Yoshiaki Nabuchi

Antibody recycling by engineered pH-dependent antigen binding improves the duration of antigen neutralization

For many antibodies, each antigen-binding site binds to only one antigen molecule during the antibodys lifetime in plasma. To increase the number of cycles of antigen binding and lysosomal degradation, we engineered tocilizumab (Actemra), an antibody against the IL-6 receptor (IL-6R), to rapidly dissociate from IL-6R within the acidic environment of the endosome (pH 6.0) while maintaining its binding affinity to IL-6R in plasma (pH 7.4). Studies using normal mice and mice expressing human IL-6R suggested that this pH-dependent IL-6R dissociation within the acidic environment of the endosome resulted in lysosomal degradation of the previously bound IL-6R while releasing the free antibody back to the plasma to bind another IL-6R molecule. In cynomolgus monkeys, an antibody with pH-dependent antigen binding, but not an affinity-matured variant, significantly improved the pharmacokinetics and duration of C-reactive protein inhibition. Engineering pH dependency into the interactions of therapeutic antibodies with their targets may enable them to be delivered less frequently or at lower doses.

Reduced elimination of IgG antibodies by engineering the variable region

Fc engineering to increase the binding affinity of IgG antibodies to FcRn has been reported to reduce the elimination of IgG antibodies. Herein, we present a novel non-FcRn-dependent approach to reduce the elimination of IgG antibodies. Pharmacokinetic studies conducted in normal mice of various humanized IgG4 antibodies, which had identical constant regions but different variable region sequences, revealed that an antibody with a lower isoelectric point (pI) has a longer half-life. These antibodies exhibited comparable binding affinity to FcRn, and with the antibodies with lower pIs, a longer half-life was also observed in beta2-microglobulin knockout mice, suggesting that differences in the pharmacokinetics were due to a non-FcRn-dependent mechanism. On the basis of our findings, we attempted to engineer the pharmacokinetic properties of a humanized anti-IL6 receptor IgG1 antibody. Selected substitutions in the variable region, without substitution in the Fc region, lowered the pI but did not reduce the biological activity and showed a significant reduction in the clearance of the antibody in cynomolgus monkey. These results suggest that lowering the pI by engineering the variable region could reduce the elimination of IgG antibodies and could provide an alternative to Fc engineering of IgG antibodies.

Prediction of human intestinal permeability using artificial membrane permeability

The purpose of the present study was to examine a correlation between the human intestinal permeability (P(eff)) and the bio-mimetic artificial membrane permeability corrected by the paracellular pathway model based on the Renkin function (P(PAMPA-PP-RF)) and to construct a prediction scheme. The effect of the unstirred water layer was incorporated to the prediction scheme. Eighteen P(eff) values of passively absorbed drugs were employed for the analysis. The correlation coefficient (CC) between the predicted and observed logP(eff) was 0.91. P(eff) of furosemide, hydrochlorothiazide and creatinine were underestimated by P(PAMPA-PP-RF). When these compounds were excluded, CC was 0.97. Without the correction for the paracellular pathway, P(eff) of small, cationic and hydrophilic compounds were underestimated. Therefore, P(PAMPA-PP-RF) was found to be an adequate in vitro surrogate for P(eff).

Identification and Multidimensional Optimization of an Asymmetric Bispecific IgG Antibody Mimicking the Function of Factor VIII Cofactor Activity

