Heavy and Light Chain Variable Single Domains of an Anti-DNA Binding Antibody Hydrolyze Both Double- and Single-stranded DNAs without Sequence Specificity

A nucleic acid-hydrolyzing antibody penetrates into cells via caveolae-mediated endocytosis, localizes in the cytosol and exhibits cytotoxicity

“…However, 3D8 scFv was not translocated from the cytosol to the nucleus, remaining in the cytosol even up to 48 h after internalization. 3D8 scFv also has a large number of cationic amino acids in the VH-and VL-CDRs (total 4 Arg and 4 Lys residues) [17]. Three-dimensional structural analysis of the surface which is exposed to CDRs of 3D8 scFv reveals two patches clustered with basic amino acids; one is on VH-CDR3 (Arg94, Lys98, and Arg99 in Kabat numbering) and the other is on VL-CDR1 (Arg27 f, Arg28, and Lys29) [17].…”

“…3D8 scFv also has a large number of cationic amino acids in the VH-and VL-CDRs (total 4 Arg and 4 Lys residues) [17]. Three-dimensional structural analysis of the surface which is exposed to CDRs of 3D8 scFv reveals two patches clustered with basic amino acids; one is on VH-CDR3 (Arg94, Lys98, and Arg99 in Kabat numbering) and the other is on VL-CDR1 (Arg27 f, Arg28, and Lys29) [17]. Therefore, it is not clear why 3D8 scFv remained in the cytosol, although it shares common features of continuous or conformational clustering of basic amino acids with other cellpenetrating anti-DNA Abs or CPPs.…”

“…Mouse-originated 3D8 scFv protein was bacterially expressed and purified by IgGsepharose affinity chromatography, according to the previously described procedures [17]. The protein concentrations were determined using extinction coefficients of 1.995 for scFv in units of mg ml -1 ·cm -1 at 280 nm, which were calculated from the amino acid sequence.…”

“…RNA cleavage assays with purified 3D8 scFv. 3D8 scFv (0.1 mM) or HW1 scFv (0.1 mM) purified from bacterial expression [17,23] was incubated with total cellular RNA (1 mg) extracted from HeLa cells, in TBS buffer (50 mM Tris-Cl, pH 7.5, 50 mM NaCl, pH 7.5) at 37 8C for 2 h prior to detection of hydrolysis. RNase A (1 U) was used as a positive control in the buffers provided by the manufacturer (New England Biolabs).…”

“…Despite the ability of anti-DNA Abs to penetrate into cells, detailed intracellular trafficking routes and cytotoxic mechanisms have not been well studied. Previously, we have reported an anti-DNA single chain variable fragment (scFv) Ab, 3D8 scFv, which binds and hydrolyzes DNA without significant sequence specificities [17]. In the present work, we investigated the endocytic pathway and intracellular trafficking of 3D8 scFv in HeLa cells, and underlying mechanism for cytotoxicity by 3D8 scFv.…”