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Identification and multidimensional optimization of an asymmetric bispecific IgG antibody mimicking the function of factor VIII cofactor activity.

Sampei Z, Igawa T, Soeda T, Okuyama-Nishida Y, Moriyama C, Wakabayashi T, Tanaka E, Muto A, Kojima T, Kitazawa T, Yoshihashi K, Harada A, Funaki M, Haraya K, Tachibana T, Suzuki S, Esaki K, Nabuchi Y, Hattori K - PLoS ONE (2013)

Bottom Line: 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.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.We expect that hBS910 (investigational drug name: ACE910) will provide significant benefit for severe hemophilia A patients.

View Article: PubMed Central - PubMed

Affiliation: Research Division, Chugai Pharmaceutical Co., Ltd., Gotemba, Shizuoka, Japan.

ABSTRACT
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.

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Improvement of pharmaceutical properties of bispecific antibodies.(A) Antibody solution profiles of hBS376 and hBS560 at different antibody concentrations, pH, and NaCl concentrations. The antibody solution under each condition was photographed and the state determined (P, precipitation; L, liquid–liquid phase separation; –, clear liquid). (B) Cation exchange chromatography of hBS560 and hBS660 before (black) and after incubation at 40°C for 2 weeks (red). Acidic peak indicating deamidation at HCDR3 increased after incubation at 40°C for 2 weeks for hBS560. No marked increase of acidic peak was observed for hBS660.
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pone-0057479-g006: Improvement of pharmaceutical properties of bispecific antibodies.(A) Antibody solution profiles of hBS376 and hBS560 at different antibody concentrations, pH, and NaCl concentrations. The antibody solution under each condition was photographed and the state determined (P, precipitation; L, liquid–liquid phase separation; –, clear liquid). (B) Cation exchange chromatography of hBS560 and hBS660 before (black) and after incubation at 40°C for 2 weeks (red). Acidic peak indicating deamidation at HCDR3 increased after incubation at 40°C for 2 weeks for hBS560. No marked increase of acidic peak was observed for hBS660.

Mentions: hBS106, a variant with improved FVIII-mimetic activity, had unexpectedly low solubility, exhibiting either precipitation or liquid–liquid phase separation [30] (supplementary Fig. S5). This lack of solubility was partially due to the positive charge cluster, since the Tyr30Glu mutation described above markedly improved the solubility. However, for hBS376, the variant whose anti-FIXa heavy chain pI was lowered, at concentrations of 4 and 40 mg/mL, precipitation and phase separation still occurred in phosphate buffer of pH 5.5 to 7.0 and NaCl of 100 mM or less (Fig. 6A).


Identification and multidimensional optimization of an asymmetric bispecific IgG antibody mimicking the function of factor VIII cofactor activity.

Sampei Z, Igawa T, Soeda T, Okuyama-Nishida Y, Moriyama C, Wakabayashi T, Tanaka E, Muto A, Kojima T, Kitazawa T, Yoshihashi K, Harada A, Funaki M, Haraya K, Tachibana T, Suzuki S, Esaki K, Nabuchi Y, Hattori K - PLoS ONE (2013)

Improvement of pharmaceutical properties of bispecific antibodies.(A) Antibody solution profiles of hBS376 and hBS560 at different antibody concentrations, pH, and NaCl concentrations. The antibody solution under each condition was photographed and the state determined (P, precipitation; L, liquid–liquid phase separation; –, clear liquid). (B) Cation exchange chromatography of hBS560 and hBS660 before (black) and after incubation at 40°C for 2 weeks (red). Acidic peak indicating deamidation at HCDR3 increased after incubation at 40°C for 2 weeks for hBS560. No marked increase of acidic peak was observed for hBS660.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585358&req=5

pone-0057479-g006: Improvement of pharmaceutical properties of bispecific antibodies.(A) Antibody solution profiles of hBS376 and hBS560 at different antibody concentrations, pH, and NaCl concentrations. The antibody solution under each condition was photographed and the state determined (P, precipitation; L, liquid–liquid phase separation; –, clear liquid). (B) Cation exchange chromatography of hBS560 and hBS660 before (black) and after incubation at 40°C for 2 weeks (red). Acidic peak indicating deamidation at HCDR3 increased after incubation at 40°C for 2 weeks for hBS560. No marked increase of acidic peak was observed for hBS660.
Mentions: hBS106, a variant with improved FVIII-mimetic activity, had unexpectedly low solubility, exhibiting either precipitation or liquid–liquid phase separation [30] (supplementary Fig. S5). This lack of solubility was partially due to the positive charge cluster, since the Tyr30Glu mutation described above markedly improved the solubility. However, for hBS376, the variant whose anti-FIXa heavy chain pI was lowered, at concentrations of 4 and 40 mg/mL, precipitation and phase separation still occurred in phosphate buffer of pH 5.5 to 7.0 and NaCl of 100 mM or less (Fig. 6A).

Bottom Line: 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.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.We expect that hBS910 (investigational drug name: ACE910) will provide significant benefit for severe hemophilia A patients.

View Article: PubMed Central - PubMed

Affiliation: Research Division, Chugai Pharmaceutical Co., Ltd., Gotemba, Shizuoka, Japan.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus