Dataset_Urabe_et_al.2017 ELE

Data sets of clearance rates and assimilation efficiencies for C and P (data_CLAEglm) and growth rates with ingestion and assimilation rates for various biochemicals in diets (data_Growth_s)

Nucleotide Excision Repair of 5-Formyluracil in Vitro Is Enhanced by the Presence of Mismatched Bases^†

5-Formyluracil (fU) is a major thymine lesion produced by reactive oxygen radicals and photosensitized oxidation. Although this residue is a potentially mutagenic lesion and is removed by several base excision repair enzymes, it is unknown whether fU is the substrate of nucleotide excision repair (NER). Here, we analyzed the binding specificity of XPC−HR23B, which initiates NER, and cell-free NER activity on fU opposite four different bases. The result of the gel mobility shift assay showed that XPC−HR23B binds the fU-containing substrates in the following order:  fU:C ≫ fU:T > fU:G > fU:A. Furthermore, in the presence of XPC−HR23B, the dual incision activity was the same as the order of the binding affinity of XPC−HR23B to fU. Therefore, it is concluded that even fU, regarded as a shape mimic of thymine, can be recognized as a substrate of NER incision, and the efficiency depends on instability of the base pair

Constant regions used in the cynomolgus monkey PK study.

Constant regions used in the cynomolgus monkey PK study.</p

Effect of the pH-dependent C5-binding property and surface-charge engineering on endogenous C5 accumulation and antibody PK in cynomolgus monkeys.

Anti-C5 antibodies were intravenously administered to cynomolgus monkeys, and the changes in plasma C5 concentration (A, C) and plasma antibody concentration (B, D) were measured. pH-dependent antibodies with improved FcRn-binding activity, 305LO1-SG407, 305LO1-SG419, 305LO5-SG115, and 305LO8-SG115, were injected at day 0 (20 mg/kg). A pH-dependent C5-binding antibody with no FcRn-binding activity, 305LO1-SG406, was injected at days 0, 3, 6, and 9 (40 mg/kg). A non-pH-dependent C5-binding antibody, CFA0322-IgG4, was injected at day 0 and 14 (40 mg/kg). Data are presented as mean ± S.D. n = 4. Data points affected by cynomolgus antibodies against the test samples were not included when calculating the mean concentrations.</p

Mutations in the variable region to improve the C5-binding property.

Mutations in the variable region to improve the C5-binding property.</p

Correlation between C5 accumulation and pI of the antibody or CIEX retention time of the IC.

The correlation between C5 accumulation and the experimental pI of each antibody (A) or the retention time of IC-1 (which consists of one C5 and one antibody) in CIEX analysis (B) was analyzed.</p

CIEX analysis of C5–antibody complexes.

When an antibody that recognizes a specific epitope in C5 is administered, two types of IC can form (A). A CIEX chromatography analysis was performed for the mixture of cynomolgus C5 and each anti-C5 antibody: 305LO1-SG407, 305LO1-SG419, 305LO5-SG115, and 305LO8-SG115 (B‒F).</p

Flow to generate the long-acting anti-C5 antibody.

The variable region of SKY59 was generated through rabbit immunization and multidimensional optimization (blue region). The constant region of SKY59 was designed by engineering a human IgG1 κ constant region (silver region).</p

Variable regions used in the cynomolgus monkey PK study.

Variable regions used in the cynomolgus monkey PK study.</p

Effect of mutations and their combinations on the C5-binding property.

C5-binding activity of the humanized antibody or each antibody variant containing single or multiple mutations was measured by Biacore. Mutations in the heavy chain (am1–am4) and the light chain (am5, am6) cumulatively improved the C5-binding affinity (A and B). The combination of an affinity-matured heavy chain (H-AM) with an affinity-matured light chain (L-AM) further enhanced the affinity (C). Non-histidine mutations found in the heavy chain (pm1, pm2) or the light chain (pm3), and their combinations did not worsen ka at pH 7.4 (D) and provided even better pH dependency (E). The crystal structure of the interaction site of SKY59 and the MG1 domain in human C5 is shown using PyMOL (Schrödinger, LLC) (F). Orange, light blue, and light green represent human C5, the heavy chain, and the light chain of SKY59, respectively. The lysine introduced in the CDR-L3 to improve pH dependency, H100c in the CDR-H3, T47, E48, K109, and H110 in human C5 are shown with thick sticks. The structure of SKY59 and the MG1 domain complex has been shown previously [17] and registered in the RCSB Protein Data Bank with PDB ID: 5B71.</p