Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTβR

Michaelson, Jennifer S.; Demarest, Stephen J.; Miller, Brian; Amatucci, Aldo; Snyder, William B.; Wu, Xiufeng; Huang, Flora; Phan, Samantha; Gao, Sharon X.; Doern, Adam; Farrington, Graham K.; Lugovskoy, Alexey A.; Joseph, Ingrid B.J.; Bailly, Véronique; Wang, Xin; Garber, Ellen A.; Browning, Jeffrey L.; Glaser, Scott

doi:10.4161/mabs.1.2.7631

Cited by 108 publications

(78 citation statements)

References 36 publications

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“…These advantages include the recruitment of effector cells, reducing systemic toxicities by targeting to disease sites and modulation of internalization properties, and synergistic effects might be seen when cell-surface receptors are targeted (32).…”

Section: Discussionmentioning

confidence: 99%

Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies

Schaefer

Regula²,

Bähner³

et al. 2011

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

390

371

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We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This “crossover” retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible “CrossMab” formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by standard techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMab CH1-CL was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.

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Section: Discussionmentioning

confidence: 99%

Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies

Schaefer

Regula²,

Bähner³

et al. 2011

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

390

371

View full text Add to dashboard Cite

show abstract

“…41 We previously reported a new and robust BsAb platform that utilizes stability-engineered scFvs appended to full length IgGs to target two tumor necrosis factor receptor family members or two distinct epitopes of IGF-1R. [42][43][44] Both BsAbs displayed IgGlike pharmaceutical properties, dual specificity and improved anti-tumor activity. Here, we describe the development of a novel BsAb targeting EGFR and IGF-1R using this platform technology.…”

Section: Introductionmentioning

confidence: 99%

A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity

Dong¹,

Sereno²,

Aivazian³

et al. 2011

mAbs

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“…To overcome these challenges, several strategies are commonly utilized, including: 1) introducing flexible linkers between the heavy-chain variable (V H ) and the light-chain variable (V L ) domains to maintain intrinsic binding and stability of the scFv, 28–30 2) introducing an additional disulfide bond between the V H and V L domains, 15,31 and 3) selecting scFvs with improved stability early in the protein engineering process. 32 The use of these engineering strategies, which have the chief objective to improve stability and binding, must be balanced against other undesirable consequences, such as lower expression levels and poor expression fidelity.…”

Section: Introductionmentioning

confidence: 99%

Characterization and analysis of scFv-IgG bispecific antibody size variants

Cao¹,

Wang²,

Chung³

et al. 2018

mAbs

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Bispecific antibodies are an emergent class of biologics that is of increasing interest for therapeutic applications. In one bispecific antibody format, single-chain variable fragments (scFv) are linked to or inserted in different locations of an intact immunoglobulin G (IgG) molecule to confer dual epitope binding. To improve biochemical stability, cysteine residues are often engineered on the heavy- and light-chain regions of the scFv to form an intrachain disulfide bond. Although this disulfide bond often improves stability, it can also introduce unexpected challenges to manufacturing or development. We report size variants that were observed for an appended scFv-IgG bispecific antibody. Structural characterization studies showed that the size variants resulted from the engineered disulfide bond on the scFv, whereby the engineered disulfide was found to be either open or unable to form an intrachain disulfide bond due to cysteinylation or glutathionylation of the cysteines. Furthermore, the scFv engineered cysteines also formed intermolecular disulfide bonds, leading to the formation of highly stable dimers and aggregates. Because both the monomer variants and dimers showed lower bioactivity, they were considered to be product-related impurities that must be monitored and controlled. To this end, we developed and optimized a robust, precise, and accurate high-resolution size-exclusion chromatographic method, using a statistical design-of-experiments methodology.

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Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTβR

Cited by 108 publications

References 36 publications

Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies

Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies

A stable IgG-like bispecific antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor demonstrates superior anti-tumor activity

Characterization and analysis of scFv-IgG bispecific antibody size variants

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