Treatment of Eye Diseases with Post-Translationally Modified Fully Human Anti-VEGF Fab

Abstract

Compositions and methods are described for delivering post-translationally modified fully human (HuP™) monoclonal antibodies ("mAbs") or antigen-binding fragments of mAbs against human vascular endothelial growth factor ("hVEGF"), such as fully human glycosylated (HuGly) anti-hVEGF antigen-binding fragments, to the retina / vitreous humor of the eye of human subjects diagnosed with ocular diseases caused by increased neovascularization, such as neovascular age-related macular degeneration ("nAMD"), also known as "wet" age-related macular degeneration ("WAMD"), age-related macular degeneration ("AMD"), and diabetic retinopathy. [Selection diagram] Figure 23

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Patent Application No. 62 / 564,095, filed Sep. 27, 2017; No. 62 / 574,657, filed Oct. 19, 2017; No. 62 / 579,682, filed Oct. 31, 2017; and No. 62 / 632,812, filed Feb. 20, 2018, each of which is incorporated herein by reference in its entirety.

[0002] (Reference to Electronically Submitted Sequence Listing) This application incorporates by reference the Sequence Listing submitted herewith as a text file entitled "Sequence_Listing_12656 - 110 - 228.TXT", created on Sep. 19, 2018, having a size of 97,512 bytes.

[0003] (1. Introduction) Compositions and methods are described for delivering a translated and post - modified fully human (HuPTM) monoclonal antibody ("mAb") or an antigen - binding fragment of an mAb against vascular endothelial growth factor ("VEGF") - for example, a fully human glycosylated (HuGly) anti - VEGF antigen - binding fragment, etc. - to the retina / vitreous humor of the eye of a human subject diagnosed with an eye disease, particularly an eye disease caused by an increase in neovascularization, such as "neovascular age - related macular degeneration" ("nAMD"), also known as "exudative age - related macular degeneration" ("WAMD" or "exudative AMD"), age - related macular degeneration ("AMD"), and diabetic retinopathy.

Background Art

[0004] (2. Background of the Invention) Age - related macular degeneration (AMD) is a degenerative retinal eye disease that causes progressive and irreversible severe loss of central vision. This disease damages the macula - the area with the highest visual acuity (VA) - and is the leading cause of blindness in Americans over 60 years of age (NIH 2008).

[0005] Age-related macular degeneration with neovascularization (nAMD), also known as "exudative" neovascular form of AMD ("WAMD" or "exudative AMD"), accounts for 15-20% of AMD cases and is characterized by abnormal neovascularization within and beneath the neurosensory retina in response to various stimuli. This abnormal blood vessel growth leads to the formation of leaky blood vessels and often bleeding, as well as distortion and destruction of the normal retinal structure. In nAMD, visual function is severely impaired, and eventually, due to inflammation and scarring, the visual function of the affected retina is permanently lost. Ultimately, due to photoreceptor cell death and scar formation, central vision is severely lost, making it impossible to read, write, recognize faces, or drive. Many patients can no longer maintain high-income jobs and carry out daily activities, resulting in a decline in the quality of life (Mitchell, 2006).

[0006] Diabetic retinopathy is an eye complication of diabetes and is characterized by non-proliferative microaneurysms, hard exudates, hemorrhages, and venous abnormalities, as well as proliferative neovascularization, preretinal or vitreous hemorrhage, and fibrovascular proliferation. Hyperglycemia induces changes in the retinal microvasculature, resulting in blurry vision, dark spots or flickering lights, and sudden vision loss (Cai and McGinnis, 2016).

[0007] Preventive treatment shows little effect, and treatment strategies mainly focus on the treatment of neovascular lesions. Available treatments for nAMD include laser photocoagulation, photodynamic therapy with verteporfin, and intravitreal (IVT) injection of drugs that are related to the stimulation of angiogenesis by vascular endothelial growth factor (VEGF) - a cytokine that is the target of intervention and aims to bind to and neutralize it. Such anti-VEGF agents used include, for example, bevacizumab (a humanized monoclonal antibody (mAb) against VEGF produced by CHO cells), ranibizumab (the Fab portion of a variant with improved affinity for bevacizumab produced in the prokaryote Escherichia coli (E. coli)), aflibercept (a recombinant fusion protein consisting of the VEGF-binding region of the extracellular domain of the human VEGF receptor fused to the Fc portion of human IgG1), or pegaptanib (a pegylated aptamer (single-stranded nucleic acid molecule) that binds to VEGF). Each of these treatment methods has a certain effect on best-corrected visual acuity; however, the effect seems to be limited in terms of vision recovery and sustainability.

[0008] Anti-VEGF IVT injection has been shown to be effective in reducing leakage and sometimes restoring vision loss. However, since these drugs are only effective for a short period, repeated injections are often required over a long period, thereby causing a significant treatment burden on patients. Long-term treatment with ranibizumab once a month or aflibercept once a month / once every eight weeks can slow down the progression of vision loss and improve vision, but none of these treatments prevent the recurrence of neovascularization (Brown, 2006; Rosenfeld, 2006; Schmidt-Erfurth, 2014). To prevent the worsening of the disease, each must be readministered. The need for repeated treatment places additional risks on patients and is inconvenient for both patients and treating physicians.

Summary of the Invention

[0009] (3. Summary of the Invention) Compositions and methods are described for delivering a translationally modified full human (HuPTM) antibody against VEGF to the retina / vitreous humor of the eye of a patient (human subject) diagnosed with an eye disease, particularly an eye disease caused by an increase in neovascularization, such as neovascular age-related macular degeneration (nAMD, also known as "exudative" AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD). Antibodies include monoclonal antibodies, polyclonal antibodies, recombinantly produced antibodies, human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, tetrameric antibodies comprising two heavy chains and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-heavy chain pairs, intrabodies, heteroconjugate antibodies, monovalent antibodies, antigen-binding fragments of full-length antibodies, and fusion proteins of the foregoing, but are not limited thereto. Such antigen-binding fragments include single domain antibodies (variable domains of heavy chain antibodies (VHH) or nanobodies), Fab, F(ab') of a full-length anti-VEGF antibody (preferably a full-length anti-VEGF monoclonal antibody (mAb)) 2, and single-chain variable fragments (scFv) (collectively referred to herein as "antigen-binding fragments"), but are not limited thereto. In a preferred embodiment, a post-translationally modified fully human antibody against VEGF is a post-translationally modified fully human antigen-binding fragment of a monoclonal antibody (mAb) against VEGF ("HuPTMFabVEGFi"). In a more preferred embodiment, HuPTMFabVEGFi is a fully human glycosylated antigen-binding fragment of an anti-VEGF mAb ("HuGlyFabVEGFi"). In alternative embodiments, full-length mAbs can be used. Delivery can be achieved by gene therapy - for example, by administering a viral vector or other DNA expression construct encoding an anti-VEGF antigen-binding fragment or mAb (or a hyperglycosylated derivative) to the suprachoroidal space, subretinal space (using a transvitreal approach or through a catheter into the suprachoroidal space), intravitreal cavity, and / or the outer surface of the sclera of the eye of a patient (human subject) diagnosed with exudative age-related macular degeneration (AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD) to create a permanent depot that continuously supplies a human PTM, such as a human glycosylation transgene product, into the eye. In a preferred embodiment, the methods provided herein can be used in patients (human subjects) diagnosed with exudative AMD.

[0010] Described herein are anti-human vascular endothelial growth factor (hVEGF) antibodies, such as anti-hVEGF antigen-binding fragments, produced by human retinal cells. Human VEGF (hVEGF) is a human protein encoded by the VEGF (VEGFA, VEGFB, VEGFC, or VEGFD) gene. An exemplary amino acid sequence of hVEGF can be found at GenBank accession number AAA35789.1. An exemplary nucleic acid sequence of hVEGF can be found at GenBank accession number M32977.1.

[0011] In one aspect, described herein is a method of treating a human subject diagnosed with neovascular age-related macular degeneration (nAMD) (also known as exudative AMD or wet AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the retina of the human subject a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human retinal cells. In a specific aspect, described herein is a method of treating a human subject diagnosed with nAMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the retina of the human subject a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human retinal cells by administering an expression vector encoding the anti-hVEGF antigen-binding fragment to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject (e.g., by suprachoroidal injection (e.g., by a suprachoroidal drug delivery device such as a microinjector with a microneedle), subretinal injection by a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure by a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space towards the posterior pole, penetrated, and injected into the subretinal space with a thin needle at the posterior pole), or a juxtascleral depot procedure (e.g., by a juxtascleral drug delivery device comprising a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface)). In a specific aspect, described herein is a method of treating a human subject diagnosed with nAMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the retina of the human subject a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human retinal cells by use of a suprachoroidal drug delivery device such as a microinjector.In a specific embodiment, described herein is a method of treating a human subject diagnosed with neovascular age-related macular degeneration (nAMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the retina of the human subject a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human retinal cells, wherein the human subject has a best corrected visual acuity (BCVA) of ≦20 / 20 and ≧20 / 400.

[0012] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the retina of the human subject a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane. In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the retina of the human subject a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane by administering an expression vector encoding the anti-hVEGF antigen-binding fragment to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject (e.g., by suprachoroidal injection (e.g., by a suprachoroidal drug delivery device such as a microinjector with a microneedle), subretinal injection by a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure by a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space towards the posterior pole, penetrated, and injected into the subretinal space with a fine needle at the posterior pole), or juxtascleral depot procedure (e.g., by a juxtascleral drug delivery device comprising a cannula whose tip can be inserted and held juxtaposed directly on the scleral surface)).In a specific embodiment, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising delivering, by use of a suprachoroidal drug delivery device such as a microinjector, a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane to the retina of the human subject. In a specific embodiment, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising delivering a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane to the retina of the human subject, wherein the human subject has a BCVA of ≦20 / 20 and ≧20 / 400.

[0013] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the eye of the human subject a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human retinal cells. In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the eye of the human subject a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human retinal cells by administering an expression vector encoding the anti-hVEGF antigen-binding fragment to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject (e.g., by suprachoroidal injection (e.g., by a suprachoroidal drug delivery device such as a microinjector with a microneedle), subretinal injection by a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure by a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space towards the posterior pole, penetrated through it, and injected into the subretinal space with a thin needle at the posterior pole), or a juxtascleral depot procedure (e.g., by a juxtascleral drug delivery device comprising a cannula that can be inserted at its tip and held directly juxtaposed to the scleral surface)). In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the eye of the human subject a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human retinal cells by using a suprachoroidal drug delivery device such as a microinjector.In a specific embodiment, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the eye of the human subject a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human retinal cells, wherein the human subject has a BCVA of ≦20 / 20 and ≧20 / 400.

[0014] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the eye of the human subject a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane. In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the eye of the human subject a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane by administering an expression vector encoding the anti-hVEGF antigen-binding fragment to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject (e.g., by suprachoroidal injection (e.g., by a suprachoroidal drug delivery device such as a microinjector with a microneedle), subretinal injection by a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure by a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space towards the posterior pole, penetrated, and injected into the subretinal space with a fine needle at the posterior pole), or a juxtascleral depot procedure (e.g., by a juxtascleral drug delivery device comprising a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface)).In certain embodiments, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering, to the eye of the human subject, a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane, by use of a suprachoroidal drug delivery device such as a microinjector. In certain embodiments, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering, to the eye of the human subject, a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane, wherein the human subject has a BCVA of ≦20 / 20 and ≧20 / 400.

[0015] In certain embodiments of the methods described herein, the antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and a light chain comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0016] In certain embodiments of the methods described herein, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16, and heavy chain CDR1-3 of SEQ ID NOs: 17-19 or SEQ ID NOs: 20, 18, and 21.

[0017] In a specific embodiment of the method described herein, the antigen-binding fragment comprises the light chain CDR1-3 of SEQ ID NOs: 14-16, and the heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the second amino acid residue of the light chain CDR3 (i.e.,

Chem.

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Chem.

Chemical formula

[0018] In a specific embodiment of the method described herein, the antigen-binding fragment comprises the light chain CDR1-3 of SEQ ID NOs: 14-16, and the heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the last amino acid residue of the heavy chain CDR1 (i.e.,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0019] In a specific embodiment of the method described herein, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16, and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the last amino acid residue of the heavy chain CDR1 (i.e., [Chemical formula] The N in it does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (PyrGlu), and the second amino acid residue of the light chain CDR3 (i.e.,

Chemical formula

Chemical formula

Chemical formula

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Chemical formula

[0020] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the eye of the human subject a therapeutically effective amount of an anti-hVEGF antibody produced by human retinal cells. In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the eye of the human subject a therapeutically effective amount of an anti-hVEGF antibody produced by human retinal cells by administering an expression vector encoding the anti-hVEGF antibody to the suprachoroidal space, the subretinal space, or the outer surface of the sclera of the eye of the human subject (e.g., by suprachoroidal injection (e.g., by a suprachoroidal drug delivery device such as a microinjector with a microneedle), subretinal injection by a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure by a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space towards the posterior pole, passed through it, and injected into the subretinal space with a thin needle at the posterior pole), or a juxtascleral depot procedure (e.g., by a juxtascleral drug delivery device comprising a cannula that can be inserted at its tip and held directly juxtaposed to the scleral surface)). In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the eye of the human subject a therapeutically effective amount of an anti-hVEGF antibody produced by human retinal cells by using a suprachoroidal drug delivery device such as a microinjector.In a specific embodiment, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (in particular, exudative AMD), comprising delivering to the eye of the human subject a therapeutically effective amount of an anti-hVEGF antibody produced by human retinal cells, wherein the human subject has a BCVA of ≦20 / 20 and ≧20 / 400.

[0021] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the eye of the human subject a therapeutically effective amount of an anti-hVEGF antibody produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane. In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the eye of the human subject a therapeutically effective amount of an anti-hVEGF antibody produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane by administering an expression vector encoding the anti-hVEGF antibody to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject (e.g., by suprachoroidal injection (e.g., by a suprachoroidal drug delivery device such as a microinjector with a microneedle), subretinal injection by a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space (surgical procedure by a subretinal drug delivery device including a catheter that can be inserted into the suprachoroidal space towards the posterior pole, penetrated, and injected into the subretinal space with a thin needle at the posterior pole), or posterior scleral near depot procedure (e.g., by a scleral near drug delivery device including a cannula whose tip can be inserted and held juxtaposed directly on the scleral surface)).In a specific embodiment, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising delivering, to the eye of the human subject by use of a suprachoroidal drug delivery device such as a microinjector, a therapeutically effective amount of an anti-hVEGF antibody produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane. In a specific embodiment, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising delivering, to the eye of the human subject, a therapeutically effective amount of an anti-hVEGF antibody produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane, wherein the human subject has a BCVA of ≦20 / 20 and ≧20 / 400.

[0022] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the retina of the human subject a therapeutically effective amount of an anti-hVEGF antibody produced by human retinal cells. In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the retina of the human subject a therapeutically effective amount of an anti-hVEGF antibody produced by human retinal cells by administering an expression vector encoding the anti-hVEGF antibody to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject (e.g., by suprachoroidal injection (e.g., by a suprachoroidal drug delivery device such as a microinjector with a microneedle), subretinal injection by a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure by a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space towards the posterior pole, penetrate it, and inject into the subretinal space with a thin needle at the posterior pole), or a juxtascleral depot procedure (e.g., by a juxtascleral drug delivery device comprising a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface)). In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the retina of the human subject a therapeutically effective amount of an anti-hVEGF antibody produced by human retinal cells by using a suprachoroidal drug delivery device such as a microinjector.In a specific embodiment, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (in particular, exudative AMD), the method comprising delivering to the retina of the human subject a therapeutically effective amount of an anti-hVEGF antibody produced by human retinal cells, wherein the human subject has a BCVA of ≦20 / 20 and ≧20 / 400.

[0023] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the retina of the human subject a therapeutically effective amount of an anti-hVEGF antibody produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane. In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the retina of the human subject a therapeutically effective amount of an anti-hVEGF antibody produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane by administering an expression vector encoding the anti-hVEGF antibody to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject (e.g., by suprachoroidal injection (e.g., by a suprachoroidal drug delivery device such as a microinjector with a microneedle), subretinal injection by a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure using a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space towards the posterior pole, penetrated through it, and injected into the subretinal space with a thin needle at the posterior pole), or juxtascleral depot procedure (e.g., by a juxtascleral drug delivery device comprising a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface)).In certain embodiments, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering, to the retina of the human subject, a therapeutically effective amount of an anti-hVEGF antibody produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane, by use of a suprachoroidal drug delivery device such as a microinjector. In certain embodiments, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering, to the retina of the human subject, a therapeutically effective amount of an anti-hVEGF antibody produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane, wherein the human subject has a BCVA of ≦20 / 20 and ≧20 / 400.

[0024] In certain embodiments of the methods described herein, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and a light chain comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0025] In certain embodiments of the methods described herein, the antibody comprises light chain CDR1-3 of SEQ ID NOs: 14-16, and heavy chain CDR1-3 of SEQ ID NOs: 17-19 or SEQ ID NOs: 20, 18, and 21.

[0026] In a specific embodiment of the method described herein, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16, and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the second amino acid residue of the light chain CDR3 (i.e.,

Chem.

Chem.

Chem.

Chem.

Chem.

Chemical formula

[0027] In a specific embodiment of the method described herein, the antigen-binding fragment comprises the light chain CDR1-3 of SEQ ID NOs: 14-16, and the heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the last amino acid residue of the heavy chain CDR1 (i.e.,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0028] In a specific embodiment of the method described herein, the antigen-binding fragment comprises the light-chain CDR1-3 of SEQ ID NOs: 14-16, and the heavy-chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the last amino acid residue of the heavy-chain CDR1 (i.e., [Chemical formula] The N) therein does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (PyrGlu), and the second amino acid residue of the light chain CDR3 (i.e.,

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

[0029] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: delivering to the eye of the human subject a therapeutically effective amount of an antigen-binding fragment of an mAb against hVEGF, wherein the antigen-binding fragment comprises an α2,6-sialylated glycan. In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: delivering to the eye of the human subject a therapeutically effective amount of an antigen-binding fragment of an mAb against hVEGF by administering an expression vector encoding the antigen-binding fragment of the mAb to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject (e.g., by suprachoroidal injection (e.g., by a suprachoroidal drug delivery device such as a microinjector with a microneedle), subretinal injection by a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure by a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space towards the posterior pole, penetrated, and injected into the subretinal space with a thin needle at the posterior pole), or a juxtascleral depot procedure (e.g., by a juxtascleral drug delivery device comprising a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface)), wherein the antigen-binding fragment comprises an α2,6-sialylated glycan. In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: delivering to the eye of the human subject a therapeutically effective amount of an antigen-binding fragment of an mAb against hVEGF by using a suprachoroidal drug delivery device such as a microinjector, wherein the antigen-binding fragment comprises an α2,6-sialylated glycan.In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (in particular, exudative AMD), comprising delivering to the eye of the human subject a therapeutically effective amount of an antigen-binding fragment of an mAb against hVEGF, wherein the antigen-binding fragment comprises an α2,6-sialylated glycan, wherein the human subject has a BCVA of ≦20 / 20 and ≧20 / 400.

[0030] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: delivering to the eye of the human subject a therapeutically effective amount of a glycosylated antigen-binding fragment of an mAb against hVEGF, wherein the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal antigens (i.e., as used herein, "detectable" means a level detectable by the standard assays described below). In a specific embodiment, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: delivering to the eye of the human subject a therapeutically effective amount of a glycosylated antigen-binding fragment of an mAb against hVEGF by administering an expression vector encoding the glycosylated antigen-binding fragment of the mAb against hVEGF to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject (e.g., by suprachoroidal injection (e.g., by a suprachoroidal drug delivery device such as a microinjector with a microneedle), subretinal injection by a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure by a subretinal drug delivery device including a catheter that can be inserted into the suprachoroidal space towards the posterior pole, penetrated through it, and injected into the subretinal space with a thin needle at the posterior pole), or a juxtascleral depot procedure (e.g., by a juxtascleral drug delivery device including a cannula that can be inserted at its tip and held directly juxtaposed to the scleral surface)), wherein the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal antigens.In a specific embodiment, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: delivering, to the eye of the human subject, a therapeutically effective amount of a glycosylated antigen-binding fragment of an mAb against hVEGF, by use of a suprachoroidal drug delivery device such as a microinjector, wherein the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal antigens. In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: delivering, to the eye of the human subject, a therapeutically effective amount of a glycosylated antigen-binding fragment of an mAb against hVEGF, wherein the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal antigens, and wherein the human subject has a BCVA of ≦20 / 20 and ≧20 / 400.

[0031] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), where the method comprises: administering to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject, an expression vector encoding an antigen-binding fragment of an mAb against hVEGF (e.g., by suprachoroidal injection, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space, or a posterior scleral depot procedure), where the antigen-binding fragment is α2,6-sialylated when expressed from the expression vector in human immortalized retinal-derived cells. In a specific embodiment, the administering step comprises the use of a suprachoroidal drug delivery device such as a microinjector. In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), where the method comprises: administering to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject, an expression vector encoding an antigen-binding fragment of an mAb against hVEGF (e.g., by suprachoroidal injection, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space), or a posterior scleral depot procedure), where the antigen-binding fragment is α2,6-sialylated when expressed from the expression vector in human immortalized retinal-derived cells, and where the human subject has a BCVA of ≤20 / 20 and ≥20 / 400. In a specific embodiment, the administering step comprises the use of a suprachoroidal drug delivery device such as a microinjector.

[0032] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), where the method comprises: administering or delivering to the retina of the human subject, via the suprachoroidal space of the eye of the human subject (e.g., by a suprachoroidal drug delivery device such as a microinjector with a micro-needle), an expression vector encoding an antigen-binding fragment of an mAb against hVEGF, where the expression of the antigen-binding fragment is α2,6-sialylated when expressed from the expression vector in human immortalized retinal-derived cells. In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), where the method comprises: administering or delivering to the retina of the human subject, via the suprachoroidal space of the eye of the human subject (e.g., by a suprachoroidal drug delivery device such as a microinjector with a micro-needle), an expression vector encoding an antigen-binding fragment of an mAb against hVEGF, where the expression of the antigen-binding fragment is α2,6-sialylated when expressed from the expression vector in human immortalized retinal-derived cells, and where the human subject has a BCVA of ≤20 / 20 and ≥20 / 400.

[0033] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), where the method comprises: administering, via the suprachoroidal space of the eye of the human subject (e.g., by a subretinal drug delivery device including a catheter that can be inserted into and passed through the suprachoroidal space), into the subretinal and / or intravitreal cavity of the human subject, an expression vector encoding an antigen-binding fragment of an mAb against hVEGF, where the expression of the antigen-binding fragment is α2,6-sialylated when expressed from the expression vector in human immortalized retinal-derived cells. In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), where the method comprises: administering, via the suprachoroidal space of the eye of the human subject (e.g., by a subretinal drug delivery device including a catheter that can be inserted into the suprachoroidal space towards the posterior pole, passed through it, and injected into the subretinal space with a thin needle at the posterior pole), into the subretinal and / or intravitreal cavity of the human subject, an expression vector encoding an antigen-binding fragment of an mAb against hVEGF, where the expression of the antigen-binding fragment is α2,6-sialylated when expressed from the expression vector in human immortalized retinal-derived cells, and where the human subject has a BCVA of ≤20 / 20 and ≥20 / 400.

[0034] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), where the method comprises: administering to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject, an expression vector encoding an antigen-binding fragment against hVEGF (e.g., by suprachoroidal injection, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space, or a posterior scleral juxtadepot procedure), where the expression of the antigen-binding fragment is α2,6-sialylated when expressed from the expression vector in human immortalized retinal-derived cells, and where the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal antigens. In a specific embodiment, the administering step comprises the use of a suprachoroidal drug delivery device such as a microinjector. In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), where the method comprises: administering to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject, an expression vector encoding an antigen-binding fragment against hVEGF (e.g., by suprachoroidal injection, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space, or a posterior scleral juxtadepot procedure), where the expression of the antigen-binding fragment is α2,6-sialylated when expressed from the expression vector in human immortalized retinal-derived cells, and where the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal antigens, and where the human subject has a BCVA of ≤20 / 20 and ≥20 / 400. In a specific embodiment, the administering step comprises the use of a suprachoroidal drug delivery device such as a microinjector.

[0035] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), wherein the method comprises: administering or delivering to the retina of the human subject, via the suprachoroidal space of the eye of the human subject (e.g., by a suprachoroidal drug delivery device such as a microinjector with a microneedle), an expression vector encoding an antigen-binding fragment against hVEGF, wherein the expression of the antigen-binding fragment is α2,6-sialylated when expressed from the expression vector in human immortalized retinal-derived cells, and wherein the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal antigens. In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), wherein the method comprises: administering or delivering to the retina of the human subject, via the suprachoroidal space of the eye of the human subject (e.g., by a suprachoroidal drug delivery device such as a microinjector with a microneedle), an expression vector encoding an antigen-binding fragment against hVEGF, wherein the expression of the antigen-binding fragment is α2,6-sialylated when expressed from the expression vector in human immortalized retinal-derived cells, and wherein the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal antigens, and wherein the human subject has a BCVA of ≤20 / 20 and ≥20 / 400.

[0036] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), wherein the method comprises: administering, via the suprachoroidal space of the eye of the human subject (e.g., by a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space towards the posterior pole, penetrate it, and inject into the subretinal space with a fine needle at the posterior pole), into the subretinal and / or intravitreal cavity of the human subject, an expression vector encoding an antigen-binding fragment against hVEGF, wherein the antigen-binding fragment is α2,6-sialylated when expressed from the expression vector in human immortalized retinal-derived cells, and wherein the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal antigens. In a specific aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), wherein the method comprises: administering, via the suprachoroidal space of the eye of the human subject (e.g., by a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space towards the posterior pole, penetrate it, and inject into the subretinal space with a fine needle at the posterior pole), into the subretinal and / or intravitreal cavity of the human subject, an expression vector encoding an antigen-binding fragment against hVEGF, wherein the antigen-binding fragment is α2,6-sialylated when expressed from the expression vector in human immortalized retinal-derived cells, and wherein the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal antigens, and wherein the human subject has a BCVA of ≤20 / 20 and ≥20 / 400.

[0037] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: administering to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF (e.g., by suprachoroidal injection, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space, or a posterior scleral juxtadepot procedure), resulting in the formation of a depot that releases the antigen-binding fragment comprising α2,6-sialylated glycans. In a specific embodiment, the administering step comprises the use of a suprachoroidal drug delivery device such as a microinjector. In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: administering to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF (e.g., by suprachoroidal injection, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space, or a posterior scleral juxtadepot procedure), resulting in the formation of a depot that releases the antigen-binding fragment comprising α2,6-sialylated glycans, wherein the human subject has a BCVA of ≤20 / 20 and ≥20 / 400. In a specific embodiment, the administering step comprises the use of a suprachoroidal drug delivery device such as a microinjector.

[0038] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: administering or delivering to the retina of the human subject, via the suprachoroidal cavity of the human subject's eye (e.g., by a suprachoroidal drug delivery device such as a microinjector with a micro-needle), a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, such that a depot is formed that releases the antigen-binding fragment comprising α2,6-sialylated glycan. In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: administering or delivering to the retina of the human subject, via the suprachoroidal cavity of the human subject's eye (e.g., by a suprachoroidal drug delivery device such as a microinjector with a micro-needle), a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, such that a depot is formed that releases the antigen-binding fragment comprising α2,6-sialylated glycan, wherein the human subject has a BCVA of ≤20 / 20 and ≥20 / 400.

[0039] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: administering to the subretinal and / or intravitreal space of the human subject via the suprachoroidal space of the human subject's eye (e.g., by a subretinal drug delivery device including a catheter that can be inserted into the suprachoroidal space toward the posterior pole, passed through it, and injected into the subretinal space with a fine needle at the posterior pole), a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, such that a depot is formed that releases the antigen-binding fragment comprising α2,6-sialylated glycans. In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: administering to the subretinal and / or intravitreal space of the human subject via the suprachoroidal space of the human subject's eye (e.g., by a subretinal drug delivery device including a catheter that can be inserted into the suprachoroidal space toward the posterior pole, passed through it, and injected into the subretinal space with a fine needle at the posterior pole), a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, such that a depot is formed that releases the antigen-binding fragment comprising α2,6-sialylated glycans, wherein the human subject has a BCVA of ≤20 / 20 and ≥20 / 400.

[0040] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: administering to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF (e.g., by suprachoroidal injection, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space, or a posterior scleral juxtadepot procedure), such that a depot is formed that releases the antigen-binding fragment, wherein the antigen-binding fragment is glycosylated but does not contain detectable NeuGc and / or α-Gal antigens. In a specific embodiment, the administering step comprises the use of a suprachoroidal drug delivery device such as a microinjector. In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: administering to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF (e.g., by suprachoroidal injection, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space, or a posterior scleral juxtadepot procedure), such that a depot is formed that releases the antigen-binding fragment, wherein the antigen-binding fragment is glycosylated but does not contain detectable NeuGc and / or α-Gal antigens, and wherein the human subject has a BCVA of ≤20 / 20 and ≥20 / 400. In a specific embodiment, the administering step comprises the use of a suprachoroidal drug delivery device such as a microinjector.

[0041] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: administering or delivering to the retina of the human subject, via the suprachoroidal space of the human subject's eye (e.g., by a suprachoroidal drug delivery device such as a microinjector with a micro needle), a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, such that a depot is formed that releases the antigen-binding fragment, wherein the antigen-binding fragment is glycosylated and does not contain detectable NeuGc and / or α-Gal antigens. In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: administering or delivering to the retina of the human subject, via the suprachoroidal space of the human subject's eye (e.g., by a suprachoroidal drug delivery device such as a microinjector with a micro needle), a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, such that a depot is formed that releases the antigen-binding fragment, wherein the antigen-binding fragment is glycosylated and does not contain detectable NeuGc and / or α-Gal antigens, and wherein the human subject has a BCVA of ≤20 / 20 and ≥20 / 400.

[0042] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: administering to the subretinal and / or intraretinal lumen of the human subject, via the suprachoroidal space of the human subject's eye (e.g., by a subretinal drug delivery device including a catheter that can be inserted into the suprachoroidal space toward the posterior pole, passed through it, and injected into the subretinal space with a fine needle at the posterior pole), a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, such that a depot that releases the antigen-binding fragment is formed, wherein the antigen-binding fragment is glycosylated but does not contain detectable NeuGc and / or α-Gal antigens. In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: administering to the subretinal and / or intraretinal lumen of the human subject, via the suprachoroidal space of the human subject's eye (e.g., by a subretinal drug delivery device including a catheter that can be inserted into the suprachoroidal space toward the posterior pole, passed through it, and injected into the subretinal space with a fine needle at the posterior pole), a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, such that a depot that releases the antigen-binding fragment is formed, wherein the antigen-binding fragment is glycosylated, does not contain detectable NeuGc and / or α-Gal antigens, and wherein the human subject has a BCVA of ≦20 / 20 and ≧20 / 400.

[0043] In one aspect of the methods described herein, the antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and a light chain comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0044] In one aspect of the methods described herein, the antigen-binding fragment further comprises tyrosine sulfation.

[0045] In certain embodiments of the methods described herein, production of the antigen-binding fragment comprising an α2,6-sialylated glycan is confirmed by transducing the recombinant nucleotide expression vector into PER.C6 or RPE cell lines in cell culture.

[0046] In certain embodiments of the methods described herein, production of the antigen-binding fragment comprising tyrosine sulfation is confirmed by transducing the recombinant nucleotide expression vector into PER.C6 or RPE cell lines in cell culture.

[0047] In certain embodiments of the methods described herein, the vector has a hypoxia-inducible promoter.

[0048] In certain embodiments of the methods described herein, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16, and heavy chain CDR1-3 of SEQ ID NOs: 17-19 or SEQ ID NOs: 20, 18, and 21.

[0049] In a specific embodiment of the methods described herein, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16, and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the second amino acid residue of the light chain CDR3 (i.e.,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0050] In a specific embodiment of the method described herein, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16, and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the last amino acid residue of the heavy chain CDR1 (i.e., [Chem.] N) in does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu). In a specific embodiment, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16, and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the 9th amino acid residue of the heavy chain CDR1 (i.e., [Chem.] M) in possesses one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu), and the 3rd amino acid residue of the heavy chain CDR2 (i.e., [Chem.] N) in possesses one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu), and the last amino acid residue of the heavy chain CDR1 (i.e., [Chem.] N) in does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu). In a specific embodiment, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16, and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the last amino acid residue of the heavy chain CDR1 (i.e., [Chem.] N) in is not acetylated. In a specific embodiment, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16, and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the 9th amino acid residue of the heavy chain CDR1 (i.e., [Chem.] M) therein has one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu), and the third amino acid residue of the heavy chain CDR2 (i.e.,

Chemical formula

Chemical formula

[0051] In a specific embodiment of the method described herein, the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14-16, and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the last amino acid residue of the heavy chain CDR1 (i.e.,

Chemical formula

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

Chem.

Chem.

Chem.

[0052] In certain aspects of the methods described herein, the antigen-binding fragment transgene encodes a leader peptide. The leader peptide may also be referred to herein as a signal peptide or a leader sequence.

[0053] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), particularly exudative AMD, comprising: administering to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF (e.g., by suprachoroidal injection, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space, or posterior scleral near depot placement), resulting in the formation of a depot that releases the antigen-binding fragment comprising α2,6-sialylated glycan; wherein the recombinant vector, when used to transduce PER.C6 or RPE cells in culture, results in the production of the antigen-binding fragment comprising α2,6-sialylated glycan in the cell culture. In a specific embodiment, the administering step comprises the use of a suprachoroidal drug delivery device such as a microinjector. In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), particularly exudative AMD, comprising: administering to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF (e.g., by suprachoroidal injection, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space, or posterior scleral near depot placement), resulting in the formation of a depot that releases the antigen-binding fragment comprising α2,6-sialylated glycan; wherein the recombinant vector, when used to transduce PER.C6 or RPE cells in culture, results in the production of the antigen-binding fragment comprising α2,6-sialylated glycan in the cell culture, and wherein the human subject has a BCVA of ≦20 / 20 and ≧20 / 400. In a specific embodiment, the administering step comprises the use of a suprachoroidal drug delivery device such as a microinjector.

[0054] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: administering or delivering to the retina of the human subject, via the suprachoroidal space of the human subject's eye (e.g., by a suprachoroidal drug delivery device such as a microinjector with a microneedle), a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, such that a depot is formed that releases the antigen-binding fragment comprising α2,6-sialylated glycan; wherein the recombinant vector, when used to transduce PER.C6 or RPE cells in culture, results in the production of the antigen-binding fragment comprising α2,6-sialylated glycan in the cell culture. In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: administering or delivering to the retina of the human subject, via the suprachoroidal space of the human subject's eye (e.g., by a suprachoroidal drug delivery device such as a microinjector with a microneedle), a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, such that a depot is formed that releases the antigen-binding fragment comprising α2,6-sialylated glycan; wherein the recombinant vector, when used to transduce PER.C6 or RPE cells in culture, results in the production of the antigen-binding fragment comprising α2,6-sialylated glycan in the cell culture, and wherein the human subject has a BCVA of ≦20 / 20 and ≧20 / 400.

[0055] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising: administering, via the suprachoroidal space of the eye of the human subject (e.g., by a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space towards the posterior pole, passed through it, and injected into the subretinal space with a fine needle at the posterior pole), a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF into the subretinal and / or intravitreal cavity of the human subject, whereby a depot is formed that releases the antigen-binding fragment comprising α2,6-sialylated glycans; wherein the recombinant vector, when used to transduce PER.C6 or RPE cells in culture, results in the production of the antigen-binding fragment comprising α2,6-sialylated glycans in the cell culture. In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising: administering, via the suprachoroidal space of the eye of the human subject (e.g., by a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space towards the posterior pole, passed through it, and injected into the subretinal space with a fine needle at the posterior pole), a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF into the subretinal and / or intravitreal cavity of the human subject, whereby a depot is formed that releases the antigen-binding fragment comprising α2,6-sialylated glycans; wherein the recombinant vector, when used to transduce PER.C6 or RPE cells in culture, results in the production of the antigen-binding fragment comprising α2,6-sialylated glycans in the cell culture, and wherein the human subject has a BCVA of ≦20 / 20 and ≧20 / 400.

[0056] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (in particular, exudative AMD), comprising: administering to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF (e.g., by suprachoroidal injection, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space, or a posterior scleral juxtadepot procedure), resulting in the formation of a depot that releases the antigen-binding fragment, wherein the antigen-binding fragment is glycosylated but does not contain detectable NeuGc and / or α-Gal antigens; and wherein the recombinant vector, when used to transduce PER.C6 or RPE cells in culture, results in the production in the cell culture of the antigen-binding fragment that is glycosylated but does not contain detectable NeuGc and / or α-Gal antigens. In a specific embodiment, the administering step comprises the use of a suprachoroidal drug delivery device, such as a microinjector.In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD): administering to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of the human subject a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF (e.g., by suprachoroidal injection, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space, or a posterior scleral near depot procedure), resulting in the formation of a depot that releases the antigen-binding fragment, where the antigen-binding fragment is glycosylated but does not contain detectable NeuGc and / or α-Gal antigens; where the recombinant vector, when used to transduce PER.C6 or RPE cells in culture, results in the production of the antigen-binding fragment that is glycosylated but does not contain detectable NeuGc and / or α-Gal antigens in the cell culture, and where the human subject has a BCVA of ≦20 / 20 and ≧20 / 400. In a specific embodiment, the administering step includes the use of a suprachoroidal drug delivery device such as a microinjector.

[0057] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: administering or delivering to the retina of the human subject, via the suprachoroidal space of the human subject's eye (e.g., by a suprachoroidal drug delivery device such as a microinjector with a microneedle), a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, such that a depot that releases the antigen-binding fragment is formed, wherein the antigen-binding fragment is glycosylated and does not contain detectable NeuGc and / or α-Gal antigens; wherein when the recombinant vector is used to transduce PER.C6 or RPE cells in culture, it results in the production of the antigen-binding fragment that is glycosylated and does not contain detectable NeuGc and / or α-Gal antigens in the cell culture. In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: administering or delivering to the retina of the human subject, via the suprachoroidal space of the human subject's eye (e.g., by a suprachoroidal drug delivery device such as a microinjector with a microneedle), a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, such that a depot that releases the antigen-binding fragment is formed, wherein the antigen-binding fragment is glycosylated and does not contain detectable NeuGc and / or α-Gal antigens; wherein when the recombinant vector is used to transduce PER.C6 or RPE cells in culture, it results in the production of the antigen-binding fragment that is glycosylated and does not contain detectable NeuGc and / or α-Gal antigens in the cell culture, and wherein the human subject has a BCVA of ≦20 / 20 and ≧20 / 400.

[0058] In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: administering to the subretinal and / or intravitreal space of the human subject via the suprachoroidal space of the human subject's eye (e.g., by a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space towards the posterior pole, penetrate it, and inject into the subretinal space with a fine needle at the posterior pole), a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, such that a depot that releases the antigen-binding fragment is formed, wherein the antigen-binding fragment is glycosylated and does not contain detectable NeuGc and / or α-Gal antigens; wherein when the recombinant vector is used to transduce PER.C6 or RPE cells in culture, it results in the production of the antigen-binding fragment that is glycosylated and does not contain detectable NeuGc and / or α-Gal antigens in the cell culture. In one aspect, described herein is a method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising: administering to the subretinal and / or intravitreal space of the human subject via the suprachoroidal space of the human subject's eye (e.g., by a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space towards the posterior pole, penetrate it, and inject into the subretinal space with a fine needle at the posterior pole), a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, such that a depot that releases the antigen-binding fragment is formed, wherein the antigen-binding fragment is glycosylated and does not contain detectable NeuGc and / or α-Gal antigens; wherein when the recombinant vector is used to transduce PER.C6 or RPE cells in culture, it results in the production of the antigen-binding fragment that is glycosylated and does not contain detectable NeuGc and / or α-Gal antigens in the cell culture, and wherein the human subject has a BCVA of ≤20 / 20 and ≥20 / 400.

[0059] In certain aspects of the methods described herein, the human subject has a BCVA that is ≤20 / 63 and ≥20 / 400.

[0060] In certain aspects of the methods described herein, the BCVA is the BCVA of the eye to be treated in the human subject.

[0061] In certain aspects of the methods described herein, delivering to the eye includes delivering to the retina, choroid, and / or vitreous humor of the eye.

[0062] In certain aspects of the methods described herein, the antigen-binding fragment includes a heavy chain that contains one, two, three, or four additional amino acids at the C-terminus.

[0063] In certain aspects of the methods described herein, the antigen-binding fragment includes a heavy chain that does not contain additional amino acids at the C-terminus.

[0064] In certain aspects, the methods described herein produce a population of antigen-binding fragment molecules, where the antigen-binding fragment molecules include a heavy chain, and where 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, or 20%, or less, of the population of antigen-binding fragment molecules includes one, two, three, or four additional amino acids at the C-terminus of the heavy chain. In certain aspects, the methods described herein produce a population of antigen-binding fragment molecules, where the antigen-binding fragment molecules include a heavy chain, and where 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, or 20%, or less, but more than 0%, of the population of antigen-binding fragment molecules includes one, two, three, or four additional amino acids at the C-terminus of the heavy chain.

[0065] In certain aspects, the methods described herein produce a population of antigen-binding fragment molecules, where the antigen-binding fragment molecules include a heavy chain, and where 0.5-1%, 0.5%-2%, 0.5%-3%, 0.5%-4%, 0.5%-5%, 0.5%-10%, 0.5%-20%, 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%-10%, 1%-20%, 2%-3%, 2%-4%, 2%-5%, 2%-10%, 2%-20%, 3%-4%, 3%-5%, 3%-10%, 3%-20%, 4%-5%, 4%-10%, 4%-20%, 5%-10%, 5%-20%, or 10%-20% of the population of antigen-binding fragment molecules includes one, two, three, or four additional amino acids at the C-terminus of the heavy chain.

[0066] The subject to whom such gene therapy is administered should be a subject responsive to anti-VEGF therapy. In certain embodiments, the method includes treating a patient diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD) and identified as responsive to treatment with an anti-VEGF antibody. In more specific embodiments, the patient is responsive to treatment with an anti-VEGF antigen-binding fragment. In certain embodiments, the patient has been shown to be responsive to treatment with an anti-VEGF antigen-binding fragment injected intravitreally prior to treatment with gene therapy. In specific embodiments, the patient has previously been treated with LUCENTIS® (ranibizumab), EYLEA® (aflibercept), and / or AVASTIN® (bevacizumab) and has been found to be responsive to one or more of LUCENTIS® (ranibizumab), EYLEA® (aflibercept), and / or AVASTIN® (bevacizumab).

[0067] The subject to which such a viral vector or other DNA expression construct is to be delivered should be responsive to an anti-hVEGF antigen-binding fragment encoded by a transgene in the viral vector or expression construct. To determine responsiveness, an anti-VEGF antigen-binding fragment transgene product (e.g., produced in cell culture, bioreactor, etc.) can be administered directly to the subject, e.g., by intravitreal injection.

[0068] Examples of HuPTMFabVEGFi encoded by a transgene, such as HuGlyFabVEGFi, include antigen-binding fragments of antibodies that bind to hVEGF, such as bevacizumab; anti-hVEGF Fab portions, such as ranibizumab; or Fab portions of such bevacizumab or ranibizumab that have been modified to include additional glycosylation sites on the Fab domain, but are not limited thereto (see, e.g., Courtois et al., 2016, mAbs 8: 99-112, which is hereby incorporated by reference in its entirety for the description of derivatives of bevacizumab that are highly glycosylated on the Fab domain of the full-length antibody).

[0069] The recombinant vector used for the delivery of the transgene should have tropism for human retinal cells or photoreceptor cells. Examples of such vectors include non-replicating recombinant adeno-associated virus vectors ("rAAV"), and in particular, those having an AAV8 capsid are preferred. However, other viral vectors may be used, including but not limited to lentiviral vectors, vaccinia virus vectors, or non-viral expression vectors referred to as "naked DNA" constructs. Preferably, the HuPTMFabVEGFi, for example, the HuGlyFabVEGFi transgene should be controlled by appropriate expression control elements, such as, for example, the CB7 promoter (chicken β-actin promoter and CMV enhancer), the RPE65 promoter, or the opsin promoter, and other expression control elements (e.g., chicken β-actin intron, murine minute virus (MVM) intron, human factor IX intron (e.g., FIX truncated intron 1), β-globin splice donor / immunoglobulin heavy chain splice acceptor intron, adenovirus splice donor / immunoglobulin splice acceptor intron, SV40 late splice donor / splice acceptor (19S / 16S) intron, and hybrid adenovirus splice donor / IgG splice acceptor intron, etc. introns, as well as polyA signals such as rabbit β-globin polyA signal, human growth hormone (hGH) polyA signal, SV40 late polyA signal, synthetic polyA (SPA) signal, and bovine growth hormone (bGH) polyA signal) that enhance the expression of the transgene driven by the vector can be included. See, for example, the literature of Powell and Rivera-Soto, 2015, Discov. Med., 19(102):49-57.

[0070] The gene therapy construct is designed such that both the heavy and light chains are expressed. More specifically, the heavy and light chains should be expressed in approximately equal amounts, in other words, the heavy and light chains should be expressed at an approximate heavy chain to light chain ratio of 1:1. The coding sequences of the heavy and light chains can be modified in a single construct separated by a linker or IRES that allows the heavy and light chains to be cleaved such that separate heavy and light chain polypeptides are expressed. For example, for specific leader sequences that can be used with the methods and compositions provided herein, see Section 5.2.4, and for specific IRESs, 2As, and other linker sequences, see Section 5.2.5.

[0071] A pharmaceutical composition suitable for suprachoroidal, subretinal, episcleral, and / or intravitreal administration comprises a suspension of a recombinant (e.g., rHuGlyFabVEGFi) vector in a formulated buffer that includes a physiologically compatible aqueous buffer, a surfactant, and any excipients.

[0072] The recombinant vector in a therapeutically effective amount should be administered subretinally and / or intravitreally (e.g., by subretinal injection via a pars plana approach (surgical procedure) or subretinal administration via the suprachoroidal space) in a volume ranging from ≧0.1 mL to ≦0.5 mL, preferably 0.1 - 0.30 mL (100 - 300 μl), and most preferably 0.25 mL (250 μl). The recombinant vector in a therapeutically effective amount should be administered to the suprachoroid in a volume of 100 μl or less, e.g., in a volume of 50 - 100 μl (e.g., by suprachoroidal injection). The recombinant vector in a therapeutically effective amount should be administered to the outer surface of the sclera in a volume of 500 μl or less, e.g., in a volume of 10 - 20 μl, 20 - 50 μl, 50 - 100 μl, 100 - 200 μl, 200 - 300 μl, 300 - 400 μl, or 400 - 500 μl (e.g., by a posterior sclera near depot procedure). Subretinal injection is a surgical procedure performed by a skilled retinal surgeon involving vitrectomy and subretinal injection of gene therapy into the retina of a subject under local anesthesia (see, e.g., Campochiaro et al., 2017, Hum Gen Ther 28(1):99 - 111, which is hereby incorporated by reference in its entirety). In a specific embodiment, subretinal administration is performed through the suprachoroidal space using a subretinal drug delivery device that includes a suprachoroidal catheter for injecting the drug into the subretinal space, e.g., a catheter that can be inserted into the suprachoroidal space towards the posterior pole, passed through it, and injected into the subretinal space with a thin needle at the posterior pole (see, e.g., Baldassarre et al., 2017, Subretinal Delivery of Cells via the Suprachoroidal Space: Janssen Trial. In: Schwartz et al. (eds.), Cellular Therapies for Retinal Disease, Springer, Cham; International Patent Application Publication WO 2016 / 040635 A1; each of these is hereby incorporated by reference in its entirety).Administration to the suprachoroid involves the administration of a drug into the suprachoroidal cavity of the eye and is typically performed using a suprachoroidal drug delivery device such as a microinjector with a microneedle (see, for example, Hariprasad, 2016, Retinal Physician 13: 20-23; Goldstein, 2014, Retina Today 9(5): 82-87, each of which is incorporated herein by reference in its entirety). Examples of suprachoroidal drug delivery devices that can be used to deposit an expression vector into the suprachoroidal cavity according to the invention described herein include, but are not limited to, suprachoroidal drug delivery devices manufactured by Clearside® Biomedical (see, for example, Hariprasad, 2016, Retinal Physician 13: 20-23) and MedOne suprachoroidal catheters. Examples of subretinal drug delivery devices that can be used to deposit an expression vector into the subretinal space via the suprachoroidal cavity according to the invention described herein include, but are not limited to, subretinal drug delivery devices manufactured by Janssen Pharmaceuticals (see, for example, International Patent Application Publication WO 2016 / 040635 A1). In a specific embodiment, administration to the outer surface of the sclera is performed by a sclera-adjacent drug delivery device that includes a cannula that can be inserted with its tip and held directly juxtaposed to the scleral surface. See Section 5.3.2 for further details on various administration modalities. The soluble transgene product should be delivered to the retina, vitreous humor, and / or aqueous humor by suprachoroidal, subretinal, sclera-adjacent, and / or intraretinal administration. Expression of the transgene product (e.g., the encoded anti-VEGF antibody) by retinal cells, such as rod cells, cone cells, retinal pigment epithelial cells, horizontal cells, bipolar cells, amacrine cells, ganglion cells, and / or Müller cells, results in delivery and maintenance of the transgene product in the retina, vitreous humor, and / or aqueous humor. The transgene product concentration should be at least 0.330 μg / mL in the vitreous humor or 0.110 μg / mL in the aqueous humor (anterior chamber) for 3 months. minA dosage to maintain is desirable; thereafter, the vitreous C of the transgene product in the range of 1.70 - 6.60 μg / mL min concentration and / or the aqueous humor C in the range of 0.567 - 2.20 μg / mL min should be maintained. However, since the transgene product is produced continuously, it may be effective to maintain a lower concentration. The concentration of the transgene product can be measured in patient samples of vitreous humor and / or aqueous humor derived from the anterior chamber of the treated eye. Alternatively, the vitreous humor concentration can be estimated and / or monitored by measuring the patient serum concentration of the transgene product - the ratio of the systemic exposure to the vitreous exposure of the transgene product is about 1:90,000 (see, for example, the vitreous humor and serum concentrations of ranibizumab reported on p.1621 and Table 5 on p.1623 of Xu L et al., 2013, Invest. Opthal. Vis. Sci. 54: 1616 - 1624, which is incorporated herein by reference in its entirety).

[0073] The present invention has several advantages over standard therapeutic procedures involving repeated intravitreal injections of high-dose boluses of VEGF inhibitors that dissipate over time to produce peak and trough levels. The continuous expression of the transgene product antibody allows for a more consistent level of antibody to be present at the site of action, as opposed to repeated injections of the antibody, resulting in fewer injections being required and, as a result, fewer clinic visits, making it less risky and more convenient for the patient. Consistent protein production reduces the likelihood of rebound edema in the retina, which can lead to better clinical outcomes. Additionally, since the microenvironment present during and after translation is different, the antibody expressed from the transgene is post-translationally modified in a different manner than the directly injected antibody. Without being bound by any particular theory, this results in antibodies with various diffusion, bioactivity, distribution, affinity, pharmacokinetic, and immunogenic properties that are "biobetters" compared to the directly injected antibody when delivered to the site of action.

[0074] Furthermore, antibodies expressed from transgenes in vivo are less likely to contain degradation products associated with antibodies produced by recombinant techniques, such as protein aggregates and protein oxides. Aggregation is a problem associated with the production and storage of proteins due to high protein concentrations, surface interactions with manufacturing equipment and containers, and purification with certain buffer systems. These conditions that promote aggregation do not exist in transgene expression in gene therapy. Oxidation, such as methionine, tryptophan, and histidine oxidation, is also associated with protein production and storage and is caused by stressed cell culture conditions, contact with metals and air, and impurities in buffers and excipients. Proteins expressed from transgenes in vivo can also oxidize under stressed conditions. However, humans and many other organisms have antioxidant defense systems that not only reduce oxidative stress but can also repair and / or reverse oxidation. Therefore, proteins produced in vivo are less likely to be in an oxidized form. Both aggregation and oxidation can affect efficacy, pharmacokinetics (clearance), and immunogenicity.

[0075] Without being bound by theory, the methods and compositions provided herein are based in part on the following principles: (i) Human retinal cells are secretory cells that possess the cellular machinery for the post-translational processing of secreted proteins, including glycosylation and tyrosine-O-sulfation, which are robust processes of retinal cells (e.g., for post-translational modifications performed by human retinal cells, see Wang et al., 2013, Analytical Biochem. 427: 20-28 and Adamis et al., 1993, BBRC 193: 631-638, which report the production of glycoproteins by retinal cells; and Kanan et al., 2009, Exp. Eye Res. 89: 559-567 and Kanan and Al-Ubaidi, 2015, Exp. Eye Res. 133: 126-131, which report the production of tyrosine-sulfated glycoproteins secreted by retinal cells, each of which is hereby incorporated by reference in its entirety). (ii) Contrary to the understanding of the prior art, anti-VEGF antigen-binding fragments, e.g., ranibizumab (and the Fab domain of full-length anti-VEGF mAbs such as bevacizumab), actually possess N-linked glycosylation sites. For example, the C H domain (TVSWN 165 SGAL) and the non-consensus asparagine (「N」) glycosylation sites in the C L domain (QSGN 158 SQE), as well as the glutamine (「Q」) residues (and the corresponding portions in the Fab of bevacizumab) that are glycosylation sites in the V H domain (Q 115 GT) and the V L domain (TFQ 100 GT) are identified in Figure 1 (e.g., for the identification of N-linked glycosylation sites in antibodies, see Valliere-Douglass et al., 2009, J. Biol. Chem. 284: 32493-32506, and Valliere-Douglass et al., 2010, J. Biol. Chem. 285: 16012-16022, each of which is hereby incorporated by reference in its entirety). (iii) Such non-standard sites typically result in low levels of glycosylation (e.g., about 1-5%) of the antibody population, but in immunoprivileged organs such as the eye, functional advantages can be prominent (see, for example, the literature of van de Bovenkamp et al., 2016, J. Immunol. 196:1435-1441). For example, Fab glycosylation can affect antibody stability, half-life, and binding properties. To determine the effect of Fab glycosylation on the affinity of the antibody for its target, any technique known to those skilled in the art, such as enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR), can be used. To determine the effect of Fab glycosylation on the half-life of the antibody, any technique known to those skilled in the art can be used, for example, by measuring the level of radioactivity in blood or organs (e.g., the eye) in a subject administered a radiolabeled antibody. To determine the effect of Fab glycosylation on antibody stability, such as the level of aggregation or protein unfolding, any technique known to those skilled in the art, such as differential scanning calorimetry (DSC), high-performance liquid chromatography (HPLC), such as size-exclusion high-performance liquid chromatography (SEC-HPLC), capillary electrophoresis, mass spectrometry, or turbidity measurement, can be used. The HuPTMFabVEGFi provided herein, such as the HuGlyFabVEGFi transgene, results in the production of Fab glycosylated at non-standard sites at 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more. In certain embodiments, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more of the Fab derived from the Fab population is glycosylated at non-standard sites. In certain embodiments, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more of the non-standard sites are glycosylated.In certain embodiments, glycosylation of the Fab at these non-standard sites is 25%, 50%, 100%, 200%, 300%, 400%, 500%, or greater than the amount of glycosylation of these non-standard sites in Fab produced in HEK293 cells. (iv) In addition to glycosylation sites, anti-VEGF Fabs such as ranibizumab (and the Fab of bevacizumab) contain tyrosine ("Y") sulfation sites inside or near the CDRs; the V H (EDTAVY 94 Y 95 ) and V L (EDFATY 86 ) tyrosine-O-sulfation sites in the domain (and corresponding sites in the Fab of bevacizumab) are identified in Figure 1 (see, for example, Yang et al., 2015, Molecules 20:2138-2164, especially p. 2154, which is incorporated in its entirety by reference for the analysis of amino acids around tyrosine residues that have undergone protein tyrosine sulfation. The "rules" can be summarized as follows: the Y residue has an E or D within +5 to -5 from the position of Y, where the -1 position of Y is a neutral or acidic charged amino acid but not a basic amino acid that inactivates sulfation, such as R, K, or H). Human IgG antibodies can exhibit several other post-translational modifications, such as N-terminal modifications, C-terminal modifications, degradation or oxidation of amino acid residues, cysteine-related variants, and glycosylation (see, for example, Liu et al., 2014, mAbs 6(5):1145-1154). (v) Glycosylation of anti-VEGF Fabs by human retinal cells, such as Fab fragments of ranibizumab or bevacizumab, results in the addition of glycans that can improve the stability and half-life of the transgene product and reduce its undesirable aggregation and / or immunogenicity (see, for example, Bovenkamp et al., 2016, J. Immunol. 196: 1435-1441 for a review of the emerging importance of Fab glycosylation). Importantly, the glycans that can be added to the HuPTMFabVEGFi provided herein, such as HuGlyFabVEGFi, are highly processed complex biantennary N-glycans containing 2,6-sialic acid (see, for example, FIG. 2 showing the glycans that can be incorporated into HuPTMFabVEGFi, such as HuGlyFabVEGFi) and bisecting GlcNAc and are not NGNA (N-glycolylneuraminic acid, Neu5Gc). Such glycans are not present in ranibizumab, which is produced in E. coli and is not glycosylated at all, nor in bevacizumab, which is produced in CHO cells that do not have the 2,6-sialyltransferase required for this post-translational modification or the bisecting GlcNAc of CHO cell products but add Neu5Gc (NGNA), a sialic acid not specific to humans (and potentially immunogenic), instead of Neu5Ac (NANA). See, for example, Dumont et al., 2015, Crit. Rev. Biotechnol. (early online edition, published online September 18, 2015, pp. 1-13, p. 5). Additionally, CHO cells can produce the immunogenic glycan α-Gal antigen, which reacts with anti-α-Gal antibodies present in most individuals and can induce anaphylaxis at high concentrations. See, for example, Bosques, 2010, Nat Biotech 28: 1153-1156. The human glycosylation pattern of the HuPTMFabVEGFi provided herein, such as HuGlyFabVEGFi, should reduce the immunogenicity of the transgene product and improve its efficacy. (vi) Tyrosine sulfation - a robust post - translational process in human retinal cells - of anti - VEGF Fabs, e.g., Fab fragments of ranibizumab or bevacizumab, can result in transgene products with increased binding affinity to VEGF. Indeed, tyrosine sulfation of Fabs of therapeutic antibodies against other targets has been shown to dramatically increase binding affinity and activity to the antigen (see, e.g., Loos et al., 2015, PNAS 112: 12675 - 12680, and Choe et al., 2003, Cell 114: 161 - 170). Such post - translational modifications do not exist in ranibizumab (which is produced in E. coli, a host that does not possess the enzymes required for tyrosine sulfation), and even in bevacizumab, a CHO - cell product, they are presented only poorly. Unlike human retinal cells, CHO cells are not secretory cells and have limited ability for post - translational tyrosine sulfation (see, e.g., Mikkelsen and Ezban, 1991, Biochemistry 30: 1533 - 1537, especially the discussion on p. 1537).

[0076] For the reasons above, the production of HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, is achieved by gene therapy - e.g., administering a viral vector or other DNA expression construct encoding HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of a patient (human subject) diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD) (e.g., by suprachoroidal injection, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space, or a posterior scleral juxtadepot procedure), creating an intravitreal permanent depot that continuously supplies a post-translationally modified fully human, e.g., glycosylated, sulfated human transgene product produced by the transduced retinal cells, which should result in a "bio-better" molecule for the treatment of exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD). The cDNA construct for Fab VEGFi should contain a signal peptide that ensures proper co-translational and post-translational processing (glycosylation and protein sulfation) by the transduced retinal cells. Such signal sequences used by retinal cells include, but are not limited to, the following: [Chemical formula] · For example, for signal peptides that can be used, see Stern et al., 2007, Trends Cell. Mol. Biol., 2:1-17 and Dalton and Barton, 2014, Protein Sci, 23: 517-525, each of which is hereby incorporated by reference in its entirety.

[0077] As an alternative to gene therapy or as an additional treatment to gene therapy, HuPTMFabVEGFi products, such as HuGlyFabVEGFi glycoproteins, can be produced in human cell lines by recombinant DNA technology and administered by intravitreal injection to patients diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD). HuPTMFabVEGFi products, such as glycoproteins, can also be administered to patients having exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD). Examples of human cell lines that can be used for the production of such recombinant glycoproteins include, but are not limited to, human embryonic kidney 293 cells (HEK293), fibrosarcoma HT-1080, HKB-11, CAP, HuH-7, and the retinal cell lines PER.C6 or RPE (for a review of human cell lines that can be used for the recombinant production of HuPTMFabVEGFi products, such as HuGlyFabVEGFi glycoproteins, see Dumont et al., 2015, Crit. Rev. Biotechnol. (Early online edition, published online September 18, 2015, pp. 1-13), "Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives", which is incorporated herein by reference in its entirety). To ensure full glycosylation, particularly sialylation and tyrosine sulfation, the cell line used for production can be enhanced by modifying the host cell to co-express α-2,6-sialyltransferase (or both α-2,3-sialyltransferase and α-2,6-sialyltransferase) and / or the TPST-1 and TPST-2 enzymes involved in tyrosine-O-sulfation in retinal cells.

[0078] Delivery of HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, to the eye / retina with delivery of other available therapies is encompassed by the methods provided herein. Additional therapies can be administered before, simultaneously with, or after the gene therapy treatment. Available therapies for exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD) that can be combined with the gene therapy provided herein include laser photocoagulation, photodynamic therapy with verteporfin, and, without limitation, intravitreal (“IVT”) injection of anti-VEGF agents including, but not limited to, pegaptanib, ranibizumab, aflibercept, or bevacizumab. Additional treatment with anti-VEGF agents, e.g., biologics, may be referred to as “rescue” therapy.

[0079] Unlike small molecule drugs, biologic agents typically contain a mixture of many variants with different modifications or forms having different potencies, pharmacokinetics, and safety profiles. It is not essential that all molecules produced in either a gene therapy or protein therapy approach be fully glycosylated and sulfated. Rather, the population of glycoproteins produced should have glycosylation (about 1% to about 10% of the population) and sulfation that includes sufficient 2,6-sialylation to exhibit potency. The goal of the gene therapy treatment provided herein is to slow or halt the progression of retinal degeneration and to slow or prevent vision loss with minimal intervention / invasive procedures. Potency can be monitored by measurement of BCVA (best corrected visual acuity), intraocular pressure, slit lamp biomicroscopy, indirect ophthalmoscopy, SD-OCT (SD-optical coherence tomography), and electroretinogram (ERG). Signs of other safety events including vision loss, infections, inflammation, and retinal detachment can also be monitored. Retinal thickness may be monitored to determine the efficacy of the treatment provided herein. Without being bound by any particular theory, retinal thickness can be used as a clinical readout, in which case the greater the decrease in retinal thickness or the longer the period to retinal thickening, the more effective the treatment. Retinal thickness can be determined, for example, by SD-OCT. SD-OCT is a three-dimensional imaging technique that uses low coherence interferometry to determine the echo time delay and amplitude of backscattered light reflected from the object of interest. Using OCT, tissue samples (e.g., the retina) can be scanned with an axial resolution of 3 to 15 μm, and SD-OCT improves axial resolution and scan speed over previous types of technology (Schuman, 2008, Trans. Am. Opthamol. Soc. 106:426-458). Retinal function can be determined, for example, by ERG. ERG is a non-invasive electrophysiological test of retinal function approved by the FDA for use in humans that examines the response of the eye's photoreceptor cells (rods and cones) and the ganglion cells to which they are connected, particularly to a flash stimulus.

[0080] In a preferred embodiment, the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal. As used herein, the phrase "detectable NeuGc and / or α-Gal" means NeuGc and / or α-Gal moieties that are detectable by standard assay methods known in the art. For example, NeuGc can be detected by HPLC according to the method for detecting NeuGc described in Hara et al., 1989, "Highly Sensitive Determination of N-Acetyl-and N-Glycolylneuraminic Acids in Human Serum and Urine and Rat Serum by Reversed-Phase Liquid Chromatography with Fluorescence Detection.", J. Chromatogr., B: Biomed. 377: 111-119, which is incorporated herein by reference. Alternatively, NeuGc can be determined by mass spectrometry.α-Gal can be detected using ELISA (see, e.g., Galili et al., 1998, "A sensitive assay for measuring alpha-Gal epitope expression on cells by a monoclonal anti-Gal antibody.", Transplantation. 65(8):1129-32), or by mass spectrometry (see, e.g., Ayoub et al., 2013, "Correct primary structure assessment and extensive glyco-profiling of cetuximab by a combination of intact, middle-up, middle-down and bottom-up ESI and MALDI mass spectrometry techniques.", Landes Bioscience. 5(5): 699-710). See also the references cited in Platts-Mills et al., 2015, "Anaphylaxis to the Carbohydrate Side-Chain Alpha-gal", Immunol Allergy Clin North Am. 35(2): 247-260.

[0081] In one aspect, also provided herein is an anti-VEGF antigen-binding fragment comprising light chain CDR1-3 of SEQ ID NOs: 14-16, and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21 (i.e., an antigen-binding fragment that immunospecifically binds to VEGF), where the second amino acid residue of the light chain CDR3 (i.e.,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0082] In one aspect, also provided herein is an anti-VEGF antigen-binding fragment comprising light chain CDR1-3 of SEQ ID NOs: 14-16, and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the last amino acid residue of heavy chain CDR1 (i.e.,

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

Chem.

[0083] In one aspect, also provided herein is an anti-VEGF antigen-binding fragment comprising light chain CDR1-3 of SEQ ID NOs: 14-16, and heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, wherein the last amino acid residue of heavy chain CDR1 (i.e.,

Chem.

Chem.

Chem.

Chem.

Chem.

[0084] The unexpected benefits of the present invention are shown in the following examples, which show that by the expression of HuPTMFabVEGFi from the rAAV8.anti-hVEGF Fab vector injected into the subretinal space, (i) subretinal neovascularization in transgenic mice, which is a model of nAMD in human subjects, is reduced; and (ii) surprisingly, retinal detachment is prevented in a transgenic mouse model of intraocular neovascular disease that develops severe proliferative retinopathy and retinal detachment caused by intraocular production of VEGF.

[0085] These examples also show that suprachoroidal administration of the rAAV8.anti-hVEGF Fab vector was as effective as subretinal injection of the vector in neutralizing VEGF-induced damage. Suprachoroidal administration allows for a rapid and easy in-hospital procedure with a low risk of complications.

[0086] Another contemplated route of administration is subretinal administration via the suprachoroidal space using a subretinal drug delivery device having a catheter that can be inserted into the suprachoroidal space toward the posterior pole, passed through it, and injected into the subretinal space with a fine needle at the posterior pole. This route of administration makes it possible to keep the vitreous intact, and thus has a lower risk of complications (less risk of complications such as gene therapy withdrawal and retinal detachment and macular hole), and without vitrectomy, the resulting blebs can spread more diffusely and more surface area of the retina can be transduced with less volume. The risk of inducing cataracts after this procedure is minimized, which is desirable for young patients. Furthermore, this procedure can deliver blebs to beneath the fovea more safely than the standard transvitreal approach, which is desirable for patients with hereditary retinal diseases and results in central vision when the target cells for transduction are in the macula. This procedure is also desirable for patients with neutralizing antibodies (Nabs) to AAV present in the systemic circulation that can affect other delivery routes. Furthermore, this method has been shown to create blebs with less release from the retinotomy site than the standard transvitreal approach.

