In view of the enhanced antigen neutralizing capability imparted by the catalytic function, the investigators propose to develop human catalytic antibodies (Abs) capable of cleaving gp120 for immunotherapy of HIV-1 infection. This goal has become feasible because of identification of serum antibodies with the catalytic activity in lupus patients and a monoclonal light chain from a multiple myeloma patient. This proposal has assembled the component technologies necessary to reach this goal, i.e., isolation of recombinant Ab fragments from the cloned immune repertoire of a lupus patient, selection strategies designed to trap the Abs with the greatest chemical reactivity combined with specificity for gp120, and detailed study of HIV-1 neutralization by the Abs. In the first specific aim, catalytically proficient gp120-cleaving Fv domains from a lupus phage display library will be isolated. The phage displayed Abs will be bound to reagents that trap serine protease sites found in the Abs, followed by selection for binding to gp120. In vitro HIV-1 infectivity studies using a T cell line and peripheral blood mononuclear cells will be carried out to identify catalysts suitable for further development as anti-HIV-1 reagents. In the second specific aim, the gp120 binding VH domains from an HIV-1 infected subject will be conjugated to catalytic VL domains to improve the specificity of the Fv constructs, and to derive catalysts with broad epitope specificity and neutralization capability. The catalytic properties of the Fv constructs with the greatest neutralizing activity will be studied in detail, including analysis of the gp120 binding affinity, catalytic rate constant, peptide bonds cleaved by the catalysts, the epitope in gp120 recognized by the catalysts, and the cleavage of polypeptides unrelated to gp120. Further study of HIV-1 neutralization activity will be done using a variety of clinical isolates, in assays designed to reveal the mechanism of neutralization and to approximate in vivo HIV-1 infection, including assay of neutralization using tonsil histoculture and SCID mouse models. The investigators expect these studies to generate catalytic Fv constructs ready for the final stages of drug development for passive immunotherapy of HIV infection.
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