In hemophilia A, routine prophylaxis with exogenous factor VIII (FVIII) requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies (FVIII inhibitors). To overcome these drawbacks, we screened asymmetric bispecific IgG antibodies to factor IXa (FIXa) and factor X (FX), mimicking the FVIII cofactor function. Since the therapeutic potential of the lead bispecific antibody was marginal, FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain for the anti-FIXa and anti-FX heavy chains through framework/complementarity determining region shuffling, and by pI engineering of the two heavy chains to facilitate ion exchange chromatographic purification of the bispecific antibody from the mixture of byproducts. Engineering to overcome low solubility and deamidation was also performed. The multidimensionally optimized bispecific antibody hBS910 exhibited potent FVIII-mimetic activity in human FVIII-deficient plasma, and had a half-life of 3 weeks and high subcutaneous bioavailability in cynomolgus monkeys. Importantly, the activity of hBS910 was not affected by FVIII inhibitors, while anti-hBS910 antibodies did not inhibit FVIII activity, allowing the use of hBS910 without considering the development or presence of FVIII inhibitors. Furthermore, hBS910 could be purified on a large manufacturing scale and formulated into a subcutaneously injectable liquid formulation for clinical use. These features of hBS910 enable routine prophylaxis by subcutaneous delivery at a long dosing interval without considering the development or presence of FVIII inhibitors. We expect that hBS910 (investigational drug name: ACE910) will provide significant benefit for severe hemophilia A patients.

Correction of Permeability with Pore Radius of Tight Junctions in Caco-2 Monolayers Improves the Prediction of the Dose Fraction of Hydrophilic Drugs Absorbed by Humans

AbstractPurpose. To improve predictions of fraction dose absorbed (Fa) for hydrophilic drugs, a correction of paracellular permeability using the pore radius of tight junctions (TJs) in Caco-2 monolayers was performed. Methods. The apparent permeability coefficient (Papp) of drugs was measured using the Caco-2 assay and the parallel artificial membrane permeation assay (PAMPA), and values were corrected with the pore radius of TJs. Results. An equation for calculating the pore radius of TJs from the Papp of lucifer yellow was obtained. The optimal pore radius of TJs in Caco-2 monolayers for predicting human Fa was calculated to be 7 Å. The correlation between the actual and predicted Fa was improved by using the Papp corrected with the pore radius of TJs. Permeability in the PAMPA, which was corrected using the pore radius and membrane potential, was well correlated with that in the Caco-2 assay. Most of the hydrophilic drugs tested in this study were absorbed mainly through the paracellular pathway. Conclusions. The results suggest the necessity of optimizing paracellular permeation for the prediction of Fa, and also the importance of the paracellular pathway to the absorption of hydrophilic drugs. This method might contribute to the setting of appropriate dosages and the development of hydrophilic drugs.

Oxidation of recombinant human parathyroid hormone: effect of oxidized position on the biological activity.

AbstractPurpose. To determine the oxidation products of recombinant human parathyroid hormone (rhPTH) treated with H2O2, the amino acid residue oxidized, and the biological activity of the oxidation products. Methods. Oxidized residues were determined by CNBr cleavage, trypsin digestion and subsequent fast atom bombardment mass spectrometry. The biological activity of each oxidized rhPTH was examined in rat osteosarcoma cell adenylate cyclase assay. Results. Three oxidized products were isolated, namely, Met at position 8 (Met8) sulfoxide, Met at position 18 (Met 18) sulfoxide and both positions Met sulfoxide. It appears that the Met8 and Met 18 oxidized forms are intermediates in the generation of the Met doubly oxidized form. All oxidized forms possessed reduced biological activity, more so for oxidation at Met8 than at Met 18. Conclusions. The region around Met8 is important for the activity of the parathyroid hormone.

Quantitative structure-intestinal permeability relationship of benzamidine analogue thrombin inhibitor.

The intestinal permeability of benzamidine analogue thrombin inhibitor is correlated with molecular volume, lipophilicity (calculated log P and IAM column capacity factor), hydrogen bond acidity/basicity and dipolarity.

