A new use for intracellular antibody expression: inactivation of human immunodeficiency virus type 1.

An article in this issue ofthe Proceedings (1) uses the intracellular expression of an antibody to inactivate the human immunodeficiency virus type 1 (HIV-1) envelope protein and to reduce the infectivity of HIV particles. This work is the latest in a series of studies in which engineered antibody genes have been introduced into a cell in order to alter its phenotype. In 1988, to test the feasibility of inactivating a protein in vivo through the inducible, intracellular expression of an antibody, antibody sequences were introduced into yeast (2). The protein target chosen for this study was alcohol dehydrogenase I (ADH I), a cytoplasmic enzyme whose activity can be selected either for or against. Heavy and light chain cDNAs encoding a neutralizing antiADH I monoclonal antibody (3) were isolated from the secreting hybridoma. The signal sequences were removed from these cDNAs, and synthetic translation initiation codons were added. These truncated cDNAs were then inserted into a single expression vector such that both were under the control of a galactoseinducible promoter. When this construct was introduced into yeast, stable heavyand light-chain polypeptides were synthesized (2). The heavy chain, light chain, and ADH I coprecipitated as a complex, indicating that the antibodies were capable of assembly and antigen binding. To test whether the antibodies neutralized ADH activity intracellularly, cells were plated under conditions that favor the growth of cells containing reduced levels of ADH, and evidence was found that the antibodies did in fact produce a limited degree of neutralization in vivo. Several extensions of this approach were suggested (2): the efficiency of neutralization might be improved by genetic selection; antigens in other cellular compartments might be neutralized by leaving the signal sequences intact or by replacing them with intracellular sorting sequences; the antibody genes might be placed under developmental control; a wide variety ofantigens might be susceptible to neutralization, including products of multigene families, nonprotein molecules, and oncogene products. In 1991, elegant studies by Hilvert and coworkers (4, 5) used intracellular antibody expression to alter cell metabolism in a different way: they expressed a catalytic antibody, or abzyme, so as to endow a yeast cell with an enzymatic activity. Specifically, they used an abzyme that catalyzes a step in aromatic amino acid synthesis, a step normally catalyzed by the product of the yeast ARO7 gene. They cloned cDNAs encoding both heavy and light chains of this antibody, and they used an expression strategy similar to that for the anti-ADH antibodies, in that signal sequences were removed and a new ATG initiation codon was added; however, rather than use the entire antibody genes, the heavy-chain gene was truncated so that an Fab fragment would be expressed. Using a high-copy expression plasmid in an aro7 mutant, Hilvert and coworkers were able to recover functional antibody from yeast (4). They found that the yeastderived antibody had binding and catalytic properties indistinguishable from the hybridoma-derived antibody, and they estimated that this functional Fab antibody accounted for :0.1% of total cellular protein. Assembly of heavy and light chains was estimated to be =60701% efficient, and immunolocalization experiments revealed a staining pattern expected of a cytoplasmic protein. However, enzymatic activity was initially not detected in vivo, perhaps because the abzyme is 104 less efficient than the natural enzyme and was not overexpressed correspondingly. A genetic selection was then successfully used by Hilvert and coworkers (5) to isolate a mutant strain of yeast in which antibody function could be detected in vivo. The antibody was shown to complement the aro7 defect in this strain, allowing the cells to grow under selective conditions. The mutation did not increase the chemical efficiency of the enzyme; rather, the mutation was suggested to improve the intracellular expression and/or stability of the antibody, among other possibilities. It will be of interest to characterize this mutation in more detail and to determine whether it may provide or suggest means of increasing the efficiency of intracellular antibody neutralization in yeast and other systems. In this issue of the Proceedings Marasco et al. (1) report a phenotypic alteration caused by intracellular antibody expression in a mammalian system. The antibody chosen for this study recognizes the envelope glycoprotein of the HIV-1. This glycoprotein, gpl20, binds to the CD4 protein present in the membrane of a subset oflymphocytes. Rather than express the heavy and light chains of the antibody independently, the authors use a single-chain antibody (6, 7), in which the heavy chain variable region is linked to the light chain variable region by means of a (Gly-Gly-Gly-Gly-Ser)3 flexible linker. This antibody is also different from those used in the yeast studies in that it contains an N-terminal signal sequence, in order to target it to the endoplasmic reticulum, where it could bind to gpl20. The authors show that the antibody is in fact expressed and can be detected in the endoplasmic reticulum. They show that the antibody and the envelope protein coprecipitate, and they provide evidence that (i) processing of the envelope protein precursor is inhibited, (ii) the function of the envelope protein is reduced, in that envelope-mediated syncytium formation was reduced by 80-90o in assays with CD4+ cells, and (iii) expression of antibody substantially reduces the infectivity of HIV-1 produced by infected cells. What are the prospects for the future use of intracellular antibody expression in research and, eventually, medicine? Two considerations provide grounds for cautious optimism. First, microinjection of various antibodies has given rise to a wide variety of phenotypic effects: for example, ras oncogene-induced cell transformation was transiently reverted by microinjection of certain anti-ras antibodies (8). Second, over the past several years a number of workers, in addition to those cited above, have engineered the expression of intracellular antibodies in various cellular compartments of several cell types. For example,