[0087] Suprachoroidal administration provides an additional route of administration that avoids the risks of endophthalmitis and retinal detachment, common side effects associated with directly injecting a therapeutic agent into the eye. (3.1 Exemplary Embodiments) (3.1.1 Set 1) A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), the method comprising delivering to the retina of the human subject a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human retinal cells. 2. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising delivering a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human retinal cells to the retina of the human subject by administering an expression vector encoding the anti-hVEGF antigen-binding fragment into the subretinal space of the eye of the human subject by a transvitreal approach or by subretinal injection via the suprachoroidal space of the eye of the human subject. 3. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), and diabetic retinopathy (DR), comprising delivering a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human retinal cells to the retina of the human subject by using a suprachoroidal drug delivery device such as a microinjector. 4. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising delivering a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane to the retina of the human subject. 5. A method for treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising delivering a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane to the retina of the human subject by administering an expression vector encoding the anti-hVEGF antigen-binding fragment into the subretinal space of the eye of the human subject by a transvitreal approach or by subretinal injection via the suprachoroidal cavity of the eye of the human subject. 6. A method for treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising delivering a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human photoreceptor cells (e.g., cone cells and / or rod cells), horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia), and / or retinal pigment epithelial cells of the outer limiting membrane to the retina of the human subject by using a suprachoroidal drug delivery device such as a microinjector. 7. The method according to any one of paragraphs 1 to 6, wherein the antigen-binding fragment is a Fab. 8. The method according to any one of paragraphs 1 to 6, wherein the antigen-binding fragment is F(ab') 2 thereof. 9. The method according to any one of paragraphs 1 to 6, wherein the antigen-binding fragment is a single-chain variable domain (scFv). 10. The method according to any one of paragraphs 1 to 6, wherein the antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and a light chain comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. 11. The method according to any one of paragraphs 1 to 6, wherein the antigen-binding fragment comprises light chain CDR1 to 3 of SEQ ID NOs: 14 to 16, and heavy chain CDR1 to 3 of SEQ ID NOs: 17 to 19 or SEQ ID NOs: 20, 18, and 21. 12. The method according to paragraph 11, wherein the second amino acid residue of the light chain CDR3 does not carry one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu). 13. The method according to paragraph 12, wherein the second amino acid residue of the light chain CDR3 is not acetylated. 14. The method according to paragraph 12 or 13, wherein the 8th and 11th amino acid residues of the light chain CDR1 each carry one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu). 15. The method according to any one of paragraphs 11 to 14, wherein the antigen-binding fragment comprises the heavy chain CDR1 of SEQ ID NO: 20, and the last amino acid residue of the heavy chain CDR1 does not carry one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu). 16. The method according to paragraph 15, wherein the last amino acid residue of the heavy chain CDR1 is not acetylated. 17. The method according to paragraph 15 or 16, wherein the 9th amino acid residue of the heavy chain CDR1 carries one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu), and the 3rd amino acid residue of the heavy chain CDR2 carries one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu). 18. The step of delivering comprises administering an expression vector encoding an anti-hVEGF antigen-binding fragment at a dose in the range of 3×10 9 genomic copies to 2.5×10 11 genomic copies. The method according to any one of paragraphs 1 to 17. 19. The step of delivering comprises administering an expression vector encoding an anti-hVEGF antigen-binding fragment at a dose of about 3×10 9 genomic copies. The method according to any one of paragraphs 1 to 17. 20. The method according to any one of paragraphs 1 to 17, wherein the delivering step comprises administering an expression vector encoding an anti-hVEGF antigen-binding fragment at a dose of about 1×10 10 genomic copies. 21. The method according to any one of paragraphs 1 to 17, wherein the delivering step comprises administering an expression vector encoding an anti-hVEGF antigen-binding fragment at a dose of about 6×10 10 genomic copies. 22. The method according to any one of paragraphs 1 to 17, wherein the delivering step comprises administering an expression vector encoding an anti-hVEGF antigen-binding fragment at a dose of about 1.6×10 11 genomic copies. 23. The method according to any one of paragraphs 1 to 17, wherein the delivering step comprises administering an expression vector encoding an anti-hVEGF antigen-binding fragment at a dose of about 2.5×10 11 genomic copies. 24. A method of treating a human subject diagnosed with neovascular age-related macular degeneration (exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), particularly exudative AMD), comprising delivering to the eye of the human subject a therapeutically effective amount of an antigen-binding fragment (Fab, F(ab') 2 , or scFv, collectively referred to herein as "antigen-binding fragment") of an mAb against hVEGF, wherein the antigen-binding fragment comprises an α2,6-sialylated glycan. 25. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), particularly exudative AMD), comprising delivering to the subretinal space of the eye of the human subject a therapeutically effective amount of an antigen-binding fragment of an mAb against hVEGF by intravitreal approach or by subretinal injection via the suprachoroidal space of the eye of the human subject, by administering an expression vector encoding the antigen-binding fragment of the mAb against hVEGF to the subretinal space of the eye of the human subject, wherein the antigen-binding fragment (Fab, F(ab') 2 , or scFv, collectively referred to herein as "antigen-binding fragment") of the mAb against hVEGF comprises an α2,6-sialylated glycan. 26. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising delivering, to the eye of the human subject, a therapeutically effective amount of an antigen-binding fragment (Fab, F(ab') 2 , or scFv, collectively referred to herein as "antigen-binding fragment") of an mAb against hVEGF, wherein the antigen-binding fragment comprises an α2,6-sialylated glycan. 27. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising delivering, to the eye of the human subject, a therapeutically effective amount of a glycosylated antigen-binding fragment of an mAb against hVEGF, wherein the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal antigens. 28. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising delivering, to the subretinal space of the eye of the human subject, a therapeutically effective amount of a glycosylated antigen-binding fragment of an mAb against hVEGF by intravitreal approach or by subretinal injection via the suprachoroidal space of the eye of the human subject, by administering an expression vector encoding the glycosylated antigen-binding fragment of the mAb against hVEGF, wherein the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal antigens. 29. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising delivering, to the eye of the human subject, a therapeutically effective amount of a glycosylated antigen-binding fragment of an mAb against hVEGF by using a suprachoroidal drug delivery device such as a microinjector, wherein the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal antigens. 30. The delivering step comprises administering a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF at a dose in the range of 3×10 9 genomic copies to 2.5×10 11 genomic copies, the method according to any one of paragraphs 24 to 29. 31. The delivering step comprises administering a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF at a dose of about 3×10 9 genomic copies, the method according to any one of paragraphs 24 to 29. 32. The delivering step comprises administering a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF at a dose of about 1×10 10 genomic copies, the method according to any one of paragraphs 24 to 29. 33. The delivering step comprises administering a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF at a dose of about 6×10 10 genomic copies, the method according to any one of paragraphs 24 to 29. 34. The delivering step comprises administering a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF at a dose of about 1.6×10 11 genomic copies, the method according to any one of paragraphs 24 to 29. 35. The delivering step comprises administering a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF at a dose of about 2.5×10 11 genomic copies, the method according to any one of paragraphs 24 to 29. 36. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR), particularly exudative AMD, comprising: administering to the subretinal space of the eye of the human subject an expression vector encoding an antigen-binding fragment against hVEGF, wherein the antigen-binding fragment is α2,6-sialylated when expressed from the expression vector in human immortalized retinal-derived cells. 37. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising: administering to the subretinal space of the eye of the human subject an expression vector encoding an antigen-binding fragment against hVEGF, wherein the expression of the antigen-binding fragment is α2,6-sialylated when expressed from the expression vector in human immortalized retinal-derived cells, and wherein the administering step comprises using a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space towards the posterior pole, passed through it, and injected into the subretinal space with a thin needle at the posterior pole. 38. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising: administering or delivering to the retina of the human patient an expression vector encoding an antigen-binding fragment against hVEGF via the suprachoroidal space of the eye of the human subject, wherein the expression of the antigen-binding fragment is α2,6-sialylated when expressed from the expression vector in human immortalized retinal-derived cells. 39. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising: administering to the subretinal space of the eye of the human subject an expression vector encoding an antigen-binding fragment against hVEGF, wherein the expression of the antigen-binding fragment is α2,6-sialylated when expressed from the expression vector in human immortalized retinal-derived cells, and wherein the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal antigens. 40. A method for treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising: administering into the subretinal space of the eye of the human subject an expression vector encoding an antigen-binding fragment against hVEGF, wherein the antigen-binding fragment is α2,6-sialylated when expressed from the expression vector in human immortalized retinal-derived cells, wherein the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal antigens, and wherein the administering step comprises using a subretinal drug delivery device including a catheter that can be inserted into the suprachoroidal space toward the posterior pole, passed through it, and injected into the subretinal space with a thin needle at the posterior pole. 41. A method for treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising: administering or delivering into the retina of the human patient an expression vector encoding an antigen-binding fragment against hVEGF via the suprachoroidal space of the eye of the human subject, wherein the antigen-binding fragment is α2,6-sialylated when expressed from the expression vector in human immortalized retinal-derived cells, and wherein the antigen-binding fragment does not contain detectable NeuGc and / or α-Gal antigens. 42. The method according to any one of paragraphs 36 to 41, wherein the expression vector encoding the antigen-binding fragment against hVEGF is administered at a dose in the range of 3×10 9 genomic copies to 2.5×10 11 genomic copies. 43. The method according to any one of paragraphs 36 to 41, wherein the expression vector encoding the antigen-binding fragment against hVEGF is administered at a dose of about 3×10 9 genomic copies. 44. The method according to any one of paragraphs 36 to 41, wherein the expression vector encoding the antigen-binding fragment against hVEGF is administered at a dose of about 1×10 10 genomic copies. 45. The expression vector encoding the antigen-binding fragment against hVEGF is 6×10 10The method according to any one of paragraphs 36 to 41, administered at a genomic copy approximate dose. 46. An expression vector encoding an antigen-binding fragment against hVEGF is about 1.6×10 11 The method according to any one of paragraphs 36 to 41, administered at a genomic copy dose. 47. An expression vector encoding an antigen-binding fragment against hVEGF is about 2.5×10 11 The method according to any one of paragraphs 36 to 41, administered at a genomic copy dose. 48. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising administering to the subretinal space of the eye of the human subject a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, resulting in the formation of a depot that releases the antigen-binding fragment containing α2,6-sialylated glycan. 49. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising administering to the subretinal space of the eye of the human subject a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, resulting in the formation of a depot that releases the antigen-binding fragment containing α2,6-sialylated glycan, wherein the administering step comprises the use of a subretinal drug delivery device including a catheter that can be inserted into the suprachoroidal space towards the posterior pole, passed through it, and injected into the subretinal space with a thin needle at the posterior pole. 50. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising administering or delivering to the retina of the human patient, via the suprachoroidal space of the eye of the human subject, a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, resulting in the formation of a depot that releases the antigen-binding fragment comprising α2,6-sialylated glycans. 51. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising administering to the subretinal space of the eye of the human subject a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, resulting in the formation of a depot that releases the antigen-binding fragment, wherein the antigen-binding fragment is glycosylated and does not contain detectable NeuGc and / or α-Gal antigens. 52. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising administering to the subretinal space of the eye of the human subject a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, resulting in the formation of a depot that releases the antigen-binding fragment, wherein the antigen-binding fragment is glycosylated and does not contain detectable NeuGc and / or α-Gal antigens, and wherein the administering step comprises the use of a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space towards the posterior pole, passed through it, and injected into the subretinal space with a thin needle at the posterior pole. 53. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly, exudative AMD), comprising administering or delivering to the retina of the human patient, via the suprachoroidal space of the eye of the human subject, a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, whereby a depot is formed that releases the antigen-binding fragment, wherein the antigen-binding fragment is glycosylated but does not contain detectable NeuGc and / or α-Gal antigens. 54. The recombinant nucleotide expression vector encoding an antigen-binding fragment against hVEGF is administered at a dose in the range of 3×10 9 genomic copies to 2.5×10 11 genomic copies, the method according to any one of paragraphs 48 to 53. 55. The recombinant nucleotide expression vector encoding an antigen-binding fragment against hVEGF is administered at a dose of about 3×10 9 genomic copies, the method according to any one of paragraphs 48 to 53. 56. The recombinant nucleotide expression vector encoding an antigen-binding fragment against hVEGF is administered at a dose of about 1×10 10 genomic copies, the method according to any one of paragraphs 48 to 53. 57. The recombinant nucleotide expression vector encoding an antigen-binding fragment against hVEGF is administered at a dose of about 6×10 10 genomic copies, the method according to any one of paragraphs 48 to 53. 58. The recombinant nucleotide expression vector encoding an antigen-binding fragment against hVEGF is administered at a dose of about 1.6×10 11 genomic copies, the method according to any one of paragraphs 48 to 53. 59. The recombinant nucleotide expression vector encoding an antigen-binding fragment against hVEGF is administered at a dose of about 2.5×10 11 genomic copies, the method according to any one of paragraphs 48 to 53. 60. The method according to any one of paragraphs 24 to 59, wherein the antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and a light chain comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. 61. The method according to any one of paragraphs 24 to 60, wherein the antigen-binding fragment further comprises tyrosine sulfation. 62. The method according to any one of paragraphs 24 to 61, wherein the production of the antigen-binding fragment comprising α2,6-sialylated glycan is confirmed by transducing the recombinant nucleotide expression vector into a PER.C6 or RPE cell line in cell culture. 63. The method according to any one of paragraphs 24 to 61, wherein the production of the antigen-binding fragment comprising tyrosine sulfation is confirmed by transducing the recombinant nucleotide expression vector into a PER.C6 or RPE cell line in cell culture. 64. The method according to any one of paragraphs 24 to 63, wherein the vector has a hypoxia-inducible promoter. 65. The method according to any one of paragraphs 24 to 64, wherein the antigen-binding fragment comprises light chain CDR1-3 of SEQ ID NOs: 14 to 16, and heavy chain CDR1-3 of SEQ ID NOs: 17 to 19 or SEQ ID NOs: 20, 18, and 21. 66. The method according to paragraph 65, wherein the second amino acid residue of the light chain CDR3 does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu). 67. The method according to paragraph 66, wherein the second amino acid residue of the light chain CDR3 is not acetylated. 68. The method according to paragraph 66 or 67, wherein the 8th and 11th amino acid residues of the light chain CDR1 each possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu). 69. The method according to any one of paragraphs 65 to 68, wherein the antigen-binding fragment comprises the heavy chain CDR1 of SEQ ID NO: 20, and the last amino acid residue of the heavy chain CDR1 does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu). 70. The method according to paragraph 69, wherein the last amino acid residue of the heavy chain CDR1 is not acetylated. 71. The method according to paragraph 69 or 70, wherein the 9th amino acid residue of the heavy chain CDR1 has one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (PyrGlu), and the 3rd amino acid residue of the heavy chain CDR2 has one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (PyrGlu). 72. The method according to any one of paragraphs 24 to 71, wherein the antigen-binding fragment transgene encodes a leader peptide. 73. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (especially exudative AMD), comprising administering a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF into the subretinal space of the eye of the human subject, resulting in the formation of a depot that releases the antigen-binding fragment containing α2,6-sialylated glycan; wherein when the recombinant vector is used to transduce PER.C6 or RPE cells in a culture medium, it results in the production of the antigen-binding fragment containing α2,6-sialylated glycan in the cell culture. 74. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (especially exudative AMD), comprising administering a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF into the subretinal space of the eye of the human subject, resulting in the formation of a depot that releases the antigen-binding fragment containing α2,6-sialylated glycan; wherein when the recombinant vector is used to transduce PER.C6 or RPE cells in a culture medium, it results in the production of the antigen-binding fragment containing α2,6-sialylated glycan in the cell culture, and wherein the administering step comprises using a subretinal drug delivery device including a catheter that can be inserted into the suprachoroidal space toward the posterior pole, passed through it, and injected into the subretinal space with a thin needle at the posterior pole. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising administering or delivering to the retina of the human patient, via the suprachoroidal space of the eye of the human subject, a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, resulting in the formation of a depot that releases the antigen-binding fragment comprising α2,6-sialylated glycans; wherein the recombinant vector, when used to transduce PER.C6 or RPE cells in culture, results in the production of the antigen-binding fragment comprising α2,6-sialylated glycans in the cell culture. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising administering to the subretinal space of the eye of the human subject a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, resulting in the formation of a depot that releases the antigen-binding fragment, wherein the antigen-binding fragment is glycosylated but does not contain detectable NeuGc and / or α-Gal antigens; wherein the recombinant vector, when used to transduce PER.C6 or RPE cells in culture, results in the production in the cell culture of the antigen-binding fragment that is glycosylated but does not contain detectable NeuGc and / or α-Gal antigens. 77. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising administering to the subretinal space of the eye of the human subject a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF, resulting in the formation of a depot that releases the antigen-binding fragment, wherein the antigen-binding fragment is glycosylated but does not contain detectable NeuGc and / or α-Gal antigens; wherein when the recombinant vector is used to transduce PER.C6 or RPE cells in culture, it results in the production of the antigen-binding fragment that is glycosylated but does not contain detectable NeuGc and / or α-Gal antigens in the cell culture, and wherein the administering step comprises the use of a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space toward the posterior pole, passed through it, and injected into the subretinal space with a fine needle at the posterior pole. 78. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising administering or delivering to the retina of the human patient a therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of an mAb against hVEGF via the suprachoroidal space of the eye of the human subject, resulting in the formation of a depot that releases the antigen-binding fragment, wherein the antigen-binding fragment is glycosylated but does not contain detectable NeuGc and / or α-Gal antigens; wherein when the recombinant vector is used to transduce PER.C6 or RPE cells in culture, it results in the production of the antigen-binding fragment that is glycosylated but does not contain detectable NeuGc and / or α-Gal antigens in the cell culture. 79. The method according to any one of paragraphs 73 to 78, wherein the recombinant nucleotide expression vector encoding the antigen-binding fragment against hVEGF is administered at a dose in the range of 3×10 9 genomic copies to 2.5×10 11 genomic copies. 80. A recombinant nucleotide expression vector encoding an antigen-binding fragment against hVEGF is administered at a dose of about 3×10 9 genomic copies, according to any one of paragraphs 73 to 78. 81. A recombinant nucleotide expression vector encoding an antigen-binding fragment against hVEGF is administered at a dose of about 1×10 10 genomic copies, according to any one of paragraphs 73 to 78. 82. A recombinant nucleotide expression vector encoding an antigen-binding fragment against hVEGF is administered at a dose of about 6×10 10 genomic copies, according to any one of paragraphs 73 to 78. 83. A recombinant nucleotide expression vector encoding an antigen-binding fragment against hVEGF is administered at a dose of about 1.6×10 11 genomic copies, according to any one of paragraphs 73 to 78. 84. A recombinant nucleotide expression vector encoding an antigen-binding fragment against hVEGF is administered at a dose of about 2.5×10 11 genomic copies, according to any one of paragraphs 73 to 78. 85. A method according to any one of paragraphs 24 to 35, wherein delivery to the eye includes delivery to the retina, choroid, and / or vitreous humor of the eye. 86. A method according to any one of paragraphs 1 to 85, wherein the antigen-binding fragment includes a heavy chain containing one, two, three, or four additional amino acids at the C-terminus. 87. A method according to any one of paragraphs 1 to 85, wherein the antigen-binding fragment includes a heavy chain that does not contain additional amino acids at the C-terminus. 88. A method according to any one of paragraphs 1 to 85, which produces a population of antigen-binding fragment molecules, wherein the antigen-binding fragment molecules include a heavy chain, and wherein 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, or 20%, or less, but more than 0% of the population of antigen-binding fragment molecules contains one, two, three, or four additional amino acids at the C-terminus of the heavy chain. 89. A method according to any one of paragraphs 1-85, which produces a population of antigen-binding fragment molecules, wherein the antigen-binding fragment molecules contain a heavy chain, and wherein 0.5-1%, 0.5%-2%, 0.5%-3%, 0.5%-4%, 0.5%-5%, 0.5%-10%, 0.5%-20%, 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%-10%, 1%-20%, 2%-3%, 2%-4%, 2%-5%, 2%-10%, 2%-20%, 3%-4%, 3%-5%, 3%-10%, 3%-20%, 4%-5%, 4%-10%, 4%-20%, 5%-10%, 5%-20%, or 10%-20% of the population of antigen-binding fragment molecules contains one, two, three, or four additional amino acids at the C-terminus of the heavy chain. 90. A method according to any one of paragraphs 1-89, wherein the human subject has a BCVA of ≦20 / 20 and ≧20 / 400. 91. A method according to any one of paragraphs 1-90, wherein the human subject has a BCVA of ≦20 / 63 and ≧20 / 400. 92. The method according to paragraph 90 or 91, wherein the BCVA is the BCVA of the eye to be treated in the human subject. 93. An antigen-binding fragment that immunospecifically binds to VEGF, wherein the antigen-binding fragment contains the light chain CDR1-3 of SEQ ID NOs: 14-16, and the heavy chain CDR1-3 of SEQ ID NOs: 20, 18, and 21, and wherein the second amino acid residue of the light chain CDR3 does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu). 94. The antigen-binding fragment according to paragraph 93, wherein the second amino acid residue of the light chain CDR3 is not acetylated. 95. The antigen-binding fragment according to paragraph 93 or 94, wherein the 8th and 11th amino acid residues of the light chain CDR1 each possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu). 96. The antigen-binding fragment according to any one of paragraphs 93-95, wherein the last amino acid residue of the heavy chain CDR1 does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu). The antigen-binding fragment according to paragraph 96, wherein the last amino acid residue of the heavy-chain CDR1 is not acetylated. 98. The antigen-binding fragment according to paragraph 96 or 97, wherein the 9th amino acid residue of the heavy-chain CDR1 has one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu), and the 3rd amino acid residue of the heavy-chain CDR2 has one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu). (3.1.2 Set 2) 1. A method for treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), the method comprising administering to the suprachoroidal space of the eye of the human subject an expression vector encoding an anti-human vascular endothelial growth factor (hVEGF) antibody. 2. The method according to paragraph 1, wherein the administering is by injecting the expression vector into the suprachoroidal space using a suprachoroidal drug delivery device. 3. The method according to paragraph 1 or 2, wherein the suprachoroidal drug delivery device is a microinjector. 4. A method for treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), the method comprising administering to the subretinal space of the eye of the human subject an expression vector encoding an anti-hVEGF antibody via the suprachoroidal space of the eye of the human subject. 5. The method according to paragraph 4, wherein the administering is by use of a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space toward the posterior pole, passed through it, and injected into the subretinal space with a thin needle at the posterior pole. 6. The method according to paragraph 5, wherein the administering comprises inserting and passing through the catheter of the subretinal drug delivery device from the suprachoroidal space. 7. A method of treating a human subject diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD), comprising administering an expression vector encoding an anti-hVEGF antibody to the outer surface of the sclera of the eye of the human subject. 8. The method according to paragraph 7, wherein the administering is by use of a sclera proximity drug delivery device comprising a cannula whose tip can be inserted and held directly juxtaposed to the scleral surface. 9. The method according to paragraph 8, wherein the administering comprises inserting the tip of the cannula and holding it directly juxtaposed to the scleral surface. 10. The method according to any one of paragraphs 1 to 9, wherein the administering delivers a therapeutically effective amount of the anti-hVEGF antibody to the retina of the human subject. 11. The method according to paragraph 10, wherein the therapeutically effective amount of the anti-hVEGF antibody is produced by human retinal cells of the retina of the human subject. 12. The method according to paragraph 10, wherein the therapeutically effective amount of the anti-hVEGF antibody is produced by human photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, and / or retinal pigment epithelial cells of the outer limiting membrane of the human subject. 13. The method according to paragraph 12, wherein the human photoreceptor cells are cone cells and / or rod cells. 14. The method according to paragraph 12, wherein the retinal ganglion cells are midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia. 15. The method according to any one of paragraphs 1 to 14, wherein the human subject has a best corrected visual acuity (BCVA) of ≤ 20 / 20 and ≥ 20 / 400. 16. The method according to any one of paragraphs 1 to 14, wherein the human subject has a BCVA of ≤ 20 / 63 and ≥ 20 / 400. 17. The method according to paragraph 15 or 16, wherein the BCVA is the BCVA of the eye of the human subject to be treated. 18. The method according to any one of paragraphs 1 to 17, wherein the anti-hVEGF antibody is an anti-hVEGF antigen-binding fragment. 19. The method according to paragraph 18, wherein the antigen-binding fragment is Fab. 20. The method according to paragraph 18, wherein the antigen-binding fragment is F(ab') 2 as described. 21. The method according to paragraph 18, wherein the antigen-binding fragment is a single-chain variable domain (scFv). 22. The method according to any one of paragraphs 1 to 21, wherein the anti-hVEGF antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and a light chain comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. 23. The method according to any one of paragraphs 1 to 21, wherein the anti-hVEGF antibody comprises light chain CDR1-3 of SEQ ID NOs: 14-16, and heavy chain CDR1-3 of SEQ ID NOs: 17-19 or SEQ ID NOs: 20, 18, and 21. 24. The method according to paragraph 22, wherein the second amino acid residue of the light chain CDR3 does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu). 25. The method according to paragraph 23, wherein the second amino acid residue of the light chain CDR3 is not acetylated. 26. The method according to paragraph 23 or 24, wherein the 8th and 11th amino acid residues of the light chain CDR1 each possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu). 27. The method according to any one of paragraphs 22 to 25, wherein the anti-hVEGF antibody comprises the heavy chain CDR1 of SEQ ID NO: 20, and the last amino acid residue of the heavy chain CDR1 does not possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu). 28. The method according to paragraph 26, wherein the last amino acid residue of the heavy chain CDR1 is not acetylated. 29. The method according to paragraph 26 or 27, wherein the 9th amino acid residue of the heavy chain CDR1 possesses one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu), and the 3rd amino acid residue of the heavy chain CDR2 possesses one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu). 30. The method according to any one of paragraphs 1 to 29, wherein the expression vector is an AAV vector. 31. The method according to paragraph 30, wherein the expression vector is an AAV8 vector.

