The Design and Characterization of Oligospecific Antibodies for Simultaneous Targeting of Multiple Disease Mediators

Dimasi, Nazzareno; Gao, Changshou; Fleming, Ryan; Woods, R. Claude; Yao, Xiao-Tao; Shirinian, Lena; Kiener, Peter A.; Wu, Herren

doi:10.1016/j.jmb.2009.08.032

Cited by 82 publications

(97 citation statements)

References 51 publications

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“…The production yield of iMab-EI was 10 mg/L as determined using a quantitative protein A binding method. 4 This expression yield is similar to the singlechain IgG1 reported by Schirrmann et al 30 However, the expression level of the iMab-EI was improved by using 2 engineering strategies. First, by cloning the iMab-EI expression cassette into a vector containing UCOEs elements, 37 which maintains the chromatin in an open state and facilitates transcription, we obtained a 3-fold improvement (30 mg/L) in the expression level (Fig.…”

Section: Expression and Purification Of The Imab-eisupporting

confidence: 82%

“…For these reasons, linkers rich in glycine and serine have been widely used for the construction of stable, bioactive fusion proteins, including antibodies. [3][4][5]41 The linkers we used for the iMab are also rich in glycine and serine and have been designed to have a longer length than previous described linkers. [3][4][5][29][30][31][32] Our hypothesis is that longer linkers would facilitate the correct assembly of the different domains of the iMab.…”

Section: Discussionmentioning

confidence: 99%

“…S1). 4 The iMab-EI expression vector was transfected into HEK293 cells. The production yield of iMab-EI was 10 mg/L as determined using a quantitative protein A binding method.…”

Section: Expression and Purification Of The Imab-eimentioning

confidence: 99%

“…2 Many existing bispecific antibody (BsAb) formats bind bivalently to each antigen, but their structures deviate substantially from that of a canonical IgG structure. [3][4][5] Some of these BsAbs have inferior pharmacokinetic properties, including rapid clearance and shorter half-life relative to their IgG counterparts, thus limiting their therapeutic applications. [6][7][8][9][10][11][12] Several solutions have been described for the generation of BsAb with an IgG chain-like sequence and structure (IgG-Bs).…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Expression and Purification Of The Imab-eisupporting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

“…S1). 4 The iMab-EI expression vector was transfected into HEK293 cells. The production yield of iMab-EI was 10 mg/L as determined using a quantitative protein A binding method.…”

Section: Expression and Purification Of The Imab-eimentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Dimasi¹,

Fleming²,

Sachsenmeier

et al. 2017

mAbs

Self Cite

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show abstract

“…24 Since then, the secondary binding site, often in an scFv format, has been fused to the C terminus/N terminus of the heavy chain, the hinge region, the C terminus/N terminus of the light chain, the CH3 domain of the heavy chain, or other regions. 25–27 …”

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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Strand Exchange Engineered Domain (Seed): A Novel Platform Designed to Generate Mono and Multispecific Protein Therapeutics

Gross

Dawson

Muda

et al. 2013

Fusion Protein Technologies for Biopharmaceuticals

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The Design and Characterization of Oligospecific Antibodies for Simultaneous Targeting of Multiple Disease Mediators

Cited by 82 publications

References 51 publications

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

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

Characterization and analysis of scFv-IgG bispecific antibody size variants

Strand Exchange Engineered Domain (Seed): A Novel Platform Designed to Generate Mono and Multispecific Protein Therapeutics

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