Genetically modified T lymphocytes for redirection of the immune function again

该项目的目的是基因工程T淋巴细胞针对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发挥效应子功能。 为了实现这一目标，我们将设计跨膜嵌合分子，其胞外区表达EGFR/ErbB配体、调蛋白（HRG）的受体结合结构域和T细胞受体（TCR）的ζ链的胞质结构域。考虑中的工作假设是这些HRG/ζ链嵌合体的EGF样结合结构域将介导肿瘤细胞表面上表达的ErbB受体的识别，从而促进在肿瘤部位的转导的T淋巴细胞的募集。 HRG是一种简单工程的方便分子，具有用于动物和人类的潜在应用。 然而，其他结合分子可用于嵌合体的细胞外组分。 嵌合蛋白质的胞质结构域来源于TCR ζ链，包括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），预期该链促进下游信号传导的激活并原位诱导特异性细胞毒性免疫应答。 预期需要共刺激分子：为此，也正在进行表达T细胞共刺激分子CD 28的HRG结合结构域和胞质结构域的嵌合构建体的工程化。 HRG/zeta和HRG/CD 28嵌合分子目前正在工程化，并将通过体外（离体）转导在T细胞系和原代分离的T淋巴细胞表面异位表达。 将对转染的T细胞进行生物化学和功能表征。将考虑通过使用不同的HRG同种型、细胞外铰链（间隔区）和跨膜结构域来优化嵌合构建体的表达和功能。将建立体外模型以测试嵌合分子激活通常在TCR与抗原相互作用之后的级联事件的能力。表达ErbB分子的肿瘤将用作靶标。 将检查响应ErbB阳性肿瘤激发的信号转导、活化标志物、淋巴因子分泌、增殖、细胞毒性功能的生化参数。进一步的开发将包括建立用于临床前测试的体内模型和响应特性的微调，例如表达不同ITAM数量和/或类型的工程化细胞内ζ链。如果获得的信息证明合理，也将考虑将逆转录病毒载体用于体内应用。

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.