Safety and Tolerability of the Adeno-Associated Virus Vector, AAV6.2FF, Expressing a Monoclonal Antibody in Murine and Ovine Animal Models

Abstract

Adeno-associated virus (AAV) vector mediated expression of therapeutic monoclonal antibodies is an alternative strategy to traditional vaccination to generate immunity in immunosuppressed or immunosenescent individuals. In this study, we vectorized a human monoclonal antibody (31C2) directed against the spike protein of SARS-CoV-2 and determined the safety profile of this AAV vector in mice and sheep as a large animal model. In both studies, plasma biochemical parameters and hematology were comparable to untreated controls. Except for mild myositis at the site of injection, none of the major organs revealed any signs of toxicity. AAV-mediated human IgG expression increased steadily throughout the 28-day study in sheep, resulting in peak concentrations of 21.4-46.7 µg/ mL, demonstrating practical scale up from rodent to large animal models. This alternative approach to immunity is worth further exploration after this demonstration of safety, tolerability, and scalability in a large animal model.

Keywords: adeno-associated virus (AAV) vector; large animal model; monoclonal antibody; safety; tolerability; vectored immunoprophylaxis.

Figure 1

Evaluation of vectorized 31C2 monoclonal antibody. (A) 6-week-old male and female BALB/c mice (n = 4) were administered 2 × 10¹⁰ VG of AAV-mAb and plasma was monitored for hIgG expression over a period of 14 weeks. (B) BALF was collected at endpoint and hIgG was quantified. Data are represented as the mean ± standard deviation.

Figure 2

Findings of the murine AAV6.2FF-31C2 safety and tolerability study. 6-week-old male and female BALB/c mice (n = 6; equal male and female mice per group) were administered either a low (1 × 10¹¹ VG), mid (2 × 10¹¹ VG) or high (6 × 10¹¹ VG) dose and sacrificed either 7-, 28- or 56-days post AAV administration. Terminal blood samples were collected at endpoint and plasma was analyzed for (A) hIgG concentration, (B) mIgG concentration and (C) the ratio of hIgG to mIgG is shown. (D) Mice were weighted on a weekly/ biweekly schedule throughout the in-life phase of the study. Plasma and whole blood samples were analyzed for (E) alanine aminotransferase, (F) aspartate aminotransferase, (G) creatine kinase, (H) creatinine, (I) white blood cells, (J) lymphocytes, IFNγ (K) and TNFα (L). A one-way ANOVA was used for analysis. Data are represented as the mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 3

Findings of the ovine AAV6.2FF-31C2 feasibility study. Three 2-week-old lambs were administered 5x10¹² VG/ kg of AAV6.2FF-31C2 by IM injections to the rump. Aged-matched controls (n = 3) were administered PBS IM. Weekly blood samples were collected and analyzed for plasma (A) hIgG concentration, (B) oIgG concentration and (C) the calculated ratio of hIgG to oIgG is displayed. (D) Sheep were weighted weekly throughout the in-life phase of the study. (E) Lung homogenates at endpoint (two sample sites per animal) were analyzed for hIgG concentration. (F) Anti-AAV6.2FF capsid antibodies were monitored weekly. Plasma and whole blood samples were analyzed for (G) creatine kinase, (H) creatinine, (I) alanine aminotransferase, (J) aspartate aminotransferase, (K) white blood cells and (L) absolute lymphocytes. The black dotted line represents the average value for the three control sheep.

Figure 4

Comparison of striated murine and ovine muscle tissue using H&E staining. Histopathological images of inflamed murine muscle tissue 28 days post IM administered of 5 × 10¹² VG/kg of AAV-31C2 at 20× (A) and 40× (B), showing lymphocytes and macrophages in the endomysium surrounding numerous myofibers. Straited muscle tissue from Sheep #8486 28 days post IM administered of 5 × 10¹² VG/kg of AAV-31C2 is seen at 40× (C). Scattered lymphocytes, plasma cells and rare macrophages were present surrounding myofibers, with accumulation seen in the endomysium and medium-size vessels.

Figure 5

Immunohistochemical staining of human IgG in murine and ovine muscle. Stained murine muscle tissue from a mouse intramuscularly injected with 5 × 10¹² VG/kg AAV6.2FF-31C2 28 days prior at (A) 4× and (C) 20× and negative control murine muscle receiving vehicle only at (B) 4× and (D) 20× Stained ovine muscle tissue from sheep #8486 28 days post injection with 5 × 10¹² VG/kg AAV6.2FF-31C2 at (E) 4× and (G) 20× and healthy negative control tissue at (F) 4× and (H) 20×.