Glycoengineering of AAV-delivered monoclonal antibodies yields increased ADCC activity

The absence of fucose on asparagine-297 of the human immunoglobulin G (IgG) heavy chain has been shown to enhance antibody-dependent cellular cytotoxicity (ADCC) activity by 10- to 100-fold compared to fucosylated antibody. Our lab is studying the use of adeno-associated virus (AAV) as a vector for the delivery of HIV-specific antibodies for therapeutic purposes. Since the antibody is produced by vector-transduced cells in vivo, current techniques of glycoengineering cannot be utilized. In order to achieve similar enhancement of ADCC with AAV-delivered antibodies, short hairpin RNAs (shRNAs) that target fucosyltransferase-8 (FUT8), were designed, tested, and cloned into AAV vectors used to deliver HIV-specific broadly neutralizing antibodies (bNAbs). Antibodies produced by our glycoengineered-AAV (GE-AAV) vectors were analyzed for fucose content and ADCC. GE-AAV constructs were able to achieve over 80% knockdown of FUT8. Results were confirmed by lectin western blot for α1-6 fucose, which revealed almost a complete absence of fucose on GE-AAV-produced antibodies. GE-AAV-produced antibodies revealed >10-fold enhancement of ADCC, while showing identical neutralization and gp140 trimer binding compared to their fucosylated counterparts. ADCC was enhanced 40- to 60-fold when combined with key Fc mutations known to enhance binding to FcγRIIIA. Our findings define a powerful approach for supercharging AAV-delivered anti-HIV antibodies. [Display omitted] Termini et al. describe the creation and validation of a novel glycoengineered-AAV vector for the delivery of anti-HIV antibodies. These AAV are capable of knocking down fucosyltransferase-8 following transduction and, when combined with antibody-Fc mutations, produce broadly neutralizing antibodies with 40- to 60-fold higher ADCC

Further improvements of ssAAV vector expression cassettes.

<p>The exact same samples were analyzed by two methods. Yields of secreted 5L7 IgG antibodies were compared by (<b>A</b>) Western Blot and quantified by (<b>B</b>) ELISA, following transfection of HEK293T cells with different ssAAV vector plasmids.</p

Levels of IgG expression from modified ssAAV vectors.

<p>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). (<b>A</b>) The conventional ssAAV-5L7 IgG2 vector (as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158009#pone.0158009.g001" target="_blank">Fig 1B</a>) 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>B</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.</p

Coomassie staining of purified antibodies and immunoadhesins.

<p>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 (<b>A</b>) non-reducing and (<b>B</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.</p

Expression of full-length antibodies from recombinant AAV vectors.

<p>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 (<b>A</b>) 4L6 IgGs or (<b>B</b>) 5L7 IgGs from co-transfection of heavy and light chain vectors (two vector approach) <i>vs</i>. 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.</p

Expression of 5L7 IgG1 after AAV-mediated transduction <i>in vitro</i>.

<p>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 (<b>A</b>) 2x10⁴ rAAV genome copies per cell (GC/cell), (<b>B</b>) 5x10³ GC/cell and (<b>C</b>) 1x10³ GC/cell. (<b>D</b>) Rhesus fibroblast cells were infected with 2x10⁵ GC/cell. AAV transduction experiments shown in (<b>A</b> + <b>D</b>) were conducted at a different time than experiments in (<b>B</b> + <b>C</b>). 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, ***<i>p</i> < 0.001, **<i>p</i> < 0.01, *<i>p</i> < 0.05.</p

Schematic illustration of AAV constructs and transgene products.

<p>Design of recombinant AAV vectors expressing antibody or antibody-like molecules. (<b>A</b>) 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 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158009#pone.0158009.ref043" target="_blank">43</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158009#pone.0158009.ref052" target="_blank">52</a>]. (<b>B</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) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158009#pone.0158009.ref016" target="_blank">16</a>]; 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.</p

