Abstract
Purpose :
Intravitreal delivery of therapeutic antibodies have revolutionized the treatment of ocular diseases, but their rapid clearance from the vitreous limits the duration of action and necessitates monthly intraocular injections. We sought to solve this problem with an entirely novel anchoring molecule which has two key molecular properties: (1) non-covalently binds the Fc region of any IgG1 based therapeutic antibody, and (2) is retained in the vitreous cavity. Because the binding between anchoring molecule and therapeutic antibody is non-covalent, endogenous immunoglobulins are able to compete with and release the free therapeutic antibody. As an initial proof-of-principle, we sought to test the efficacy of this novel approach to sustained antibody delivery in the rats.
Methods :
An anchoring molecule composed of an IgG1 binding single-chain variable fragment cross-linked to agarose beads was chosen for our initial testing. Cell toxicity was studied using an MTT assay in h-RPE cells. In vitro release kinetics were assessed by first incubating the anchoring molecule with bevacizumab and then exposing the bevacizumab-bound anchoring molecule to various concentrations of endogenous immunoglobulins from rat, rabbit or human. In vivo efficacy was tested in Long Evans rats, which were intravitreally injected with bevacizumab alone (control) or bevacizumab-bound anchoring molecule (treatment). At 1, 7, 14, 21, 28, 35, and 42 days post-injection, eyes were enucleated and free and bound bevacizumab were measured using ELISA.
Results :
The anchoring molecule did not show cellular toxicity in h-RPE cells (p=0.316 at 72h). In vitro release test confirmed that polyclonal human (p<0.001), rat (p=0.033) and rabbit (p=0.006) IgG immunoglobulins were able to release free bevacizumab from the anchoring molecule. In vivo experiments demonstrated that at 42 days, free and bound bevacizumab concentrations in the presence of the anchoring molecule were over 200-fold greater than free bevacizumab in the control (7.69±0.12 and 16.41±1.06 mcg/ml vs 0.038±0.003 mcg/ml, respectively; p<0.001).
Conclusions :
We have the first evidence that an anchoring molecule is capable of retaining and releasing a therapeutic antibody in the vitreous cavity. Future studies will optimize the anchoring molecule and offers to radically reshape patient care by increasing clinical efficacy of any intravitreal therapeutic antibody
This is a 2020 ARVO Annual Meeting abstract.