Takaaki Hatanaka

Discovery and Characterization of a Peptide Motif That Specifically Recognizes a Non-native Conformation of Human IgG Induced by Acidic pH Conditions

In therapeutic antibody preparation, acidic pH conditions are generally used for elution from Protein A affinity column of IgG or for its viral inactivation. Exposing IgG to low pH conditions induces conformational changes, leading to its functional damage or loss, although the mechanisms have not been fully elucidated. In this study using random peptide T7 phage display libraries, we isolated a unique and novel peptide motif that specifically recognized the non-native conformer (acid conformer) of human IgG that was generated by the low pH treatment, but not the native conformer. We examined the generation conditions and biochemical properties of acid conformer using the peptide motif as an affinity ligand. The acid conformer was easily generated at acidic pH (<pH 3.0) and at moderate temperatures (20-40 °C). The conformer was present in a monomeric form functionally maintaining antigen or Fc receptor binding, but showed a tendency to aggregate with a long incubation time at neutral pH (>25 °C). The peptides isolated here could contribute to the elucidation of the mechanisms of antibody dysfunction or aggregation during acid exposure as well as storage of human IgG.

Human IgA-binding Peptides Selected from Random Peptide Libraries AFFINITY MATURATION AND APPLICATION IN IGA PURIFICATION

Background: Pharmaceutical application of human IgA requires a highly specific IgA purification system. Results: A peptide affinity ligand for IgA was designed and optimized for affinity/specificity using randomized phage libraries and mutational studies. Conclusion: The designed IgA-binding peptide has high affinity and specificity for human IgA. Significance: This IgA-binding peptide can be used for specific purification of human IgA. Phage display system is a powerful tool to design specific ligands for target molecules. Here, we used disulfide-constrained random peptide libraries constructed with the T7 phage display system to isolate peptides specific to human IgA. The binding clones (A1–A4) isolated by biopanning exhibited clear specificity to human IgA, but the synthetic peptide derived from the A2 clone exhibited a low specificity/affinity (Kd = 1.3 μm). Therefore, we tried to improve the peptide using a partial randomized phage display library and mutational studies on the synthetic peptides. The designed Opt-1 peptide exhibited a 39-fold higher affinity (Kd = 33 nm) than the A2 peptide. An Opt-1 peptide-conjugated column was used to purify IgA from human plasma. However, the recovered IgA fraction was contaminated with other proteins, indicating nonspecific binding. To design a peptide with increased binding specificity, we examined the structural features of Opt-1 and the Opt-1-IgA complex using all-atom molecular dynamics simulations with explicit water. The simulation results revealed that the Opt-1 peptide displayed partial helicity in the N-terminal region and possessed a hydrophobic cluster that played a significant role in tight binding with IgA-Fc. However, these hydrophobic residues of Opt-1 may contribute to nonspecific binding with other proteins. To increase binding specificity, we introduced several mutations in the hydrophobic residues of Opt-1. The resultant Opt-3 peptide exhibited high specificity and high binding affinity for IgA, leading to successful isolation of IgA without contamination.

Expression of the extracellular region of the human interleukin-4 receptor α chain and interleukin-13 receptor α1 chain by a silkworm–baculovirus system

The receptor binding to interleukin (IL)-13 is composed of the IL-13 receptor alpha1 chain (IL-13Ralpha1) and the IL-4 receptor alpha chain (IL-4Ralpha). In order to investigate the interaction of IL-13 with IL-13Ralpha1 and IL-4Ralpha, the DNA fragments coding the extracellular regions of human IL-13Ralpha1 and the IL-4Ralpha (containing a cytokine receptor homologous region) were fused with mouse Fc and expressed by a silkworm-baculovirus system. The expressed receptors were successfully purified by affinity chromatography using protein A, and the Fc region was removed by thrombin digestion. After further purification with anion-exchange chromatography, these receptors were used to investigate the ligand-receptor interaction. Size exclusion chromatography and SPR analysis revealed that mixture of IL-13 and IL-13Ralpha1 showed predominant affinity to IL-4Ralpha, although neither detectable affinity of IL-13 nor IL-13Ralpha1 was observed against IL-4Ralpha. Combining these data with the moderate affinity of IL-13 to IL-13Ralpha1, this indicates that IL-13 first binds to IL-13Ralpha1 and recruits consequently to IL-4R.

