmic domain of the chimeric protein, derived from the TCR zeta chain, encompasses the primary signal transducer of the TCR. Throughout its immuno-specific tyrosine activation motifs (ITAM's), this chain is expected to promote the activation of downstream signaling and to induce a specific cytotoxic immune response in situ. A requirement for co-stimulatory molecules is anticipated: To this end, the engineering of chimeric constructs expressing the HRG binding domains and cytoplasmic domains of the T cell co-stimulatory molecule, CD28, is also being undertaken. HRG/zeta and HRG/CD28 chimeric molecules are currently being engineered and will be ectopically expressed by in vitro (ex vivo) transduction on the surface of T cell lines and primary isolated T lymphocytes. Transfected T cells will be characterized biochemically and functionally. Consideration has been given to optimizing expression and functionality of the chimeric constructs by using different HRG isoforms, extracellular hinges (spacers), and transmembrane domains. An in vitro model has being established to measure the ability of the chimeric molecules to activate the cascade of events that normally follows the interaction of the TCR with the antigen. Tumors expressing ErbB molecules will be used as targets. Biochemical parameters of signal transduction, activation markers, lymphokine secretion, proliferation, cytotoxic function in response to ErbB-positive tumor challenges have been examined in vitro. Retroviral vectors have been engineered for in vivo applications. A animal model for pre-clinical testing is being developed for fine-tuning of response characteristics, such as the engineering intracellular zeta chains expressing different ITAM number and/or types.