Treatment of ocular diseases with fully human post-translationally modified Anti-VEGF fab

Abstract

To provide a method of treating a human subject diagnosed with neovascular age-related macular degeneration (nAMD).SOLUTION: Provided are compositions and methods for the delivery of the antigen-binding fragment of a fully human post-translationally modified monoclonal antibody (mAb) against VEGF, e.g., a fully human-glycosylated anti-VEGF antigen-binding fragment of an anti-VEGF mAb to the retina / vitreal humour in the eye(s) of subjects diagnosed with nAMD. Such antigen-binding fragments include a Fab, F(ab')2, or scFv (single-chain variable fragment) of an anti-VEGF mAb.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] No. 6,313,933, filed Apr. 15, 2016, each of which is incorporated herein by reference in its entirety. No. 62 / 323,285, filed on Jan. 5, 2017; and U.S. Provisional Application No. 62 / 442,802, filed on Jan. 5, 2017. No. 62 / 450,438, filed January 25, 2017, and U.S. Provisional Application No. 62 / 450,438, filed February 17, 2017. This application claims the benefit of U.S. Provisional Application No. 62 / 460,428, filed on Oct. 13, 2007.

[0002] (Reference to electronically submitted sequence listing) This application includes a file named "Sequence_Listing_12656-083- 228.TXT" and is included with the present application as a text file having a size of 21,394 bytes. The submitted sequence listing is incorporated by reference.

[0003] (1. Introduction) Post-translationally modified fully human (HuPTM) monoclonal antibodies ("mAbs") or vascular endothelial proliferation Antigen-binding fragment of a mAb against VEGF ("VEGF") - e.g., fully human glycosylated (HuGly) Anti-VEGF antigen-binding fragments, etc., can be used to treat eye diseases caused by increased angiogenesis, e.g. Neovascular age-related macular degeneration ("WAMD"), also known as wet age-related macular degeneration ("WAMD"), The eyes of human subjects diagnosed with age-related macular degeneration ("AMD"), age-related macular degeneration ("AMD"), and diabetic retinopathy ( DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Compositions and methods for delivering a retinal or vitreous humor of a subject to the method of the present invention are described. [Background technology]

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

[0005] "Wet" neovascular form of AMD (WAMD) is also known as neovascular age-related macular degeneration (nAMD). It is known that the retina reacts to various stimuli in and beneath the optic nerve retina, accounting for 15-20% of AMD cases. This abnormal vascular growth leads to the formation of leaky ducts and often bleeding. In AMD, visual function is severely impaired. Eventually, inflammation and scarring can lead to permanent loss of visual function in the affected retina. Cell death and scarring can result in severe loss of central vision and impaired ability to read, write, recognize faces, or navigate. Many patients are no longer able to maintain gainful employment or carry out daily activities. They report being unable to do what they want and experience reduced quality of life as a result (Mitchell, 2006).

[0006] Diabetic retinopathy is a diabetic eye complication that can manifest as microaneurysms, severe exudation, hemorrhage, and and non-proliferative forms of venous abnormalities, as well as neovascularization, preretinal or vitreous hemorrhage, and proliferative forms. Hyperglycemia induces microvascular changes in the retina, The patient may experience blurred vision, dark spots or blinking lights, and sudden loss of vision (Cai and McGin nis references, 2016).

[0007] Preventive treatments have shown little efficacy, and therapeutic strategies have focused primarily on treating neovascular lesions. Available treatments for nAMD include laser photocoagulation, photocoagulation with verteporfin, and Mechanical therapy and vascular endothelial growth factor ("VEGF") - involved in stimulating angiogenesis and a target for intervention Intravitreal (IV) administration of drugs aimed at binding and neutralizing cytokines that cause Such anti-VEGF drugs that are used include, for example, bevacizumab (CHO cell Ranibizumab, a humanized monoclonal antibody (mAb) against VEGF produced in human cells (Affinity-enhanced bevacizumab antibodies produced in the prokaryotic organism Escherichia coli (E. coli) aflibercept (human VEGF receptor fused to the Fc portion of human IgG1) a recombinant fusion protein consisting of the VEGF-binding region of the extracellular domain of There are also pegylated aptamers (single-stranded nucleic acid molecules) that bind to VEGF. Each of these therapies have some effect on best corrected visual acuity; however, these effects appear to be limited to the restoration of visual acuity and It is limited in its effectiveness and persistence.

[0008] IVT injections of anti-VEGF have proven effective in reducing leakage and sometimes reversing vision loss. However, these drugs are only effective for a short period of time and may not be effective for long periods of time. Repeated injections are often required, resulting in a significant therapeutic burden for patients. Long-term treatment with monthly ranibizumab or monthly / every 8 week aflibercept can slow the progression of vision loss and improve vision, whereas these treatments neither prevent recurrent neovascularization (Brown 2006; Rosenfeld 2006; Schmidt-Erfurth, 2014). Each must be re-administered to prevent disease progression. Repeated treatments may pose additional risks to the patient. This is inconvenient for both the patient and the treating physician. Summary of the Invention

[0009] (3. SUMMARY OF THEINVENTION) Antigen binding of a post-translationally modified fully human (HuPTM) monoclonal antibody (mAb) against VEGF A fully human glycosylated anti-VEGF antigen binding fragment of an anti-VEGF mAb ("HuPTMFabVEGFi"), e.g. The combined fragment ("HuGlyFabVEGFi") was used to treat eye diseases caused by increased angiogenesis, e.g. For example, the eye(s) of a patient (human subject) diagnosed with nAMD, also known as "wet" AMD. Compositions and methods for delivering such antigen-binding fragments to the retina / vitreous humor are described. The antibodies include Fab, F(ab')2, or scFv (single-chain variable fragment) antibodies of anti-VEGF mAbs (collectively referred to herein as "anti-VEGF antibodies"). In an alternative embodiment, a full length mAb can be used. Gene therapy - for example, the production of anti-VEGF antigen-binding fragments or mAbs (or hyperglycosylation Viral vectors or other DNA expression constructs encoding the glycosylated derivatives into the subretinal and / or intraretinal space within the eye(s) of a patient (human subject) diagnosed with nAMD. to continuously provide the eye with human PTMs, e.g., human glycosylated transgene products. Delivery can be accomplished by creating a permanent depot. The method is for use in a patient (human subject) diagnosed with AMD or diabetic retinopathy. It is also possible.

[0010] Herein, anti-human vascular endothelial growth factor (hVEGF) antibodies produced by human retinal cells are The present invention describes antibodies, such as anti-hVEGF antigen-binding fragments. Human VEGF (hVEGF) is produced by the VEGFA gene. An exemplary amino acid sequence for hVEGF is available under GenBank Accession No. An exemplary nucleic acid sequence for hVEGF can be found in GenBank Accession No. M32977. 1. can be found.

[0011] In one embodiment, the present invention relates to a method for treating a human retinal edema diagnosed with neovascular age-related macular degeneration (nAMD). A method of treating a subject, comprising administering to the retina of the human subject a therapeutically effective amount of a therapeutically effective amount of a human retinal cell-produced The method comprises delivering an anti-hVEGF antigen-binding fragment to a mammalian cell.