The stability and degradation pathway of recombinant human parathyroid hormone : Deamidation of asparaginyl residue and peptide bond cleavage at aspartyl and asparaginyl residues

AbstractPurpose. The stability of recombinant human parathyroid hormone (rhPTH) was examined under acidic to alkaline conditions; its degradation pathways were elucidated from resultant products. Methods. Degradation assay was performed in the pH range 2 to 10 at 40, 50 and 60°C. The approximate molecular mass and pI values of the degradation products were estimated by electrophoresis. FAB-MS peptide mapping and amino acid composition analysis were used to determine these structures. The amount of each respective product was determined by HPLC. Results. At pH2, eight degradation products were found: l-30rhPTH, l-74rhPTH, l-71rhPTH, l-56rhPTH, l-45rhPTH, 46-84rhPTH, 31-84rhPTH and Asp76-rhPTH; these were mainly as a consequence of peptide bond cleavage of the amide bond of Asp. At pH9, five products were found: isoAsp16-rhPTH, Aspl6-rhPTH, Asp57-rhPTH, Asp76-rhPTH, 17-84rhPTH; the main degradation pathway was deamidation of Asn via a cyclic imide intermediate. Degradation products resulting from cleavage at Asp were increased in proportion to the extent that pH was lowered below 5. As pH was increased above 5, so were products resulting from deamidation of Asn. Correspondingly, levels of intact rhPTH were at a peak at pH5. Conclusions. Degradation of rhPTH under acidic conditions predominantly occurs by cleavage at Asp, whereas, above pH5, deamidation of Asn is the more prominent. rhPTH is most stable at pH5.

Ion Mobility and Collision-Induced Dissociation Analysis of Carbonic Anhydrase 2

Folding analysis of the zinc protein, carbonic anhydrase 2 (CA2), was performed using electrospray ionization ion mobility spectrometry coupled with collision-induced dissociation (ESI IMS/CID). Multiply protonated ions with a bimodal charge state distribution were observed indicating the presence of at least two folding states for gas-phase CA2 ions as was described in a previous study (Nabuchi, Y.; Murao, N.; Asoh, Y.; Takayama, M. Anal. Chem. 2007, 79, 8342-8349). In the IMS driftgram, several ions with different mobility were observed for each multiply charged ion, and this suggests that CA2 ions consist of several components with different folding states. IMS/CID spectra were acquired against precursor ions separated by mobility. The CID spectra gave several characteristic product ions including those from the N- and C-terminal region of CA2. A shift to larger charge number for the most abundant of the several product ions was observed for ions having a larger drift time. This charge number shift indicates that the folding state of the ion is more unfolded. Furthermore, differences in the production of an ion corresponding to the N-terminal side fragment gave information about the unfolding process of CA2.

Transport of NG-Nitro-L-Arginine Across Intestinal Brush Border Membranes by Na+-Dependent and Na+ Independent Amino Acid Transporters

AbstractPurpose. To clarify the transport mechanism of NG-nitro-L-arginine (L-NNA), a potent NO-synthase inhibitor, across intestinal brush border membranes (BBM). Methods. Dog intestinal BBM vesicles were used. Results. The time course of L-NNA uptake showed a Na+-dependent overshoot phenomenon. Concentration-dependence curves of L-NNA initial uptake were saturable in the presence and absence of Na+, indicating participation of Na+-dependent and Na+-independent carrier-mediated transport systems. The calculated kinetic parameters of L-NNA initial uptake indicate that the former is a low-affinity high-capacity system and the latter is a high-affinity low-capacity one, similar to those in neutral amino acid transport. Neutral and basic amino acids showed cis-inhibitory and trans-stimulatory effects on L-NNA uptake in the presence or absence of Na+. NG-Nitro-L-arginine methyl ester, another potent NO-synthase inhibitor, also had both effects, which were smaller than with amino acids. Conclusions. The present study clearly indicates that transport of L-NNA across the intestinal BBM occurs in the same manner as neutral amino acid transport. However, it is affected by both neutral and basic amino acids in the presence or absence of Na+ differently from that across plasma membranes of nonepithelial cells, because B0, + and b0, + amino acid transporters function partly in L-NNA transport across intestinal BBM.