Genetically targeted adenovirus vector directed to CD40-expressing cells

Abstract

The success of gene therapy depends on the specificity of transgene delivery by therapeutic vectors. The present study describes the use of an adenovirus (Ad) fiber replacement strategy for genetic targeting of the virus to human CD40, which is expressed by a variety of diseased tissues. The tropism of the virus was modified by the incorporation into its capsid of a protein chimera comprising structural domains of three different proteins: the Ad serotype 5 fiber, phage T4 fibritin, and the human CD40 ligand (CD40L). The tumor necrosis factor-like domain of CD40L retains its functional tertiary structure upon incorporation into this chimera and allows the virus to use CD40 as a surrogate receptor for cell entry. The ability of the modified Ad vector to infect CD40-positive dendritic cells and tumor cells with a high efficiency makes this virus a prototype of choice for the derivation of therapeutic vectors for the genetic immunization and targeted destruction of tumors.

FIG. 1.

Expression of recombinant forms of human sCD40L protein in E. coli. Recombinant proteins corresponding to the TNF-like domain of either the native human CD40L, sCD40L (lane 1), or its mutated, cysteine-free forms, CD40L/AV and CD40L/VA (lanes 2 and 3, respectively), were expressed in E. coli by using plasmid vector pET20b(+). All of these molecules were designed to contain amino-terminal six-His tags. The proteins were purified from cleared bacterial lysates by affinity chromatography on Ni-NTA-Sepharose, denatured in a sample buffer, and analyzed by SDS-PAGE.

FIG. 2.

Specific binding of sCD40L proteins to CD40-expressing cells. 293 (A) or 293.CD40 (B) cells were probed with either anti-CD40 MAb G28.5 or one of the three recombinant proteins comprising the TNF-like domain of human CD40L (white profiles). sCD40L corresponds to the wild-type CD40L protein, while sCD40L/AV and sCD40L/VA are cysteine-free mutants. The bound proteins were detected with an FITC-conjugated secondary Ab. Control samples were incubated with the secondary Ab only (black profiles). The cells were sorted on a FACScan flow cytometer.

FIG. 3.

Analysis of trimerization of recombinant FF/CD40L chimeras. FF/CD40L proteins (lanes 3 and 4) expressed in either E. coli (A) or 293T/17 cells (B) were analyzed by SDS-PAGE or Western blotting, respectively. Wild-type Ad5 fiber protein expressed in insect cells was used as a control (lanes 1 and 2). Samples comprising IMAC-purified proteins (A) or cleared cell lysates (B) were loaded on the gels in amounts normalized by total protein contents. Lanes 1 and 3 contain samples which were fully denatured by boiling in a sample buffer prior to loading on the gels. Samples in lanes 2 and 4 were not denatured and thus contain proteins in their native configurations.

FIG. 4.

Incorporation of FF/CD40L proteins into Ad particles. Samples of the modified Ad vector, Ad5LucFF/CD40L (lanes 2 and 4), or the unmodified control, Ad5Luc1 (lanes 1 and 3), containing 10¹⁰ viral particles were resolved by SDS-PAGE with (lanes 1 and 2) or without (lanes 3 and 4) boiling in a sample buffer. Proteins electroblotted onto a PVDF membrane were detected with antifiber MAb 4D2.

FIG. 5.

Structural integrity of the TNF-like domain of CD40L incorporated into the FF/CD40L chimera. A control virus containing unmodified Ad5 fibers, Ad5Luc1, or CD40-targeted Ad5LucFF/CD40L was incubated with the conformation-specific anti-CD40 MAb 2B3 adsorbed on an ELISA plate. Bound virions were detected with a polyclonal anti-Ad2 Ab. The amounts of each virus used are shown below the graph (number of particles). OD₄₉₀, optical density at 490 nm.

FIG. 6.

Receptor specificity and efficiency of transduction by a CD40-targeted Ad vector. CD40-negative 293 cells and CD40-positive 293.CD40 cells were transduced with Ad5LucFF/CD40L or untargeted Ad5Luc1 (control) at an MOI of 10 viral particles/cell. Prior to infection, the cells in the samples shown by the gray and white bars were preincubated with the recombinant Ad5 fiber knob and with sCD40L (each used at 100 μg/ml), respectively. Black bars correspond to luciferase activity in cells infected in the absence of inhibitors. Luciferase activity (relative light units) detected in the lysates of infected cells is the average of three measurements. Error bars indicate standard deviations.

FIG. 7.

Integration of mutant F5Δ and FF/CD40L proteins into mosaic Ad5Luc.FF/CD40L.F5Δ virions. Aliquots of 10¹⁰ viral particles of Ad5Luc1 (lane 1), Ad5Luc.FF/CD40L (lane 2), and Ad5Luc.FF/CD40L.F5Δ (lane 3) were fully denatured, and viral proteins were separated by SDS-PAGE. The wild-type (F5) and mutant (F5Δ) Ad5 fibers as well as the FF/CD40L chimera were identified on the blots with MAb 4D2.

FIG. 8.

Comparison of the gene transfer and cell-binding profiles of Ad5 vectors. (A) Transduction of 293 and 293.CD40 cells with Ad5Luc1, Ad5Luc.FF/CD40L, or Ad5Luc.FF/CD40L.F5Δ was done essentially as described in the legend to Fig. 6, except that no inhibition of infection was attempted. (B) Radiolabeled viruses were allowed to bind to cells under conditions which prevented their internalization. After unbound virus was removed, the cell-associated radioactivity was measured with a scintillation counter. All of the measurements were done in triplicate. part/cell, particles per cell.

FIG. 9.

CD40-mediated transduction of human cells with Ad5Luc.FF/CD40L. (A) The expression of CAR and CD40 by humanmonocyte-derived DCs and T24 bladder cancer cells was assessed by flow cytometry with relevant antibodies (see Materials and Methods for details). Of note, the control peak on the left panel overlaps the RmcB-generated signal and thus is not seen. (B) The infectivities of the targeted vectors, Ad5Luc.FF/CD40L and Ad5Luc.FF/CD40L.F5Δ, and that of the control vector, Ad5Luc1, were compared. Gray and white bars show samples blocked with 100 μg each of the Ad5 fiber knob and sCD40L/ml, respectively, prior to addition of the virus. Black bars correspond to luciferase activity in cells infected in the absence of inhibitors. Error bars indicate standard deviations from the average reporter activity seen in three identical samples.