[0012] In one embodiment, provided herein is a method of treating a human subject diagnosed with nAMD, comprising: administering a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human photoreceptor cells to the retina of a human subject; The method includes delivering the

[0013] In one embodiment, provided herein is a method of treating a human subject diagnosed with nAMD, comprising: administering to the eye of a human subject a therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human retinal cells; The method includes delivering the

[0014] In one embodiment, provided herein is a method of treating a human subject diagnosed with nAMD, comprising: A therapeutically effective amount of an anti-hVEGF antigen-binding fragment produced by human photoreceptor cells is delivered to the eye of a human subject. The method is described, comprising:

[0015] In certain embodiments of the methods described herein, the antigen-binding fragment is selected from the group consisting 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. include.

[0016] In certain embodiments of the methods described herein, the antigen-binding fragment comprises a light chain CD4A fragment having a sequence identical to that of SEQ ID NOs: 14-16. R1 to R3, and heavy chain CDRs 1 to 3 of SEQ ID NOs: 17 to 19 or SEQ ID NOs: 20, 18, and 21.

[0017] In one embodiment, the present invention relates to a human subject diagnosed with neovascular age-related macular degeneration (nAMD). The present invention relates to a method of treating a subject's eye comprising administering to the eye of the subject a therapeutically effective amount of human retinal cells produced by the human retinal cells. Such methods are described, comprising delivering an anti-hVEGF antibody.

[0018] In one embodiment, provided herein is a method of treating a human subject diagnosed with nAMD, the method comprising: and delivering to the eye of the subject a therapeutically effective amount of an anti-hVEGF antibody produced in human photoreceptor cells. The method is described below.

[0019] In one embodiment, provided herein is a method of treating a human subject diagnosed with nAMD, the method comprising: The present invention aims to deliver a therapeutically effective amount of an anti-hVEGF antibody produced in human retinal cells to the retina of a subject. The method is described, comprising:

[0020] In one embodiment, provided herein is a method of treating a human subject diagnosed with nAMD, the method comprising: The method includes delivering a therapeutically effective amount of an anti-hVEGF antibody produced in human photoreceptor cells to the retina of a subject. The method will now be described.

[0021] In certain embodiments of the methods described herein, the antibody comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3. The present invention includes 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:2 or SEQ ID NO:4.

[0022] In certain embodiments of the methods described herein, the antibody comprises light chain CDRs 1-3 of SEQ ID NOs: 14-16; and heavy chain CDRs 1 to 3 of SEQ ID NOs: 17 to 19 or SEQ ID NOs: 20, 18, and 21.

[0023] In one embodiment, the present invention relates to a method for treating a human retinal edema diagnosed with neovascular age-related macular degeneration (nAMD). 1. A method of treating a subject, comprising administering to the eye of a human subject a therapeutically effective amount of a mAb against hVEGF, The methods are described, comprising delivering an antigen-binding fragment that comprises a sialylated glycan.

[0024] In one embodiment, provided herein is a method of treating a human subject diagnosed with nAMD, comprising: A method for delivering a therapeutically effective amount of a glycosylated antigen-binding fragment of a mAb against hVEGF to the eye of a human subject. wherein the antigen-binding fragment does not contain NeuGc.

[0025] In one embodiment, the term "nAMD" or "age-related macular degeneration (AMD)" is used herein to describe diabetic retinopathy. The method of claim 1, further comprising administering to a human subject a therapeutically effective amount of hVEGF to the subretinal space of the eye of the human subject. and administering to the subject an expression vector encoding an antigen-binding fragment of a mAb that binds the antigen. Expression of the fusion fragment from the expression vector in human immortalized retina-derived cells was found to be α2, The method is described above, in which the 6-sialylated

[0026] In one embodiment, the present invention relates to a method for treating a human subject diagnosed with nAMD or AMD or diabetic retinopathy. 20. A method of treating an elephant comprising administering to the subretinal space within the eye of said human subject an antigen-binding fragment against hVEGF. administering an expression vector encoding an antigen-binding fragment thereof, wherein expression of the antigen-binding fragment is in a human Upon expression from the expression vector in cells derived from dead retina, α2,6-sialylated and wherein the antigen-binding fragment does not contain NeuGc.

[0027] In one embodiment, provided herein is a method of treating a human subject diagnosed with nAMD, comprising: and administering to the subretinal space within the eye of a human subject a therapeutically effective amount of an antigen-binding fragment of a mAb against hVEGF. and administering a recombinant nucleotide expression vector that results in the expression of α2,6-sialylated glycans. The method further comprises forming a depot that releases the antigen-binding fragment comprising the antibody.

[0028] In one embodiment, provided herein is a method of treating a human subject diagnosed with nAMD, comprising: and administering to the subretinal space within the eye of a human subject a therapeutically effective amount of an antigen-binding fragment of a mAb against hVEGF. and administering a recombinant nucleotide expression vector to the depot which then releases the antigen-binding fragment. wherein the antigen-binding fragment is glycosylated, but not NeuGc The method is described below, which does not include:

[0029] In certain embodiments of the methods described herein, the antigen-binding fragment is selected from the group consisting 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. include.

[0030] In certain embodiments of the methods described herein, the antigen-binding fragment further comprises tyrosine sulfation. include.

[0031] In certain embodiments of the methods described herein, the antigen comprises α2,6-sialylated glycans. The production of the binding fragments was carried out by transfecting the recombinant nucleotide expression vector into PER.C6 or RPE cell lines in cell culture. This is confirmed by transducing the target gene.

[0032] In certain embodiments of the methods described herein, the antigen-binding fragment contains tyrosine sulfation. The production is carried out by transducing the recombinant nucleotide expression vector into PER.C6 or RPE cell lines in cell culture. This is confirmed by entering

[0033] In certain embodiments of the methods described herein, the vector comprises a hypoxia-inducible promoter. Yes.

[0034] In certain embodiments of the methods described herein, the antigen-binding fragment comprises a light chain CD4A fragment having a sequence identical to that of SEQ ID NOs: 14-16. R1 to R3, and heavy chain CDRs 1 to 3 of SEQ ID NOs: 17 to 19 or SEQ ID NOs: 20, 18, and 21.

[0035] In some embodiments of the methods described herein, the antigen-binding fragment transgene comprises a leader peptide. A leader peptide is herein defined as a signal peptide or a leader peptide. It may also be called an array.

[0036] In one embodiment, provided herein is a method of treating a human subject diagnosed with nAMD, comprising: and administering to the subretinal space within the eye of a human subject a therapeutically effective amount of an antigen-binding fragment of a mAb against hVEGF. and administering a recombinant nucleotide expression vector that results in the expression of α2,6-sialylated glycans. forming a depot that releases said antigen-binding fragment comprising said recombinant vector; When used to transduce PER.C6 or RPE cells in culture, The method also provides for the production of said antigen-binding fragments comprising 1,6-sialylated glycans. .

[0037] In one embodiment, provided herein is a method of treating a human subject diagnosed with nAMD, comprising: and administering to the subretinal space within the eye of a human subject a therapeutically effective amount of an antigen-binding fragment of a mAb against hVEGF. The recombinant nucleotide expression vector that results in glycosylated NeuG forming a depot that releases said antigen-binding fragment free of c; If the vector is used to transduce PER.C6 or RPE cells in culture, the cell culture resulting in the production of said antigen-binding fragment that is glycosylated but does not contain NeuGc in culture. The method will now be described.

[0038] In certain embodiments of the methods described herein, delivery to the eye comprises delivery to the retina, choroid, and / or ocular layer of the eye. or into the vitreous humor.

[0039] In certain embodiments of the methods described herein, the antigen-binding fragment has 1, 2, 3, or 4 amino acids at the C-terminus. The heavy chain contains two additional amino acids.

