A novel anti-NGF PEGylated Fab' provides analgesia with lower risk of adverse effects

Abstract

Nerve growth factor (NGF) has emerged as a key driver of pain perception in several chronic pain conditions, including osteoarthritis (OA), and plays an important role in the generation and survival of neurons. Although anti-NGF antibodies improve pain control and physical function in patients with clinical chronic pain conditions, anti-NGF IgGs are associated with safety concerns such as effects on fetal and postnatal development and the risk of rapidly progressive osteoarthritis. To overcome these drawbacks, we generated a novel anti-NGF PEGylated Fab' antibody. The anti-NGF PEGylated Fab' showed specific binding to and biological inhibitory activity against NGF, and analgesic effects in adjuvant-induced arthritis model mice in a similar manner to an anti-NGF IgG. In collagen-induced arthritis model mice, the anti-NGF PEGylated Fab' showed higher accumulation in inflamed foot pads than the anti-NGF IgG. In pregnant rats and non-human primates, the anti-NGF PEGylated Fab' was undetectable in fetuses, while the anti-NGF IgG was detected and caused abnormal postnatal development. The PEGylated Fab' and IgG also differed in their ability to form immune complexes in vitro. Additionally, while both PEGylated Fab' and IgG showed analgesic effects in sodium monoiodoacetate-induced arthritic model rats, their effects on edema were surprisingly quite different. While the anti-NGF IgG promoted edema over time, the anti-NGF PEGylated Fab' did not. The anti-NGF PEGylated Fab' (ASP6294) may thus be a potential therapeutic candidate with lower risk of adverse effects for various diseases in which NGF is involved such as OA and chronic back pain.

Keywords: NGF; PEGylated antibody; arthritis; fab; immune complex; pain; placental transfer.

Figure 1.

Generation and characterization of a novel anti-NGF Fab’-PEG. (a) Scheme showing how the Fab’-PEG was generated. (b) Inhibitory activity of NGF/TrkA binding in competitive ELISA. (c) The inhibitory activity of NGF induced a calcium influx in TrkA-expressing HEK293 cells. (d) The inhibitory activity of NGF induced TrkA phosphorylation in TrkA-expressing HEK293 cells. Values indicate percent activity, with the effect of NGF alone (without antibody) representing 100%, and the basal activity without NGF or maximum concentration of anti-NGF Fab’-PEG defined as 0%. (e) Binding activity of the anti-NGF Fab’-PEG to human NGF, BDNF, NT-3 and NT-4. Values indicate absorbance at 450 nm. Data are presented as mean ± SD.

Figure 2.

Analgesic effect and distribution of anti-NGF Fab’-PEG in arthritis models. (a) Analgesic effect of the anti-NGF Fab’-PEG and anti-NGF IgG in a murine AIA model. The number of rearing events was measured as an indicator of pain. Data are expressed as the mean ± SEM of 7–8 mice in each group. ### P < .001, statistically significant compared to the sham group (Student’s t-test). * P < .05, ** P < .01, *** P < .001, statistically significant compared to the vehicle group (Dunnett’s multiple comparisons test). (B, C, D) Biofluorescence imaging of the anti-NGF Fab’-PEG and anti-NGF IgG distribution in the inflamed paws of the murine CIA model. The images were captured and analyzed using the IVIS Spectrum in vivo imaging system (B, 2 hours after administration in CIA mice) and the fluorescence intensities were plotted at each time point. Change in fluorescence intensity (c) and fluorescence intensity 2 hours after administration (d). Data are expressed as the mean ± SEM of 4 mice in each group. ## P < .01, statistically significant compared to the anti-NGF IgG-Normal group. ** P < .01, statistically significant compared to the anti-NGF IgG-CIA group (Student’s t-test).

Figure 3.

Comparison of placental transfer of anti-NGF Fab’-PEG and anti-NGF IgG in rats and NHPs. (a) Scheme of the study design used to evaluate placental transfer in pregnant rats and the concentration of anti-NGF IgG or anti-NGF Fab’-PEG in plasma from rat dams and fetuses. Data are expressed as individual values and the mean ± SEM of 3 dams or 9 fetuses in each group. (b) Scheme of the study design used to evaluate placental transfer in pregnant cynomolgus monkeys and the concentration of anti-NGF IgG or anti-NGF Fab’-PEG in serum from cynomolgus monkey dams and fetuses. Data are expressed as individual values and the mean of 2 dams or 2 fetuses in each group. % values represent the proportion of the concentration of anti-NGF IgG in fetuses to dams.

Figure 4.

Comparison of the immune complex-forming ability and knee edema induced by anti-NGF Fab’-PEG and anti-NGF IgG in a rat MIA model. (a) DLS analysis was used to detect the particle size of the immune complex formed by control IgG, anti-NGF IgG and anti-NGF Fab’-PEG. The volume particle size distributions of complexes formed by antibody alone (red) or a mixture of antibody and NGF (green) were measured using Zetasizer Nano. (b) Difference in immune complex formation between the anti-NGF IgG and anti-NGF Fab’-PEG. (c) Analgesic effects of the anti-NGF IgG or Fab’-PEG determined via assessment of hind paw weight-bearing in MIA rats. Data are presented as mean ± SEM of 8 rats in each group. ### P < .001 statistically significant compared to the sham group (Student’s t-test). ** P < .01 or *** P < .001, statistically significant compared to the vehicle group (Dunnett’s multiple comparisons test). (d) Knee diameter was assessed in MIA model rats on day 0, 2, 4, 8, 14, 21 and 28 post MIA injection. Rats were injected with 1 mg of MIA or saline (Sham) into the right knee. Anti-NGF IgG or PBS (Vehicle) was intravenously administered once (on day 2), or anti-NGF Fab’-PEG was intravenously administered 4 times (on day 2, 10, 18 and 26) after MIA injection. Knee width was determined by subtracting the value for the left side from that for the right. Data are presented as the mean ± SEM of 6 rats in each group.