Targeting and Internalization Mechanism of LFA-1

描述（由申请人提供）：本提案的长期目标是了解整合素的内化机制及其用于将药物靶向至特定细胞的效用。该提案的短期目标是研究LFA-1在T细胞上的内化机制，并利用该机制将药物靶向白细胞。核心假设是ICAM-1衍生肽（cIBR）可以结合LFA-1并被其内化。因此，细胞毒性药物（甲氨蝶呤，MTX）将与cIBR肽连接，得到MTX-cIBR缀合物;该缀合物将具有双重功能：（a）通过抑制细胞粘附信号抑制T细胞活化和（B）通过内化MTX诱导T细胞死亡。MTX-cIBR缀合物对表达LFA-1的T细胞具有选择性毒性，但对LFA-1缺陷型KB上皮细胞不具有选择性毒性。MTX cIBR缀合物在胶原诱导的关节炎（CIA）小鼠模型中比单独MTX更好地抑制类风湿性关节炎。因此，第一个目的是评价MTX-cIBR偶联物抑制关节炎的机制（特异性目的1）。将阐明MTX-cIBR缀合物在CIA小鼠模型中将T细胞活化从Th 1应答改变为Th 2应答的机制。此外，还将确定MTX-cIBR对CIA小鼠模型中T细胞定型的长期动力学影响。其次，MTX-cIBR或FITC-cIBR缀合物的内化将通过细胞质中的LFA-1释放;或者，其可以（a）保留在内体中，（B）再循环至细胞表面，或（c）靶向其他膜隔室。因此，将确定这些缀合物的内化过程和定位（具体目标2）。第三，MTX-cIBR的内化也可以通过还原叶酸载体（RFC）或膜叶酸结合蛋白（mFBP）。在特定目标3中，将使用野生型和RFC缺陷型小鼠白血病T细胞比较MTX-cIBR。此外，将在存在和不存在叶酸的情况下，在mFBP过表达白血病小鼠细胞中比较该缀合物与MTX。最后，LFA-1内化的确切机制和参与该过程的其他蛋白质的身份尚未得到很好的理解。在特定目标4中，cIBR肽将被修饰并与活化T细胞上的LFA-1交联;交联将与与LFA-1相互作用的蛋白质一起沿着分离。将鉴定这些分离的蛋白质。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to understand the mechanism of internalization of integrins and their utility for targeting drugs to specific cells. The short-term objective of this proposal is to study the mechanism of internalization of LFA-1 on T cells and to utilize this mechanism for targeting drugs to leukocytes. The central hypothesis is that an ICAM-1-derived peptide (cIBR) can bind to and be internalized by LFA-1. Therefore, a cytotoxic drug (methotrexate, MTX) will be linked with cIBR peptide to give MTX-cIBR conjugate; this conjugate will have dual functions: (a) to inhibit T-cell activation via inhibition of the cell adhesion signal and (b) to induce T-cell death by internalizing MTX. The MTX-cIBR conjugate has selective toxicity for LFA-1-expressing T cells but not LFA-1-deficient KB epithelial cells. MTXcIBR conjugate suppressed rheumatoid arthritis in the collagen-induced arthritis (CIA) mouse model better than MTX alone. Therefore, the first objective is to evaluate the mechanism of suppression of arthritis by MTX-cIBR conjugate (Specific Aim 1). The mechanism of MTX-cIBR conjugate in altering the activation of T cells from Th1 to Th2 response in the CIA mouse model will be elucidated. In addition, the long-term kinetic effects of MTX-cIBR on the T-cell commitment in the CIA mouse model will be determined. Secondly, the internalization of MTX-cIBR or FITC-cIBR conjugates will be released by LFA-1 in the cytoplasm; alternatively, it could (a) remain with the endosome, (b) be recycled to the cell surface, or (c) be targeted to other membrane compartments. Therefore, the internalization process and localization of these conjugates will be determined (Specific Aim 2). Thirdly, the internalization of MTX-cIBR may also be via reduced folate carrier (RFC) or membrane folate binding protein (mFBP). In Specific Aim 3, MTX-cIBR will be compared using wild type and RFC-deficient mouse leukemia T cells. In addition, this conjugate will be compared with MTX in mFBP over-expressing leukemia mouse cells in the presence and absence of folic acid. Finally, the exact mechanism of the LFA-1 internalization and the identity of other proteins that are involved in this process are not well understood. In Specific Aim 4, cIBR peptide will be modified and cross-linked to LFA-1 on activated T cells; the crosslink will be isolated along with proteins that interact with LFA-1. These isolated proteins will be identified.