[0040] In certain embodiments of the methods described herein, the antigen-binding fragment comprises an additional amino acid at the C-terminus. The heavy chain does not include

[0041] In some embodiments of the methods described herein, a population of antigen-binding fragment molecules is produced, wherein wherein the antigen-binding fragment molecules comprise a heavy chain, and 5%, 10%, or 20% of the population of antigen-binding fragment molecules are It includes 1, 2, 3, or 4 additional amino acids at the C-terminus of the heavy chain.

[0042] Subjects receiving such gene therapy should be those who are responsive to anti-VEGF therapy. In certain embodiments, the method comprises the step of determining whether a patient has been diagnosed with nAMD and is responsive to treatment with an anti-VEGF antibody. In a more specific embodiment, the patient is treated with In one embodiment, the patient is responsive to treatment with an anti-VEGF antigen-binding fragment. It has been shown that patients respond to treatment with intravitreal injection of anti-VEGF antigen-binding fragments prior to treatment. In a specific embodiment, the patient has previously received LUCENTIS® (ranibizumab). , EYLEA® (aflibercept), and / or AVASTIN® (bevacizumab) and the LUCENTIS® (ranibizumab), EYLEA® ( ) (aflibercept), and / or AVASTIN® (bevacizumab) It has been found to react with

[0043] The subject to which such a viral vector or other DNA expression construct is delivered is the virus. Anti-hVEGF antigen encoded by a transgene in a vector or expression construct To determine reactivity, the anti-VEGF antigen-binding fragment transgene was The product (e.g., produced in a cell culture, a bioreactor, etc.) can be administered, e.g., by intravitreal injection. It can be administered directly to a subject.

[0044] The HuPTMFabVEGFi encoded by the transgene, e.g., HuGlyFabVEGFi, Antigen-binding fragments of antibodies that bind to hVEGF, such as, but not limited to, bevacizumab; anti-hVEGF F The ab portion, e.g., ranibizumab; or the Fab domain may contain additional glycosylation sites. The Fab portion of such bevacizumab or ranibizumab may be engineered to describes derivatives of bevacizumab that are hyperglycosylated on the Fab domain of the full-length antibody. See Courtois et al., 2016, mAb, which is incorporated by reference in its entirety. (see bs 8:99-112).

[0045] The recombinant vector used to deliver the transgene has affinity for human retinal cells or photoreceptor cells. Such vectors include non-replicating recombinant adeno-associated virus vectors. ("rAAV"), with viral vectors having an AAV8 capsid being particularly preferred. However, the vectors may include, but are not limited to, lentivirus vectors, vaccinia virus vectors, etc. These vectors include non-viral expression vectors, also called "naked DNA" constructs. Other viral vectors can be used, including those described herein. Preferably, HuPTMFabVEGFi, e.g. The uGlyFabVEGFi transgene may be coupled to suitable expression control elements, such as the CB7 promoter, to name a few. Motor (chicken β-actin promoter and CMV enhancer), RPE65 promoter or should be controlled by the opsin promoter and driven by a vector Other expression control elements that enhance expression of the transgene (e.g., chicken β-actin intron, minute virus of mice (MVM) intron, human factor IX intron (e.g., F IX truncated intron 1, β-globin splice donor / immunoglobulin Heavy chain splice acceptor intron, adenovirus splice donor / immunoglobulin Rinsplice acceptor intron, SV40 late splice donor / splice accessor (19S / 16S) intron and hybrid adenovirus splice donor / IgG Introns, such as splice acceptor introns, and polyA signals, e.g. Heron β-globin polyA signal, human growth hormone (hGH) polyA signal, SV40 late polyA signal signal, synthetic polyA (SPA) signal, and bovine growth hormone (bGH) polyA signal) See, e.g., Powell and Rivera-Soto, 2015, Discov. Med., 19(102):49-57. Please refer to.

[0046] The gene therapy construct is designed to express both the heavy and light chains. Specifically, the heavy and light chains should be expressed in approximately equal amounts; The heavy and light chains are expressed in an approximately 1:1 ratio of heavy to light chains. The coding sequences are sequenced such that the heavy and light chains are cleaved such that separate heavy and light chain polypeptides are expressed. In a single construct separated by a possible linker or IRES For example, specific examples of compounds that can be used in the methods and compositions provided herein include See Section 5.2.4 for leader sequences and Section 5.2.5 for specific IRES, 2A, and other linker sequences. See Section 5.2.5 for more information.

[0047] Pharmaceutical compositions suitable for subretinal and / or intraretinal administration include aqueous solutions containing physiologically compatible aqueous buffers, Recombinant (e.g., rHuGlyFabVEGFi) vector in a formulation buffer containing surfactant and optional excipients. Contains a suspension of acetone.

[0048] A therapeutically effective dose of the recombinant vector is ≧0.1 mL to ≦0.5 mL, preferably 0.1 to 0.30 mL (1 Injection volumes ranging from 0.00 to 300 μl, and most preferably in volumes of 0.25 mL (250 μl). The subretinal injection should be administered subretinal and / or intraretinal. The procedure was performed by an experienced retinal surgeon, involving selective vitrectomy and injection of gene therapy into the retina. (See, e.g., Campochi et al., supra, which is incorporated herein by reference in its entirety.) See Aro et al., 2016, Hum Gen Ther Sep 26 epub:doi:10.1089 / hum.2016.117 Subretinal and / or intraretinal administration results in the delivery of soluble transgene products to the retina, vitreous humor, and The transgene product (e.g., an antibody encoding an anti-Valkyrin antibody) should be delivered to the retina and / or aqueous humor. EGF antibody) retinal cells, such as rod cells, cone cells, retinal pigment epithelial cells, horizontal cells, bipolar retina, by expression by cells, amacrine cells, ganglion cells, and / or Müller cells; The transgene product is delivered and maintained in the vitreous humor and / or aqueous humor. or 0.110 μg / mL in the aqueous humor (anterior chamber of the eye). min Transgenesis in A dose that maintains product concentrations for 3 months is desirable; thereafter, a range of 1.70 to 6.60 μg / mL is recommended. Transgene product in vitreous C min Concentration and / or chamber C in the range of 0.567 to 2.20 μg / mL min dark However, because the transgene product is produced continuously, It may be effective to maintain the concentration of the transgene product in the vitreous of the treated eye. Alternatively, vitreous humor concentrations can be measured in patient samples of vitreous humor and / or anterior chamber. Estimating and / or monitoring by measuring the patient's serum concentration of the transgene product. - the ratio of systemic exposure to the transgene product to vitreous exposure is approximately 1:90,000 ( See, for example, Xu L et al., 2013, Invest. Glasses reported in Table 5 of Opthal. Vis. Sci. 54: 1616-1624, p. 1621 and p. 1623 (see ranibizumab concentrations in body fluids and serum).

[0049] The present invention provides a method for treating recurrent VEGF inhibitor therapy that dissipates over time and produces peak and trough levels. It has several advantages over standard nursing care procedures involving high-dose bolus ocular injections. The sustained expression of gene product antibodies, as opposed to repeated antibody injections, allows for a more targeted response depending on the site of action. This allows for a more stable level of antibodies to be present, meaning fewer injections need to be given. This means fewer hospital visits, which is less risky and more convenient for patients. In addition, antibodies expressed from transgenes are subject to different microenvironments during and after translation. Therefore, antibodies undergo post-translational modifications in a manner that differs from that of directly injected antibodies. This allows antibodies to have different diffusion, bioactivity, distribution, affinity, and drug The resulting antibody will have the kinetic and immunogenic properties required to be delivered directly to the site of action. It is a "biobetter" compared to injected antibodies.