Gp10 based-thioetherification (10BASEd-T) on a displaying library peptide of bacteriophage T7

The site-specific introduction of a haloacetamide derivative into a designated cysteine on a displaying peptide on a capsid protein (gp10) of bacteriophage T7 has been achieved. This easiest gp10-based thioetherification (10BASEd-T) is carried out in one-pot without side reactions or loss of phage infectivity.

Pharmacophore Generation from a Drug-like Core Molecule Surrounded by a Library Peptide via the 10BASEd-T on Bacteriophage T7

We have achieved site-specific conjugation of several haloacetamide derivatives into designated cysteines on bacteriophage T7-displayed peptides, which are fused to T7 capsid protein gp10. This easiest gp10 based-thioetherification (10BASEd-T) undergoes almost quantitatively like a click reaction without side reaction or loss of phage infectivity. The post-translational modification yield, as well as the site-specificity, is quantitatively analyzed by a fluorescent densitometric analysis after gel electrophoresis. The detailed structure of the modified peptide on phage is identified with tandem mass spectrometry. Construction of such a peptide-fused phage library possessing non-natural core structures will be useful for future drug discovery. For this aim, we propose a novel concept of pharmacophore generation from a drug-like molecule (i.e., salicylic acid) conjugated with surrounding randomized peptides. By using the hybrid library, streptavidin-specific binders are isolated through four rounds of biopanning.

Regulation of T cell response by blocking the ICOS signal with the B7RP-1-specific small antibody fragment isolated from human antibody phage library

A costimulatory signal is required for the full activation of T cells, in addition to the antigen-specific signal via the T-cell receptor. The inducible costimulator, ICOS is one of the costimulatory molecules that play an essential role in this process, particularly in the expansion or the development of effector T cells. As blocking of the interaction between ICOS and its ligand, B7RP-1, suppresses the T-cell response, it can be applied to the treatment of allograft rejection or autoimmune diseases. Here, we isolated four scFv clones that were specific to human B7RP-1 by biopanning a human antibody phage library. We found that three of these clones inhibited the interaction between ICOS-Fc and B7RP-1-Fc. These inhibitory clones not only recognized B7RP-1 molecules expressed on B cells, as assessed by FACS, but also exhibited inhibitory activity in a proliferation assay of T cells stimulated with anti-CD3 mAb and B7RP-1-Fc. Finally, the suppression effect of the scFv on the allogenic immune response was examined using a mixed lymphocyte reaction assay, which demonstrated a successful inhibition of the allogenic reaction, in spite of the high dose needed for complete inhibition (360 nM).

IgY-binding peptide screened from a random peptide library as a ligand for IgY purification

Chicken egg yolk immunoglobulin (IgY) is a functional substitute for mammalian IgG for antigen detection. Traditional IgY purification methods involve multi‐step procedures resulting in low purity and recovery of IgY. In this study, we developed a simple IgY purification system using IgY‐specific peptides identified by T7 phage display technology. From disulfide‐constrained random peptide libraries constructed on a T7 phage, we identified three specific binding clones (Y4‐4, Y5‐14, and Y5‐55) through repeated biopanning. The synthetic peptides showed high binding specificity to IgY‐Fc and moderate affinity for IgY‐Fc (Kd: Y4‐4 = 7.3 ± 0.2 μM and Y5‐55 = 4.4 ± 0.1 μM) by surface plasmon resonance analysis. To evaluate the ability to purify IgY, we performed immunoprecipitation and affinity high‐performance liquid chromatography using IgY‐binding peptides; the result indicated that these peptides can be used as affinity ligands for IgY purification. We then used a peptide‐conjugated column to purify IgY from egg yolks pre‐treated using an optimized delipidation technique. Here, we report the construction of a cost‐effective, one‐step IgY purification system, with high purity and recovery.