Targeting and Internalization Mechanism of LFA-1

描述(由申请人提供)：这项建议的长期目标是了解整合素内化的机制及其对特定细胞靶向药物的效用。这项建议的短期目标是研究LFA-1在T细胞上内化的机制，并利用这一机制将药物靶向白细胞。中心假设是ICAM-1衍生的多肽(Cibr)可以与LFA-1结合并被LFA-1内化。因此，细胞毒性药物(甲氨蝶呤，MTX)将与Cibr多肽相连，得到MTX-Cibr结合物；这种结合物具有双重功能：(A)通过抑制细胞黏附信号来抑制T细胞的激活；(B)通过内化MTX来诱导T细胞死亡。MTX-Cibr结合物对表达LFA-1的T细胞具有选择性毒性，而对LFA-1缺陷的KB上皮细胞无选择性毒性。MTXcIBR结合物对胶原诱导性关节炎(CIA)小鼠模型中类风湿性关节炎的抑制作用优于单独使用MTX。因此，第一个目标是评估MTX-Cibr结合物(特异性目标1)抑制关节炎的机制。MTX-Cibr结合物在CIA小鼠模型中改变T细胞活化从Th1反应到Th2反应的机制将被阐明。此外，将确定MTX-Cibr在CIA小鼠模型中对T细胞承诺的长期动力学效应。其次，MTX-Cibr或FITC-Cibr结合物的内化将由LFA-1在细胞质中释放；或者，它可以(A)留在内吞体内，(B)循环到细胞表面，或(C)靶向其他膜间隔。因此，将确定这些偶联物的内化过程和本地化(具体目标2)。第三，MTX-Cibr的内化也可能通过还原型叶酸载体(RFC)或膜叶酸结合蛋白(MFBP)实现。在具体目标3中，MTX-Cibr将使用野生型和RFC缺陷的小鼠白血病T细胞进行比较。此外，在叶酸存在和不存在的情况下，该结合物将与MTX在mFBP过表达的白血病小鼠细胞中进行比较。最后，LFA-1内化的确切机制以及参与这一过程的其他蛋白质的身份还不是很清楚。在特定的目标4中，Cibr多肽将被修饰并与激活的T细胞上的LFA-1交联；这种交联将与与LFA-1相互作用的蛋白质一起被分离出来。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.