Design and production of novel tetravalent bispecific antibodies

Coloma, M. J.; Morrison, Sherie L.

doi:10.1038/nbt0297-159

Cited by 211 publications

(149 citation statements)

References 27 publications

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“…S2). The scFv is in the orientation VH-(G 4 S) 4 -VL and has VH 44 and VL 100 residues mutated to cysteine, which form an interdomain disulfide in the scFv designed to improve stability of the scFv. 22 The transient expression levels of Bs4Ab-VA in human embryonic kidney (HEK)293 cells was 150 mg/L, which is similar to that of the parental anti-VEGF and anti-Ang2 antibodies.…”

Section: Examples Of Antibodies Derived From the Bs4ab Technologymentioning

confidence: 99%

“…[1][2][3][4][5][6][7][8][9][10][11][12] Bispecific molecules described thus far can be divided into five classes based on their molecular format: 1) BsAb with IgG1-like structure, which are monovalent for each antigen; 2) BsAb prepared by appending binding domains to the IgG, which are bivalent for each antigen; 3) BsAb prepared using antibody fragments, which are monovalent for each antigen and often lack the Fc region; 4) bispecific fusion proteins, which are formed of an antibody fragment genetically linked to a protein to add additional functionality or specificity; and 5) bispecific conjugates, which are prepared by chemical conjugation of antibody fragments. While some such bispecific molecules have demonstrated manufacturing robustness, in vivo drug-like properties, suitable pharmacokinetics (PK) and efficacy have been achieved, platforms that yield multifunctional bispecific antibodies with different spatial configurations and flexibility suitable for a variety of target and disease applications are still needed.…”

Section: Introductionmentioning

confidence: 99%

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Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties

Bezabeh¹,

Fleming²,

Fazenbaker³

et al. 2016

mAbs

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By simultaneous binding two disease mediators, bispecific antibodies offer the opportunity to broaden the utility of antibody-based therapies. Herein, we describe the design and characterization of Bs4Ab, an innovative and generic bispecific tetravalent antibody platform. The Bs4Ab format comprises a full-length IgG1 monoclonal antibody with a scFv inserted into the hinge domain. The Bs4Ab design demonstrates robust manufacturability as evidenced by MEDI3902, which is currently in clinical development. To further demonstrate the applicability of the Bs4Ab technology, we describe the molecular engineering, biochemical, biophysical, and in vivo characterization of a bispecific tetravalent Bs4Ab that, by simultaneously binding vascular endothelial growth factor and angiopoietin-2, inhibits their function. We also demonstrate that the Bs4Ab platform allows Fc-engineering similar to that achieved with IgG1 antibodies, such as mutations to extend half-life or modulate effector functions.

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Section: Examples Of Antibodies Derived From the Bs4ab Technologymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties

Bezabeh¹,

Fleming²,

Fazenbaker³

et al. 2016

mAbs

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show abstract

“…Morrison reported that the anti-DNS scFv possessed high affinity (K D ¼ 1.4 Â 10 À8 M À1 ) for DNS hapten. 35,36 Our result showing that DNS-FITC was retained by B16/DNS cells for at least 10 h indicates that the DNS hapten is stably attached to the DNS receptors. The valency of the DNS-imaging probe was also important for in vivo imaging.…”

Section: Discussionmentioning

confidence: 81%

A membrane antibody receptor for noninvasive imaging of gene expression

Roffler

Wang²,

Yu³

et al. 2005

Gene Ther

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Monitoring gene expression is important to optimize gene therapy protocols and ensure that the proper tissue distribution is achieved in clinical practice. We developed a noninvasive imaging system based on the expression of artificial antibody receptors to trap hapten-labeled imaging probes. Functional membrane-bound anti-dansyl antibodies (DNS receptor) were stably expressed on melanoma cells in vitro and in vivo. A bivalent (DNS) 2 -diethylenetriaminepentaacetic 111 Indium probe specifically bound to cells that expressed DNS receptors but not control scFv receptors.Importantly, the 111 In probe preferentially localized to DNS receptors but not control receptors on tumors in mice as assessed by gamma camera imaging. By 48 h after intravenous injection, the uptake of the probe in tumors expressing DNS receptors was 72 times greater than the amount of probe in the blood. This targeting strategy may allow noninvasive assessment of the location, extent and persistence of gene expression in living animals and in the clinic. Gene Therapy (2006) 13, 412-420.

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“…The third class of molecules are appended IgGs with symmetric architecture in which the second binding site is fused to either the IgG heavy or light chain in a symmetrical fashion. 12-15 …”

Section: Introductionmentioning

confidence: 99%

A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture

Wang¹,

Vemulapalli²,

Cao³

et al. 2018

mAbs

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Immunoglobulin G–like bispecific antibodies with asymmetric architecture are among the most widely used bispecific antibody formats for diagnostic and therapeutic applications. The primary technical challenge for this format is how to achieve correctly paired assembly of four unique polypeptide chains. Advances in protein engineering and process development are being used to overcome these challenges and are driving a corresponding demand for sensitive analytical tools to monitor and control mispaired species. Here, we report a systematic approach for analysis and characterization of mispairing in asymmetric bispecific antibodies. This approach consists of three orthogonal components, the first of which is a liquid chromatography (LC)-mass spectrometry (MS)–based method to measure the mass of intact antibodies. This method is used for fast analysis of mispairing and requires minimal method development, which makes it an ideal choice for early-stage development. The second component is a hydrophobic interaction chromatography (HIC)–based mispairing method that is suitable for lot release testing. The HIC method is robust and quality control friendly, and offers great linearity, precision, and accuracy. The third component is a two-dimensional LC-MS method for on-line chromatographic peak identification, which not only expedites this task but also reduces the risk of undesirable modifications during conventional fraction collection. These three methods dovetail to form the foundation of a complementary toolbox for analysis and characterization of mispairing in asymmetric bispecific antibodies and provide guidance and support for process development throughout the drug development life cycle.

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Design and production of novel tetravalent bispecific antibodies

Cited by 211 publications

References 27 publications

Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties

Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties

A membrane antibody receptor for noninvasive imaging of gene expression

A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture

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