A strategy for high antibody expression with low anti-drug antibodies using AAV9 vectors

Abstract

Introduction: Use of adeno-associated virus (AAV) vectors is complicated by host immune responses that can limit transgene expression. Recent clinical trials using AAV vectors to deliver HIV broadly neutralizing antibodies (bNAbs) by intramuscular administration resulted in poor expression with anti-drug antibodies (ADA) responses against the bNAb.

Methods: Here we compared the expression of, and ADA responses against, an anti-SIV antibody ITS01 when delivered by five different AAV capsids. We first evaluated ITS01 expression from AAV vectors three different 2A peptides. Rhesus macaques were selected for the study based on preexisiting neutralizing antibodies by evaluating serum samples in a neutralization assay against the five capsids used in the study. Macaques were intramuscularly administered AAV vectors at a 2.5x10^12 vg/kg over eight administration sites. ITS01 concentrations and anti-drug antibodies (ADA) were measured by ELISA and a neutralization assay was conducted to confirm ex vivo antibody potency.

Results: We observed that ITS01 expressed three-fold more efficiently in mice from AAV vectors in which heavy and light-chain genes were separated by a P2A ribosomal skipping peptide, compared with those bearing F2A or T2A peptides. We then measured the preexisting neutralizing antibody responses against three traditional AAV capsids in 360 rhesus macaques and observed that 8%, 16%, and 42% were seronegative for AAV1, AAV8, and AAV9, respectively. Finally, we compared ITS01 expression in seronegative macaques intramuscularly transduced with AAV1, AAV8, or AAV9, or with the synthetic capsids AAV-NP22 or AAV-KP1. We observed at 30 weeks after administration that AAV9- and AAV1-delivered vectors expressed the highest concentrations of ITS01 (224 µg/mL, n=5, and 216 µg/mL, n=3, respectively). The remaining groups expressed an average of 35-73 µg/mL. Notably, ADA responses against ITS01 were observed in six of the 19 animals. Lastly, we demonstrated that the expressed ITS01 retained its neutralizing activity with nearly the same potency of purified recombinant protein.

Discussion: Overall, these data suggest that the AAV9 capsid is a suitable choice for intramuscular expression of antibodies in nonhuman primates.

Keywords: AAV (adeno-associated virus); HIV - human immunodeficiency virus; SIV - simian immunodeficiency virus; anti-drug antibodies (ADA); antibody.

Figure 1

Schematic and characterization of ITS01 expressed from an AAV transfer plasmid. (A) Schematic of an the AAV vector encoding ITS01 from 5’ ITR to 3’ ITR. ITS01 expression is driven by a CASI promoter and utilizes a 2A peptide to generate both the heavy and light chains. (B) Coomassie-stained SDS-PAGE of purified ITS01 antibodies generated with the indicated 2A peptide. (C) SIVsmE660-coated ELISA plates were incubated with indicated concentrations of purified recombinant ITS01 antibody. ITS01 binding was determined using an HRP-conjugated, anti-human Fc secondary antibody and binding activity was measured at an absorbance of 450 nm. ITS01 antibody made using F2A, P2A, and T2A peptide is indicated. rh-CD4-Ig is a positive control protein. Error bars indicate standard deviation (S.D.).

Figure 2

ITS01 expression from AAV9 vectors using different 2A peptides in NSG mice. (A) Three groups of four (F2A) or eight (P2A and T2A) NSG mice each were administered a fixed dose of 5×10¹⁰ vector genomes (vg) AAV9 vectors (about 2.5×10¹² vg/kg) encoding ITS01 using the indicated 2A peptide (F2A – red, P2A – blue, T2A – green) in a 25 µL volume in the left gastrocnemius muscle. Blood draws were performed over eight weeks and plasma samples were analyzed by gp120 ELISA to determine ITS01 concentrations. (B) Comparison of ITS01 concentrations at week 8 post AAV administration. Each dot represents an individual animal. Mean is represented by the black bar. Error bars indicate S.D. Statistical comparison of the 2A peptide groups was determined by a one-way ANOVA with Tukey’s multiple comparison test. Statistical significance is defined as *** indicates P value ≤ 0.001; n.s., indicates not significant.

Figure 3

Pre-screening rhesus macaques for AAV neutralizing antibodies. (A) To screen for neutralizing antibodies, we utilized a cellular assay similar the HIV-1 TZM-bl neutralization assay. 360 rhesus macaque serum samples were diluted 1:5 in cell culture medium and mixed at a 1:1 ratio (final serum dilution of 1:10) with AAV vectors encoding the firefly luciferase reporter. After 60 minutes, 3×10⁴ cells were added to the serum/virus mixture and incubated for 24 hours. Neutralization was determined as the absence of luciferase production. We observed that 8%, 16%, and 42% of the samples were negative for neutralizing antibodies against AAV1, AAV8, and AAV9 capsids, respectively. (B) 19 rhesus macaque samples for the NHP study were screened for neutralizing responses as in (A) except that AAV-NP22 and AAV-KP1 vectors were included. Three macaques that were negative for each capsid were selected to be used in the study, except for AAV-NP22, which only had two macaques without pre-existing neutralizing antibodies. The third macaque selected, r18027, is identified with about a 50% neutralizing response to AAV-NP22. The fifth macaque selected for the AAV8 group, rh2813, had 10% neutralizing activity at a 1:10 dilution.

Figure 4

Comparison of ITS01 concentrations from rhesus macaques with different AAV vectors. (A) Quantification of ITS01 concentration from AAV vectors with AAV1, AAV8, AAV9, AAV-NP22, and AAV-KP1 capsids. Five groups of three or five macaques each were inoculated with 2.5×10¹² vg/kg of the indicated AAV vector in eight injection sites per macaque. ITS01 concentrations for the AAV1, AAV8, AAV9, AAV-NP-22, and AAV-KP1 groups were measured over the course of 30 weeks by SIVsmE660 gp120 ELISA. (B) The average ITS01 concentrations for each group are represented. (C) The average area under the curve (AUC) as for each capsid group is represented with each individual icon representing an animal from that capsid group. Error bars indicate S.D. in (A, B).

Figure 5

Low ADA responses against expressed ITS01 in all groups. ADA responses were measured by ELISA. ITS01-coated plates were incubated with diluted serum samples from the incubated timepoints. Anti-ITS01 antibodies were detected using a human, anti-lambda secondary. (A–E) The absorbance values for each macaque from the study are graphed based on capsid group. Error bars indicate S.D. (F) Average absorbance values for the five groups. Overall, 13 of 19 macaques have ADA responses around the absorbance values of the pre-administration time points.

Figure 6

ITS01 from macaque serum retains its neutralizing activity in ex vivo neutralization assays. Neutralization activity of serum samples from 30 weeks post AAV administration for the AAV1 (left) and AAV9 (right) macaques against SIVsmE660.A8 pseudovirus was assessed by TZM-bl neutralization activity. A serum sample from before AAV vector administration was used as a negative control and the same serum sample with a known concentration of ITS01 was used as a positive control. Error bars indicate S.D.