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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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Isoelectric point engineering to facilitate purification of the target bispecific antibody.(A) Isoelectric points of target bispecific (squares) antibodies and homodimeric byproducts (anti-FIXa monospecific antibodies (circles) and anti-FX monospecific antibodies (diamonds)) determined by cIEF. (B) Cation exchange purification chromatogram of the target bispecific antibody of hBS560 from its homodimeric byproducts with step-wise elution with different NaCl concentrations. Peak 1, anti-FX homodimeric antibody; Peak 2, target bispecific antibody; Peak 3, anti-FIXa homodimeric antibody. Each peak area of peak 1, peak 2 and peak 3 was 9.9%, 85.7% and 4.4%, respectively.
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pone-0057479-g005: Isoelectric point engineering to facilitate purification of the target bispecific antibody.(A) Isoelectric points of target bispecific (squares) antibodies and homodimeric byproducts (anti-FIXa monospecific antibodies (circles) and anti-FX monospecific antibodies (diamonds)) determined by cIEF. (B) Cation exchange purification chromatogram of the target bispecific antibody of hBS560 from its homodimeric byproducts with step-wise elution with different NaCl concentrations. Peak 1, anti-FX homodimeric antibody; Peak 2, target bispecific antibody; Peak 3, anti-FIXa homodimeric antibody. Each peak area of peak 1, peak 2 and peak 3 was 9.9%, 85.7% and 4.4%, respectively.

Mentions: Having a common light chain reduces the number of pairs of heavy and light chains to three, and engineering the CH3 domain enables preferential secretion of heterodimerized heavy chains. However, it is still difficult to completely prevent miss-paired homodimerization in large-scale production. Therefore, a downstream purification process to remove homodimeric byproducts is essential for pharmaceutical development. Ion exchange chromatography (IEC) is the major purification process by which to remove impurities after Protein A purification. The retention of IgG antibodies by IEC is determined by the electrostatic charge of the antibody molecule, which can be measured as its pI. Therefore, the pIs of hBS128 and hBS228, variants with improved pharmacokinetics, and pIs of their homodimeric byproducts were determined by cIEF (Fig. 5A). For both hBS128 and hBS228, the pIs of the bispecific antibody and the homodimeric byproducts were very close to each other, indicating that purification of the bispecific antibody hBS128 or hBS228 from the mixture of homodimeric byproducts is not feasible.


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)

Isoelectric point engineering to facilitate purification of the target bispecific antibody.(A) Isoelectric points of target bispecific (squares) antibodies and homodimeric byproducts (anti-FIXa monospecific antibodies (circles) and anti-FX monospecific antibodies (diamonds)) determined by cIEF. (B) Cation exchange purification chromatogram of the target bispecific antibody of hBS560 from its homodimeric byproducts with step-wise elution with different NaCl concentrations. Peak 1, anti-FX homodimeric antibody; Peak 2, target bispecific antibody; Peak 3, anti-FIXa homodimeric antibody. Each peak area of peak 1, peak 2 and peak 3 was 9.9%, 85.7% and 4.4%, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057479-g005: Isoelectric point engineering to facilitate purification of the target bispecific antibody.(A) Isoelectric points of target bispecific (squares) antibodies and homodimeric byproducts (anti-FIXa monospecific antibodies (circles) and anti-FX monospecific antibodies (diamonds)) determined by cIEF. (B) Cation exchange purification chromatogram of the target bispecific antibody of hBS560 from its homodimeric byproducts with step-wise elution with different NaCl concentrations. Peak 1, anti-FX homodimeric antibody; Peak 2, target bispecific antibody; Peak 3, anti-FIXa homodimeric antibody. Each peak area of peak 1, peak 2 and peak 3 was 9.9%, 85.7% and 4.4%, respectively.
Mentions: Having a common light chain reduces the number of pairs of heavy and light chains to three, and engineering the CH3 domain enables preferential secretion of heterodimerized heavy chains. However, it is still difficult to completely prevent miss-paired homodimerization in large-scale production. Therefore, a downstream purification process to remove homodimeric byproducts is essential for pharmaceutical development. Ion exchange chromatography (IEC) is the major purification process by which to remove impurities after Protein A purification. The retention of IgG antibodies by IEC is determined by the electrostatic charge of the antibody molecule, which can be measured as its pI. Therefore, the pIs of hBS128 and hBS228, variants with improved pharmacokinetics, and pIs of their homodimeric byproducts were determined by cIEF (Fig. 5A). For both hBS128 and hBS228, the pIs of the bispecific antibody and the homodimeric byproducts were very close to each other, indicating that purification of the bispecific antibody hBS128 or hBS228 from the mixture of homodimeric byproducts is not feasible.

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