Genetically modified T lymphocytes for redirection of the immune function again

本项目的目的是通过基因工程将T淋巴细胞定向于表达表皮生长因子受体(EGFR)/ErbB家族表面分子异二聚体的肿瘤，并对其发挥效应功能。为了实现这一目标，我们将设计跨膜嵌合分子，其细胞外区域表达EGFR/ErbB配体的受体结合域、heregulins （HRG）和T细胞受体（TCR）的zeta链的细胞质域。目前正在考虑的工作假设是，这些HRG/zeta链嵌合体的egf样结合域会介导对肿瘤细胞表面表达的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是一种方便的简单工程分子，在动物和人类中具有潜在的应用前景。然而，其他结合分子可以用于嵌合体的细胞外成分。嵌合蛋白的细胞质结构域来源于TCR的zeta链，包含了TCR的主要信号转导分子。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诱导特异性细胞毒性免疫反应。预计对共刺激分子的需求：为此，表达HRG结合域和T细胞共刺激分子CD28的细胞质域的嵌合构建的工程也正在进行中。HRG/zeta和HRG/CD28嵌合分子目前正在进行工程设计，并将通过体外（离体）转导在T细胞系和原代分离的T淋巴细胞表面进行异位表达。转染后的T细胞将被生物化学和功能表征。将考虑通过使用不同的HRG异构体、细胞外铰链（间隔器）和跨膜结构域来优化嵌合结构体的表达和功能。将建立一个体外模型来测试嵌合分子激活通常跟随TCR与抗原相互作用的级联事件的能力。表达ErbB分子的肿瘤将被用作靶标。在erbb阳性肿瘤挑战下，信号转导、激活标志物、淋巴因子分泌、增殖、细胞毒功能的生化参数将被检查。进一步的开发将包括建立用于临床前测试的体内模型和对反应特性的微调，例如表达不同ITAM数量和/或类型的工程细胞内zeta链。如果获得的信息证明逆转录病毒载体是合理的，也将考虑用于体内应用。

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 transducing molecule of the TCR. Through 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 will be 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 will be established to test 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, lymphokines secretion, proliferation, cytotoxic function in response to ErbB-positive tumor challenges will be examined. Further development will include the establishment of an in vivo model for pre-clinical testing and the fine-tuning of response characteristics, such as the engineering intracellular zeta chains expressing different ITAM number and/or types. A retroviral vector will also be considered for in vivo applications if justified by the information acquired.