A Novel Angiopoietin-2 Selective Fully Human Antibody with Potent Anti-Tumoral and Anti-Angiogenic Efficacy and Superior Side Effect Profile Compared to Pan-Angiopoietin-1/-2 Inhibitors

Thomas, Markus; Kienast, Yvonne; Scheuer, Werner; Bähner, Monika; Kaluza, Klaus; Gassner, Christian; Herting, Frank; Brinkmann, Ulrich; Seeber, Stefan; Kavlie, Anita; Welschof, Martin; Ries, Stefan; Weidner, K. Michael; Regula, Jörg T.; Klein, Christian

doi:10.1371/journal.pone.0054923

Cited by 63 publications

(61 citation statements)

References 52 publications

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“…Subsequently, the ability of the identified antibodies to interfere with ANG2-and ANG1-mediated TIE2 phosphorylation was examined. LC06 has been described previously (44). LC10 showed a dose-dependent interference with ANG2-stimulated TIE2 phosphorylation, with an EC50 value of 3 nM.…”

Section: Methodsmentioning

confidence: 92%

Angiopoietin 2 mediates microvascular and hemodynamic alterations in sepsis

et al. 2013

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Septic shock is characterized by increased vascular permeability and hypotension despite increased cardiac output. Numerous vasoactive cytokines are upregulated during sepsis, including angiopoietin 2 (ANG2), which increases vascular permeability. Here we report that mice engineered to inducibly overexpress ANG2 in the endothelium developed sepsis-like hemodynamic alterations, including systemic hypotension, increased cardiac output, and dilatory cardiomyopathy. Conversely, mice with cardiomyocyte-restricted ANG2 overexpression failed to develop hemodynamic alterations. Interestingly, the hemodynamic alterations associated with endothelial-specific overexpression of ANG2 and the loss of capillary-associated pericytes were reversed by intravenous injections of adeno-associated viruses (AAVs) transducing cDNA for angiopoietin 1, a TIE2 ligand that antagonizes ANG2, or AAVs encoding PDGFB, a chemoattractant for pericytes. To confirm the role of ANG2 in sepsis, we i.p. injected LPS into C57BL/6J mice, which rapidly developed hypotension, acute pericyte loss, and increased vascular permeability. Importantly, ANG2 antibody treatment attenuated LPS-induced hemodynamic alterations and reduced the mortality rate at 36 hours from 95% to 61%. These data indicate that ANG2-mediated microvascular disintegration contributes to septic shock and that inhibition of the ANG2/ TIE2 interaction during sepsis is a potential therapeutic target.

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Section: Methodsmentioning

confidence: 92%

Angiopoietin 2 mediates microvascular and hemodynamic alterations in sepsis

et al. 2013

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“…Construction, expression and purification of Bs4Ab-VA, anti-VEGF and anti-Ang2 antibodies and isotype control antibody VH and VL sequences of anti-Ang2 LCO6 27 and anti-VEGF 28 were retrieved from USA patent application 2010/0111967. Amino acid sequences for the light and heavy chains of the Bs4Ab-VA used herein are shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

“…We sought to determine whether the Bs4Ab design can be used to concurrently block VEGF and Ang2. The resulting molecule, Bs4Ab-VA, was prepared using an anti-Ang2 binding site from the antibody LCO6, 27 and an anti-VEGF binding site from bevacizumab. 28 In particular, the scFv domain of Bs4Ab-VA is from LCO6 and the Fab domain is from bevacizumab (Fig.…”

Section: Examples Of Antibodies Derived From the Bs4ab Technologymentioning

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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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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“…Here, we transfected HEK cells with plasmids in 1:1:1:1 ratios to co-express the 4 quadroma or KiH chains and analyzed the products by mass spectrometry for a comparative assessment. The underlying chains originate from anti-Ang-2 antibody LC06 (heavy chain denoted A and light chain a), 27 and bevacizumab 28 (Avastin Ò , heavy chain denoted B and light chain b), which are both isotype IgG1 with kappa C L . LC06 and bevacizumab are both HC allotype G1m1,17, and VH germlines: IGHV1-2-02 (99%) and IGHV3-23-04 (77%), and VL germlines: IGLV3-21-02 (97%) and IGKV1-16-01 or IGKV1-39-01 (88%), respectively.…”

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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A Novel Angiopoietin-2 Selective Fully Human Antibody with Potent Anti-Tumoral and Anti-Angiogenic Efficacy and Superior Side Effect Profile Compared to Pan-Angiopoietin-1/-2 Inhibitors

Cited by 63 publications

References 52 publications

Angiopoietin 2 mediates microvascular and hemodynamic alterations in sepsis

Angiopoietin 2 mediates microvascular and hemodynamic alterations in sepsis

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

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

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