September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
What is the biologically relevant KD for VEGF binding to ranibizumab in the eye? A comparison of in-vivo and in-vitro estimates.
Author Affiliations & Notes
  • Norman Mazer
    Clinical Pharmacology, Roche Innovation Center Basel, Basel, Switzerland
  • Dietmar Schwab
    Clinical Pharmacology, Roche Innovation Center Basel, Basel, Switzerland
  • Laurence Hutton-Smith
    Wolfson Centre of Mathematical Biology, University of Oxford, Oxford, United Kingdom
  • Helen M Byrne
    Wolfson Centre of Mathematical Biology, University of Oxford, Oxford, United Kingdom
  • Eamonn A Gaffney
    Wolfson Centre of Mathematical Biology, University of Oxford, Oxford, United Kingdom
  • Philip Maini
    Wolfson Centre of Mathematical Biology, University of Oxford, Oxford, United Kingdom
  • Guido Hartmann
    Neuroscience Ophthalmology and Rare Diseases, Roche Innovation Center Basel, Basel, Switzerland
  • Joerg Moelleken
    Large Molecule Research, Roche Innovation Center Penzberg, Penzberg, Germany
  • Christian Gassner
    Large Molecule Research, Roche Innovation Center Penzberg, Penzberg, Germany
  • Joerg Thomas Regula
    Large Molecule Research, Roche Innovation Center Penzberg, Penzberg, Germany
  • Footnotes
    Commercial Relationships   Norman Mazer, Roche (E); Dietmar Schwab, Roche (E); Laurence Hutton-Smith, None; Helen Byrne, None; Eamonn Gaffney, None; Philip Maini, None; Guido Hartmann, Roche (E); Joerg Moelleken, Roche (E); Christian Gassner, Roche (E); Joerg Regula, Roche (E)
  • Footnotes
    Support  Funding provided to Wolfson Centre of Mathematical Biology by EPSRC and MRC (Grant code EP/L016044/1). Additional funding provided by Roche Pharma Research and Early Development.
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 3327. doi:
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      Norman Mazer, Dietmar Schwab, Laurence Hutton-Smith, Helen M Byrne, Eamonn A Gaffney, Philip Maini, Guido Hartmann, Joerg Moelleken, Christian Gassner, Joerg Thomas Regula; What is the biologically relevant KD for VEGF binding to ranibizumab in the eye? A comparison of in-vivo and in-vitro estimates.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3327.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The high affinity binding of anti-VEGF antibodies to VEGF molecules within the eye is central to their mechanism of action in the treatment of neovascular and vascular permeability disorders of the retina. The strength of this binding is inversely related to the magnitude of the equilibrium dissociation constant, KD. It is presently unclear what the biologically relevant values of KD are that characterize VEGF binding to its antibodies in ocular media. We compare our recent estimate of the in-vivo KD value for VEGF binding to ranibizumab, a Fab fragment, with in-vitro estimates obtained using different experimental methods.

Methods : An estimate of the in-vivo KD for VEGF binding to ranibizumab was derived from a pharmacokinetic-pharmacodynamic model of the time-course of aqueous humor levels of free VEGF following intra-vitreal injection of ranibizumab in patients with neovascular AMD (Hutton-Smith L et al., submitted to Mol Pharmaceutics 2015). In-vitro estimates of KD for the binding of ranibizumab to human VEGF121 and VEGF165 were obtained at 25 °C using the Kinetic Exclusion Assay (KinExA®) and Surface Plasmon Resonance (BIAcore®), and preliminary results with Isothermal Titration Calorimetry (ITC). In-vitro estimates of KD at 37 °C were obtained by multiplying the 25 °C values by the ratio KD (37 °C)/ KD (25 °C) measured with BIAcore. The enthalpy of binding (ΔH) was estimated from this ratio using the Van’t Hoff equation.

Results : The in-vivo (37 °C) KD value (range) was estimated to be 21.3 nM (18.1 – 27.4 nM). The in-vitro KD values at 25 °C and 37 °C are given in the Table. ΔH was estimated to be -28.9 kcal/mole.

Conclusions : There is a considerable disparity between the in-vivo estimate of KD and the in-vitro estimates obtained by KinExA and BIAcore; whereas the preliminary in-vitro estimate obtained by ITC is comparable to the in-vivo estimate. As all methods and models are subject to assumptions and uncertainties the biologically relevant value of KD for intravitreal VEGF binding to ranibizumab and presumably other anti-VEGF antibodies has not yet been resolved and warrants further experimental investigation. Caution should be exercised while making assessments/predictions based on in-vitro or in-vivo KD values.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

 

In-vitro estimates (range) of KD at 25 and 37 °C for binding of ranibizumab to VEGF isoforms using KinExA, BIAcore and ITC.

In-vitro estimates (range) of KD at 25 and 37 °C for binding of ranibizumab to VEGF isoforms using KinExA, BIAcore and ITC.

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