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Wesley M Jackson, Livia W Brier, Amy Twite, Mavish Mahomed, Ricardo Lamy, Matilda F Chan; A Novel Long-Acting Drug Conjugate for Extended Anti-VEGF Therapy. Invest. Ophthalmol. Vis. Sci. 2019;60(9):114.
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Anti-VEGF therapy is critical for treatment of wet AMD and other retinal neovascular diseases, and current anti-VEGF drugs require administration by intravitreal injection every 1-3 months. We have previously shown how a drug consisting of multiple copies (i.e., valency) of anti-VEGF proteins conjugated to single-chain hyaluronic acid (HyA) biopolymers can substantially extend its intravitreal half-life. In this project, we tested the binding affinity of a novel anti-VEGF antibody and verified its compatibility with the multivalent conjugation technology.
We obtained a novel single-domain (VHH) antibody using phage display against anti-VEGF A121. Antibody affinity maturation was performed by subsequent yeast against the same antigen. Finally, we expressed and purified the VHH with the highest Anti-VEGF binding affinity. Using published methods, we conjugated 30-50 the anti-VEGF VHH to HyA (~800 kDa) and measured their VEGF-A binding affinity using biolayer interferometry. For comparison, we also generated multivalent conjugates using anti-VEGF designed ankyrin repeat proteins (DARPins) and single-chain antibodies (scFv). We measured the intravitreal half-lives of multivalent anti-VEGF conjugates using a rabbit model. In each eye, we injected 50 μL of a fluorescently tagged protein (600 μg/mL), either unconjugated or as a multivalent conjugate. At various times after injection, we measured the concentration of the anti-VEGF protein in the vitreous and the retina by fluorescence imaging (PerkinElmer IVIS) and/or mass spectrometry. We fit the intravitreal concentrations to exponential curves to determine the half-life of each treatment.
The novel anti-VEGF VHH obtained via phage display and affinity maturation exhibited high binding affinity to VEGF-A, which was comparable to existing anti-VEGF drugs. In the rabbit model, the intravitreal half-life of the multivalent anti-VEGF VHH conjugates was also equivalent to our previous findings (i.e., up to 6-fold longer than the corresponding unconjugated anti-VEGF protein: p<0.005, t-tests).
Multivalent protein conjugation is an effective strategy to enable less frequent administration of anti-VEGF therapy. Multivalent anti-VEGF VHH conjugates exhibited slower intravitreal diffusivity and no reduction in bioactivity. Therefore, we have validated the use of a novel anti-VEGF VHH antibody for use with this technology.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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