Pergamon 0161~5890(95)00153-O zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Molecular Immunology, Vol. 33, No. 415, 439450, pp. 1996 Copyright 0 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0161-5890196 $15.00 + 0.00 RECOMBINANT HUMAN Fab ANTIBODY FRAGMENTS TO HIV-l REV AND TAT REGULATORY PROTEINS: DIRECT SELECTION FROM A COMBINATORIAL PHAGE DISPLAY LIBRARY GLENN R. PILKINGTON,*$ LINGXUN DUAN,_F MINGHUA ZHU,? WALTER KEIL* and ROGER J. POMERANTZT *Intracel Corporation, Cambridge, MA 02139, U.S.A.; TThe Dorrance H. Hamilton Laboratories, Molecular Retrovirology Section, Division of Infectious Diseases, Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, U.S.A. (First received 20 March 1995; accepted in revisedform 25 October 1995) Abstract-A human Fab phage display library has been produced from peripheral blood lymphocytes of an individual who was asymptomatic after 10 years of infection with human immunodeficiency virus type-l (HIV-l). The library was panned against the HIV-l Rev and Tat regulatory proteins and several clones, producing Fab binding to these proteins, were isolated (3 to Rev and 4 to Tat) with binding constants varying from 10-6M to lo-*M. DNA sequencing demonstrated two unique anti- Rev Fab clones, but the four anti-Tat Fab comprised only two unique IgG, heavy chain Fd fragments, illustrating redundancy of light chains. Peptide mapping of the epitopes recognized by these Fab indicated that three of the anti-Tat Fab were directed to the functional domain between amino acid residues 22-33 of the Tat molecule, and that binding was inhibited by reduction of this cysteine-rich region with dithiothreitol. The anti-Rev Fab were directed to sites adjacent to the Rev basic nucleolar localization sequence (residues 52-64) and to the Rev activation domain (residues 75-88). Binding constants were of a similar order to that of an anti-Rev single-chain Fv fragment (SFv) used successfully for intracellular immunization, and as such intracellular effects with the human anti-Tat and anti-Rev Fab are not precluded. These newly described human antibody fragments to HIV-l regulatory proteins may be critical moieties for gene therapeutic protocols, to control HIV-l rep- lication in human cells. Copyright 0 1996 Elsevier Science Ltd. Key words: Fab, HIV- 1, Rev, Tat, regulatory proteins, phage display library. INTRODUCTION Cloning of human antibodies from long-term asympto- matic human immunodeficiency virus type- 1 (HIV- 1) infected-individuals could provide another potential modality for treatment of patients who would otherwise $Author to whom correspondence should be addressed. Abbreviations: Fd, antibody fragment Fd constituting domains VH and CH,; Fab, antibody fragment Fab constituting domains V,, CHlr V, and C,; SFv, single-chain Fv antibody fragment constituting domains V, and VL linked by a syn- thetic linker; HAMA, human anti-mouse antibody; CDR, complementarity determining region; FR, framework region; Rev, phosphoprotein regulator of HIV-l gene expression; Tat, transactivator of HIV-l gene expression; ~24, major capsid protein of HIV-l virus; gp41, trans- membrane portion of the HIV-l envelope glycoprotein; gpl20, external portion of the HIV-l envelope glycoprotein. Genbank accession numbers, rev9V,, L38560, revl6/20V,, L38561, rev16/20DeletedLC, L38562, tatl04V,, L38563, tat104/107VH, L38564, tatl07V,, L38565, tat16/3lV,, L38566, tatl6V,, L38567, tat3lV,, L38568, rev9.33V,, U33321. succumb to acquired immune deficiency syndrome (AIDS). The role of antibody based immunity in HIV-l infections is suggested by the fact that the presence of autologous neutralizing antibodies to the HIV-l virus, in the serum of HIV-l-infected mothers, is significantly associated with non-infection in 5&60% of fetuses (Bry- son et zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLK al., 1993; Scarlatti et al., 1993). In addition, the use of antibody genes in concert with specialized vectors allowing antibody expression at localized sites of viral replication and assembly within the infected human cell, as proposed by Baltimore (1988), may prove to be a potent form of gene therapy and prophylaxis. To this end. encouraging results have already been obtained using a single-chain Fv (SFv) fragment generated from a mouse monoclonal antibody which binds to the activation domain of the Rev protein of HIV-l (Duan et al., 1994a, 1994b). Results demonstrated a greater than a 95% reduction in HIV-l p24 antigen expression and syncytia formation, along with a sequestration of Rev in the cyto- plasm of HIV- 1 -infected cells expressing intracellular SFv, as compared to control HIV-l-infected cells. Clini- cal testing, however, might be preferable using DNA encoding a human antibody to Rev in order to obviate 439