Fig 1.
Schematic illustration of AAV constructs and transgene products.
Design of recombinant AAV vectors expressing antibody or antibody-like molecules. (A) Self-complementary AAV (scAAV) containing an expression cassette for a single-chain fragment variable immunoadhesin (scFvi). Upon expression, the scFvi dimerizes to form a mature immunoadhesin with a MW of approximately 120 kDa. The expression cassette is flanked by AAV2 inverted terminal repeats (ITRs); the 5' ITR is truncated to form double-stranded AAV genomes [43,52]. (B) Two strategies for achieving expression of full-length antibodies. The first approach, called the two vector approach, requires two scAAV vectors, one encoding IgG heavy chain and one the light chain. The second strategy, called the one vector approach, utilizes one single-stranded AAV (ssAAV) vector only, with heavy and light chains of IgG expressed from one open reading frame. The two polypeptide chains are separated by a 2A peptide from foot-and-mouth-disease virus (F2A) that mediates cleavage and a furin peptide that allows removal of redundant amino acids at the heavy chain C-terminus following furin enzyme-dependent cleavage. Thus, the heavy chain C-terminus is believed to attain an authentic sequence. The light chain N-terminus is believed to gain an authentic sequence following signal peptide (SP)-mediated cleavage. The transgene cassette is flanked by AAV2 ITRs to form single-stranded AAV genomes. The full-length authentic IgG has a MW of ≥ 150 kDa. Abbreviations: 5'ITRΔtrs, 5' inverted terminal repeat devoid of the terminal resolution site; Short CMV, a shortened variant of the immediate early CMV promoter (CMV) [16]; SV40 intron, an intron from simian virus 40; SP, signal peptide; VL, variable light domain; L, serine-glycine linker peptide; VH, variable heavy domain; H, hinge region; CH, constant heavy domain; CL, constant light domain; pA, polyadenylation signal; Furin, cleavage sequence for the cellular protease furin.
Fig 2.
Expression of full-length antibodies from recombinant AAV vectors.
Levels of expressed IgG or immunoadhesin were analyzed by Western Blot after transfection of HEK293T cells with equal amounts of plasmid DNA (0.5 μg + 0.5 μg or 1 μg). Comparison of secreted (A) 4L6 IgGs or (B) 5L7 IgGs from co-transfection of heavy and light chain vectors (two vector approach) vs. transfection of bicistronic vectors (one vector approach). The two vector approach yielded slightly higher levels of secreted antibodies than the one vector approach. IgG1 versions of the 4L6 and 5L7 full-length antibodies expressed better than IgG2 versions.
Fig 3.
Variations to ssAAV vector design.
Schematic illustration of modifications to the basic bicistronic ssAAV vector. (A) Variants of ssAAV vector comprise constructs with modifications to the Furin/F2A cleavage site and the 3' untranslated region (UTR); the changes include the addition of SGSG peptide and V5 peptide, deletion of furin peptide, and inclusion of a post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE). Abbreviations: ITR, inverted terminal repeat of AAV2; 5'ITRΔtrs, 5' inverted terminal repeat devoid of the terminal resolution site; Short CMV, a shortened variant of the immediate early CMV promoter; SV40 intron, an intron from simian virus 40; SP, signal peptide; VL, variable light domain; L, serine-glycine linker peptide; VH, variable heavy domain; H, hinge region; CH, constant heavy domain; CL, constant light domain; pA, polyadenylation signal; Furin, cleavage sequence for the cellular protease furin; F2A, 2A peptide from foot-and-mouth-disease virus. (B) Amino acid sequences of the Furin/F2A cleavage site in the different ssAAV vector constructs. Amino acids were colored to illustrate the range of specific sequences: encoded sequence of the heavy chain C-terminus (black), Furin peptide (red/orange), V5 peptide (gray), SGSG peptide (blue), F2A peptide (green) and the first amino acid (M, Methionine) of the light chain signal peptide (purple). Cleavage sites are indicated by arrows and are numbered in the order that cleavage is believed to occur: F2A self-cleavage (1.), Furin enzyme-mediated removal (2.), carboxypeptidase enzyme-mediated cleavage of basic amino acids (3.). The underlined amino acids represent the heavy chain C-terminus after the final cleavage within the secretory pathway. In B-cells, the heavy chain genes encode the amino acids Pro-Gly-Lys (PGK) at the C-terminus, however, the secreted IgG lacks the terminal Lys (K) due to removal by carboxypeptidases [31]. The ssAAV-4L6 IgG1 vector construct containing the appropriate Furin peptide was utilized in Fuchs et al. [17].
Fig 4.
Levels of IgG expression from modified ssAAV vectors.
Levels of expressed 5L7 IgG2 were analyzed by Western Blot after transfection of HEK 293T cells with equal amounts of bicistronic ssAAV vectors (1 μg). (A) The conventional ssAAV-5L7 IgG2 vector (as illustrated in Fig 1B) was modified by addition or deletion of peptides (SGSG, V5, Furin) and compared to each other. While all modifications improve expression of the antibody, only the SGSG version of the vector mediates correct F2A-Furin cleavage comparable to the conventional bicistronic ssAAV vector. (B) Demonstration of Furin-mediated cleavage of the F2A peptide remaining on the IgG heavy chain. Deletion of Furin peptide prevents removal of redundant amino acids from the heavy chain C-terminus following F2A cleavage.
Fig 5.
Further improvements of ssAAV vector expression cassettes.
The exact same samples were analyzed by two methods. Yields of secreted 5L7 IgG antibodies were compared by (A) Western Blot and quantified by (B) ELISA, following transfection of HEK293T cells with different ssAAV vector plasmids.
Fig 6.
Coomassie staining of purified antibodies and immunoadhesins.
Purity and integrity of purified IgGs and immunoadhesins was verified by coomassie staining following large-scale transfection of HEK293T cells with ssAAV-IgG and scAAV-immunoadhesin vector plasmids. SDS-PAGE (1 μg of purified protein per lane) and staining under (A) non-reducing and (B) reducing conditions. Both conditions confirmed the expected size and composition of the tested proteins. The immunoadhesins and full-length IgG versions of 4L6 and 5L7 have unusually long heavy chain CDR3 regions compared to polyclonal rhesus IgG heavy chains, thus, heavy chains of 4L6 and 5L7 have a considerably higher MW.
Fig 7.
Expression of 5L7 IgG1 after AAV-mediated transduction in vitro.
AAV vectors were encapsidated with AAV1 wild-type (wt) capsid or AAV1 mutant capsids (Y445F and/or Y731F); in the case of ssAAV, we utilized the modified ssAAV vector construct containing both SGSG and WPRE. Purified AAV virus particles were then used for transduction. HEK293T cells were infected with (A) 2x104 rAAV genome copies per cell (GC/cell), (B) 5x103 GC/cell and (C) 1x103 GC/cell. (D) Rhesus fibroblast cells were infected with 2x105 GC/cell. AAV transduction experiments shown in (A + D) were conducted at a different time than experiments in (B + C). Levels of secreted antibody were measured by ELISA following the time of transduction. Values are depicted as mean ± SD (n = 3/group); ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.