Intracellular Antibodies (Intrabodies) for Gene Therapy of Infectious Diseases

Abstract

Intracellular antibodies (intrabodies) represent a new class of neutralizing molecules with a potential use in gene therapy. Intrabodies are engineered single-chain antibodies in which the variable domain of the heavy chain is joined to the variable domain of the light chain through a peptide linker, preserving the affinity of the parent antibody. Intrabodies are expressed inside cells and directed to different subcellular compartments where they can exert their function more effectively. The effects of intrabodies have been investigated using structural, regulatory, and enzymatic proteins of the human immunodeficiency virus (HIV-1) as targets. These intrabodies have demonstrated their versatility by controlling early as well as late events of the viral life cycle. In this article, we review studies of the use of intrabodies as research tools and therapeutic agents against HIV-1.

T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery

“…[56][57][58][59][60] To further improve cellular efficacy of the intrabody, a KDEL endoplasmic retention signal has been included, which provides entrapment of newly synthesized and recycled CCR5 as well and maintains cellular compartmentalization of the CCR5 intrabody. 16,25 As shown in Figure 1b and c, CCR5 levels are substantially reduced (X95%) in lymphocyte and myeloid cell lines and, most importantly, primary CD4 + T cells. Moreover, the CCR5 intrabody targeting effect is restricted to CCR5 as seen in the lack of CXCR4 disruption, Figure 1d, and protection to X4-tropic viruses, Figure 2c.…”

“…Together, the findings from the PM1 and primary CD4 + T-cell HIV-1 challenge studies demonstrate the therapeutic efficacy of CAD-R5 delivery and specificity of CCR5 intrabody-mediated protection. 25). Two months after the establishment of the NOD/SCID-hu thy/liv mice, all mice were sub-lethally irradiated to reduce endogenous human cells in the thy/ liv graft, and the next day 2.5 Â 10 5 transduced HLAmismatched CD34 + cells (80% tNGFR positive) were directly injected into the graft.…”

“…36,37 Our previous findings using a neomycin PM1 cell line selected for high levels of CCR5 intrabody transgene integration demonstrated a 7-day survival advantage after R5-tropic viral exposure. 16,25 To further investigate if CCR5 intrabody expression provides both survival and cell expansion, we established assays to determine if small numbers of PM1 CAD-R5-transduced cells have a selective survival and growth advantage during an ongoing HIV-1 infection.…”

“…17,18,61 A possible explanation for the enhanced efficacy of the CCR5 intrabody in our studies could lie in the coupling of the KDEL sequence with a high-affinity intrabody. 16,25 The use of the KDEL allows efficient intrabody trapping of both recycled and newly expressed receptor in the endoplasmic reticulum, thereby enhancing CCR5 loss at the cell surface.…”

“…The CCR5 intrabody is specific for the N-terminal extracellular domain of CCR5 and engineered with the Lys-Asp-Glu-Leu (KDEL) endoplasmic reticulum retention signal. 16,25 We determined that expression of the CCR5 intrabody from the CAD-R5 vector resulted in almost complete disruption of CCR5 cell surface expression and protected both T-cell lines and primary CD4 + T cells from robust infection with free R5-tropic viruses. Furthermore, intrabody-expressing CD4 + T cells, obtained from non-obese diabetic/severely combined immunodeficient (NOD/SCID)-human thymus/liver (hu thy/liv) mice transplanted with CAD-R5-transduced CD34 + cells, were protected from high-titer R5-tropic HIV-1 challenge in tissue culture.…”