Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library

Atwell, S.; Ridgway, John Brady; Wells, James A.; Carter, Paul

doi:10.1006/jmbi.1997.1116

Cited by 234 publications

(240 citation statements)

References 42 publications

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“…We used the "knobs-into-holes" approach to generate an anti-IGF-IR/anti-HER2 hybrid IgG (41)(42)(43). A "knob" mutant was created by replacing a threonine with tyrosine (T366Y) in the CH3 domain of trastuzumab.…”

Section: Bi-ab Cotargets Igf-ir and Her2mentioning

confidence: 99%

Superior Antitumor Activity of a Novel Bispecific Antibody Cotargeting Human Epidermal Growth Factor Receptor 2 and Type I Insulin-like Growth Factor Receptor

Chen¹,

Zhang²,

Li³

et al. 2014

Molecular Cancer Therapeutics

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The humanized anti-HER2 monoclonal antibody (mAb) trastuzumab (Herceptin; Genentech) effectively inhibits human epidermal growth factor receptor 2 (HER2)-positive breast tumors. However, many patients responding to treatment often develop resistance. Cross-talk between type I insulin-like growth factor receptor (IGF-IR) and HER2 and elevated IGF-IR signaling have been implicated in tumor cell resistance to trastuzumab therapy. Previously, we reported that the anti-IGF-IR mAb m590 inhibits proliferation and migration of breast cancer MCF-7 cells in vitro. Here, we generated a "knobs-into-holes" bispecific antibody (Bi-Ab) against HER2 and IGF-IR by engineering trastuzumab and m590. We compared the effects of Bi-Ab treatment in vitro and in SKOV-3 HER2-and IGF-IR-overexpressing cancer xenograft mouse model with those of m590 and trastuzumab treatment alone or in combination. Bi-Ab effectively inhibited proliferation of HER2-and IGF-IR-overexpressing ovarian cancer SKOV-3 cells in vitro by ablating receptor phosphorylation and downstream PI3K/Akt and mitogen-activated protein kinase signaling. Bi-Ab more effectively inhibited cancer growth in SKOV-3 HER2-and IGF-IR-overexpressing cancer xenograft mouse model than m590 and trastuzumab alone or in combination. Mice bearing SKOV-3 HER2-and IGF-IR-overexpressing xenografts showed extensive and sustainable tumor regression when treated with Bi-Ab. Our results suggest that Bi-Ab has superior antitumor activity compared with monospecific antibodies, and cotargeting HER2 and IGF-IR may be clinically beneficial in minimizing the acquired resistance to trastuzumab therapy. Mol Cancer Ther; 13(1); 90-100. Ó2013 AACR.

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Section: Bi-ab Cotargets Igf-ir and Her2mentioning

confidence: 99%

Superior Antitumor Activity of a Novel Bispecific Antibody Cotargeting Human Epidermal Growth Factor Receptor 2 and Type I Insulin-like Growth Factor Receptor

Chen¹,

Zhang²,

Li³

et al. 2014

Molecular Cancer Therapeutics

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“…These two variants of EFab have a bulky hydrophobic residue (tryptophan or isoleucine) introduced at a position in the heavy chain that would prevent homodimerization (E1: L7W, E2: S10I), and smaller residues were introduced on the opposing light chain positions to make room for the bulky side chain (E1: L22G, E2: T121G), similar to the knobs-into-holes strategy in the CH3 domain. 5 …”

Section: Resultsmentioning

confidence: 99%

“…The first solution to heavy chain heterodimerization was developed in the 1990s (“knobs-into-holes”), and several other solutions have been published since. 5–7 Solutions to the light chain pairing problem, on the other hand, are not as well advanced and may require individual optimization. 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.…”

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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“…20 and 21). For example, production of homogeneous full-length IgG-like BsAb has been achieved by the so-called "knobs-into-holes" engineering for efficient Ig CH3 domain heterodimerization plus the use of a common LC shared by two antibodies of different specificities (33)(34)(35). Although functional IgG-like BsAb were formed with high yield (Ͼ95%), an obvious drawback of this method is that the inclusion of multiple mutations in the CH3 domains might pose an immunogenic risk in a therapeutic setting.…”

Section: Discussionmentioning

confidence: 99%

Single Variable Domain-IgG Fusion

Shen¹,

Vil²,

Jimenez³

et al. 2006

Journal of Biological Chemistry

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Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library

Cited by 234 publications

References 42 publications

Superior Antitumor Activity of a Novel Bispecific Antibody Cotargeting Human Epidermal Growth Factor Receptor 2 and Type I Insulin-like Growth Factor Receptor

Superior Antitumor Activity of a Novel Bispecific Antibody Cotargeting Human Epidermal Growth Factor Receptor 2 and Type I Insulin-like Growth Factor Receptor

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

Single Variable Domain-IgG Fusion

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