[0050] Additionally, antibodies expressed from transgenes in vivo are not recombinantly produced antibodies. Less likely to contain body-relevant degradation products, such as protein aggregates and protein oxides High protein concentrations, surface interactions with manufacturing equipment and vessels, and specific buffers Aggregation due to liquid purification is a problem associated with protein production and storage. These conditions promote aggregation but are not conducive to transgene expression in gene therapy. Oxidation, e.g., methionine, tryptophan, and histidine oxidation, also occurs in proteins. associated with protein production and storage, stressful cell culture conditions, exposure to metals and air Contact with the skin and impurities in buffers and excipients. Proteins expressed from the plant can also be oxidized under stressful conditions. Humans and many other organisms have an antioxidant defense system that protects against oxidative stress. Not only does it reduce oxidation, but it also sometimes repairs and / or reverses oxidation. Proteins produced in vivo are unlikely to be in an oxidized form. Both aggregation and oxidation , which can affect efficacy, pharmacokinetics (clearance), and immunogenicity.

[0051] Without being bound by theory, the methods and compositions provided herein are based in part on the following: It is based on the following principles: (i) Human retinal cells show a robust process for glycosylation of secreted proteins and retinal cells. These secretory cells have the cellular machinery for post-translational processing, including tyrosine-O-sulfation, which is involved in (For example, Wang et al., 2013, Analyt ical Biochem. 427: 20-28 and Adamis et al., 1993, BBRC 193: 631-638; and retina Kanan et al., 2, reported the production of secreted tyrosine sulfated glycoproteins by cells. 009, Exp. Eye Res. 89: 559-567 and Kanan and Al-Ubaidi, 2015, Exp. Eye Res. 133:126-131. Each of these is a post-translational modification carried out by human retinal cells. (The entirety of the description is incorporated by reference.) (ii) Contrary to current understanding, anti-VEGF antigen-binding fragments, e.g., ranibizumab (and The Fab domain of full-length anti-VEGF mAbs such as vacizumab does indeed contain N-linked glycosylation sites. For example, C of ranibizumab H Domain (TVSWN 165 SGAL) and C L Domain (QSGN 158 SQE) Non-consensus asparagine ("N") glycosylation sites in, as well as V H Domain(Q 115 G T) and V L Domain(TFQ 100 Glutamine ("Q") residues, which are glycosylation sites in and the corresponding moiety in the Fab of bevacizumab) (e.g., Valliere-Douglass et al., 2009, J. Biol. Chem. 284: 32493-32506, and Valliere-D See ouglass et al., 2010, J. Biol. Chem. 285: 16012-16022. are incorporated by reference in their entireties for identifying N-linked glycosylation sites in antibodies. (This is the case.) (iii) Such non-canonical sites typically exhibit low levels of glycosylation in antibody populations (e.g. , about 1-5%), but only in immunoprivileged organs, e.g. The functional benefits in the eye can be significant (e.g., van de Bovenkamp et al., 2016, J. Immunol. 196:1435-1441). For example, glycosylation of Fab can improve antibody stability. The effect of Fa on the affinity of the antibody for its target can be influenced. To determine the effect of glycosylation, any technique known to those skilled in the art can be used, for example, enzyme-linked immunosorbent assays. 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 one of skill in the art can be used. , e.g., in the blood or organs (e.g., the eye) of subjects administered a radiolabeled antibody. The stability of Fab glycosylation can be measured by measuring the level of radioactivity. For example, to determine the effect of antibodies on levels of aggregation or protein unfolding. This can be done by any technique known to those skilled in the art, for example, differential scanning calorimetry (DSC), high performance liquid chromatography, etc. High performance liquid chromatography (HPLC), e.g., size exclusion high performance liquid chromatography (SEC-HPLC), Capillary electrophoresis, mass spectrometry, or turbidity measurements can be used. HuPTMFabVEGFi, e.g., HuGlyFabVEGFi transgenes, are 0.5% to 1% at non-canonical sites. , 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more glycosylated In one embodiment, 0.5%, 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%, 30%, 31%, 32 %, 5%, 6%, 7%, 8%, 9%, or 10% or more of the Fabs are glycosylated at non-canonical sites. In some embodiments, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7% , 8%, 9%, or 10% or more of the non-canonical sites are glycosylated. In an embodiment, glycosylation of Fab at these non-canonical sites is achieved in HEK293 cells. The amount of glycosylation at these non-canonical sites in Fabs produced with 00%, 200%, 300%, 400%, 500% or more greater. (iv) In addition to glycosylation sites, anti-VEGF Fabs such as ranibizumab (and F of bevacizumab) ab) contains a tyrosine ("Y") sulfation site within or near the CDR; V of ranibizumab H ( EDTAVY 94 Y 95 ) and V L (EDFATY 86 ) domain (and bevacizumab See FIG. 1, which identifies the corresponding site in the Fab of the antibody (e.g., protein tyrosine The analysis of amino acids surrounding sulfated tyrosine residues is incorporated herein by reference in its entirety. See Yang et al., 2015, Molecules 20:2138-2164, especially p. 2154. The "rule" can be summarized as follows: Y residues that have E or D within +5 to -5 from the position of Y The -1 position of Y is a neutral or acidic charged amino acid, but the basic amino acid that disables sulfation is Human IgG antibodies contain many other post-translational modifications, e.g. For example, N-terminal modifications, C-terminal modifications, degradation or oxidation of amino acid residues, cysteine-related variants, and and glycosylation (see, e.g., Liu et al., 2014, mAbs 6(5):1145-1154). I want to. (v) Anti-VEGF Fab, e.g., Fab fragments of ranibizumab or bevacizumab, by human retinal cells Glycosylation of the nucleotides enhances stability, half-life and prevents unwanted aggregation and cleavage of the transgene product. and / or result in the addition of glycans that may reduce immunogenicity (e.g., new Fab glycosylation For a review of the new importance, see Bovenkamp et al., 2016, J. Immunol. 196: 1435-1441. Importantly, the HuPTMFabVEGFi provided herein, e.g., HuGlyFabVEGFi The glycans that can be added to the glycan complex are highly processed complex-type glycans containing 2,6-sialic acid. Biantennary N-glycans (e.g., HuPTMFabVEGFi, e.g., HuGly See FIG. 2, which shows glycans that can be incorporated into FabVEGFi) and bisecting Glc Such glycans are NAc and not NGNA. The glycosylated form is not glycosylated, and bevacizumab (required to carry out this post-translational modification) Neither 2,6-sialyltransferase nor the bisecting GlcNac produced by CHO cells is present. However, these cells are also present in CHO cells that produce immunogenic NGNA. For example, Dumont et al., 2015, Crit. Rev. Biotechnol. (Early online edition) , published online on September 18, 2015, pp. 1-13, p. 5). The human glycosylation pattern of the HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, is This should reduce the immunogenicity and improve efficacy of (vi) Immunotherapy of human retinal cells with anti-VEGF Fab, e.g., Fab fragments of ranibizumab or bevacizumab. Tyrosine sulfation, a robust post-translational process in VEGF, is a transgene that conferred increased affinity for VEGF. Indeed, tyrosine sulfation of the Fab of therapeutic antibodies against other targets can result in the It has been shown that this dramatically increases the affinity and activity for antigens (see, for example, Loos et al., 2003). See, e.g., Bruno et al., 2015, PNAS 112: 12675-12680, and Choe et al., 2003, Cell 114: 161-170. Such post-translational modifications are mediated by ranibizumab, which inhibits the enzyme required for tyrosine sulfation. bevacizumab produced in CHO cells was not present in Escherichia coli (a host that does not have Unlike human retinal cells, CHO cells are not secretory cells. However, posttranslational tyrosine sulfation is limited (e.g., Mikkelsen and Ezban, 1999). 91, Biochemistry 30: 1533-1537, see especially the discussion on p. 1537).