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Figure 7

[0095] (Figure 7B) Adult C57BL / 6 mice were given 10 10An empty AAV8 vector of genomic copies (GC) or 1×10 8 ~1×10 10 Subretinal injection of AAV8 - anti - VEGFfab at a dose of GC was administered. Seven days later, the mice were euthanized, the eyes were enucleated and frozen until assayed. The eyes were homogenized in lysis buffer and the AAV8 - anti - VEGFfab protein was measured by ELISA. Bars represent the mean (±SEM) with n = for each bar or n≧ for each bar.

[0096]

Figure 8

[0097] (Fig. 8B) The area of NV per retina was measured using image analysis. Bars indicate mean (±SEM). *p < 0.05; **p < 0.01 for differences from the empty vector by ANOVA with Bonferroni correction for multiple comparisons.

[0098]

Figure 9

[0099] (Fig. 9C) Hoechst - stained eye sections from eyes injected with 3×10 9 GC of AAV8 - anti - VEGFfab showed no retinal detachment, while sections from the uninjected contralateral eyes showed complete retinal detachment.

[0100] (Fig. 9D) The pie chart shows a dose - dependent decrease in exudative retinal detachment in eyes injected with AAV8 - anti - VEGFfab.

[0101] (Figure 9E) The p-values were determined by Fisher's test to show whether there was a difference from the empty vector group regarding the absence of detachment, the presence of partial detachment, or the presence of complete detachment for various doses. According to one-way ANOVA with Bonferroni correction for multiple comparisons, the mean (±SEM) retinal detachment rate was significantly lower for eyes injected with 3×10 9 or 1×10 10 GC of AAV8 - anti - VEGFfab than for any other group (*p = 0.002, **p = 0.001).

[0102]

Figure 10

[0103] (Figure 10B) Eye sections from mice injected with 3×10 9 GC of AAV8 - anti - VEGFfab and stained with Hoechst showed attached retina in the injected eye and complete detachment in the other eye (B, left two panels). Eye sections from mice injected with 3×10 9 GC of empty vector showed complete retinal detachment in each eye (B, right two panels).

[0104] (Figure 10C) Nine of the 10 eyes injected with AAV8 - anti - VEGFfab had no retinal detachment, while 8 of the 10 fellow eyes had complete retinal detachment (by Fisher's exact test, p < 0.001). In contrast, 7 of the 8 eyes injected with the empty vector and the fellow eyes had complete retinal detachment (p = 1.0). A significant prevention of retinal detachment was seen in the eyes injected with AAV8 - anti - VEGFfab compared to the eyes injected with the empty vector (by Fisher's exact test, p = 0.001).

[0105] (Figure 10D) In the eyes injected with AAV8 - anti - VEGFfab (n = 10), the mean (±SEM) retinal detachment (RD) rate per eye was significantly smaller than the mean (±SEM) retinal detachment (RD) rate of the fellow eyes (*p < 0.001) or the mean (±SEM) retinal detachment (RD) rate of the eyes injected with the empty vector (n = 8; †by one - way ANOVA with Bonferroni correction, p = 0.001).

[0106]

Figure 11

[0107]

Figure 12

[0108]

Figure 13

[0109]

Figure 14

[0110]

Figure 15

[0111]

Figure 16

[0112]

Figure 17

[0113]

Figure 18

[0114]

Figure 19

[0115]

Figure 20

[0116]

Figure 21

[0117]

Figure 22

[0118]

Figure 23

[0119]

Figure 24

[0120]

Figure 25

[0121]

Figure 26

Mode for Carrying Out the Invention

[0122] (5. Detailed Description of the Invention) Compositions and methods are described for the delivery of a post-translationally modified fully human (HuPTM) antibody against VEGF to the retina / vitreous humor of the eye of a patient (human subject) diagnosed with an eye disease, particularly an eye disease caused by an increase in neovascularization, such as neovascular age-related macular degeneration (nAMD, also known as "exudative" AMD), dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD). Antibodies include monoclonal antibodies, polyclonal antibodies, recombinantly produced antibodies, human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-heavy chain pairs, intrabodies, heteroconjugate antibodies, monovalent antibodies, antigen-binding fragments of full-length antibodies, and fusion proteins of the foregoing, but are not limited thereto. Such antigen-binding fragments include single domain antibodies (variable domains of heavy chain antibodies (VHH) or nanobodies), full-length anti-VEGF antibodies (preferably Fab, F(ab') of a full-length anti-VEGF monoclonal antibody (mAb)) 2and scFv (single-chain variable fragment) (collectively referred to herein as "antigen-binding fragment"), but not limited thereto. In a preferred embodiment, the post-translationally modified fully human antibody against VEGF is a post-translationally modified fully human antigen-binding fragment of a monoclonal antibody (mAb) against VEGF ("HuPTMFabVEGFi"). In a more preferred embodiment, HuPTMFabVEGFi is a glycosylated fully human antigen-binding fragment of an anti-VEGF mAb ("HuGlyFabVEGFi"). For the compositions and methods that can be used in accordance with the present invention described herein, each of which is incorporated herein by reference in its entirety, see International Patent Application Publication WO / 2017 / 180936 (International Patent Application PCT / US2017 / 027529 filed on April 14, 2017), and International Patent Application Publication WO / 2017 / 181021 (International Patent Application PCT / US2017 / 027650 filed on April 14, 2017). In alternative embodiments, full-length mAbs can be used. Delivery can be by gene therapy - for example, a viral vector or other DNA expression construct encoding an anti-VEGF antigen-binding fragment or mAb (or a highly glycosylated derivative) is administered to the suprachoroidal space, subretinal space (by a transvitreal approach or using a catheter through the suprachoroidal space), intravitreal cavity, and / or the outer surface of the sclera (i.e., juxtascleral administration) of the eye of a patient (human subject) diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD) to create an intraocular, persistent depot that supplies a human PTM, for example, a human glycosylation-introducing gene product. See, for example, the administration modes described in Section 5.3.2. In a preferred embodiment, the methods provided herein are used in patients (human subjects) diagnosed with exudative AMD.

[0123] The subject to whom such gene therapy is administered should be responsive to anti-VEGF therapy. In certain embodiments, the method includes treating a patient diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD) and identified as responsive to treatment with an anti-VEGF antibody. In more specific embodiments, the patient is responsive to treatment with an anti-VEGF antigen-binding fragment. In certain embodiments, the patient has been shown to be responsive to treatment with an anti-VEGF antigen-binding fragment injected intravitreally prior to treatment with gene therapy. In specific embodiments, the patient has previously been treated with LUCENTIS® (ranibizumab), EYLEA® (aflibercept), and / or AVASTIN® (bevacizumab) and has been found to be responsive to one or more of LUCENTIS® (ranibizumab), EYLEA® (aflibercept), and / or AVASTIN® (bevacizumab).

[0124] The subject to whom such viral vector or other DNA expression construct is delivered should be responsive to an anti-VEGF antigen-binding fragment encoded by the transgene in the viral vector or expression construct. To determine responsiveness, an anti-hVEGF antigen-binding fragment transgene product (produced, for example, in cell culture, bioreactor, etc.) can be administered directly to the subject, for example, by intravitreal injection.

[0125] HuPTMFabVEGFi encoded by the introduced gene, for example, HuGlyFabVEGFi, includes antigen-binding fragments of antibodies that bind to hVEGF, such as bevacizumab; anti-hVEGF Fab portions, such as ranibizumab; or Fab portions of such bevacizumab or ranibizumab modified to include additional glycosylation sites on the Fab domain, but are not limited thereto (for example, for the description of derivatives of bevacizumab that are highly glycosylated on the Fab domain of the full-length antibody, see the literature of Courtois et al., 2016, mAbs 8: 99-112, which is hereby incorporated by reference in its entirety).

[0126] The recombinant vector used for the delivery of the transgene should have tropism for human retinal cells or photoreceptor cells. Such vectors include, but are not limited to, non-replicating recombinant adeno-associated virus vectors ("rAAV"), and in particular, those having an AAV8 capsid are preferred. However, other viral vectors may be used, including, but not limited to, lentiviral vectors, vaccinia virus vectors, or non-viral expression vectors called "naked DNA" constructs. Preferably, the HuPTMFabVEGFi, for example, the HuGlyFabVEGFi transgene should be controlled by appropriate expression control elements, such as, for example, the CB7 promoter (chicken β-actin promoter and CMV enhancer), the RPE65 promoter, or the opsin promoter, and other expression control elements (e.g., chicken β-actin intron, murine minute virus (MVM) intron, human factor IX intron (e.g., FIX truncated intron 1), β-globin splice donor / immunoglobulin heavy chain splice acceptor intron, adenovirus splice donor / immunoglobulin splice acceptor intron, SV40 late splice donor / splice acceptor (19S / 16S) intron, and hybrid adenovirus splice donor / IgG splice acceptor intron, etc.) to enhance the expression of the transgene driven by the vector, as well as polyA signals (e.g., rabbit β-globin polyA signal, human growth hormone (hGH) polyA signal, SV40 late polyA signal, synthetic polyA (SPA) signal, and bovine growth hormone (bGH) polyA signal). See, for example, Powell and Rivera-Soto, 2015, Discov. Med., 19(102):49-57.

[0127] In a preferred embodiment, the gene therapy construct is designed such that both the heavy chain and the light chain are expressed. More specifically, the heavy chain and the light chain should be expressed in approximately equal amounts, in other words, the heavy chain and the light chain are expressed at an approximately 1:1 heavy chain to light chain ratio. The coding sequences of the heavy chain and the light chain can be modified in a single construct separated by a linker or IRES that allows the heavy chain and the light chain to be cleaved such that separate heavy chain and light chain polypeptides are expressed. For example, for specific leader sequences that can be used with the methods and compositions provided herein, see Section 5.2.4, and for specific IRESs, 2As, and other linker sequences, see Section 5.2.5.

[0128] A pharmaceutical composition suitable for suprachoroidal, subretinal, episcleral, and / or intravitreal administration comprises a suspension of a recombinant (e.g., rHuGlyFabVEGFi) vector in a formulated buffer comprising a physiologically compatible aqueous buffer, a surfactant, and any excipients.

[0129] The therapeutically effective amount of the recombinant vector should be administered subretinally and / or intravitreally (e.g., by subretinal injection via a transvitreal approach (surgical procedure) or subretinal administration via the suprachoroidal space) in a volume ranging from ≥0.1 mL to ≤0.5 mL, preferably in a volume of 0.1 - 0.30 mL (100 - 300 μl), and most preferably in a volume of 0.25 mL (250 μl). The therapeutically effective amount of the recombinant vector should be administered to the suprachoroid in a volume of 100 μl or less, e.g., in a volume of 50 - 100 μl (e.g., by suprachoroidal injection). The therapeutically effective amount of the recombinant vector should be administered to the outer surface of the sclera in a volume of 500 μl or less, e.g., in a volume of 500 μl or less, e.g., in a volume of 10 - 20 μl, 20 - 50 μl, 50 - 100 μl, 100 - 200 μl, 200 - 300 μl, 300 - 400 μl, or 400 - 500 μl. Subretinal injection is a surgical procedure performed by a skilled retinal surgeon involving partial vitrectomy and injection of gene therapy into the retina in a subject under local anesthesia (see, e.g., Campochiaro et al., 2017, Hum Gen Ther 28(1):99 - 111, which is incorporated herein by reference in its entirety). In a specific embodiment, subretinal administration is performed through the suprachoroidal space using a subretinal drug delivery device that includes a catheter that can be inserted into the suprachoroidal space towards the posterior pole, passed through it, and injected into the subretinal space with a fine needle at the posterior pole (see, e.g., Baldassarre et al., 2017, Subretinal Delivery of Cells via the Suprachoroidal Space: Janssen Trial. In: Schwartz et al. (eds.), Cellular Therapies for Retinal Disease, Springer, Cham; International Patent Application Publication WO 2016 / 040635 A1; each of these is incorporated herein by reference in its entirety).Administration to the suprachoroid involves the administration of a drug into the suprachoroidal cavity of the eye and is typically performed using a suprachoroidal drug delivery device such as a microinjector with a microneedle (see, for example, Hariprasad, 2016, Retinal Physician 13: 20-23; Goldstein, 2014, Retina Today 9(5): 82-87, each of which is incorporated herein by reference in its entirety). Examples of suprachoroidal drug delivery devices that can be used to deposit an expression vector into the suprachoroidal cavity according to the invention described herein include, but are not limited to, suprachoroidal drug delivery devices manufactured by Clearside® Biomedical (see, for example, Hariprasad, 2016, Retinal Physician 13: 20-23). Examples of subretinal drug delivery devices that can be used to deposit an expression vector into the subretinal space via the suprachoroidal cavity according to the invention described herein include, but are not limited to, subretinal drug delivery devices manufactured by Janssen Pharmaceuticals (see, for example, International Patent Application Publication WO 2016 / 040635 A1). In a specific embodiment, administration to the outer surface of the sclera is performed using a sclera-proximal drug delivery device that includes a cannula that can be inserted with its tip and held in direct juxtaposition to the scleral surface. See Section 5.3.2 for further details on various administration modes. The soluble transgene product should be delivered to the retina, vitreous humor, and / or aqueous humor by suprachoroidal, subretinal, sclera-proximal, and / or intraretinal administration. Expression of the transgene product (e.g., the encoded anti-VEGF antibody) by retinal cells such as rod cells, cone cells, retinal pigment epithelial cells, horizontal cells, bipolar cells, amacrine cells, ganglion cells, and / or Müller cells results in delivery and maintenance of the transgene product in the retina, vitreous humor, and / or aqueous humor. The concentration of the transgene product is at least 0.330 μg / mL in the vitreous humor or 0.110 μg / mL in the aqueous humor (anterior chamber) for 3 months. minA dosage that maintains it is desirable; thereafter, the vitreous C of the transgene product in the range of 1.70 to 6.60 μg / mL min concentration and / or the aqueous humor C in the range of 0.567 to 2.20 μg / mL min concentration should be maintained. However, since the transgene product is produced continuously, it may be effective to maintain a lower concentration. The concentration of the transgene product can be measured in patient samples of vitreous humor and / or aqueous humor derived from the anterior chamber of the treated eye. Alternatively, the vitreous humor concentration can be estimated and / or monitored by measuring the patient serum concentration of the transgene product - the ratio of the systemic exposure to the vitreous exposure of the transgene product is about 1:90,000 (see, for example, the vitreous humor and serum concentrations of ranibizumab reported in Xu L et al., 2013, Invest. Opthal. Vis. Sci. 54: 1616 - 1624, p.1621, and Table 5 on p.1623, which are incorporated herein by reference in their entirety).

[0130] The present invention has several advantages over standard treatment regimens involving repeated intravitreal injections of high-dose boluses of VEGF inhibitors that dissipate over time to produce peak and trough levels. The continuous expression of the transgene product antibody allows for a more consistent level of antibody to be present at the site of action, as opposed to repeated injections of the antibody, resulting in fewer injections being required and, as a result, fewer clinic visits, which is less risky and more convenient for the patient. Consistent protein production reduces the likelihood of rebound edema in the retina, which can lead to better clinical outcomes. Additionally, since the microenvironment present during and after translation is different, the antibody expressed from the transgene is post-translationally modified in a different manner than the directly injected antibody. Without being bound by any particular theory, this results in antibodies with various diffusion, bioactivity, distribution, affinity, pharmacokinetic, and immunogenic properties such that the antibody delivered to the site of action is a "biobetter" compared to the directly injected antibody.

[0131] Furthermore, antibodies expressed from transgenes in vivo are less likely to contain degradation products associated with antibodies produced by recombinant techniques, such as protein aggregates and protein oxides. Aggregation is a problem associated with the production and storage of proteins due to high protein concentrations, surface interactions with manufacturing equipment and containers, and purification with certain buffer systems. These conditions that promote aggregation do not exist in transgene expression in gene therapy. Oxidation, such as methionine, tryptophan, and histidine oxidation, is also associated with protein production and storage and is caused by stressed cell culture conditions, contact with metals and air, and impurities in buffers and excipients. Proteins expressed from transgenes in vivo can also oxidize under stressed conditions. However, humans and many other organisms have antioxidant defense systems that not only reduce oxidative stress but can also repair and / or reverse oxidation. Therefore, proteins produced in vivo are less likely to be in an oxidized form. Both aggregation and oxidation can affect efficacy, pharmacokinetics (clearance), and immunogenicity.

