Improved Breadth and Potency of an HIV-1-Neutralizing Human Single-chain Antibody by Random Mutagenesis and Sequential Antigen Panning

• Published in: Journal of molecular biology 2004-01, Vol.335 (1), p.209-219
• Main Authors: Zhang, Mei-Yun, Shu, Yuuei, Rudolph, Donna, Prabakaran, Ponraj, Labrijn, Aran F., Zwick, Michael B., Lal, Renu B., Dimitrov, Dimiter S.
• Format: Article
• Language: English
• Subjects: antibody; Antibody Affinity; Antibody Specificity; CD4 Antigens - immunology; Drug Evaluation, Preclinical - methods; gp140; HIV; HIV Antibodies - genetics; HIV Antibodies - immunology; HIV Antigens - immunology; HIV Envelope Protein gp120 - immunology; HIV-1 - immunology; Human immunodeficiency virus 1; Humans; Immunoglobulin Fragments - genetics; Immunoglobulin Fragments - immunology; inhibitors; Inhibitory Concentration 50; Mutagenesis; Mutation; Peptide Library; phage display
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2003.09.055

Several human monoclonal antibodies can neutralize a range of human immunodeficiency virus type 1 (HIV-1) primary isolates but their potency and related ability to suppress generation of HIV-1 escape mutants is significantly lower than the activity of antiretroviral drugs currently in clinical use. Recently, a human Fab, X5, was identified and found to neutralize primary isolates from different clades. Further improvement of the potency and breadth of HIV-1 neutralization by this antibody could be critical for its potential use in the treatment of HIV-1-infected patients. However, increasing potency of an antibody by selection from libraries may lead to a decrease in the breadth of neutralization. In an attempt to solve this problem, we subjected a random mutagenesis library of the scFv X5 to sequential rounds of selection on non-homologous HIV-1 envelope glycoproteins (Envs) dubbed sequential antigen panning (SAP). By using SAP, we identified two scFv antibodies, m6 and m9, that were tested with a panel of 33 diverse primary HIV-1 infectious isolates in an assay based on a reporter cell-line expressing high levels of CD4, CCR5 and CXCR4. The IC 50 was less than 50 μg/ml for 21 (m6) and 19 (m9) out of 29 isolates from group M (subtypes A–C, F, G and CRF-01AE) and one isolate from group N; three isolates from group O were not significantly inhibited at 50 μg/ml. The average IC 50 values for the two antibodies were significantly ( p