[0052] For the above reasons, the production of HuPTMFabVEGFi, e.g. HuGlyFabVEGFi, is of great value in gene therapy - e.g. into the subretinal space within the eye(s) of a patient (human subject) diagnosed with nAMD, For example, a viral vector or other DNA expression construct encoding HuGlyFabVEGFi may be administered. The post-translationally modified retinal cells produced by the transduced retinal cells are continuously delivered to the eye. For example, a permanent data set that provides a fully human transgene product that is human glycosylated and sulfated can be used. This will be achieved by creating a “biologically superior” molecule for the treatment of nAMD. The cDNA construct for Fab VEGFi was used to transduce retinal cells. Proper co-translational and post-translational processing (glycosylation and protein sulfation) is ensured by The signal peptide used by retinal cells should be included to identify the signal peptide that is responsible for the transcription of the retinal Null arrays include, but are not limited to: [ka] (For examples of signal peptides that can be used, see, for example, See Stern et al., 2007, Trends Cell. Mol. Biol., 2:1-17, incorporated herein by reference. and Dalton and Barton, 2014, Protein Sci, 23: 517-525).

[0053] Alternatively, or as an additional treatment to gene therapy, HuPTMFabVEGFi products, e.g. HuG The glycoprotein of lyFabVEGFi is produced in a human cell line using recombinant DNA technology and injected intravitreally. The HuPTMFabVEGFi product, e.g., The glycoprotein can also be administered to patients with AMD or diabetic retinopathy. Some of the human cell lines that can be used for the production of recombinant glycoproteins include , including but 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 (e.g., HuPTMFabVEGFi products, e.g. Human cell lines that can be used for recombinant production of, for example, HuGlyFabVEGFi glycoproteins. For a review, see Dumont et al., 2015, Crit. Rev. Biotechnol. (Early online, published online on September 18, 2015, pp. 1-13) Human cell lines for pharmaceutical manufacturing: history, status, and future prospects iopharmaceutical manufacturing: history, status, and future perspectives). To ensure complete glycosylation, particularly sialic acid addition and tyrosine sulfation, The cell lines used for production were characterized as having α-2,6-sialyltransferase (or α-2,3- and α-2,6-sialyltransferase) and / or tyrosine-O- in retinal cells By engineering host cells to co-express the TPST-1 and TPST-2 enzymes that contribute to sulfation, It can be further enhanced.

[0054] HuPTFabVEGFi, e.g. HuGlyFabVEGFi, to the eye / retina along with delivery of other available therapeutic agents The combination of delivery of the additional treatment is encompassed by the methods provided herein. The gene therapy methods provided herein can be administered prior to, simultaneously with, or after the placement of the gene. Available treatments for nAMD that can be combined with include, but are not limited to, Levitra. laser photocoagulation, photodynamic therapy with verteporfin, and other similar therapeutic approaches. anti-VEGF agents, including pegaptanib, ranibizumab, aflibercept, or bevacizumab Additional treatments with anti-VEGF drugs, such as biologics, are This is sometimes called "rescue" treatment.

[0055] Unlike small molecule drugs, biologics usually have different efficacy, pharmacokinetic, and safety profiles. The gene includes a mixture of many variants with different modifications or forms having the same sequence. Or all molecules produced in a protein therapeutic approach are fully glycosylated. It is not essential that the glycoproteins be sulfated. Rather, the population of glycoproteins produced is , glycosylation containing sufficient 2,6-sialylation to show efficacy (about 1% to about 10% of the population The purpose of the gene therapy treatments provided herein is to treat retinal degeneration. Slow or stop the progression of vision loss with minimal intervention / invasive procedures The efficacy was evaluated by measuring BCVA (best corrected visual acuity), intraocular pressure, and stroke. Littlamp biomicroscopy, indirect ophthalmoscopy, SD-OCT (SD-optical coherence tomography), retina Can be monitored by electrogram (ERG). Vision loss, including retinal detachment Signs of infection, inflammation, and other safety events can also be monitored. Retinal thickness may be monitored to determine the effectiveness of the treatment provided herein. Without being bound by any particular theory, retinal thickness can be used as a clinical reading. Here, the greater the decrease in retinal thickness or the longer the period until retinal thickness increases, the better the treatment. The retinal thickness can be determined, for example, by SD-OCT, which uses low-coherence Interferometry is used to measure the echo time delay and amplitude of backscattered light reflected from an object of interest. OCT is a three-dimensional imaging technique that determines the axial resolution of tissue samples with an axial resolution of 3–15 μm. The ability to scan layers of material (e.g., the retina) makes SD-OCT more axially accurate than previous forms of technology. Improve lateral resolution and scanning speed (Schuman, 2008, Trans. Am. Opthamol Soc. 106:426-458). For example, retinal function can be determined by ERG. ERG is a method for determining the function of the retina. It is a non-invasive electrophysiological test of retinal function approved by the FDA for use in The light-sensitive cells of the eye (rods and cones) and the ganglion cells to which they connect, especially The researchers then examined their responses to flash stimuli.