[0132] Without being bound by theory, the methods and compositions provided herein are based in part on the following principles: (i) Human retinal cells are secretory cells that possess the cellular machinery for the post-translational processing of secreted proteins, including glycosylation and tyrosine-O-sulfation, which are robust processes of retinal cells (for example, for post-translational modifications performed by human retinal cells, see the literature of Wang et al., 2013, Analytical Biochem. 427: 20-28 and Adamis et al., 1993, BBRC 193: 631-638, which report the production of glycoproteins by retinal cells; and the literature of Kanan et al., 2009, Exp. Eye Res. 89: 559-567 and Kanan and Al-Ubaidi, 2015, Exp. Eye Res. 133: 126-131, which report the production of tyrosine-sulfated glycoproteins secreted by retinal cells. These are each incorporated by reference in their entirety). (ii) Contrary to the understanding of the prior art, anti-VEGF antigen-binding fragments, for example, ranibizumab (and the Fab domain of full-length anti-VEGF mAbs such as bevacizumab) actually possess N-linked glycosylation sites. For example, the C H domain (TVSWN 165 SGAL) and the non-consensus asparagine ("N") glycosylation sites in the C L domain (QSGN 158 SQE), as well as the glutamine ("Q") residues (and the corresponding portions in the Fab of bevacizumab) that are glycosylation sites in the V H domain (Q 115 GT) and the V L domain (TFQ 100 GT) are identified in Figure 1 (for example, for the identification of N-linked glycosylation sites in antibodies, see the literature of Valliere-Douglass et al., 2009, J. Biol. Chem. 284: 32493-32506, and the literature of Valliere-Douglass et al., 2010, J. Biol. Chem. 285: 16012-16022. These are each incorporated by reference in their entirety). (iii) Such non-standard sites typically result in low levels of glycosylation (e.g., about 1-5%) of the antibody population, but in immune-privileged organs such as the eye, functional advantages can be prominent (see, e.g., van de Bovenkamp et al., 2016, J. Immunol. 196:1435-1441). For example, Fab glycosylation can affect antibody stability, half-life, and binding properties. To determine the effect of Fab glycosylation on the affinity of the antibody for its target, any technique known to those skilled in the art, such as enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR), can be used. To determine the effect of Fab glycosylation on the half-life of the antibody, any technique known to those skilled in the art can be used, such as by measuring the level of radioactivity in blood or organs (e.g., the eye) in a subject administered a radiolabeled antibody. To determine the effect of Fab glycosylation on antibody stability, such as the level of aggregation or protein unfolding, any technique known to those skilled in the art, such as differential scanning calorimetry (DSC), high-performance liquid chromatography (HPLC), such as size-exclusion high-performance liquid chromatography (SEC-HPLC), capillary electrophoresis, mass spectrometry, or turbidimetry, can be used. The HuPTMFabVEGFi provided herein, such as the HuGlyFabVEGFi transgene, results in the production of Fab that is glycosylated at non-standard sites at 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more. In certain embodiments, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more of the Fab derived from the Fab population is glycosylated at non-standard sites. In certain embodiments, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more of the non-standard sites are glycosylated. In certain embodiments, the glycosylation of Fab at these non-standard sites is 25%, 50%, 100%, 200%, 300%, 400%, 500%, or more than that of the glycosylation of these non-standard sites in Fab produced in HEK293 cells. (iv) In addition to the glycosylation sites, anti-VEGF Fabs such as ranibizumab (and the Fab of bevacizumab) contain tyrosine ("Y") sulfation sites inside or near the CDRs; the V H (EDTAVY 94 Y 95 ) and V L (EDFATY 86 ) tyrosine-O-sulfation sites in the domain (and the corresponding sites in the Fab of bevacizumab). See Figure 1 which identifies them (for example, for the analysis of the amino acids around tyrosine residues that have undergone protein tyrosine sulfation, see Yang et al., 2015, Molecules 20:2138-2164, especially p. 2154, which is hereby incorporated by reference in its entirety. The "rules" can be summarized as follows: the Y residue has an E or D within +5 to -5 from the position of Y, in which case the -1 position of Y is a neutral or acidic charged amino acid but not a basic amino acid that inactivates sulfation, such as R, K, or H). Human IgG antibodies can exhibit several other post-translational modifications, such as N-terminal modifications, C-terminal modifications, degradation or oxidation of amino acid residues, cysteine-related variants, and glycosylation (see, for example, Liu et al., 2014, mAbs 6(5):1145-1154). (v) Glycosylation of anti-VEGF Fabs by human retinal cells, such as Fab fragments of ranibizumab or bevacizumab, results in the addition of glycans that can improve the stability and half-life of the transgene product and reduce its undesirable aggregation and / or immunogenicity (see, for example, Bovenkamp et al., 2016, J. Immunol. 196: 1435-1441 for a review of the emerging importance of Fab glycosylation). Importantly, the glycans that can be added to the HuPTMFabVEGFi provided herein, such as HuGlyFabVEGFi, are highly processed complex biantennary N-glycans containing 2,6-sialic acid (see Figure 2 showing the glycans that can be incorporated into HuPTMFabVEGFi, such as HuGlyFabVEGFi) and bisecting GlcNAc and are not NGNA (N-glycolylneuraminic acid, Neu5Gc). Such glycans are not present in ranibizumab (which is produced in E. coli and is not glycosylated at all) or bevacizumab (which does not have the 2,6-sialyltransferase required for this post-translational modification or the bisecting GlcNAc of CHO cell products, but instead adds Neu5Gc (NGNA), a sialic acid not specific to humans (and potentially immunogenic), in place of Neu5Ac (NANA) and is produced in CHO cells). See, for example, Dumont et al., 2015, Crit. Rev. Biotechnol. (early online edition, published online September 18, 2015, pp. 1-13, p. 5). Furthermore, CHO cells can produce the immunogenic glycan α-Gal antigen, which reacts with anti-α-Gal antibodies present in most individuals and can induce anaphylaxis at high concentrations. See, for example, Bosques, 2010, Nat Biotech 28: 1153-1156. The human glycosylation pattern of the HuPTMFabVEGFi provided herein, such as HuGlyFabVEGFi, should reduce the immunogenicity of the transgene product and improve its efficacy. (vi) Tyrosine sulfation - a robust post - translational process in human retinal cells - of anti - VEGF Fabs, such as Fab fragments of ranibizumab or bevacizumab, can result in transgene products with increased binding affinity to VEGF. Indeed, tyrosine sulfation of Fabs of therapeutic antibodies against other targets has been shown to dramatically increase antigen - binding affinity and activity (see, e.g., Loos et al., 2015, PNAS 112: 12675 - 12680, and Choe et al., 2003, Cell 114: 161 - 170). Such post - translational modifications are absent in ranibizumab (which is produced in E. coli, a host that does not possess the enzymes required for tyrosine sulfation) and are only poorly presented, at best, in bevacizumab, a CHO - cell product. Unlike human retinal cells, CHO cells are not secretory cells and have limited ability for post - translational tyrosine sulfation (see, e.g., Mikkelsen and Ezban, 1991, Biochemistry 30: 1533 - 1537, especially the discussion on p. 1537).

[0133] For the reasons above, the production of HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, is achieved by gene therapy - e.g., administering a viral vector or other DNA expression construct encoding HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, to the suprachoroidal space, subretinal space, or outer surface of the sclera of the eye of a patient (human subject) diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (especially exudative AMD) (e.g., by suprachoroidal injection, subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space, or a posterior scleral juxtadepot procedure), creating an intravitreal permanent depot that continuously supplies the post-translationally modified fully human, e.g., glycosylated, sulfated human transgene product produced by the transduced retinal cells, which should result in a "bio-better" molecule for the treatment of exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (especially exudative AMD). The cDNA construct for Fab VEGFi should contain a signal peptide that ensures proper co-translational and post-translational processing (glycosylation and protein sulfation) by the transduced retinal cells. Such signal sequences used by retinal cells include, but are not limited to, the following:

Chemical formula

[0134] As an alternative to gene therapy or as an additional treatment to gene therapy, HuPTMFabVEGFi products, such as HuGlyFabVEGFi glycoproteins, are produced in human cell lines by recombinant DNA technology and can be administered by intravitreal injection to patients diagnosed with exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD). HuPTMFabVEGFi products, such as glycoproteins, can also be administered to patients having exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD). Human cell lines that can be used for the production of such recombinant glycoproteins include, by way of example, but are not limited to, human embryonic kidney 293 cells (HEK293), fibrosarcoma HT-1080, HKB-11, CAP, HuH-7, and the retinal cell line PER.C6 or RPE (for a review of human cell lines that can be used for the recombinant production of HuPTMFabVEGFi products, such as HuGlyFabVEGFi glycoproteins, see Dumont et al., 2015, Crit. Rev. Biotechnol. (Early Online Edition, published online September 18, 2015, pp. 1-13), "Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives", which is incorporated herein by reference in its entirety). To ensure full glycosylation, particularly sialylation and tyrosine sulfation, the cell line used for production can be enhanced by modifying the host cell to co-express α-2,6-sialyltransferase (or both α-2,3-sialyltransferase and α-2,6-sialyltransferase) and / or the TPST-1 and TPST-2 enzymes involved in tyrosine-O-sulfation in retinal cells.

[0135] Delivery of HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, to the eye / retina with delivery of other available therapies is encompassed by the methods provided herein. Additional therapies can be administered before, simultaneously with, or after the gene therapy treatment. Available therapies for exudative AMD, dry AMD, retinal vein occlusion (RVO), diabetic macular edema (DME), or diabetic retinopathy (DR) (particularly exudative AMD) that can be combined with the gene therapy provided herein include laser photocoagulation, photodynamic therapy with verteporfin, and, without limitation, intravitreal (“IVT”) injection of anti-VEGF agents including, but not limited to, pegaptanib, ranibizumab, aflibercept, or bevacizumab. Additional treatment with anti-VEGF agents, e.g., biologics, may be referred to as “rescue” therapy.

[0136] Unlike small molecule drugs, biologic agents typically contain a mixture of many variants with different modifications or forms having different potencies, pharmacokinetics, and safety profiles. It is not essential that all molecules produced in either a gene therapy or protein therapy approach be fully glycosylated and sulfated. Rather, the population of glycoproteins produced should have glycosylation (about 1% to about 10% of the population) and sulfation that includes sufficient 2,6-sialylation to exhibit potency. The purpose of the gene therapy treatment provided herein is to slow or halt the progression of retinal degeneration and to slow or prevent vision loss with minimal intervention / invasive procedures. Potency can be monitored by measurement of BCVA (best corrected visual acuity), intraocular pressure, slit lamp biomicroscopy, indirect ophthalmoscopy, SD-OCT (SD-optical coherence tomography), electroretinogram (ERG). Signs of other safety events, including vision loss, infections, inflammation, and retinal detachment, can also be monitored. Retinal thickness may be monitored to determine the efficacy of the treatment provided herein. Without being bound by any particular theory, retinal thickness can be used as a clinical readout, in which case the greater the decrease in retinal thickness or the longer the period to retinal thickening, the more effective the treatment. Retinal thickness can be determined, for example, by SD-OCT. SD-OCT is a three-dimensional imaging technique that uses low coherence interferometry to determine the echo time delay and amplitude of backscattered light reflected from the object of interest. Using OCT, tissue samples (e.g., the retina) can be scanned with an axial resolution of 3 to 15 μm, and SD-OCT improves axial resolution and scan speed over previous types of technology (Schuman, 2008, Trans. Am. Opthamol. Soc. 106:426-458). Retinal function can be determined, for example, by ERG. ERG is a non-invasive electrophysiological test of retinal function approved by the FDA for use in humans that examines the response of the eye's photoreceptor cells (rods and cones) and the ganglion cells to which they are connected, particularly to a flash stimulus.

[0137] (5.1 N-glycosylation, tyrosine sulfation, and O-glycosylation) The amino acid sequence (primary sequence) of the anti-VEGF antigen-binding fragment of HuPTMFabVEGFi, such as HuGlyFabVEGFi, used in the methods described herein contains at least one site where N-glycosylation or tyrosine sulfation occurs. In certain embodiments, the amino acid sequence of the anti-VEGF antigen-binding fragment contains at least one N-glycosylation site and at least one tyrosine sulfation site. Such sites are described in detail below. In certain embodiments, the amino acid sequence of the anti-VEGF antigen-binding fragment contains at least one O-glycosylation site, which may be additional to one or more N-glycosylation sites and / or tyrosine sulfation sites present in the amino acid sequence.

[0138] (5.1.1 N-glycosylation) (Reverse glycosylation site) It is known in the art that the standard N-glycosylation sequence is Asn-X-Ser (or Thr), where X can be any amino acid other than Pro. However, recently it has been shown that asparagine (Asn) residues of human antibodies can be glycosylated in the context of the reverse consensus motif Ser (or Thr)-X-Asn, where X can be any amino acid other than Pro. See Valliere-Douglass et al., 2009, J. Biol. Chem. 284:32493-32506; and Valliere-Douglass et al., 2010, J. Biol. Chem. 285:16012-16022. As disclosed herein, contrary to the understanding in the prior art, an anti-VEGF antigen-binding fragment for use according to the methods described herein, e.g., ranibizumab, contains some of such reverse consensus sequences. Accordingly, the methods described herein include the use of an anti-VEGF antigen-binding fragment that contains at least one N-glycosylation site (also referred to herein as a "reverse N-glycosylation site") having the sequence Ser (or Thr)-X-Asn, where X can be any amino acid other than Pro.

[0139] In certain embodiments, the methods described herein include the use of an anti-VEGF antigen-binding fragment that contains one, two, three, four, five, six, seven, eight, nine, ten, or more than ten N-glycosylation sites having the sequence Ser (or Thr)-X-Asn, where X can be any amino acid other than Pro. In certain embodiments, the methods described herein include the use of an anti-VEGF antigen-binding fragment that contains one, two, three, four, five, six, seven, eight, nine, ten, or more than ten reverse N-glycosylation sites, and one, two, three, four, five, six, seven, eight, nine, ten, or more than ten non-consensus N-glycosylation sites (defined herein below).

[0140] In a specific embodiment, the anti-VEGF antigen-binding fragment comprising one or more reverse N-glycosylation sites used in the methods described herein is ranibizumab, which comprises the light and heavy chains of SEQ ID NO: 1 and SEQ ID NO: 2, respectively. In another specific embodiment, the anti-VEGF antigen-binding fragment comprising one or more reverse N-glycosylation sites used in the methods described herein comprises the Fab of bevacizumab, which comprises the light and heavy chains of SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0141] (Non-consensus glycosylation site) In addition to reverse N-glycosylation sites, it has recently been shown that glutamine (Gln) residues of human antibodies can be glycosylated in the context of the non-consensus motif Gln-Gly-Thr. See Valliere-Douglass et al., 2010, J. Biol. Chem. 285:16012-16022. Surprisingly, anti-VEGF antigen-binding fragments for use according to the methods described herein, such as ranibizumab, contain some of such non-consensus sequences. Accordingly, the methods described herein include the use of anti-VEGF antigen-binding fragments comprising at least one N-glycosylation site comprising the sequence Gln-Gly-Thr (also referred to herein as a "non-consensus N-glycosylation site").

[0142] In certain embodiments, the methods described herein include the use of anti-VEGF antigen-binding fragments comprising one, two, three, four, five, six, seven, eight, nine, ten, or more than ten N-glycosylation sites comprising the sequence Gln-Gly-Thr.

[0143] In a specific embodiment, the anti-VEGF antigen-binding fragment containing one or more non-consensus N-glycosylation sites used in the methods described herein is ranibizumab (including the light and heavy chains of SEQ ID NO: 1 and SEQ ID NO: 2, respectively). In another specific embodiment, the anti-VEGF antigen-binding fragment containing one or more non-consensus N-glycosylation sites used in the method includes the Fab of bevacizumab (including the light and heavy chains of SEQ ID NO: 3 and SEQ ID NO: 4, respectively).

[0144] (Modified N-glycosylation site) In certain embodiments, a nucleic acid encoding an anti-VEGF antigen-binding fragment is modified to include one, two, three, four, five, six, seven, eight, nine, ten, or more N-glycosylation sites (including canonical N-glycosylation consensus sequences, reverse N-glycosylation sites, and non-consensus N-glycosylation sites) compared to that typically associated with HuGlyFabVEGFi (e.g., compared to the number of N-glycosylation sites associated with the anti-VEGF antigen-binding fragment in its unmodified state). In a specific embodiment, the introduction of glycosylation sites is achieved by insertion of an N-glycosylation site (including canonical N-glycosylation consensus sequences, reverse N-glycosylation sites, and non-consensus N-glycosylation sites) anywhere in the primary structure of the antigen-binding fragment, provided that the introduction does not affect the binding of the antigen-binding fragment to its antigen VEGF. The introduction of glycosylation sites can be achieved, for example, by adding a new amino acid to the primary structure of the antigen-binding fragment or the antibody from which the antigen-binding fragment is derived (i.e., adding the glycosylation site in whole or in part), or by mutating an existing amino acid in the antigen-binding fragment or the antibody from which the antigen-binding fragment is derived to generate an N-glycosylation site (i.e., mutating selected amino acids of the antigen-binding fragment / antibody to form an N-glycosylation site rather than adding an amino acid to the antigen-binding fragment / antibody). One of ordinary skill in the art will recognize that the amino acid sequence of a protein can be readily modified using approaches known in the art, such as recombinant approaches that include modification of the nucleic acid encoding the protein.

[0145] In a specific embodiment, the anti-VEGF antigen-binding fragment used in the methods described herein is modified such that it can be highly glycosylated when expressed in retinal cells. See Courtois et al., 2016, mAbs 8:99-112, which is hereby incorporated by reference in its entirety. In a specific embodiment, the anti-VEGF antigen-binding fragment is ranibizumab (comprising the light and heavy chains of SEQ ID NO: 1 and SEQ ID NO: 2, respectively). In another specific embodiment, the anti-VEGF antigen-binding fragment is the Fab of bevacizumab (comprising the light and heavy chains of SEQ ID NO: 3 and SEQ ID NO: 4, respectively).

[0146] (N-glycosylation of anti-VEGF antigen-binding fragment) Unlike small molecule drugs, biologic agents typically contain a mixture of many variants with different modifications or forms having different potencies, pharmacokinetics, and safety profiles. It is not essential that all molecules produced in either a gene therapy or protein therapy approach be fully glycosylated and sulfated. Rather, the population of glycoproteins produced should have sufficient glycosylation (including 2,6-sialylation) and sulfation to exhibit potency. The purpose of the gene therapy treatment provided herein is to slow or halt the progression of retinal degeneration and to slow or prevent vision loss with minimal intervention / invasive procedures.

[0147] In a specific embodiment, an anti-VEGF antigen-binding fragment, such as ranibizumab, used according to the methods described herein can be glycosylated at 100% of its N-glycosylation sites when expressed in retinal cells. However, one of ordinary skill in the art will recognize that not all N-glycosylation sites of the anti-VEGF antigen-binding fragment need to be N-glycosylated in order to obtain the advantages of glycosylation. Rather, the advantages of glycosylation can be achieved when only a percentage of the N-glycosylation sites are glycosylated and / or when only a percentage of the expressed antigen-binding fragments are glycosylated. Thus, in certain embodiments, the anti-VEGF antigen-binding fragment used according to the methods described herein is glycosylated at 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100% of its available N-glycosylation sites when expressed in retinal cells. In certain embodiments, when expressed in retinal cells, 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 100% of the anti-VEGF antigen-binding fragment used according to the methods described herein is glycosylated at at least one of its available N-glycosylation sites.

[0148] In a specific embodiment, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites present in the anti-VEGF antigen-binding fragment used according to the methods described herein are glycosylated with an Asn residue (or other related residue) present in the N-glycosylation site when the anti-VEGF antigen-binding fragment is expressed in retinal cells. That is, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites of the resulting HuGlyFabVEGFi are glycosylated.

[0149] In another specific embodiment, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites present in the anti-VEGF antigen-binding fragment used according to the methods described herein are glycosylated with the same linked glycan linked to the Asn residue (or other related residue) present in the N-glycosylation site when the anti-VEGF antigen-binding fragment is expressed in retinal cells. That is, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites of the resulting HuGlyFabVEGFi are the same linked glycan.

[0150] When an anti-VEGF antigen-binding fragment, such as ranibizumab, used according to the methods described herein is expressed in retinal cells, the N-glycosylation sites of the antigen-binding fragment can be glycosylated with a variety of different glycans. The N-glycans of the antigen-binding fragment are characterized in the art. For example, in Bondt et al., 2014, Mol. & Cell. Proteomics 13.11:3029-3039 (the disclosure of which regarding N-glycans associated with Fab is incorporated herein by reference in its entirety), the glycans associated with Fab are characterized, and the Fab and Fc portions of the antibody contain different glycosylation patterns, and it has been shown that Fab glycans have more galactosylation, sialylation, and bisecting (e.g., in the case of bisecting GlcNAc) than Fc glycans, but less fucosylation. Similar to the Bondt reference, in Huang et al., 2006, Anal. Biochem. 349:197-207 (the disclosure of which regarding N-glycans associated with Fab is incorporated herein by reference in its entirety), it was found that most of the glycans of Fab were sialylated. However, in the Fab of the antibody investigated by Huang (which was produced in a mouse cell background), the sialic acid residues identified were N-glycolylneuraminic acid ("Neu5Gc" or "NeuGc") (which is not natural to humans), rather than N-acetylneuraminic acid ("Neu5Ac", the major human sialic acid). Furthermore, in Song et al., 2014, Anal. Chem. 86:5661-5666 (the disclosure of which regarding N-glycans associated with Fab is incorporated herein by reference in its entirety), a library of N-glycans associated with a commercial antibody is described.

[0151] Importantly, when anti-VEGF antigen-binding fragments, such as ranibizumab, used according to the methods described herein are expressed in human retinal cells, the need for in vitro production in prokaryotic host cells (e.g., E. coli) or eukaryotic host cells (e.g., CHO cells) is avoided. Instead, as a result of the methods described herein (e.g., the use of retinal cells to express anti-hVEGF antigen-binding fragments), the N-glycosylation sites of the anti-VEGF antigen-binding fragments are advantageously modified with glycans relevant and beneficial to human therapy. Such advantages cannot be achieved when CHO cells or E. coli are utilized for antibody / antigen-binding fragment production, for example, because CHO cells (1) do not express 2,6-sialyltransferase and thus cannot add 2,6-sialic acid during N-glycosylation, and (2) can add Neu5Gc rather than Neu5Ac as sialic acid; and E. coli do not naturally contain the components required for N-glycosylation. Thus, in one embodiment, the anti-VEGF antigen-binding fragment expressed in retinal cells that gives rise to HuGlyFabVEGFi used in the therapeutic methods described herein is glycosylated in a manner in which the protein is N-glycosylated in human retinal cells, e.g., retinal pigment epithelial cells, but not in the manner in which the protein is N-glycosylated in CHO cells. In another embodiment, the anti-VEGF antigen-binding fragment expressed in retinal cells that gives rise to HuGlyFabVEGFi used in the therapeutic methods described herein is glycosylated in a manner in which the protein is N-glycosylated in human retinal cells, e.g., retinal pigment epithelial cells, where such glycosylation is not naturally possible using a prokaryotic host cell, e.g., E. coli.

[0152] In certain embodiments, the HuGlyFabVEGFi used according to the methods described herein, such as ranibizumab, contains one, two, three, four, five, or more different N-glycans associated with the Fab of a human antibody. In specific embodiments, the N-glycans associated with the Fab of a human antibody are N-glycans described in Bondt et al., 2014, Mol. & Cell. Proteomics 13.11:3029-3039, Huang et al., 2006, Anal. Biochem. 349:197-207, and / or Song et al., 2014, Anal. Chem. 86:5661-5666. In certain embodiments, the HuGlyFabVEGFi used according to the methods described herein, such as ranibizumab, does not contain detectable NeuGc and / or α-Gal antigens.

[0153] In a specific embodiment, the HuGlyFabVEGFi used according to the methods described herein, such as ranibizumab, is predominantly glycosylated with glycans containing 2,6-linked sialic acid. In certain embodiments, the HuGlyFabVEGFi containing 2,6-linked sialic acid is polysialylated, i.e., contains multiple sialic acids. In certain embodiments, each N-glycosylation site of the HuGlyFabVEGFi contains a glycan containing 2,6-linked sialic acid, i.e., 100% of the N-glycosylation sites of the HuGlyFabVEGFi contain a glycan containing 2,6-linked sialic acid. In another specific embodiment, at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites of the HuGlyFabVEGFi used according to the methods described herein are glycosylated with glycans containing 2,6-linked sialic acid. In another specific embodiment, at least 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 99% of the N-glycosylation sites of the HuGlyFabVEGFi used according to the methods described herein are glycosylated with glycans containing 2,6-linked sialic acid. In another specific embodiment, at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the antigen-binding fragments expressed in retinal cells (i.e., the antigen-binding fragments that give rise to HuGlyFabVEGFi, such as ranibizumab) according to the methods described herein are glycosylated with glycans containing 2,6-linked sialic acid. In another specific embodiment, at least 10% - 20%, 20% - 30%, 30% - 40%, 40% - 50%, 50% - 60%, 60% - 70%, 70% - 80%, 80% - 90%, or 90% - 99% of the antigen-binding fragments (i.e., Fab that gives rise to HuGlyFabVEGFi, such as ranibizumab) expressed in retinal cells according to the methods described herein are glycosylated with glycans containing 2,6-linked sialic acid.In another specific embodiment, the sialic acid is Neu5Ac. According to such an embodiment, if only a percentage of the N-glycosylation sites of HuGlyFabVEGFi are 2,6-sialylated or polysialylated, the remaining N-glycosylation can contain different N-glycans or can contain no N-glycans at all (i.e., remain non-glycosylated).

