Polyreactive anti-DNA monoclonal antibodies and a derived peptide as vectors for the intracytoplasmic and intranuclear translocation of macromolecules

Abstract

Naturally occurring polyreactive anti-DNA mAbs derived from a nonimmunized (NZB x NZW)F1 mouse with spontaneous lupus erythematosus penetrated and accumulated in the nuclei of a variety of cultured cells. These mAbs and their F(ab')2 and Fab' fragments, covalently coupled to fluorescein, peroxidase, or a 15-mer polynucleotide, also translocated to the cell nuclei. A 30-amino acid peptide corresponding to the combined sequences of the complementary-determining regions 2 and 3 of the heavy chain variable region of one mAb was able to penetrate into the cytoplasm and nucleus of cells of several lines. This peptide recognized DNA and was strongly polyreactive. Streptavidin-peroxidase conjugates complexed with the N-biotinylated peptide were rapidly translocated into cells. Similarly, peroxidase or anti-peroxidase polyclonal antibodies covalently coupled to the N-cysteinylated peptide through an heterobifunctional maleimide cross-linker were also rapidly internalized and frequently accumulated in nuclei. The peptide carrying 19 lysine residues at its N-terminal was highly effective in transfecting 3T3 cells with a plasmid containing the luciferase gene. Thus, penetrating mAbs and derived peptides are versatile vectors for the intracellular delivery of proteins and genes.

Figure 1

Kinetic of penetration of mAb F4.1 (50 μg/ml) into 3T3 cells cultured at 37°C or 4°C. At indicated time, cells were trypsinized, counted, washed, and lyzed, and IgG2a mAb in lysates was quantified by ELISA by reference to a standard curve established with F4.1 mAb.

Figure 2

Transfer of various macromolecules by mAbs or their fragments or by a derived peptide. (A) HEp-2 cells cultured overnight with J20.8 F(ab′)₂ fragments coupled to PO (50 μg/ml). (B) PtK2 cells cultured with biotinylated P3 (20 μg/ml) for 1 h and after fixation incubated with streptavidin-PO (5 μg/ml). (C) Biotinylated P3 (1.4 μg) was incubated with streptavidin-PO (10 μg) in 35 μl of PBS for 20 min at room temperature, diluted to 250 μl with complete medium, and added to PtK2 cell culture for 2 h. After fixation, PO was detected with ME-DAB. Arrow shows a dividing cell.

Figure 3

Intranuclear translocation of a fluorescent nucleotide coupled to J20.8 Fab in mouse thymocytes. Cells were cultured for 2 h in the presence of the conjugated nucleotide (50 μg/ml), washed, fixed, and examined under confocal microscope. (Left) Fluorescence. (Right) Phase contrast.

Figure 4

Amino acid sequences of the V_H genes of various mAbs deduced from their nucleotide sequences. Dashes indicate identity with the J20.8 mAb sequence; blanks indicate either that the V_H does not have an amino acid at that position or a sequence was not obtained for that position.

Figure 5

Luciferase expression in 3T3 cells transfected with the pCMVL gene. Cells were incubated with 3 μg of plasmid complexed with 18 μg of K₁₉-P3 (open bar), 10 equivalents of PEI nitrogen per DNA phosphate (shaded bar), or 18 μg of 19-residue polylysine (solid bar). Experiments were done in triplicate and the results show the average ± SEM of five experiments.