[0056] The unexpected benefit of the present invention is that HVEGF from rAAV8.anti-hVEGF Fab vectors injected into the subretinal space Expression of uPTMFabVEGFi has the potential to (i) develop a transgenic model of nAMD in human subjects; (ii) reduced subretinal neovascularization in mice; and, surprisingly, (iii) suppressed ocular production of VEGF. Transvascular ocular disease resulting in severe proliferative retinopathy and retinal detachment caused by The following examples demonstrate that retinal detachment was prevented in a transgenic mouse model. For example: ILLUSTRATIVE EMBODIMENTS 1. A method of treating a human subject diagnosed with nAMD, comprising administering to the retina of the human subject a therapeutically effective amount of the method comprising delivering an anti-hVEGF antigen-binding fragment produced by human retinal cells of said Law. 2. A method of treating a human subject diagnosed with nAMD, comprising administering to the retina of the human subject a therapeutically effective amount of The method comprises delivering an anti-hVEGF antigen-binding fragment produced by human photoreceptor cells. 3. The method of paragraph 1 or 2, wherein the antigen-binding fragment is a Fab. 4. The method of paragraph 1 or 2, wherein the antigen-binding fragment is F(ab')2. 5. The method of paragraph 1 or 2, wherein the antigen-binding fragment is a single-chain variable domain (scFv). 6. The antigen-binding fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3, and 3. The method of paragraph 1 or 2, comprising a light chain comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4. 7. The antigen-binding fragment comprises light chain CDRs 1 to 3 of SEQ ID NOs: 14 to 16 and SEQ ID NOs: 17 to 19 or SEQ ID NO: 20. 3. The method of paragraph 1 or 2, comprising heavy chain CDRs 1-3 of: 8. A method for treating a human subject diagnosed with neovascular age-related macular degeneration (nAMD), comprising: a therapeutically effective amount of a mAb against hVEGF administered to an antigen comprising an α2,6-sialylated glycan; Binding fragments (Fab, F(ab')2, or scFv, collectively referred to herein as "antigen-binding fragments") The method comprising delivering 9. A method for treating a human subject diagnosed with nAMD, comprising administering to the eye of the human subject a therapeutically effective amount of h delivering a glycosylated antigen-binding fragment of a mAb against VEGF, The method, wherein the fragment does not contain NeuGc. 10. A method for treating a human subject diagnosed with nAMD or AMD or diabetic retinopathy, comprising: administering an expression vector encoding an antigen-binding fragment against hVEGF into the subretinal space in the subject's eye wherein expression of the antigen-binding fragment comprises expression in a human immortalized retina-derived cell. The method as described above, wherein α2,6-sialylation is performed upon expression from the present vector. 11. A method for treating a human subject diagnosed with nAMD or AMD or diabetic retinopathy, comprising: administering an expression vector encoding an antigen-binding fragment against hVEGF into the subretinal space in the subject's eye wherein expression of the antigen-binding fragment comprises expression in a human immortalized retina-derived cell. Upon expression from the present vector, α2,6-sialylated and the antigen-binding fragment does not contain NeuGc. No, said method. 12. A method of treating a human subject diagnosed with nAMD, comprising administering to the subretinal space in the eye of the human subject A therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of a mAb against hVEGF. and administering a vector to the subject, thereby releasing the antigen-binding fragment containing α2,6-sialylated glycans. forming a depot. 13. A method of treating a human subject diagnosed with nAMD, comprising administering a therapeutic agent to the subretinal space in the eye of the human subject. A therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of a mAb against hVEGF. the administration of a vector to form a depot that releases the antigen-binding fragment, wherein the antigen-binding fragment is glycosylated but does not contain NeuGc. 14. 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 sequence The method according to any one of paragraphs 8 to 13, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Law. 15. The method of any one of paragraphs 8 to 14, wherein the antigen-binding fragment further comprises tyrosine sulfation. Law. 16. Production of the antigen-binding fragment containing α2,6-sialylated glycans by recombinant human IgG in cell culture. The expression vector was then transduced into PER.C6 or RPE cell lines to confirm the expression of the nucleotide. The method according to any one of paragraphs 8 to 15. 17. The production of the antigen-binding fragment containing tyrosine sulfation is carried out by incubating the recombinant nucleotide sequence in cell culture. Paragraphs 8 to 15, in which the expression vector is transduced into PER.C6 or RPE cell line to confirm the above. 3. The method according to claim 1 , 18. The method according to any one of paragraphs 8 to 17, wherein the vector has a hypoxia-inducible promoter. method. 19. The antigen-binding fragment comprises light chain CDRs 1 to 3 of SEQ ID NOs: 14 to 16, and light chain CDRs 1 to 3 of SEQ ID NOs: 17 to 19 or sequence The method of any one of paragraphs 8 to 18, comprising heavy chain CDRs 1 to 3 of numbers 20, 18, and 21. 20. Any one of paragraphs 8 to 19, wherein the antigen-binding fragment transgene encodes a leader peptide. The method described herein is as follows. 21. A method of treating a human subject diagnosed with nAMD, comprising administering a therapeutic agent to the subretinal space in the eye of the human subject. A therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of a mAb against hVEGF. and administering a vector to the subject, thereby releasing the antigen-binding fragment containing α2,6-sialylated glycans. forming a depot, wherein the recombinant vector is administered to PER.C6 or RPE cells in culture; When used to transduce cells, the cells contain α2,6-sialylated glycans in the cell culture. The method results in the production of said antigen-binding fragment. 22. A method for treating a human subject diagnosed with nAMD, comprising administering a therapeutic agent to the subretinal space in the eye of the human subject. A therapeutically effective amount of a recombinant nucleotide expression vector encoding an antigen-binding fragment of a mAb against hVEGF. the administration of a vector to form a depot that releases the antigen-binding fragment, wherein the antigen-binding fragment is glycosylated but does not contain NeuGc; When used to transduce PER.C6 or RPE cells in culture, The method results in the production of said antigen-binding fragment that is methylated but does not contain NeuGc. 23. The method of claim 1 or 2, wherein delivery to the eye includes delivery to the retina, choroid, and / or vitreous humor of the eye. 2. The method described in 2. 24. The antigen-binding fragment comprises a heavy chain that contains one, two, three, or four additional amino acids at the C-terminus. 24. The method of any one of paragraphs 1 to 23, comprising: 25. Any of paragraphs 1 to 23, wherein the antigen-binding fragment comprises a heavy chain that does not include additional amino acids at the C-terminus. Or one of the methods described. 26. Producing a population of antigen-binding fragment molecules, wherein the antigen-binding fragment molecules comprise a heavy chain, and 5%, 10%, or 20% of the population of antigen-binding fragment molecules have one, two, three, or four C-terminal heavy chain residues. 24. The method of any one of paragraphs 1 to 23, comprising an additional amino acid of [Brief description of the drawings]

[0057] (4. BRIEF DESCRIPTION OF THE DRAWINGS) [Figure 1] Amino acid sequence of ranibizumab (top) showing the five distinct residues in bevacizumab Fab (bottom). The start of the variable and constant heavy (VH and CH) and light (VL and VC) chains are indicated with arrows (→) and the CDRs are underlined. Non-consensus glycosylation sites ("G sites") and tyrosine-O-sulfation sites ("Y sites") are indicated.

[0058] [Diagram 2] Glycans capable of binding to HuGlyFabVEGFi (adopted from Bondt et al., 2014, Mol & Cell Proteomics 13.1: 3029-3039).

[0059] [Diagram 3] Amino acid sequences of hyperglycosylated variants of ranibizumab (top) and bevacizumab Fab (bottom). The start of the variable and constant heavy chains (VH and CH) and light chains (VL and VC) are indicated with arrows (→) and the CDRs are underlined. Non-consensus glycosylation sites ("G sites") and tyrosine-O-sulfation sites ("Y sites") are indicated. The four hyperglycosylated variants are indicated with an asterisk (*).

[0060] [Figure 4]Dose-dependent reduction in neovascularization area in Rho / VEGF mice receiving subretinal injections of Vector 1. Rho / VEGF mice were subretinal injected with the indicated doses of Vector 1 or control (PBS or empty vector at 1x1010 GC / eye), and the area of ​​retinal neovascularization was quantified 1 week later. The number of mice / group is indicated above each bar. * indicates p-values ​​between 0.0019 and 0.0062; ** indicates p-values ​​<0.0001.

[0061] [Diagram 5] Reduced incidence and severity of retinal detachment in Tet / opsin / VEGF mice given subretinal injections of Vector 1. Tet / opsin / VEGF mice were subretinal injected with the indicated doses of Vector 1 or control (PBS or empty vector at 1x1010 GC / eye). After 10 days, expression of VEGF was induced by addition of doxycycline to the drinking water, and 4 days later the eyes were evaluated for the presence of complete retinal detachment, partial detachment, or the absence of detachment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] 5. Detailed Description of the Invention Fully human post-translationally modified (HuPTM) antigen-binding fragments of monoclonal antibodies (mAbs) against VEGF ("HuPTMFabVEGFi"), e.g., a fully human glycosylated antigen-binding fragment of an anti-VEGF mAb ("HuG lyFabVEGFi"), an ocular disease caused by increased angiogenesis, such as "wet" AM. Retinal / vitreous humor in the eye(s) of a patient (human subject) diagnosed with nAMD, also known as D Such antigen-binding fragments include those of anti-VEGF mAbs. Fab, F(ab')2, or scFv (single chain variable fragment) (collectively referred to herein as "antigen-binding fragments" In an alternative embodiment, full length mAbs can be used. Delivery can be by gene transfer. Subretinal and / or intraretinal therapy, for example, in the eye(s) of a patient (human subject) diagnosed with nAMD. delivers an anti-VEGF antigen-binding fragment or mAb (or a hyperglycosylated derivative) to the intraretinal space. The human PTM gene is then continuously delivered to the eye by administering a viral vector or other DNA expression construct that expresses the gene. , for example, by creating a permanent depot that supplies human glycosylated transgene products. The methods provided herein can also be used to diagnose AMD or diabetic retinopathy. The method can be used in a patient (human subject).

