Preclinical immunogenicity testing using anti-drug antibody analysis of GX-G3, Fc-fused recombinant human granulocyte colony-stimulating factor, in rat and monkey models.

Human granulocyte colony-stimulating factor (G-CSF, this study used Fc-fused recombinant G-CSF; GX-G3) is an important glycoprotein that stimulates the proliferation of granulocytes and white blood cells. Thus, G-CSF treatment has been considered as a crucial regimen to accelerate recovery from chemotherapy-induced neutropenia in cancer patients suffering from non-myeloid malignancy or acute myeloid leukemia. Despite the therapeutic advantages of G-CSF treatment, an assessment of its immunogenicity must be performed to determine whether the production of anti-G-CSF antibodies causes immune-related disorders. We optimized and validated analytical tools by adopting validation parameters for immunogenicity assessment. Using these validated tools, we analyzed serum samples from rats and monkeys injected subcutaneously with GX-G3 (1, 3 or 10 mg/kg once a week for 4 weeks followed by a 4-week recovery period) to determine immunogenicity response and toxicokinetic parameters with serum concentration of GX-G3. Several rats and monkeys were determined to be positive for anti-GX-G3 antibodies. Moreover, the immunogenicity response of GX-G3 was lower in monkeys than in rats, which was relevant to show less inhibition of toxicokinetic profiles in monkeys, at least 1 mg/kg administrated group, compared to rats. These results suggested the establishment and validation for analyzing anti-GX-G3 antibodies and measurement of serum levels of GX-G3 and anti-GX-G3 antibodies, which was related with toxicokinetic profiles. Taken together, this study provides immunogenicity assessment which is closely implicated with toxicokinetic study of GX-G3 in 4-week repeated administrated toxicological studies.

Characterization of kinesthetic motor imagery compared with visual motor imageries.

Motor imagery (MI) is the only way for disabled subjects to robustly use a robot arm with a brain-machine interface. There are two main types of MI. Kinesthetic motor imagery (KMI) is proprioceptive (OR somato-) sensory imagination and Visual motor imagery (VMI) represents a visualization of the corresponding movement incorporating the visual network. Because these imagery tactics may use different networks, we hypothesized that the connectivity measures could characterize the two imageries better than the local activity. Electroencephalography data were recorded. Subjects performed different conditions, including motor execution (ME), KMI, VMI, and visual observation (VO). We tried to classify the KMI and VMI by conventional power analysis and by the connectivity measures. The mean accuracies of the classification of the KMI and VMI were 98.5% and 99.29% by connectivity measures (alpha and beta, respectively), which were higher than those by the normalized power (p < 0.01, Wilcoxon paired rank test). Additionally, the connectivity patterns were correlated between the ME-KMI and between the VO-VMI. The degree centrality (DC) was significantly higher in the left-S1 at the alpha-band in the KMI than in the VMI. The MI could be well classified because the KMI recruits a similar network to the ME. These findings could contribute to MI training methods.

Development, validation, and application of ELISA for detection of anti-HD105 antibodies in pre-clinical safety evaluation using monkeys.

Unwanted immunogenicity of protein therapeutics can result in severe side effects and should be assessed in animals before applying the treatment to humans. Monkeys are the most relevant choice for pre-clinical toxicity testing of antibody-based therapeutics. To assess the immunogenicity of HD105, a novel antibody therapeutic that targets both vascular endothelial growth factor and Delta-like-ligand 4, a bridging enzyme-linked immunosorbent assay was developed as an anti-drug antibody (ADA) assay and validated for use in pre-clinical studies using non-human primates. This method was found to have suitable assay sensitivity, intra- and inter-assay precision, confirmation, drug tolerance, recovery, and sample stability for measuring ADA in monkey serum samples. The results showed that ADA elevation occurred following repeated doses of HD105, and that ADA production was negatively associated with serum HD105 concentration. These results suggest that intravenous administration of HD105 induces production of ADA in monkeys and that the detection of ADA may be negatively influenced by free HD105 in serum.