Investigating the Immunogenic Potential of Variations in Host Cell Protein Levels in Clinical-Grade AAV8 Products

Abstract

Purpose: Adeno-associated virus (AAV) formulations for gene therapy contain manufacturing-associated impurities such as residual host cell protein (HCP). The aim of this study was to investigate whether high levels of HCP in AAV formulations are associated with increased inflammation and reduced ocular tolerability.

Methods: Three lots of clinical-grade AAV8 vector were analyzed for the presence of manufacturing-associated impurities. The HCP component of these impurities was characterized using mass spectrometry. Lots were then compared regarding their capacity to induce a cytokine response in primary human plasmacytoid dendritic cells (pDCs) and THP-1 cells. Furthermore, the results of an ocular safety study in healthy nonhuman primates were analyzed post hoc to investigate the influence of HCP levels on clinical signs of inflammation and chorioretinal atrophy (CRA) development.

Results: Vector lots displayed up to a ∼40-fold variation in HCP levels. Human galactin-3-binding protein was the only major HCP contaminant. Stimulation of human pDCs and THP-1 cells with a high HCP lot did not result in an increased cytokine response. High HCP also did not exacerbate clinical signs of inflammation. However, on retinal imaging, CRA lesions were significantly larger in high HCP-treated eyes (P = 0.001-0.048).

Conclusions: HCP impurities were of low complexity, but pronounced variations in their abundance were observed between lots. High HCP levels were not overtly immunogenic in vivo and in vitro. However, despite statistical limitations, they seemed to be associated with increased CRA. Thus, a negative effect of high HCP levels on retinal tolerability could not be ruled out.

Figure 1.

Evaluation of immune response to AAV8 lots containing different HCP levels in pDCs (a–c) and PMA-differentiated THP-1 cells (d, e). (a–c) Fold increase of cytokine release of IP-10, MIP-1β, and TNF-α after stimulation of pDCs with AAV8-RK-hPDE6A vector lots (MOI: 1:1 × 10⁶ vg) carrying different HCP contents (L1: 36.9 ng/mL, H1: 1433.7 ng/mL), relative to medium-only control. (d, e) Fold increase of cytokine release of IL-8 and MIP-1β over baseline for the TLR-4 agonist LPS and lot H1 relative to a medium-only control. Error bars indicate the standard deviations between replicate assays. Statistical significance was determined using an unpaired Student’s t-test. *P ≤ 0.05.

Figure 2.

Multimodal imaging of CRA at the injection site. Representative lesion examples for low (a–c) versus high (d–f) HCP levels. CRA at the injection site (>) was recorded using (a, d) FAF, (b, e) FA, and (c, f) ICGA. Findings related to the subretinal injection included mechanical displacement of pigment (*). Qualitatively, there was no obvious difference between CRA in eyes treated with a low HCP (L1) versus a high HCP (H2) lot.

Figure 3.

Clinical findings after subretinal injection of AAV8 lots containing different HCP levels in nonhuman primates, color-coded by AAV dose level. (a–c) Retinal imaging and (d, e) slit-lamp exam. (a–c) Inflammatory and atrophic changes were detected at the injection site 13 weeks after subretinal delivery of AAV in a subset of treated animals. The injection site lesion size was quantified using (a) FAF, (b) FA, and (c) ICGA. There was a statistically significant difference between L1- and H2-treated eyes. This was supported by a trend toward larger lesion size in animals treated with lot H2. Error bars: mean ± SD. *P ≤ 0.05, **P ≤ 0.01. (d, e) Peak clinical inflammatory activity after treatment was determined by quantifying the number of cells in the ACC and VC using the Standard of Uveitis Nomenclature (SUN) grading scheme. There was no difference in overt inflammation between L1- and H2-treated eyes. Error bars: median ± interquartile range. ns, not significant. Statistical significance was determined using the Wilcoxon rank-sum test. L1: vector lot with low HCP (37 ng/mL). H2: vector lot with high HCP (582 ng/mL).