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内化的机制及其靶向药物到特定细胞的效用。本课题的短期目标是研究LFA-1在T细胞上的内化机制，并利用这一机制将药物靶向到白细胞。核心假设是icam -1衍生肽（cIBR）可以结合并被LFA-1内化。因此，细胞毒性药物（甲氨蝶呤，MTX）将与cIBR肽结合，得到MTX-cIBR偶联物；这种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；(b)通过内化MTX诱导t细胞死亡。MTX-cIBR偶联物对表达lfa -1的T细胞具有选择性毒性，但对缺乏lfa -1的KB上皮细胞没有选择性毒性。MTXcIBR偶联物对胶原诱导关节炎（CIA）小鼠模型类风湿关节炎的抑制作用优于单用MTX。因此，第一个目的是评估MTX-cIBR偶联物抑制关节炎的机制（Specific Aim 1）。在CIA小鼠模型中，MTX-cIBR偶联物改变T细胞从Th1到Th2反应的激活机制将被阐明。此外，MTX-cIBR对CIA小鼠模型中t细胞承诺的长期动力学效应将被确定。其次，MTX-cIBR或FITC-cIBR偶联物的内在化将由LFA-1在细胞质中释放；或者，它可以(a)留在核内体中，(b)再循环到细胞表面，或(c)靶向其他膜室。因此，这些共轭物的内化过程和定位将被确定（具体目标2）。第三，MTX-cIBR也可能通过叶酸载体（RFC）或膜叶酸结合蛋白（mFBP）内化。在Specific Aim 3中，MTX-cIBR将使用野生型和rfc缺陷小鼠白血病T细胞进行比较。此外，该偶联物将在叶酸存在和不存在的情况下与MTX在mFBP过表达的白血病小鼠细胞中进行比较。最后，LFA-1内化的确切机制和参与这一过程的其他蛋白质的身份尚不清楚。在Specific Aim 4中，cIBR肽将被修饰并与活化T细胞上的LFA-1交联；与LFA-1相互作用的蛋白质将被分离。这些分离的蛋白质将被鉴定。

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.