[0063] Subjects receiving such gene therapy should be those who are responsive to anti-VEGF therapy. In certain embodiments, the method comprises the step of determining whether a patient has been diagnosed with nAMD and is responsive to treatment with an anti-VEGF antibody. In a more specific embodiment, the patient is treated with In one embodiment, the patient is responsive to treatment with an anti-VEGF antigen-binding fragment. It has been shown that patients respond to treatment with intravitreal injection of anti-VEGF antigen-binding fragments prior to treatment. In a specific embodiment, the patient has previously received LUCENTIS® (ranibizumab). , EYLEA® (aflibercept), and / or AVASTIN® (bevacizumab) and the LUCENTIS® (ranibizumab), EYLEA® ( ) (aflibercept), and / or AVASTIN® (bevacizumab) It has been found to react with

[0064] The subject to which such a viral vector or other DNA expression construct is delivered is the virus. Anti-VEGF antigen binding protein encoded by a transgene in a vector or expression construct. To determine reactivity, the antibody should react with the anti-hVEGF antigen-binding fragment transgene. The product (e.g., produced in a cell culture, a bioreactor, etc.) can be administered, e.g., by intravitreal injection. It can be administered directly to a subject.

[0065] The HuPTMFabVEGFi encoded by the transgene, e.g., HuGlyFabVEGFi, Antigen-binding fragments of antibodies that bind to hVEGF, such as, but not limited to, bevacizumab; anti-hVEGF F The ab portion, e.g., ranibizumab; or the Fab domain may contain additional glycosylation sites. The Fab portion of such bevacizumab or ranibizumab may be engineered to describes derivatives of bevacizumab that are hyperglycosylated on the Fab domain of the full-length antibody. See Courtois et al., 2016, mAb, which is incorporated by reference in its entirety. (see bs 8:99-112).

[0066] The recombinant vectors used for delivery of the transgenes are parenteral vectors for human retinal cells or photoreceptor cells. Such vectors should be compatible with non-replicating recombinant adeno-associated virus vectors. In particular, vectors having an AAV8 capsid can be included. However, the vectors include, but are not limited to, lentivirus vectors, vaccinia virus vectors, Non-viral expression vectors, also called "naked DNA" constructs Other viral vectors including the HuPTMFabVEGFi vector can be used. For example, the HuGlyFabVEGFi transgene can be fused to suitable expression control elements, such as , CB7 promoter (chicken β-actin promoter and CMV enhancer), RPE65 The vector should be controlled by the opsin promoter or the opsin promoter. Other expression control elements that enhance expression of the transgene driven by β-actin intron, minute virus of mice (MVM) intron, human factor IX intron ron (e.g., FIX truncated intron 1), β-globin splice donor / immunoglobulin heavy chain splice acceptor intron, adenovirus splice donor / immunoglomer Brin splice acceptor intron, SV40 late splice donor / splice acceptor Sceptor (19S / 16S) intron and hybrid adenovirus splice donor / Ig Introns, such as the G splice acceptor intron, and polyA signals, e.g. Heron β-globin polyA signal, human growth hormone (hGH) polyA signal, SV40 late polyA signal signal, synthetic polyA (SPA) signal, and bovine growth hormone (bGH) polyA signal) See, for example, Powell and Rivera-Soto, 2015, Discov. Med., 19(10 2):49-57.

[0067] The gene therapy construct is designed to express both the heavy and light chains. Specifically, the heavy and light chains should be expressed in approximately equal amounts; The heavy and light chains are expressed in an approximately 1:1 ratio of heavy to light chains. The coding sequences are sequenced such that the heavy and light chains are cleaved such that separate heavy and light chain polypeptides are expressed. In a single construct separated by a possible linker or IRES For example, specific examples of compounds that can be used in the methods and compositions provided herein include See Section 5.2.4 for leader sequences and Section 5.2.5 for specific IRES, 2A, and other linker sequences. See Section 5.2.5 for more information.

[0068] Pharmaceutical compositions suitable for subretinal and / or intraretinal administration include aqueous solutions containing physiologically compatible aqueous buffers, Recombinant (e.g., rHuGlyFabVEGFi) vector in a formulation buffer containing surfactant and optional excipients. Contains a suspension of acetone.

[0069] A therapeutically effective dose of the recombinant vector is ≧0.1 mL to ≦0.5 mL, preferably 0.1 to 0.30 mL (1 Injection volumes ranging from 0.00 to 300 μl, and most preferably in volumes of 0.25 mL (250 μl). The subretinal injection should be administered subretinal and / or intraretinal. The procedure was performed by an experienced retinal surgeon, involving selective vitrectomy and injection of gene therapy into the retina. (See, e.g., Campochi et al., supra, which is incorporated herein by reference in its entirety.) See Aro et al., 2016, Hum Gen Ther Sep 26 epub:doi:10.1089 / hum.2016.117 Subretinal and / or intraretinal administration results in the delivery of soluble transgene products to the retina, vitreous humor, and The transgene product (e.g., an antibody encoding an anti-Valkyrin antibody) should be delivered to the retina and / or aqueous humor. EGF antibody) retinal cells, such as rod cells, cone cells, retinal pigment epithelial cells, horizontal cells, bipolar retina, by expression by cells, amacrine cells, ganglion cells, and / or Müller cells; The transgene product is delivered and maintained in the vitreous humor and / or aqueous humor. or 0.110 μg / mL in the aqueous humor (anterior chamber of the eye). min Transgenesis in A dose that maintains product concentrations for 3 months is desirable; thereafter, a range of 1.70 to 6.60 μg / mL is recommended. Transgene product in vitreous C min Concentration and / or chamber C in the range of 0.567 to 2.20 μg / mL min dark However, because the transgene product is produced continuously, It may be effective to maintain the concentration of the transgene product in the vitreous of the treated eye. Alternatively, vitreous humor concentrations can be measured in patient samples of vitreous humor and / or anterior chamber. Estimating and / or monitoring by measuring the patient's serum concentration of the transgene product. - the ratio of systemic exposure to the transgene product to vitreous exposure is approximately 1:90,000 ( See, for example, Xu L et al., 2013, Invest. Glasses reported in Table 5 of Opthal. Vis. Sci. 54: 1616-1624, p. 1621 and p. 1623 (see ranibizumab concentrations in body fluids and serum).

[0070] The present invention provides a method for treating recurrent VEGF inhibitor therapy that dissipates over time and produces peak and trough levels. It has several advantages over standard nursing care procedures involving high-dose bolus ocular injections. The sustained expression of gene product antibodies, as opposed to repeated antibody injections, allows for a more targeted response depending on the site of action. This allows for a more stable level of antibodies to be present, meaning fewer injections need to be given. This means fewer hospital visits, which is less risky and more convenient for patients. In addition, antibodies expressed from transgenes are subject to different microenvironments during and after translation. Therefore, antibodies undergo post-translational modifications in a manner that differs from that of directly injected antibodies. This allows antibodies to have different diffusion, bioactivity, distribution, affinity, and drug The resulting antibody will have the kinetic and immunogenic properties required to be delivered directly to the site of action. It is "biologically superior" compared to injected antibodies.