[0154] When HuGlyFabVEGFi is 2,6-polysialylated, it contains multiple sialic acid residues, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 sialic acid residues. In certain embodiments, when HuGlyFabVEGFi is polysialylated, it contains 2 - 5, 5 - 10, 10 - 20, 20 - 30, 30 - 40, or 40 - 50 sialic acid residues. In certain embodiments, when HuGlyFabVEGFi is polysialylated, it is of the 2,6-linked type (sialic acid) n (where n can be any number from 1 to 100) is included.

[0155] In a specific embodiment, the HuGlyFabVEGFi used according to the methods described herein, for example, ranibizumab, is predominantly glycosylated with glycans containing bisecting GlcNAc. In certain embodiments, each N-glycosylation site of the HuGlyFabVEGFi contains a glycan containing bisecting GlcNAc, i.e., 100% of the N-glycosylation sites of the HuGlyFabVEGFi contain a glycan containing bisecting GlcNAc. In another specific embodiment, at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the N-glycosylation sites of the HuGlyFabVEGFi used according to the methods described herein are glycosylated with glycans containing bisecting GlcNAc. In another specific embodiment, at least 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-99% of the N-glycosylation sites of the HuGlyFabVEGFi used according to the methods described herein are glycosylated with glycans containing bisecting GlcNAc. In another specific embodiment, at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the antigen-binding fragments expressed in retinal cells (i.e., antigen-binding fragments that give rise to HuGlyFabVEGFi, e.g., ranibizumab) according to the methods described herein are glycosylated with glycans containing bisecting GlcNAc. In another specific embodiment, at least 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-99% of the antigen-binding fragments expressed in retinal cells (i.e., antigen-binding fragments that give rise to HuGlyFabVEGFi, e.g., ranibizumab) according to the methods described herein are glycosylated with glycans containing bisecting GlcNAc.

[0156] In certain embodiments, the HuGlyFabVEGFi used according to the methods described herein, for example, ranibizumab, is highly glycosylated, i.e., in addition to N-glycosylation obtained from natural N-glycosylation sites, the HuGlyFabVEGFi contains glycans at N-glycosylation sites engineered to be present in the amino acid sequence of the antigen-binding fragment that gives rise to the HuGlyFabVEGFi. In certain embodiments, the HuGlyFabVEGFi used according to the methods described herein, for example, ranibizumab, is highly glycosylated but does not contain detectable NeuGc and / or α-Gal antigens.

[0157] Assays for determining the glycosylation pattern of antibodies, including antigen-binding fragments, are known in the art. For example, hydrazinolysis can be used to analyze glycans. First, the polysaccharide is released from its associated protein by incubation with hydrazine (the Ludger Liberate Hydrazinolysis Glycan Release Kit, Oxfordshire, UK can be used). The nucleophilic hydrazine attacks the glycosidic bond between the polysaccharide and the carrier protein, enabling the release of the bound glycan. The N-acetyl group is lost and must be reconstituted by N-acetylation during this process. The glycan can also be released using enzymes, such as glycosidases or endoglycosidases like PNGase F and Endo H, which cleave completely with fewer side reactions than hydrazine. The released glycan can be purified on a carbon column and then labeled at the reducing end with the fluorescent substance 2-aminobenzamide. The labeled polysaccharide can be separated on a GlycoSep-N column (GL Sciences) according to the HPLC protocol of Royle et al., Anal Biochem 2002, 304(1):70-90. The resulting fluorescence chromatogram shows the length of the polysaccharide and the number of repeating units. Structural information can be collected by collecting individual peaks and then performing MS / MS analysis. Thereby, the monosaccharide composition and the sequence of the repeating units can be confirmed, and furthermore, the homogeneity of the polysaccharide composition can be identified. Specific peaks of low or high molecular weight can be analyzed by MALDI-MS / MS, and the results can be used to confirm the glycan sequence. Each peak in the chromatogram corresponds to a polymer consisting of a specific number of repeating units, such as a glycan and its fragments, such as sugar residues. Thus, the chromatogram enables the measurement of the length distribution of polymers, such as glycans. The elution time is an indicator of the polymer length, while the fluorescence intensity correlates with the molar abundance of each polymer, such as a glycan.As other methods for evaluating glycans associated with antigen-binding fragments, methods described in the literature of Bondt et al., 2014, Mol. & Cell. Proteomics 13.11:3029-3039, Huang et al., 2006, Anal. Biochem. 349:197-207, and / or Song et al., 2014, Anal. Chem. 86:5661-5666 may be mentioned.

[0158] The uniformity or non-uniformity of the glycan pattern associated with an antibody (including antigen-binding fragments) relates to both the length or size of the glycan and the number of glycans present across glycosylation sites, and thus can be evaluated using methods known in the art, for example, methods for measuring the length or size of the glycan and the hydrodynamic radius. HPLC, such as size exclusion, normal phase, reverse phase, and anion exchange HPLC, and capillary electrophoresis enable the measurement of the hydrodynamic radius. When there are a large number of glycosylation sites in a protein, the variation in the hydrodynamic radius becomes larger compared to carriers with fewer glycosylation sites. However, when analyzing a single glycan chain, these can be more uniform as their length is more controlled. The length of the glycan can be measured by hydrazinolysis, SDS PAGE, and capillary gel electrophoresis. Furthermore, uniformity can also mean that the pattern of specific glycosylation site usage varies over a wider / narrower range. These factors can be measured by glycopeptide LC-MS / MS.

[0159] (Advantages of N-glycosylation) N-glycosylation confers many advantages to the HuGlyFabVEGFi used in the methods described herein. Such advantages cannot be achieved by the production of antigen-binding fragments in E. coli, as E. coli do not naturally possess the components required for N-glycosylation. Further, some advantages cannot be achieved, for example, through antibody production in CHO cells, as CHO cells lack the components required for the addition of specific glycans (e.g., 2,6-sialic acid and bisecting GlcNAc), and as CHO cells can add glycans that are not typical for humans, such as Neu5Gc. See, for example, Song et al., 2014, Anal. Chem. 86:5661-5666. Thus, due to the discovery described herein that anti-VEGF antigen-binding fragments, such as ranibizumab, contain non-standard N-glycosylation sites (including both reverse glycosylation sites and non-consensus glycosylation sites), methods have been realized for expressing such anti-VEGF antigen-binding fragments in a manner that results in their glycosylation (and, as a result, an improvement in the advantages associated with the antigen-binding fragments). In particular, the expression of anti-VEGF antigen-binding fragments in human retinal cells results in the production of HuGlyFabVEGFi (e.g., ranibizumab) that contains beneficial glycans that would otherwise not be associated with either the antigen-binding fragment or its parent antibody.

[0160] Non-standard glycosylation sites typically result in low levels of glycosylation (e.g., about 1-5%) in an antibody population, but in immune-privileged organs such as the eye, functional advantages can be significant (see, e.g., van de Bovenkamp et al., 2016, J. Immunol. 196:1435-1441). For example, Fab glycosylation can affect antibody stability, half-life, and binding properties. To determine the effect of Fab glycosylation on the affinity of an antibody for its target, any technique known to those of skill in the art can be used, such as an enzyme-linked immunosorbent assay (ELISA), or surface plasmon resonance (SPR). To determine the effect of Fab glycosylation on the half-life of an antibody, any technique known to those of skill in the art can be used, such as by measuring the level of radioactivity in blood or an organ (e.g., the eye) in a subject administered a radiolabeled antibody. To determine the effect of Fab glycosylation on antibody stability, such as the level of aggregation or protein unfolding, any technique known to those of skill in the art can be used, such as differential scanning calorimetry (DSC), high performance liquid chromatography (HPLC), such as size exclusion high performance liquid chromatography (SEC-HPLC), capillary electrophoresis, mass spectrometry, or turbidity measurement. The HuGlyFabVEGFi transgene provided herein results in the production of antigen-binding fragments that are glycosylated at non-standard sites at 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more. In certain embodiments, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more of the antigen-binding fragments from a population of antigen-binding fragments are glycosylated at non-standard sites. In certain embodiments, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more of the non-standard sites are glycosylated. In certain embodiments, glycosylation of the antigen-binding fragments at these non-standard sites is 25%, 50%, 100%, 200%, 300%, 400%, 500%, or more than that of the glycosylation of these non-standard sites in antigen-binding fragments produced in HEK293 cells.

[0161] The presence of sialic acid on HuGlyFabVEGFi used in the methods described herein can affect the clearance rate of HuGlyFabVEGFi, e.g., the clearance rate from the vitreous humor. Thus, the sialic acid pattern of HuGlyFabVEGFi can be used to generate a therapeutic agent having an optimized clearance rate. Methods for assessing the clearance rate of antigen-binding fragments are known in the art. See, e.g., Huang et al., 2006, Anal. Biochem. 349:197-207.

[0162] In another specific embodiment, an advantage conferred by N-glycosylation is a reduction in aggregation. Occupied N-glycosylation sites can shield amino acid residues that are prone to aggregation, resulting in a decrease in aggregation. Such N-glycosylation sites can be inherent to the antigen-binding fragment used herein or can be artificially created in the antigen-binding fragment used herein and, when expressed, e.g., when expressed in retinal cells, result in a less aggregation-prone HuGlyFabVEGFi. Methods for assessing antibody aggregation are known in the art. See, e.g., Courtois et al., 2016, mAbs 8:99-112, which is hereby incorporated by reference in its entirety.

[0163] In another specific embodiment, an advantage conferred by N-glycosylation is a reduction in immunogenicity. Such N-glycosylation sites can be inherent to the antigen-binding fragment used herein or can be artificially created in the antigen-binding fragment used herein and, when expressed, e.g., when expressed in retinal cells, result in a HuGlyFabVEGFi that is less immunogenic.

[0164] In another specific embodiment, the advantage conferred by N-glycosylation is protein stability. N-glycosylation of proteins is well known to confer stability to the protein, and methods for assessing the stability of proteins due to N-glycosylation are known in the art. See, for example, the literature of Sola and Griebenow, 2009, J Pharm Sci., 98(4): 1223-1245.

[0165] In another specific embodiment, the advantage conferred by N-glycosylation is a change in binding affinity. The presence of N-glycosylation sites in the variable domains of antibodies is known in the art to be able to increase the affinity of the antibody for its antigen. See, for example, the literature of Bovenkamp et al., 2016, J. Immunol. 196:1435-1441. Assays for measuring the binding affinity of antibodies are known in the art. See, for example, the literature of Wright et al., 1991, EMBO J. 10:2717-2723; and the literature of Leibiger et al., 1999, Biochem. J. 338:529-538.

[0166] (5.1.2 Tyrosine Sulfation) Tyrosine sulfation occurs at Y residues that have glutamic acid (E) or aspartic acid (D) within positions +5 to -5 of tyrosine (Y), where the -1 position of Y is a neutral or acidic charged amino acid, but not a basic amino acid that inactivates sulfation, such as arginine (R), lysine (K), or histidine (H). Surprisingly, anti-VEGF antigen-binding fragments for use according to the methods described herein, such as ranibizumab, contain tyrosine sulfation sites (see Figure 1). Accordingly, the methods described herein include the use of anti-VEGF antigen-binding fragments that contain at least one tyrosine sulfation site, such as HuPTMFabVEGFi, and such anti-VEGF antigen-binding fragments can be tyrosine-sulfated when expressed in retinal cells.

[0167] Importantly, tyrosine-sulfated antigen-binding fragments, such as ranibizumab, cannot be produced in Escherichia coli, which do not naturally possess the enzymes required for tyrosine sulfation. Furthermore, CHO cells have insufficient tyrosine sulfation - the cells are not secretory and have limited capacity for post-translational tyrosine sulfation. See, for example, Mikkelsen and Ezban, 1991, Biochemistry 30: 1533-1537. Advantageously, the methods provided herein require the expression of anti-VEGF antigen-binding fragments, such as HuPTMFabVEGFi, such as ranibizumab, in retinal cells that are secretory and actually have the ability to tyrosine sulfate. See Kanan et al., 2009, Exp. Eye Res. 89: 559-567 and Kanan and Al-Ubaidi, 2015, Exp. Eye Res. 133: 126-131, which report the production of tyrosine-sulfated glycoproteins secreted by retinal cells.

[0168] Tyrosine sulfation is advantageous for several reasons. For example, tyrosine sulfation of the antigen-binding fragment of a therapeutic antibody to a target has been shown to dramatically increase the binding affinity and activity to the antigen. See, for example, Loos et al., 2015, PNAS 112: 12675-12680, and Choe et al., 2003, Cell 114: 161-170. Assays for detecting tyrosine sulfation are known in the art. See, for example, Yang et al., 2015, Molecules 20:2138-2164.

[0169] (5.1.3 O-glycosylation) O-glycosylation involves the addition of N-acetyl-galactosamine to serine or threonine residues by an enzyme. Amino acid residues present in the hinge region of an antibody have been shown to be O-glycosylated. In certain embodiments, an anti-VEGF antigen-binding fragment used according to the methods described herein, e.g., ranibizumab, includes all or a portion of its hinge region and can thus be O-glycosylated when expressed in human retinal cells. The potential for O-glycosylation confers an additional advantage to the HuPTMFabVEGFi provided herein, e.g., HuGlyFabVEGFi, as compared to antigen-binding fragments produced in E. coli, for example, because E. coli do not naturally contain a mechanism corresponding to that used in human O-glycosylation (instead, O-glycosylation in E. coli has only been shown when the bacteria are engineered to contain a specific O-glycosylation machinery. See, for example, Faridmoayer et al., 2007, J. Bacteriol. 189:8088-8098). Because they carry a glycan, O-glycosylated HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, share advantageous properties with N-glycosylated HuGlyFabVEGFi (discussed above).

[0170] (5.2 Constructs and Formulations) For use in the present method, provided herein is a viral vector or other DNA expression construct encoding an anti-VEGF antigen-binding fragment or a hyperglycosylated derivative of an anti-VEGF antigen-binding fragment. The viral vectors and other DNA expression constructs provided herein include any suitable method for delivering a transgene to a target cell (e.g., a retinal pigment epithelial cell). Means for delivering a transgene include viral vectors, liposomes, other lipid-containing complexes, other polymeric complexes, synthetically modified mRNA, unmodified mRNA, small molecules, non-bioactive molecules (e.g., gold particles), polymeric molecules (e.g., dendrimers), naked DNA, plasmids, phages, transposons, cosmids, or episomes. In some embodiments, the vector is a targeted vector, e.g., a vector targeted to retinal pigment epithelial cells.

[0171] In some embodiments, the present disclosure provides a nucleic acid for use, wherein the nucleic acid encodes a HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, operably linked to a promoter selected from the group consisting of a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, an MMT promoter, an EF-1α promoter, a UB6 promoter, a chicken β-actin promoter, a CAG promoter, an RPE65 promoter, and an opsin promoter.

[0172] In certain embodiments, provided herein is a recombinant vector comprising one or more nucleic acids (e.g., polynucleotides). The nucleic acid can include DNA, RNA, or a combination of DNA and RNA. In certain embodiments, the DNA includes one or more of the sequences selected from the group consisting of a promoter sequence, a sequence of a gene of interest (transgene, e.g., an anti-VEGF antigen-binding fragment), an untranslated region, and a termination sequence. In certain embodiments, the viral vector provided herein includes a promoter operably linked to a gene of interest.

[0173] In certain embodiments, the nucleic acids (e.g., polynucleotides) and nucleic acid sequences disclosed herein can be codon-optimized by any codon-optimization technique known to those of skill in the art (see, e.g., the review by Quax et al., 2015, Mol Cell 59:149-161).

[0174] In a specific embodiment, the construct described herein comprises the following components: (1) an AAV2 inverted terminal repeat adjacent to the expression cassette; (2) control elements comprising a) a CB7 promoter comprising a CMV enhancer / chicken β-actin promoter, b) a chicken β-actin intron, and c) a rabbit β-globin polyA signal; and (3) nucleic acid sequences encoding the heavy and light chains of an anti-VEGF antigen-binding fragment separated by a self-cleaving 2A peptide (F2A) linker that ensures expression of equal amounts of the heavy and light chain polypeptides.

[0175] (5.2.1 mRNA) In certain embodiments, the vectors provided herein are modified mRNAs encoding a target gene (e.g., a transgene, e.g., an anti-VEGF antigen-binding fragment moiety). Synthesis of modified and unmodified mRNAs for delivery of a transgene to retinal pigment epithelial cells is taught, for example, in Hansson et al., J. Biol. Chem., 2015, 290(9):5661-5672, which is hereby incorporated by reference in its entirety. In certain embodiments, provided herein is a modified mRNA encoding an anti-VEGF antigen-binding fragment moiety.

[0176] (5.2.2 Viral vector) Viral vectors include adenovirus, adeno-associated virus (AAV, e.g., AAV8), lentivirus, helper-dependent adenovirus, herpes simplex virus, poxvirus, hemagglutinin virus of Japan (HVJ), alphavirus, vaccinia virus, and retroviral vectors. Retroviral vectors include mouse leukemia virus (MLV) and human immunodeficiency virus (HIV)-based vectors. Alphavirus vectors include Semliki Forest virus (SFV) and Sindbis virus (SIN). In certain embodiments, the viral vectors provided herein are recombinant viral vectors. In certain embodiments, the viral vectors provided herein are modified to be replication-deficient in humans. In certain embodiments, the viral vector is a hybrid vector, e.g., an AAV vector encapsulated in a "helperless" adenovirus vector. In certain embodiments, provided herein is a viral vector comprising a viral capsid from a first virus and a viral envelope protein from a second virus. In a specific embodiment, the second virus is vesicular stomatitis virus (VSV). In a more specific embodiment, the envelope protein is the VSV-G protein.

[0177] In certain embodiments, the viral vectors provided herein are HIV-based viral vectors. In certain embodiments, the HIV-based vectors provided herein comprise at least two polynucleotides, wherein the gag and pol genes are derived from the HIV genome and the env gene is derived from another virus.

[0178] In certain embodiments, the viral vectors provided herein are herpes simplex virus-based viral vectors. In certain embodiments, the herpes simplex virus-based vectors provided herein do not contain one or more immediate early (IE) genes and are thus modified to be non-cytopathic.

[0179] In certain embodiments, the viral vectors provided herein are MLV-based viral vectors. In certain embodiments, the MLV-based vectors provided herein contain up to 8 kb of heterologous DNA in place of viral genes.

[0180] In certain embodiments, the viral vectors provided herein are lentivirus-based viral vectors. In certain embodiments, the lentiviral vectors provided herein are derived from human lentiviruses. In certain embodiments, the lentiviral vectors provided herein are derived from non-human lentiviruses. In certain embodiments, the lentiviral vectors provided herein are packaged in a lentiviral capsid. In certain embodiments, the lentiviral vectors provided herein contain one or more of the following elements: long terminal repeats, primer binding sites, polypurine tracts, att sites, and capsid-forming sites.

[0181] In certain embodiments, the viral vectors provided herein are alphavirus-based viral vectors. In certain embodiments, the alphavirus vectors provided herein are recombinant replication-deficient alphaviruses. In certain embodiments, the alphavirus replicon in the alphavirus vectors provided herein is targeted to specific cell types by presenting a functional heterologous ligand on the virion surface.

[0182] In certain embodiments, the viral vectors provided herein are AAV-based viral vectors. In preferred embodiments, the viral vectors provided herein are AAV8-based viral vectors. In certain embodiments, the AAV8-based viral vectors provided herein retain tropism for retinal cells. In certain embodiments, the AAV-based vectors provided herein encode the AAV rep gene (required for replication) and / or the AAV cap gene (required for synthesis of the capsid protein). Multiple AAV serotypes have been identified. In certain embodiments, the AAV-based vectors provided herein comprise components derived from one or more serotypes of AAV. In certain embodiments, the AAV-based vectors provided herein comprise capsid components derived from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAVrh10. In preferred embodiments, the AAV-based vectors provided herein comprise components derived from one or more of the AAV8, AAV9, AAV10, AAV11, or AAVrh10 serotypes.

[0183] Provided in certain embodiments is an AAV8 vector comprising a viral genome comprising an expression cassette for the expression of a viral capsid having an amino acid sequence of a transgene and an AAV8 capsid protein under the control of a regulatory element and adjacent to an ITR, or having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the amino acid sequence of the AAV8 capsid protein (SEQ ID NO: 48) while retaining the biological function of the AAV8 capsid. In certain embodiments, the encoded AAV8 capsid has an amino acid sequence of SEQ ID NO: 48 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions and retaining the biological function of the AAV8 capsid. FIG. 18 provides a comparative alignment of the amino acid sequences of capsid proteins of different AAV serotypes having potential amino acids that can be substituted at specific positions in the aligned sequences based on a comparison of columns labeled SUBS. Thus, in specific embodiments, the AAV8 vector comprises an AAV8 capsid variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions identified in the SUBS column of FIG. 18 that are not present at that position in the native AAV8 sequence.

[0184] In certain embodiments, the AAV used in the methods described herein is Anc80 or Anc80L65 as described in Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety. In certain embodiments, the AAV used in the methods described herein includes one of the following amino acid insertions: LGETTRP or LALGETTRP as described in U.S. Patent Nos. 9,193,956; 9,458,517; and 9,587,282 and U.S. Patent Application Publication No. 2016 / 0376323, each of which is incorporated by reference in its entirety herein. In certain embodiments, the AAV used in the methods described herein is AAV.7m8 as described in U.S. Patent Nos. 9,193,956; 9,458,517; and 9,587,282 and U.S. Patent Application Publication No. 2016 / 0376323, each of which is incorporated by reference in its entirety herein. In certain embodiments, the AAV used in the methods described herein is any AAV disclosed in U.S. Patent No. 9,585,971, such as AAV-PHP.B. In certain embodiments, the AAV used in the methods described herein is an AAV disclosed in any of the following patents and patent applications, each of which is incorporated by reference in its entirety herein: U.S. Patent Nos. 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9,458,517; and 9,587,282, U.S. Patent Application Publication Nos. 2015 / 0374803; 2015 / 0126588; 2017 / 0067908; 2013 / 0224836; 2016 / 0215024; 2017 / 0051257; and International Patent Applications PCT / US2015 / 034799; PCT / EP2015 / 053335.

[0185] AAV8-based viral vectors are used in some of the methods described herein. The nucleic acid sequences of AAV-based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in U.S. Patent No. 7,282,199 B2, U.S. Patent No. 7,790,449 B2, U.S. Patent No. 8,318,480 B2, U.S. Patent No. 8,962,332 B2, and International Patent Application PCT / EP2014 / 076466, each of which is incorporated herein by reference in its entirety. In one aspect, provided herein is an AAV (e.g., AAV8)-based viral vector encoding a transgene (e.g., an anti-VEGF antigen-binding fragment). In a specific embodiment, provided herein is an AAV8-based viral vector encoding an anti-VEGF antigen-binding fragment. In a more specific embodiment, provided herein is an AAV8-based viral vector encoding ranibizumab.

