In view of the enhanced antigen neutralizing capability imparted by the catalytic function, we propose to isolate antibodies (Abs) for passive immunotherapy of HIV-1. This goal has become feasible because of the recent acquisition of the following information: a subset of Abs expresses peptidase activity; synthesis of peptidase Abs, including gp120 cleaving Abs incresed in lupus mice; the peptidase activity is encoded by a germline gene encoding the variable (V) region of the light chain subunit; and, the catalytic site is structurally similar to the active sites found in non-Ab serine proteases.
In Aim 1, the specificity of the gp120 cleaving Abs in lupus mice will be enhanced by immunization with gp120, a B cell epitope derived from the CD4 binding site of gp120, and if needed, intact HIV-1 particles. Recombinant Fv constructs with peptidase activity will be selected from phage display libraries (constructed by Core B) using gp120 analogs designed to bind covalently to the serine protease-like site found in certain Ab light chains (covalently reactive analogs, CRAs). The CRAs to be employed include derivatives of the whole gp120 molecule and the gp120 epitope. The essential features of the CRAs include: the presence of an electrophilic ester group reactive with nucelophilic Ser residues, a phosphonate structure mimicking the tetrahedral transition structure, a basic flanking residue reactive with the germline peptidase sites, and additional flanking residues derived from gp120 to permit high affinity gp120 recognition by the Abs.
In Aim 2, the CRAs will be employed as immunogens to permit the selective recruitment and somatic maturation of the catalyst gene(s) for synthesis of gp120-cleaving Abs. Improvements in the catalytic turnover are predicted over the course of clonal selection because superior CRA binders are likely to be superior transition state stabilizers. This will be evident as increased binding of the Abs to the CRAs relative to the unmodified gp120. As in aim 1, the best catalysts will be isolated from Fv phage display libraries using CRA selections. Cleavage of the gp120 B cell epitope, monomer gp120 in solution, and native gp120 expressed on the HIV surface will be studied by electrophoresis, HPLC and radioassay methods. The catalytic Ab responses in autoimmune and non-autoimmune mice will be compared to determine whether the regulatory factors limiting catalytic Ab synthesis by the healthy immune system are surmounted by the CRA immunizations. In vitro HIV-1 infectivity studies will be carrie dout by Core C to compare the HIV neutralizing activity of catalytic and noncatalytic Abs. The desired result from these studies is that we will have in hand catalytically efficient Fv constructs derived from autoimmune mice with the capability of specifically cleaving gp120 and potently neutralizing the infectivity of HIV-1. In addition, if the gp120 CRAs provoke a catalytic Ab response in non-autoimmune mice, these reagents can be considered as prototypes for a prophylactic vaccine capable of eliciting catalytic immunity to HIV.
| Taguchi, Hiroaki; Keck, Zhenyong; Foung, Steven K H et al. (2004) Antibody light chain-catalyzed hydrolysis of a hepatitis C virus peptide. Bioorg Med Chem Lett 14:4529-32 |
| Paul, Sudhir; Karle, Sangeeta; Planque, Stephanie et al. (2004) Naturally occurring proteolytic antibodies: selective immunoglobulin M-catalyzed hydrolysis of HIV gp120. J Biol Chem 279:39611-9 |
| Karle, S; Nishiyama, Y; Taguchi, H et al. (2003) Carrier-dependent specificity of antibodies to a conserved peptide determinant of gp120. Vaccine 21:1213-8 |
| Planque, Stephanie; Taguchi, Hiroaki; Burr, Gary et al. (2003) Broadly distributed chemical reactivity of natural antibodies expressed in coordination with specific antigen binding activity. J Biol Chem 278:20436-43 |
| Paul, Sudhir; Planque, Stephanie; Zhou, Yong-Xin et al. (2003) Specific HIV gp120-cleaving antibodies induced by covalently reactive analog of gp120. J Biol Chem 278:20429-35 |
| Bangale, Yogesh; Karle, Sangeeta; Planque, Stephanie et al. (2003) VIPase autoantibodies in Fas-defective mice and patients with autoimmune disease. FASEB J 17:628-35 |
| Taguchi, Hiroaki; Burr, Gary; Karle, Sangeeta et al. (2002) A mechanism-based probe for gp120-Hydrolyzing antibodies. Bioorg Med Chem Lett 12:3167-70 |
| Zhou, Yong-Xin; Karle, Sangeeta; Taguchi, Hiroaki et al. (2002) Prospects for immunotherapeutic proteolytic antibodies. J Immunol Methods 269:257-68 |
| Nishiyama, Yasuhiro; Taguchi, Hiroaki; Luo, Jin-Quan et al. (2002) Covalent reactivity of phosphonate monophenyl esters with serine proteinases: an overlooked feature of presumed transition state analogs. Arch Biochem Biophys 402:281-8 |
| Paul, S; Tramontano, A; Gololobov, G et al. (2001) Phosphonate ester probes for proteolytic antibodies. J Biol Chem 276:28314-20 |
