A Novel Antibody Engineering Strategy for Making Monovalent Bispecific Heterodimeric IgG Antibodies by Electrostatic Steering Mechanism

Zhi, Liu; Leng, Esther; Gunasekaran, Kannan; Pentony, Martin; Shen, Min; Howard, Monique L.; Stoops, Janelle; Manchulenko, Kathy; Razinkov, Vladimir I.; Liu, Hua; Fanslow, William C.; Hu, Zhonghua; Sun, Nancy; Hasegawa, Haruki; Clark, Rutilio H.; Foltz, Ian N.; Yan, Wenqiang

doi:10.1074/jbc.m114.620260

Cited by 66 publications

(61 citation statements)

References 46 publications

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“…by use of linkers, in vitro assembly of KiH half antibodies, 16 or HC-LC interface engineering. 17,18,19 Our solution, the CrossMabs, uses KiH for the HC heterodimerization and a domain exchange of HC and LC domains in one arm of the antibody to avoid LC mispairing. 20 Two antibodies of this type, both directed simultaneously against angiopoietin-2 (Ang2) and vascular endothelial growth factor A (VEGF-A) are currently being clinically investigated in oncology.…”

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

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(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.

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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

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“…8,9 In order to drive correct assembly of LC:HC pairs in a bispecific antibody, the interface between the chains might be engineered so that steric clashes or repelling charges prevent incorrect assembly. 10,11 The light chain of an antibody makes contact with the heavy chain in the variable and the constant domain and both contacts, VL:VH and CL:CH1, contribute to recognition and engagement. Consequently, point mutations in the constant domains are not sufficient to steer each light chain towards correct pairing, and further engineering of the variable domains is necessary.…”

Section: Introductionmentioning

confidence: 99%

“…Consequently, point mutations in the constant domains are not sufficient to steer each light chain towards correct pairing, and further engineering of the variable domains is necessary. 11 Therefore, we sought an alternative strategy to enable correct pairing.…”

Section: Introductionmentioning

confidence: 99%

EFab domain substitution as a solution to the light-chain pairing problem of bispecific antibodies

Cooke

Arndt

Quan

et al. 2018

mAbs

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Bispecific antibody therapeutics can expand the functionality of a conventional monoclonal antibody drug because they can bind multiple antigens. However, their great potential is counterbalanced by the challenges faced in their production. The classic asymmetric bispecific containing an Fc requires the expression of four unique chains – two light chains and two heavy chains; each light chain must pair with its correct heavy chain, which then must heterodimerize to form the full bispecific. The light-chain pairing problem has several solutions, some of which require engineering and optimization for each bispecific pair. Here, we introduce a technology called EFab Domain Substitution, which replaces the Cϵ2 of IgE for one of the CL/CH1 domains into one arm of an asymmetric bispecific to encourage the correct pairing of the light chains. EFab Domain Substitution provides very robust correct pairing while maintaining antibody function and is effective for many variable domains. We report its effect on the biophysical properties of an antibody and the crystal structure of the EFab domain substituted into the adalimumab Fab (PDB ID 6CR1).

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“…[26][27][28] Although these approaches generate IgG-Bs, the deviation from the IgG chain sequence and structure remains a substantial issue, [6][7][8][9][10][11][12] and novel antibody engineering solutions are thus still needed.…”

Section: Introductionmentioning

confidence: 99%

“…The iMab design offers a unique engineering solution to overcome some of the limitations of the described previously IgG-Bs, [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] such as the potential formation of half-antibodies and homodimers, which can be difficult to eliminate and require complex multi-step purification methods.…”

Section: Introductionmentioning

confidence: 99%

Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

Dimasi¹,

Fleming²,

Sachsenmeier

et al. 2017

mAbs

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We developed an IgG1 domain-tethering approach to guide the correct assembly of 2 light and 2 heavy chains, derived from 2 different antibodies, to form bispecific monovalent antibodies in IgG1 format. We show here that assembling 2 different light and heavy chains by sequentially connecting them with protease-cleavable polypeptide linkers results in the generation of monovalent bispecific antibodies that have IgG1 sequence, structure and functional properties. This approach was used to generate a bispecific monovalent antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor that: 1) can be produced and purified using standard IgG1 techniques; 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies.

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A Novel Antibody Engineering Strategy for Making Monovalent Bispecific Heterodimeric IgG Antibodies by Electrostatic Steering Mechanism

Cited by 66 publications

References 46 publications

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

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

EFab domain substitution as a solution to the light-chain pairing problem of bispecific antibodies

Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

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