[0186] In certain embodiments, single-stranded AAV (ssAAV) can be used as described above. In certain embodiments, self-complementary vectors, such as scAAV, can be used (see, for example, Wu, 2007, Human Gene Therapy, 18(2):171-82; McCarty et al., 2001, Gene Therapy, Vol 8, Number 16, Pages 1248-1254; and U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety).

[0187] In one embodiment, the viral vector used in the methods described herein is an adenovirus-based viral vector. A recombinant adenoviral vector can be used to transfer into an anti-VEGF antigen-binding fragment. The recombinant adenovirus can be a first-generation vector that has an E1 deletion, may or may not have an E3 deletion, and has an expression cassette inserted into either deletion region. The recombinant adenovirus can be a second-generation vector that includes a complete or partial deletion of the E2 and E4 regions. The helper-dependent adenovirus retains only the adenovirus inverted terminal repeats and the packaging signal (φ). The transgene may or may not have a stuffer sequence inserted between the packaging signal and the 3' ITR to maintain the genome at a wild-type size of approximately 36 kb. An exemplary protocol for producing an adenoviral vector can be found in Alba et al., 2005, "Gutless adenovirus: last generation adenovirus for gene therapy", Gene Therapy 12:S18-S27, which is incorporated herein by reference in its entirety.

[0188] In one embodiment, the viral vector used in the methods described herein is a lentivirus-based vector. A recombinant lentiviral vector can be used to transfer into an anti-VEGF antigen-binding fragment. A construct can be made using four plasmids: a plasmid containing the Gag / pol sequence, a plasmid containing the Rev sequence, a plasmid containing the envelope protein (i.e., VSV-G), and a Cis plasmid containing the packaging element and the anti-VEGF antigen-binding fragment gene.

[0189] For the production of lentiviral vectors, four plasmids are co-transfected into cells (i.e., HEK293 cells), where polyethyleneimine or calcium phosphate can be used as transfection agents, in particular. Subsequently, the lentivirus is harvested from the supernatant (since the lentivirus needs to bud from the cells to become active, cell harvesting is not required / should not be done). The supernatant is filtered (0.45 μm), and then magnesium chloride and benzonase are added. Further downstream processes can vary, and the use of TFF and column chromatography is the most GMP-compliant process. In other processes, ultracentrifugation is used, with or without column chromatography. Exemplary protocols for the production of lentiviral vectors can be found in Lesch et al., 2011, "Production and purification of lentiviral vector generated in 293T suspension cells with baculoviral vectors", Gene Therapy 18:531-538, and Ausubel et al., 2012, "Production of CGMP-Grade Lentiviral Vectors", Bioprocess Int. 10(2):32-43, both of which are incorporated herein by reference in their entirety.

[0190] In a specific embodiment, the vector for use in the methods described herein is a vector encoding an anti-VEGF antigen-binding fragment (e.g., ranibizumab) such that when the vector is introduced into a relevant cell (e.g., in vivo or in vitro retinal cells), a variant of the glycosylated and / or tyrosine-sulfated anti-VEGF antigen-binding fragment is expressed by the cell. In a specific embodiment, the expressed anti-VEGF antigen-binding fragment comprises a glycosylation and / or tyrosine-sulfation pattern as described in Section 5.1 above.

[0191] (5.2.3 Promoters and Modulators of Gene Expression) In certain embodiments, the vectors provided herein include components that regulate gene delivery or gene expression (e.g., "expression control elements"). In certain embodiments, the vectors provided herein include components that regulate gene expression. In certain embodiments, the vectors provided herein include components that affect binding or targeting to cells. In certain embodiments, the vectors provided herein include components that affect the intracellular localization of polynucleotides (e.g., transgenes) after uptake. In certain embodiments, the vectors provided herein include components that can be used as detectable or selectable markers, for example, to detect or select cells that have taken up a polynucleotide.

[0192] In certain embodiments, the viral vectors provided herein include one or more promoters. In certain embodiments, the promoter is a constitutive promoter. In certain embodiments, the promoter is an inducible promoter. An inducible promoter may be preferred that can switch the on and off of transgene expression as desired for therapeutic efficacy. Such promoters include, for example, hypoxia-inducible promoters as well as drug-inducible promoters, such as promoters induced by rapamycin and related agents. Hypoxia-inducible promoters include promoters having HIF binding sites. For example, for teachings of hypoxia-inducible promoters, see Schodel et al., 2011, Blood 117(23):e207-e217 and Kenneth and Rocha, 2008, Biochem J. 414:19-29, each of which is incorporated by reference. Further, hypoxia-inducible promoters that may be used in the construct include the erythropoietin promoter and the N-WASP promoter (for teachings of hypoxia-inducible promoters, see Tsuchiya, 1993, J. Biochem. 113:395 regarding the disclosure of the erythropoietin promoter, and Salvi, 2017, Biochemistry and Biophysics Reports 9:13-21 regarding the disclosure of the N-WASP promoter, both of which are incorporated by reference). Alternatively, the construct may include a drug-inducible promoter, such as a promoter that can be induced by administration of rapamycin and related analogs (for example, for its disclosure of drug-inducible promoters, see International Patent Application Publications WO94 / 18317, WO 96 / 20951, WO 96 / 41865, WO 99 / 10508, WO 99 / 10510, WO 99 / 36553, and WO 99 / 41258, and International Patent US 7,067,526 (disclosing rapamycin analogs), each of which is incorporated by reference herein). In certain embodiments, the promoter is a hypoxia-inducible promoter.In certain embodiments, the promoter comprises a hypoxia-inducible factor (HIF) binding site. In certain embodiments, the promoter comprises a HIF-1α binding site. In certain embodiments, the promoter comprises a HIF-2α binding site. In certain embodiments, the HIF binding site comprises an RCGTG motif. For details regarding the location and sequence of the HIF binding site, see, for example, Schodel et al., Blood, 2011, 117(23):e207-e217, which is incorporated herein by reference in its entirety. In certain embodiments, the promoter comprises a binding site for a hypoxia-inducible transcription factor other than the HIF transcription factor. In certain embodiments, the viral vectors provided herein comprise one or more IRES sites that are preferentially translated under hypoxic conditions. For teachings regarding hypoxia-inducible gene expression and factors involved therein, see, for example, Kenneth and Rocha, Biochem J., 2008, 414:19-29, which is incorporated herein by reference in its entirety.

[0193] In certain embodiments, the promoter is the CB7 promoter (see Dinculescu et al., 2005, Hum Gene Ther 16: 649-663, which is hereby incorporated by reference in its entirety). In some embodiments, the CB7 promoter includes other expression control elements that enhance the expression of the transgene driven by the vector. In certain embodiments, other expression control elements include the chicken β-actin intron and / or the rabbit β-globin polyA signal. In certain embodiments, the promoter includes a TATA box. In certain embodiments, the promoter includes one or more elements. In certain embodiments, one or more promoter elements can be inverted or moved relative to each other. In certain embodiments, the elements of the promoter are arranged to function cooperatively. In certain embodiments, the elements of the promoter are arranged to function independently. In certain embodiments, the viral vectors provided herein include one or more promoters selected from the group consisting of the CMV immediate early gene promoter, the SV40 early promoter, the long terminal repeat of Rous sarcoma virus (RS), and the rat insulin promoter. In certain embodiments, the vectors provided herein include one or more long terminal repeat (LTR) promoters selected from the group consisting of AAV, MLV, MMTV, SV40, RSV, HIV-1, and HIV-2 LTR. In certain embodiments, the vectors provided herein include one or more tissue-specific promoters (e.g., a retinal pigment epithelial cell-specific promoter). In certain embodiments, the viral vectors provided herein include the RPE65 promoter. In certain embodiments, the vectors provided herein include the VMD2 promoter.

[0194] In certain embodiments, the viral vectors provided herein include one or more regulatory elements other than a promoter. In certain embodiments, the viral vectors provided herein include an enhancer. In certain embodiments, the viral vectors provided herein include a repressor. In certain embodiments, the viral vectors provided herein include an intron or a chimeric intron. In certain embodiments, the viral vectors provided herein include a polyadenylation sequence.

[0195] (5.2.4 Signal Peptide) In certain embodiments, the vectors provided herein include components that regulate the delivery of a protein. In certain embodiments, the viral vectors provided herein include one or more signal peptides. A signal peptide may also be referred to herein as a “leader sequence” or a “leader peptide.” In certain embodiments, the signal peptide enables the transgene product (e.g., an anti-VEGF antigen-binding fragment moiety) to achieve proper packaging (e.g., glycosylation) within the cell. In certain embodiments, the signal peptide enables the transgene product (e.g., an anti-VEGF antigen-binding fragment moiety) to achieve proper localization within the cell. In certain embodiments, the signal peptide enables the transgene product (e.g., an anti-VEGF antigen-binding fragment moiety) to achieve secretion from the cell. Examples of signal peptides for use in connection with the vectors and transgenes provided herein can be found in Table 1. Table 1. Signal Peptides for Use with the Vectors Provided herein [Table 1]

[0196] (5.2.5 Polycistronic Message - IRES and F2A Linker) Internal ribosome entry site. A single construct can be modified to encode both a heavy chain and a light chain separated by a cleavable linker or an IRES such that the separate heavy and light chain polypeptides are expressed by the transduced cell. In certain embodiments, the viral vectors provided herein provide a polycistronic (e.g., bicistronic) message. For example, the viral construct can encode a heavy chain and a light chain separated by an internal ribosome entry site (IRES) element (see, e.g., Gurtu et al., 1996, Biochem. Biophys. Res. Comm. 229(1):295-8, which is incorporated herein by reference in its entirety, for an example of the use of an IRES element to make a bicistronic vector). The IRES element bypasses the ribosome scanning model and initiates translation at an internal site. The use of IRES in AAV is described, for example, in Furling et al., 2001, Gene Ther 8(11): 854-73, which is incorporated herein by reference in its entirety. In certain embodiments, the bicistronic message is included within the viral vector with limitations regarding the size of the polynucleotides therein. In certain embodiments, the bicistronic message is included within an AAV virus-based vector (e.g., an AAV8-based vector).

[0197] Furin-F2A linker. In other embodiments, the viral vectors provided herein encode a heavy chain and a light chain separated by a cleavable linker, such as a self-cleaving furin / F2A (F / F2A) linker (Fang et al., 2005, Nature Biotechnology 23: 584-590, and Fang, 2007, Mol Ther 15: 1153-9, each of which is incorporated herein by reference in its entirety).

[0198] For example, the furin-F2A linker is incorporated into the expression cassette to separate the heavy chain coding sequence from the light chain coding sequence, with the structure: Leader-heavy chain-Furin site-F2A site-Leader-light chain-polyA A construct having the same can be generated.

[0199] Amino acid sequence [Chemical formula] The F2A site having the same self-processes to effect "cleavage" between the last G amino acid residue and the P amino acid residue. Further linkers that can be used include: [Chemical formula] but are not limited thereto.

[0200] When the ribosome encounters the F2A sequence in the open reading frame, the peptide bond is skipped, resulting in termination of translation or continuation of translation of the downstream sequence (light chain). This self-processing sequence generates a series of additional amino acids at the C-terminus of the heavy chain. However, such additional amino acids are then cleaved by the host cell's furin at the furin site located immediately upstream of the F2A site and downstream of the heavy chain sequence, and further cleaved by carboxypeptidase. The resulting heavy chain may or may not have 1, 2, 3, or more additional amino acids at the C-terminus, depending on the sequence of the furin linker used and the carboxypeptidase that cleaves the linker in vivo (see, e.g., Fang et al., 17 April 2005, Nature Biotechnol. Advance online publication; Fang et al., 2007, Molecular Therapy 15(6):1153-1159; Luke, 2012, Innovations in Biotechnology, Chapter 8, 161-186). Furin linkers that can be used include a run of four basic amino acids, such as RKRR, RRRR, RRKR, or RKKR. Once this linker is cleaved by carboxypeptidase, additional amino acids may remain such that 0, 1, 2, 3, or 4 additional amino acids, such as R, RR, RK, RKR, RRR, RRK, RKK, RKRR, RRRR, RRKR, or RKKR, may remain on the C-terminus of the heavy chain. In certain embodiments, no additional amino acids remain once the linker is cleaved by carboxypeptidase. In certain embodiments, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, or 20%, or less, but more than 0%, of a population of antibodies, such as antigen-binding fragments, produced by a construct for use in the methods described herein have 1, 2, 3, or 4 additional amino acids remaining on the C-terminus of the heavy chain after cleavage.In certain embodiments, 0.5 to 1%, 0.5% to 2%, 0.5% to 3%, 0.5% to 4%, 0.5% to 5%, 0.5% to 10%, 0.5% to 20%, 1% to 2%, 1% to 3%, 1% to 4%, 1% to 5%, 1% to 10%, 1% to 20%, 2% to 3%, 2% to 4%, 2% to 5%, 2% to 10%, 2% to 20%, 3% to 4%, 3% to 5%, 3% to 10%, 3% to 20%, 4% to 5%, 4% to 10%, 4% to 20%, 5% to 10%, 5% to 20%, or 10% to 20% of a population of antibodies, such as antigen-binding fragments, produced by a construct for use in the methods described herein have 1, 2, 3, or 4 amino acids remaining on the C-terminus of the heavy chain after cleavage. In certain embodiments, the hinge linker has the sequence R-X-K / R-R such that the additional amino acids on the C-terminus of the heavy chain are R, RX, RXK, RXR, RXKR, or RXRR, where X is any amino acid, such as alanine (A). In certain embodiments, the additional amino acids may not remain on the C-terminus of the heavy chain.

[0201] In certain embodiments, the expression cassettes described herein are included within viral vectors with size restrictions on the polynucleotides therein. In certain embodiments, the expression cassette is included within an AAV virus-based vector (e.g., an AAV8-based vector).

[0202] (5.2.6 Untranslated Regions) In certain embodiments, the viral vectors provided herein include one or more untranslated regions (UTRs), such as 3' and / or 5' UTRs. In certain embodiments, the UTRs are optimized for the desired level of protein expression. In certain embodiments, the UTRs are optimized for the half-life of the transgene mRNA. In certain embodiments, the UTRs are optimized for the stability of the transgene mRNA. In certain embodiments, the UTRs are optimized for the secondary structure of the transgene mRNA.

[0203] (5.2.7 Inverted Terminal Repeats) In certain embodiments, the viral vectors provided herein include one or more inverted terminal repeat (ITR) sequences. The ITR sequences can be used to package a recombinant gene expression cassette into the virion of the viral vector. In certain embodiments, the ITRs are derived from AAV, such as AAV8 or AAV2 (see, e.g., Yan et al., 2005, J. Virol., 79(1):364-379; U.S. Patent No. 7,282,199 B2, U.S. Patent No. 7,790,449 B2, U.S. Patent No. 8,318,480 B2, U.S. Patent No. 8,962,332 B2, and International Patent Application PCT / EP2014 / 076466, each of which is incorporated herein by reference in its entirety).

[0204] (5.2.8 Transgene) Examples of HuPTMFabVEGFi encoded by the transgene, such as HuGlyFabVEGFi, include antigen-binding fragments of antibodies that bind VEGF, such as bevacizumab; anti-VEGF Fab moieties, such as ranibizumab; or Fab moieties of such bevacizumab or ranibizumab modified to include additional glycosylation sites on the Fab domain, but are not limited thereto (see, e.g., Courtois et al., 2016, mAbs 8: 99-112, which is incorporated herein by reference in its entirety for the description of derivatives of bevacizumab that are highly glycosylated on the Fab domain of the full-length antibody).

[0205] In certain embodiments, the vectors provided herein encode an anti-VEGF antigen-binding fragment transgene. In specific embodiments, the anti-VEGF antigen-binding fragment transgene is controlled by appropriate expression control elements for expression in retinal cells. In certain embodiments, the anti-VEGF antigen-binding fragment transgene comprises the Fab portion of bevacizumab comprising the light and heavy chain cDNA sequences (SEQ ID NOs: 10 and 11, respectively). In certain embodiments, the anti-VEGF antigen-binding fragment transgene comprises the ranibizumab light and heavy chain cDNA sequences (SEQ ID NOs: 12 and 13, respectively). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes a bevacizumab Fab comprising a light chain and a heavy chain having amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequences set forth in SEQ ID NOs: 3 and 4, respectively. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 3. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 3 and a heavy chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 4. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes a highly glycosylated ranibizumab comprising a light chain and a heavy chain having amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequences set forth in SEQ ID NOs: 1 and 2, respectively.In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 1. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 2. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 1 and a heavy chain having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 2.

[0206] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes a highly glycosylated bevacizumab Fab comprising a light chain and a heavy chain of SEQ ID NOs: 3 and 4, having one or more of the following mutations: L118N (heavy chain), E195N (light chain), or Q160N or Q160S (light chain). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes a highly glycosylated ranibizumab comprising a light chain and a heavy chain of SEQ ID NOs: 1 and 2, having one or more of the following mutations: L118N (heavy chain), E195N (light chain), or Q160N or Q160S (light chain). The sequence of the antigen-binding fragment transgene cDNA can be found, for example, in Table 2. In certain embodiments, the sequence of the antigen-binding fragment transgene cDNA is obtained by replacing the signal sequence of SEQ ID NOs: 10 and 11 or SEQ ID NOs: 12 and 13 with one or more of the signal sequences listed in Table 1.

[0207] In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment and comprises the nucleotide sequences of the six bevacizumab CDRs. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment and comprises the nucleotide sequences of the six ranibizumab CDRs. In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising ranibizumab heavy chain CDR1-3 (SEQ ID NOs: 20, 18, and 21). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising ranibizumab light chain CDR1-3 (SEQ ID NOs: 14-16). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising bevacizumab heavy chain CDR1-3 (SEQ ID NOs: 17-19). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a light chain variable region comprising bevacizumab light chain CDR1-3 (SEQ ID NOs: 14-16). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising ranibizumab heavy chain CDR1-3 (SEQ ID NOs: 20, 18, and 21) and a light chain variable region comprising ranibizumab light chain CDR1-3 (SEQ ID NOs: 14-16). In certain embodiments, the anti-VEGF antigen-binding fragment transgene encodes an antigen-binding fragment comprising a heavy chain variable region comprising bevacizumab heavy chain CDR1-3 (SEQ ID NOs: 17-19) and a light chain variable region comprising bevacizumab light chain CDR1-3 (SEQ ID NOs: 14-16).

[0208] In certain...

Claims

**Claim 1** A pharmaceutical composition for treating neovascular age-related macular degeneration (nAMD) in a human subject, comprising an AAV8 expression vector encoding an anti-human vascular endothelial growth factor (hVEGF) antibody or an antigen-binding fragment thereof that immunospecifically binds to hVEGF, wherein the treatment comprises administering the expression vector into the suprachoroidal space of the eye of the human subject, and the anti-hVEGF antibody or the antigen-binding fragment thereof that immunospecifically binds to hVEGF comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, and a light chain comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

3. **Claim 2** The pharmaceutical composition according to claim 1, wherein the administration is by injecting the expression vector into the suprachoroidal space using a suprachoroidal drug delivery device, optionally wherein the suprachoroidal drug delivery device is a microinjector. **Claim 3** By the administration, a therapeutically effective amount of the anti-hVEGF antibody or the antigen-binding fragment thereof that immunospecifically binds to hVEGF is delivered to the retina of the human subject, and optionally the therapeutically effective amount of the anti-hVEGF antibody or the antigen-binding fragment thereof that immunospecifically binds to hVEGF is: (i) human retinal cells of the human subject; or (ii) produced by human photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, and / or retinal pigment epithelial cells of the outer limiting membrane of the human subject; Optionally, the human photoreceptor cells are cone cells and / or rod cells, or the retinal ganglion cells are midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia. The pharmaceutical composition according to claim 1 or claim 2. **Claim 4** The human subject has (i) ≤ 20 / 20 and ≥ 20 / 400; or (ii) ≤ 20 / 63 and ≥ 20 / 400 best corrected visual acuity (BCVA), and optionally the BCVA is the BCVA of the eye to be treated in the human subject. The pharmaceutical composition according to any one of claims 1, 2, or 3. **Claim 5** The antigen-binding fragment that immunospecifically binds to hVEGF is: (i) Fab; (ii) F(ab') 2 ; or (iii) single-chain variable domain (scFv) The pharmaceutical composition according to any one of claims 1, 2, 3, or 4. **Claim 6** The pharmaceutical composition according to any one of claims 1 to 5, wherein the anti-hVEGF antibody or its antigen-binding fragment that immunospecifically binds to hVEGF comprises CDR1 to 3 of the light chain of SEQ ID NOs: 14 to 16, and CDR1 to 3 of the heavy chain of SEQ ID NOs: 17 to 19 or SEQ ID NOs: 20, 18, and 21.

7. The second amino acid residue of the light chain CDR3 is (a) free of one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu); or (b) not acetylated, The pharmaceutical composition according to claim 6.

8. The pharmaceutical composition according to claim 6 or 7, wherein the 8th and 11th amino acid residues of the light chain CDR1 each possess one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu), and optionally the last amino acid residue of the heavy chain CDR1 is not acetylated.

9. The pharmaceutical composition according to claim 6 or 7, wherein the anti-hVEGF antibody or its antigen-binding fragment that immunospecifically binds to hVEGF comprises CDR1 of the heavy chain of SEQ ID NO: 20, and the last amino acid residue of the heavy chain CDR1 is free of one or more of the following chemical modifications: oxidation, acetylation, deamidation, and pyroglutamylation (pyroGlu).

10. The pharmaceutical composition according to claim 8 or 9, wherein the 9th amino acid residue of the heavy chain CDR1 possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamylation (pyroGlu), and the 3rd amino acid residue of the heavy chain CDR2 possesses one or more of the following chemical modifications: acetylation, deamidation, and pyroglutamylation (pyroGlu).

11. The AAV8 expression vector comprises an expression cassette adjacent to the AAV2 inverted terminal repeat (ITR), and the expression cassette (i) the CB7 promoter consisting of the chicken β-actin promoter and the CMV enhancer; (ii) the chicken β-actin intron; (iii) the heavy chain of the antigen-binding fragment that immunospecifically binds to hVEGF and comprises the amino acid sequence of SEQ ID NO: 2; a self-cleaving 2A peptide (F) / F2A linker; and the light chain of the antigen-binding fragment that immunospecifically binds to hVEGF and comprises the amino acid sequence of SEQ ID NO: 1 encoding nucleotide sequence; and (iv) the rabbit β-globin poly(A) signal The pharmaceutical composition according to any one of claims 1 to 10, comprising

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