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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 therapeutic potential of the bispecific antibody.(A) Improving FVIII-mimetic activity of the bispecific antibody. Effect of hBS1 (circles), hBS106 (squares), and hBS910 (diamonds) on FX activation in the presence of FIXa, FX, and synthetic phospholipid is shown. The Y-axis indicates the absorbance at 405 nm of the chromogenic substrate assay (in many cases, the bars depicting s.d. are shorter than the height of the symbols). (B) Improving pharmacokinetics of the bispecific antibody. Time profiles of plasma concentration of hBS106 (circles), hBS128 (squares), and hBS228 (diamonds) in mice after subcutaneous injection at a dose of 1 mg/kg are shown. All the data were collected in triplicate and are expressed as mean ± s.d.
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pone-0057479-g004: Improvement of therapeutic potential of the bispecific antibody.(A) Improving FVIII-mimetic activity of the bispecific antibody. Effect of hBS1 (circles), hBS106 (squares), and hBS910 (diamonds) on FX activation in the presence of FIXa, FX, and synthetic phospholipid is shown. The Y-axis indicates the absorbance at 405 nm of the chromogenic substrate assay (in many cases, the bars depicting s.d. are shorter than the height of the symbols). (B) Improving pharmacokinetics of the bispecific antibody. Time profiles of plasma concentration of hBS106 (circles), hBS128 (squares), and hBS228 (diamonds) in mice after subcutaneous injection at a dose of 1 mg/kg are shown. All the data were collected in triplicate and are expressed as mean ± s.d.

Mentions: Although the humanized bispecific antibody hBS1 enhanced FX activation dose-dependently, demonstrating FVIII-mimetic activity, its therapeutic potential was marginal and its FVIII-mimetic activity needed to be improved for therapeutic application. Therefore, we explored mutations in the CDRs of hBS1 to improve the FVIII-mimetic activity, and we identified several effective mutations in the CDRs of the three chains. Following extensive studies to identify effective combinations of mutations that additively or synergistically improved FVIII-mimetic activity, we successfully generated hBS106. hBS106 demonstrated marked improvement of FVIII-mimetic activity over hBS1, including maximum activity, in the enzymatic assay (Fig. 4A).


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 therapeutic potential of the bispecific antibody.(A) Improving FVIII-mimetic activity of the bispecific antibody. Effect of hBS1 (circles), hBS106 (squares), and hBS910 (diamonds) on FX activation in the presence of FIXa, FX, and synthetic phospholipid is shown. The Y-axis indicates the absorbance at 405 nm of the chromogenic substrate assay (in many cases, the bars depicting s.d. are shorter than the height of the symbols). (B) Improving pharmacokinetics of the bispecific antibody. Time profiles of plasma concentration of hBS106 (circles), hBS128 (squares), and hBS228 (diamonds) in mice after subcutaneous injection at a dose of 1 mg/kg are shown. All the data were collected in triplicate and are expressed as mean ± s.d.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057479-g004: Improvement of therapeutic potential of the bispecific antibody.(A) Improving FVIII-mimetic activity of the bispecific antibody. Effect of hBS1 (circles), hBS106 (squares), and hBS910 (diamonds) on FX activation in the presence of FIXa, FX, and synthetic phospholipid is shown. The Y-axis indicates the absorbance at 405 nm of the chromogenic substrate assay (in many cases, the bars depicting s.d. are shorter than the height of the symbols). (B) Improving pharmacokinetics of the bispecific antibody. Time profiles of plasma concentration of hBS106 (circles), hBS128 (squares), and hBS228 (diamonds) in mice after subcutaneous injection at a dose of 1 mg/kg are shown. All the data were collected in triplicate and are expressed as mean ± s.d.
Mentions: Although the humanized bispecific antibody hBS1 enhanced FX activation dose-dependently, demonstrating FVIII-mimetic activity, its therapeutic potential was marginal and its FVIII-mimetic activity needed to be improved for therapeutic application. Therefore, we explored mutations in the CDRs of hBS1 to improve the FVIII-mimetic activity, and we identified several effective mutations in the CDRs of the three chains. Following extensive studies to identify effective combinations of mutations that additively or synergistically improved FVIII-mimetic activity, we successfully generated hBS106. hBS106 demonstrated marked improvement of FVIII-mimetic activity over hBS1, including maximum activity, in the enzymatic assay (Fig. 4A).

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