A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens

Moore, Gregory L.; Bautista, Cristina; Pong, Erik; Nguyen, Duc-Hanh T.; Jacinto, Jonathan; Eivazi, Araz; Muchhal, Umesh S.; Karki, Sher; Chu, Seung Y.; Lazar, Greg A.

doi:10.4161/mabs.3.6.18123

Cited by 94 publications

(86 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Unclassified

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: 99%

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

Labrijn

Meesters

Goeij

et al. 2013

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

267

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: 99%

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

Labrijn

Meesters

Goeij

et al. 2013

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

267

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

“…Alternatively, replacing Fab with scFv or BiTE as the binding entity has been applied to overcome LC mispairing, resulting in scFv-knobs-into-holes (KIH)-Fc or BiTE-KIH-Fc. 4 Meanwhile, a number of engineering strategies in C H 3 domains have also significantly enhanced HC heterodimerization based on steric [36][37][38] or electrostatic complementarity. 39,40 Among them, the most successful and widely applied route is knobs-into-holes, in which a "knob" is created by replacing T366 with a bulky residue W on one HC, and the corresponding "hole" is made by triple mutations of T366S, L368A and Y407V on the partner HC.…”

Section: Introductionmentioning

confidence: 99%

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

Lee

Tran

et al. 2015

mAbs

View full text Add to dashboard Cite

(2015) Production of bispecific antibodies in "knobs-into-holes" using a cell-free expression system , mAbs, 7:1, 231-242, DOI: 10.4161/19420862.2015.989013 To link to this article: https://doi.org/10. 4161/19420862.2015 Bispecific antibodies have emerged in recent years as a promising field of research for therapies in oncology, inflammable diseases, and infectious diseases. Their capability of dual target recognition allows for novel therapeutic hypothesis to be tested, where traditional mono-specific antibodies would lack the needed mode of target engagement. Among extremely diverse architectures of bispecific antibodies, knobs-into-holes (KIHs) technology, which involves engineering C H 3 domains to create either a "knob" or a "hole" in each heavy chain to promote heterodimerization, has been widely applied. Here, we describe the use of a cell-free expression system (Xpress CF) to produce KIH bispecific antibodies in multiple scaffolds, including 2-armed heterodimeric scFv-KIH and one-armed asymmetric BiTE-KIH with tandem scFv. Efficient KIH production can be achieved by manipulating the plasmid ratio between knob and hole, and further improved by addition of prefabricated knob or hole. These studies demonstrate the versatility of Xpress CF in KIH production and provide valuable insights into KIH construct design for better assembly and expression titer.

show abstract

A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens

Cited by 94 publications

References 41 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

Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system

Contact Info

Product

Resources

About