Generating Bispecific Human IgG1 and IgG2 Antibodies from Any Antibody Pair

Strop, Pavel; Ho, Wei-Hsien; Boustany, Leila M.; Abdiche, Yasmina; Lindquist, Kevin C.; Farias, Santiago; Rickert, Mathias; Appah, Charles Takeshi; Pascua, Edward; Radcliffe, Teresa; Sutton, Janette; Chaparro‐Riggers, Javier; Chen, Wei; Casas, Meritxell Galindo; Chin, Sherman M.; Wong, Oi Kwan; Liu, Shuhui; Vergara, German J.; Shelton, Dave; Rajpal, Arvind; Pons, Jaume

doi:10.1016/j.jmb.2012.04.020

Cited by 131 publications

(119 citation statements)

References 29 publications

Supporting

Mentioning

118

Contrasting

Order By: Relevance

“…Mutations promoting H chain heterodimerization by various methods have been described previously by others (7,10,11,40,41). In those cases, however, multiple mutations in at least one of the H chains were required.…”

Section: Discussionmentioning

confidence: 95%

Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange

Labrijn

Meesters

Goeij

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

274

298

View full text Add to dashboard Cite

The promise of bispecific antibodies (bsAbs) to yield more effective therapeutics is well recognized; however, the generation of bsAbs in a practical and cost-effective manner has been a formidable challenge. Here we present a technology for the efficient generation of bsAbs with normal IgG structures that is amenable to both antibody drug discovery and development. The process involves separate expression of two parental antibodies, each containing single matched point mutations in the CH3 domains. The parental antibodies are mixed and subjected to controlled reducing conditions in vitro that separate the antibodies into HL half-molecules and allow reassembly and reoxidation to form highly pure bsAbs. The technology is compatible with standard large-scale antibody manufacturing and ensures bsAbs with Fcmediated effector functions and in vivo stability typical of IgG1 antibodies. Proof-of-concept studies with HER2×CD3 (T-cell recruitment) and HER2×HER2 (dual epitope targeting) bsAbs demonstrate superior in vivo activity compared with parental antibody pairs.immunotherapy | pharmacokinetics | anti-tumor

show abstract

Section: Discussionmentioning

confidence: 95%

Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange

Labrijn

Meesters

Goeij

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

274

298

View full text Add to dashboard Cite

show abstract

“…A number of alternative approaches for the same purpose have been developed recently. 9,10,11,12,13,14 Upon application of one of these methods alone, however, there still remains a mixture of 4 compounds except for cases where the chain association issue is circumvented by a common LC approach. 15 The LC mispairing problem is more difficult to address because a total of 4 possible pairings of heavy and light chains have to be considered.…”

Section: Introductionmentioning

confidence: 99%

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry

Schaefer

Völger

Lorenz

et al. 2015

mAbs

View full text Add to dashboard Cite

(2016) Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry, mAbs, 8:1, 49-55, DOI: 10.1080/19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Keywords: bispecific antibody, CrossMab, chain pairing, ESI-QTOF mass spectrometry, knobs-into-holes, quadromaAbbreviations: Ang2, angiopoietin-2; bsAbs, bispecific antibodies; ESI, electrospray ionization; GuA HCl, guanidine hydrochloride; HEK, human embryonic kidney; HC, heavy chain; IgG1, immunoglobulin G1; ISCID, ion source collision induced dissociation; KiH, knobs-into-holes; LC, light chain; MS, mass spectrometry; TCEP, tris(2-carboxyethyl)phosphine; UHR, ultrahigh-resolution; VEGF-A, vascular endothelial growth factor A; QTOF, quadrupole time-of-flightThe quadroma antibody represents the first attempt to produce a bispecific heterodimeric IgG antibody by somatic fusion of 2 hybridoma cells each expressing monoclonal antibodies with distinctive specificities. However, because of random heavy and light chain pairing, the desired functional bispecific antibody represents only a small fraction of the protein produced. Subsequently, the knobs-into-holes (KiH) approach was developed to enforce correct heavy chain heterodimerization. Assuming equimolar expression of 4 unmodified chains comprising 2 heavy and 2 light chains, the statistical distribution of all paired combinations can be calculated. With equimolar expression as the goal, we transfected HEK cells with 1:1:1:1 plasmid ratios and analyzed the protein A affinity-purified antibodies from the quadroma and KiH approaches qualitatively and quantitatively with regard to the estimated relative amounts of the products using electrospray quadrupole time-of-flight mass spectrometry. Our results show that all expected species are formed, and that, within the methodological limits, the species distribution in the mixtures corresponds approximately to the statistical distribution.

show abstract

“…20 Similarly, electrostatic interactions have been used to promote heterodimerization between two heavy chains. 21,22 By mutating charged groups in the Fc region or introducing new charged groups in the hinge region, one heavy chain is enriched with positively charged functionality and vice versa. Homodimerization is thus minimized due to electrostatic repulsion, and the desired heterodimerization is maximized due to electrostatic attraction.…”

Section: Introductionmentioning

confidence: 99%

A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture

Wang¹,

Vemulapalli²,

Cao³

et al. 2018

mAbs

View full text Add to dashboard Cite

Immunoglobulin G–like bispecific antibodies with asymmetric architecture are among the most widely used bispecific antibody formats for diagnostic and therapeutic applications. The primary technical challenge for this format is how to achieve correctly paired assembly of four unique polypeptide chains. Advances in protein engineering and process development are being used to overcome these challenges and are driving a corresponding demand for sensitive analytical tools to monitor and control mispaired species. Here, we report a systematic approach for analysis and characterization of mispairing in asymmetric bispecific antibodies. This approach consists of three orthogonal components, the first of which is a liquid chromatography (LC)-mass spectrometry (MS)–based method to measure the mass of intact antibodies. This method is used for fast analysis of mispairing and requires minimal method development, which makes it an ideal choice for early-stage development. The second component is a hydrophobic interaction chromatography (HIC)–based mispairing method that is suitable for lot release testing. The HIC method is robust and quality control friendly, and offers great linearity, precision, and accuracy. The third component is a two-dimensional LC-MS method for on-line chromatographic peak identification, which not only expedites this task but also reduces the risk of undesirable modifications during conventional fraction collection. These three methods dovetail to form the foundation of a complementary toolbox for analysis and characterization of mispairing in asymmetric bispecific antibodies and provide guidance and support for process development throughout the drug development life cycle.

show abstract

Generating Bispecific Human IgG1 and IgG2 Antibodies from Any Antibody Pair

Cited by 131 publications

References 29 publications

Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange

Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry

A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture

Contact Info

Product

Resources

About