No abstract

Studies of naturally occurring polymorphisms of the CCR5 gene have shown that deletion of the functional receptor or reduced expression of the gene can have beneficial effects in preventing HIV-1 infection or delaying disease. Because these polymorphisms are found in otherwise healthy people, strategies that aim to prevent or limit expression of CCR5 should be beneficial in the treatment of HIV-1 disease. To test this approach we have developed a CCR5-specific single-chain antibody that was expressed intracellularly and retained in the endoplasmic reticulum. This CCR5-intrabody efficiently blocked surface expression of human and rhesus CCR5 and thus prevented cellular interactions with CCR5-dependent HIV-1 and simian immunodeficiency virus envelope glycoprotein. Intrabody-expressing cells were shown to be highly refractory to challenge with R5 HIV-1 viruses or infected cells. These results suggest that gene therapy approaches that deliver this intracellular antibody could be of benefit to infected individuals. Because the antibody reacts with a conserved primate epitope on CCR5 this strategy can be tested in nonhuman lentivirus models of HIV-1 disease.

The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human scFv (single chain antibody fragment) libraries using a short linker (GGSSRSS) or a long linker (GGSSRSSSSGGGGSGGGG). In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final scFv products that are used for cloning.

The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human Fab (fragment antigen binding) antibody libraries. In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final Fab products that are used for cloning.

Bacteriophage display of combinatorial antibody libraries is one means by which monoclonal antibodies of a desired specificity can be selected without the use of conventional hybridoma technology. Phage display is the expression of proteins and peptides on the surface of a bacteriophage particle, and a combinatorial antibody library is a collection of rearranged variable‐region gene segments that have been randomly combined through the use of recombinant molecular biology techniques to yield a large ‘library’ of antibodies.