Our objective is to develop a rational approach to specific desensitization of HLA class I-sensitized patients by means of anti-idiotypic antibodies (Ab-2). The project is divided into two parts: I) characterization of the memory B cell response to HLA-A2 and II) development of therapeutic anti- idiotypic reagents. The first section, comprising the bulk of the work to be done, consists of 4 inter-related components: 1) production of mg quantities of soluble HLA class I antigens by transfer into a class I """"""""null"""""""" mutant human lymphoblastoid cell of HLA-A2 alpha chain genes encoding a culture supernatant; 2) coupling of purified HLA-A2 to solid supports for affinity purification of A2-specific IgG antibodies and B cells from A2-sensitized patients; 3) analysis using HLA-A2 subtype gene transferent LCL, mouse monoclonal antibodies to HLA-A2 antigens, and polyclonal anti-idiotypic antibodies (Ab-2) of the epitope and idiotope specificity of the antibody response to HLA-A2; and 4) in vitro monitoring of anti-HLA-A2 memory B cell responses in transfused patients, and leukapheresis of selected A2-sensitized patients to provide sufficient B cells for in vitro testing of Ab-2 preparations. The information generated by part I of the project will be used to develop therapeutic Ab-2 preparations. The goal of part II will be to generate mouse monoclonal antibodies with specificity for """"""""public"""""""" or cross-reactive idiotopes of A2-specific memory B cells. These Ab-2 will be tested as is, or coupled to ricin-A chain and tested for efficacy as immunotoxins using EBV-transformed, HLA-A2-specific memory B cell lines, and bulk cultures of HLA-A2-specific B cells and T helper cells for sensitized patients.
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