Long-Term Delivery of an Anti-SIV Monoclonal Antibody With AAV

Long-term delivery of anti-HIV monoclonal antibodies using adeno-associated virus (AAV) holds promise for the prevention and treatment of HIV infection. We previously reported that after receiving a single administration of AAV vector coding for anti-SIV antibody 5L7, monkey 84-05 achieved high levels of AAV-delivered 5L7 IgG1 which conferred sterile protection against six successive, escalating dose, intravenous challenges with highly infectious, highly pathogenic SIVmac239, including a final challenge with 10 animal infectious doses (1). Here we report that monkey 84-05 has successfully maintained 240-350 μg/ml of anti-SIV antibody 5L7 for over 6 years. Approximately 2% of the circulating IgG in this monkey is this one monoclonal antibody. This monkey generated little or no anti-drug antibodies (ADA) to the AAV-delivered antibody for the duration of the study. Due to the nature of the high-dose challenge used and in order to rule out a potential low-level infection not detected by regular viral loads, we have used ultrasensitive techniques to detect cell-associated viral DNA and RNA in PBMCs from this animal. In addition, we have tested serum from 84-05 by ELISA against overlapping peptides spanning the whole envelope sequence for SIVmac239 (PepScan) and against recombinant p27 and gp41 proteins. No reactivity has been detected in the ELISAs indicating the absence of naturally arising anti-SIV antibodies; moreover, the ultrasensitive cell-associated viral tests yielded no positive reaction. We conclude that macaque 84-05 was effectively protected and remained uninfected. Our data show that durable, continuous antibody expression can be achieved after one single administration of AAV and support the potential for lifelong protection against HIV from a single vector administration

High concordance of ELISA and neutralization assays allows for the detection of antibodies to individual AAV serotypes

Prescreening of participants in clinical trials that use adeno-associated virus (AAV) vectors is required to identify naive participants, as preexisting neutralizing antibodies can limit the efficacy of AAV gene therapies. The presence of antibodies to individual AAV serotypes is typically detected by neutralization assay. To streamline the screening process, we compared an ELISA-based screening method with a neutralization assay for the detection of antibodies against AAV1, AAV8, and AAV9 in a collection of 50 rhesus macaque sera and 20 human sera. We observed a high level of concordance between the two assays (Pearson r > 0.8) for all three serotypes in both sample sets. We thus investigated pre- vs post-vector inoculation sera samples from rhesus macaques that received AAV1 or AAV8 vector inoculations for cross-reactive anti-AAV antibodies. All 12 macaques seroconverted to the vector they received, but many also reacted to the other serotypes. Our results validate an easy-to-use ELISA for reliable detection of antibodies to individual serotypes of AAV. Our results also demonstrate that an antibody response post-AAV inoculation may partially cross-react with other AAV serotypes. Overall, these results suggest that either assay can be used by academic labs for prescreening samples for preexisting anti-AAV antibodies. [Display omitted] As more studies deploy novel AAV gene therapies, the need has grown for identifying hosts negative for anti-AAV capsid antibodies. Gardner et al. compare and show a high degree of correlation between an ELISA and neutralization assay to identify hosts negative for binding and neutralizing antibodies against AAV capsids

Use of a Gamma-2 Herpesvirus as a Vector to Deliver Antibodies to Rhesus Monkeys

The gamma-2 herpesvirus of rhesus monkeys, rhesus monkey rhadinovirus (RRV), persists principally in B cells of its host. We constructed recombinant strains of RRV expressing the rhesus monkey-derived anti-SIV monoclonal antibodies 4L6 and 5L7 and compared the RRV-mediated in vivo delivery of these antibodies in rhesus monkeys with previous studies that utilized intramuscular delivery with adeno-associated virus (AAV) vector. Recombinant RRV-4L6 and RRV-5L7 were both shown to stably produce the antibodies in persistently infected B cell lines in culture. Two RRV-negative rhesus monkeys were experimentally infected with recombinant RRV-4L6 and two with recombinant RRV-5L7. Following infection, the appearance of the delivered antibody was readily detected in all four animals. However, the levels of the delivered antibody were considerably lower than what has been typically observed following intramuscular AAV delivery. Furthermore, three of the four monkeys had an antibody response to the delivered antibody as had been observed previously with intramuscular AAV delivery of these same antibodies. We conclude that this recombinant herpesvirus has no inherent advantage over AAV for delivery of potentially therapeutic monoclonal antibodies in a rhesus monkey model