Recombinant human antibodies against the reverse transcriptase of human immunodeficiency virus type-1

Abstract

Inhibitory antibodies to the reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1) can be used to block the life cycle of the virus. We have isolated five different human single chain Fv (ScFv) antibodies specific for HIV-1 RT from an antibody phage display library. Three of these antibodies inhibited the RNA-dependent DNA polymerase (RDDP) activity of RT and one of the three (F-6) inhibited also its DNA-dependent DNA polymerase (DDDP) activity. Unexpectedly, F-6 binds to the carboxyl terminus of the large subunit of RT, which contains the ribonuclease H (RNase H) domain, and not the polymerase domain of the protein. Moreover, this binding did not inhibit the RNase H enzymatic activity. To further characterize F-6 antibody, two cyclic synthetic peptides based on the amino acids sequences of the CDR3 of F-6 were synthesized. Peptide F-6CDRH3, with the sequence of CDR3 of the heavy chain, inhibited the RDDP activity of RT while peptide F-6CDRL3, with the sequence of CDR3 of the light chain, had no effect on this activity of RT. These results indicate that some of the effects of F-6 are mediated by the CDR3 of the heavy chain. The antibodies identified here will be further tested as intrabodies for their capacity to protect human cells from HIV-1 infection.

Introduction

In recent years, many attempts have been made to render CD4⁺ cells resistant to human immunodeficiency virus (HIV) infection by blocking the key steps in the life cycle of the virus after entry to the target cells [1], [2], [3], [4], [5], [6]. One potential approach to prevent HIV replication is to express recombinant antibodies (intrabodies) directed against HIV enzymes inside the cells in order to inhibit their enzymatic activities and so block the life cycle of the virus [7], [8]. Such treatments may be used in combination with the current available chemotherapies against HIV to obtain a more efficient method of suppressing the virus. The reverse transcriptase (RT) enzyme of HIV is an attractive target for inhibition since RT is crucial for the early stages of HIV life cycle and its inhibition is likely to lead to an abortive infection. As in all retroviruses, RT copies the viral genomic single-stranded RNA into double-stranded DNA, which is subsequently integrated into the cellular DNA by the viral enzyme integrase [9]. Blocking the life cycle of HIV at this early stage of reverse transcription prevents the viral DNA synthesis and, thus, also the subsequent integration of the viral genome into the cellular DNA [10] leading to an abortive infection.

A number of murine antibodies directed against HIV-1 RT were previously isolated [11], [12], [13], [14], [15], [16], [17], [18]. Some of them were expressed inside human cells, efficiently blocking HIV replication in these cells [10], [19]. However, cells that express foreign proteins, such as murine antibodies, are likely to elicit in vivo immune reactions, leading to the rejection of the transfected cells. Even after humanization of the mouse antibodies used for transfection of the cells, such a rejection will not be necessarily prevented [20]. The best approach to overcome these obstacles should rely on the use of human antibodies against HIV-1 enzymes. Unfortunately, only two such antibodies have so far been reported. Both of these were not specific enough and inhibited other DNA polymerases as well, possibly due to recognition of structures common to different DNA polymerases [21].

In order to isolate new human, recombinant anti-HIV RT intrabodies, we have screened a human semisynthetic phage display library containing a large repertoire of human single chain Fv (ScFv) antibodies with randomized amino acid sequences both in their heavy chain and light chain CDR3s [22]. Such ScFv antibodies, when isolated from large phage display libraries, may posses binding affinities as high as those of natural antibodies and therefore will be of a potential use for therapeutic intervention [23]. In this manuscript, we describe the isolation and characterization of five different ScFv antibodies directed against HIV-1 RT. Three of these inhibited in vitro the RNA-dependent DNA polymerase (RDDP) activity of HIV-1 RT and one of them inhibited also the DNA-dependent DNA polymerase (DDDP) activity of this enzyme. A synthetic cyclic peptide derived from the CDR3 amino acid sequence of the heavy chain of one of these inhibitory ScFv antibodies was also capable of inhibitivy of HIV-1 RT.