A general strategy for generating intact, full-length IgG antibodies that penetrate into the cytosol of living cells

Abstract

These authors contributed equally to this work.Keywords: cell-penetrating antibody, cytosolic protein targeting, cellular internalization, endosomal release, IgG antibody, intracellular trafficking, next-generation antibodyAbbreviations: IgG, immunoglobulin G; VL, light chain variable domain; VH, heavy chain variable domain; LC, light chain; HC, heavy chain; CDR, complementarity-determining region.Full-length IgG antibodies cannot cross cell membranes of living cells; this limits their use for direct targeting of cytosolic proteins. Here, we describe a general strategy for the generation of intact, full-length IgG antibodies, herein called cytotransmabs, which internalize into living cells and localize in the cytosol. We first generated a humanized light chain variable domain (VL) that could penetrate into the cytosol of living cells and was engineered for association with various subtypes of human heavy chain variable domains (VHs). When light chains with humanized VL were coexpressed with 3 heavy chains (HCs), including 2 HCs of the clinically approved adalimumab (Humira Ò ) and bevacizumab (Avastin Ò ), all 3 purified IgG antibodies were internalized into the cytoplasm of living cells. Cytotransmabs primarily internalized into living cells by the clathrin-mediated endocytic pathway through interactions with heparin sulfate proteoglycan that was expressed on the cell surface. The cytotransmabs escaped into the cytosol from early endosomes without being further transported into other cellular compartments, like the lysosomes, endoplasmic reticulum, Golgi apparatus, and nucleus. Furthermore, we generated a cytotransmab that co-localized with the targeted cytosolic protein when it was incubated with living cells, demonstrating that the cytotransmab can directly target cytosolic proteins. Internalized cytotransmabs did not show any noticeable cytotoxicity and remained in the cytosol for more than 6 h before being degraded by proteosomes. These results suggest that cytotransmabs, which efficiently enter living cells and reach the cytosolic space, will find widespread uses as research, diagnostic, and therapeutic agents.

Humanization of a phosphothreonine peptide-specific chicken antibody by combinatorial library optimization of the phosphoepitope-binding motif

“…The C-terminal biotinylated pT231 peptide ( 224 KKVAVVR(pT 231 ) PPKSPSSAK 240 -biotin) derived from the human tau protein [5], the non-phosphorylated T231-peptide ( 224 KKVAVVR(T 231 ) PPKSPSSAK 240 ), and off-target pT52-peptide ( 44 SEKQLSQA(pT 52 ) AAATNHTTDNGK 64 -biotin) derived from human lysyl-tRNA synthetase [16] were synthesized with 95% purity (Peptron, Korea). DNAs encoding the initial humanized sequences of VH and VL of the huPT0 clone ( Fig.…”

“…The plasmids encoding HC and LC, respectively, were transiently co-transfected into HEK293F cells to express the IgG antibody [16,20]. Both huPT3 and huPT9 IgG antibodies were well-expressed in the correctly assembled form (Fig.…”

“…The huPT3 and huPT9 humanized Fab antibodies were converted into the conventional human IgG1 (IgG1 with VH3-Vl3) by subcloning the VH and VL sequences into the human IgG1 and CL constant domains, respectively [16]. The plasmids encoding HC and LC, respectively, were transiently co-transfected into HEK293F cells to express the IgG antibody [16,20].…”

“…Antibodies were produced by transient transfection of the plasmids into HEK293F cells using the Freestyle 293-F expression and media system (Invitrogen), as described in previous studies [16,20]. Antibodies were purified from the culture supernatants using a CaptivA™ PriMAB protein A column (Repligen, USA) following the manufacturer's protocol.…”

“…The two isolated Fab clones (huPT3 and huPT9) were reformatted into IgG1 through sub-cloning the HC (VHeCH1) gene into the modified pcDNA 3.4 vector (Invitrogen) carrying the human IgG1 Fc (hinge-CH 2 -CH 3 ) gene and LC (VL-CL) gene into the pcDNA 3.4 vector, as described previously [16,20]. Antibodies were produced by transient transfection of the plasmids into HEK293F cells using the Freestyle 293-F expression and media system (Invitrogen), as described in previous studies [16,20].…”