[0071] Additionally, antibodies expressed from transgenes in vivo are not recombinantly produced antibodies. Less likely to contain body-relevant degradation products, such as protein aggregates and protein oxides High protein concentrations, surface interactions with manufacturing equipment and vessels, and specific buffers Aggregation due to liquid purification is a problem associated with protein production and storage. These conditions promote aggregation but are not conducive to transgene expression in gene therapy. Oxidation, e.g., methionine, tryptophan, and histidine oxidation, also occurs in proteins. associated with protein production and storage, stressful cell culture conditions, exposure to metals and air Contact with the skin and impurities in buffers and excipients. Proteins expressed from the plant can also be oxidized under stressful conditions. Humans and many other organisms have an antioxidant defense system that protects against oxidative stress. Not only does it reduce oxidation, but it also sometimes repairs and / or reverses oxidation. Proteins produced in vivo are unlikely to be in an oxidized form. Aggregation and oxidation can affect efficacy, This may affect drug pharmacokinetics (clearance), and immunogenicity.

[0072] Without being bound by theory, the methods and compositions provided herein are based in part on the following: It is based on the following principles: (i) Human retinal cells show a robust process for glycosylation of secreted proteins and retinal cells. These secretory cells have the cellular machinery for post-translational processing, including tyrosine-O-sulfation, which is involved in (For example, Wang et al., 2013, Analyt ical Biochem. 427: 20-28 and Adamis et al., 1993, BBRC 193: 631-638; and retina Kanan et al., 2, reported the production of secreted tyrosine sulfated glycoproteins by cells. 009, Exp. Eye Res. 89: 559-567 and Kanan and Al-Ubaidi, 2015, Exp. Eye Res. 133:126-131. Each of these is a post-translational modification carried out by human retinal cells. (The entirety of the description is incorporated by reference.) (ii) Contrary to current understanding, anti-VEGF antigen-binding fragments, e.g., ranibizumab (and The Fab domain of full-length anti-VEGF mAbs such as vacizumab does indeed contain N-linked glycosylation sites. For example, C of ranibizumab H Domain (TVSWN 165 SGAL) and C L Domain (QSGN 158 SQE) Non-consensus asparagine ("N") glycosylation sites in, as well as V H Domain(Q 115 G T) and V L Domain(TFQ 100 Glutamine ("Q") residues, which are glycosylation sites in and the corresponding moiety in the Fab of bevacizumab) (e.g., Valliere-Douglass et al., 2009, J. Biol. Chem. 284: 32493-32506, and Valliere-D See ouglass et al., 2010, J. Biol. Chem. 285: 16012-16022. are incorporated by reference in their entireties for identifying N-linked glycosylation sites in antibodies. (This is the case.) (iii) Such non-canonical sites typically exhibit low levels of glycosylation in antibody populations (e.g. , approximately 1-5%), but confers functional benefits in immune-privileged organs, such as the eye. can be significant (see, e.g., van de Bovenkamp et al., 2016, J. Immunol. 196:1435-1 For example, glycosylation of Fab affects antibody stability, half-life, and binding. The effect of Fab glycosylation on the affinity of the antibody for its target. To determine the amount of β-amino acid in the sample, any technique known to those skilled in the art can be used, such as enzyme-linked immunosorbent assay (ELISA). A) or surface plasmon resonance (SPR) can be used. To determine the effect of glycosylation, any technique known to one of skill in the art may be used, for example, using radiolabeled antibodies. The level of radioactivity in the blood or organs (e.g., the eye) of the administered subject The stability of Fab glycosylation, e.g., antibody aggregation or To determine the effect on the level of protein unfolding, any method known to those skilled in the art can be used. Any technique, such as differential scanning calorimetry (DSC), high performance liquid chromatography (HPLC), For example, size-exclusion high performance liquid chromatography (SEC-HPLC), capillary electrophoresis, Mass spectrometry or turbidity measurements can be used. For example, the HuGlyFabVEGFi transgene was found to be capable of expressing 0.5%, 1%, 2%, 3%, 4%, and 5% of non-canonical sites. %, 6%, 7%, 8%, 9%, or 10% or more glycosylated Fab. In some embodiments, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7% of the Fab population , 8%, 9%, or 10% or more of the Fabs are glycosylated at non-canonical sites In some embodiments, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or In one embodiment, 10% or more of the non-canonical sites are glycosylated. However, glycosylation of Fab at these non-canonical sites was not observed in Fabs produced in HEK293 cells. The amount of glycosylation at these non-canonical sites was 25%, 50%, 100%, 200%, 300%, and %, 400%, 500% or more greater. (iv) In addition to glycosylation sites, anti-VEGF Fabs such as ranibizumab (and F of bevacizumab) ab) contains a tyrosine ("Y") sulfation site within or near the CDR; V of ranibizumab H ( EDTAVY 94 Y 95 ) and V L (EDFATY 86 ) domain (and bevacizumab See FIG. 1, which identifies the corresponding site in the Fab of the antibody (e.g., protein tyrosine The analysis of amino acids surrounding sulfated tyrosine residues is incorporated herein by reference in its entirety. See Yang et al., 2015, Molecules 20:2138-2164, especially p. 2154. The "rule" can be summarized as follows: Y residues that have E or D within +5 to -5 from the position of Y The -1 position of Y is a neutral or acidic charged amino acid, but the basic amino acid that disables sulfation is Human IgG antibodies contain many other post-translational modifications, e.g. For example, N-terminal modifications, C-terminal modifications, degradation or oxidation of amino acid residues, cysteine-related variants, and and glycosylation (see, e.g., Liu et al., 2014, mAbs 6(5):1145-1154). I want to. (v) Anti-VEGF Fab, e.g., Fab fragments of ranibizumab or bevacizumab, by human retinal cells Glycosylation of the nucleotides enhances stability, half-life and prevents unwanted aggregation and cleavage of the transgene product. and / or result in the addition of glycans that reduce immunogenicity (e.g., new Fab glycosylation For a review of the importance of Importantly, the HuPTMFabVEGFi provided herein, e.g., HuGlyFabVEGFi The glycans that can be added are highly processed complex-type glycans containing 2,6-sialic acid. Ternary N-glycans (e.g., glycans that can be incorporated into HuPTMFabVEGFi, e.g., HuGlyFabVEGFi) glycan) and bisecting GlcNAc, but not NGNA. Such glycans are not glycosylated in ranibizumab (produced in E. coli). ) and bevacizumab (the 2,6-sialyltransferase required to carry out this post-translational modification). Although these cells do not have the enzyme or the bisecting GlcNac produced by CHO cells, It is also absent in the Dum Ont et al., 2015, Crit. Rev. Biotechnol. (Early online, September 18, 2015) See, for example, US Pat. No. 6,399,341, published in the same journal, pp. 1-13, p. 5. For example, the human glycosylation pattern of HuGlyFabVEGFi reduces the immunogenicity of the transgene product. , which should improve effectiveness. (vi) Immunotherapy of human retinal cells with anti-VEGF Fab, e.g., Fab fragments of ranibizumab or bevacizumab. Tyrosine sulfation, a robust post-translational process in VEGF, is a transgene that conferred increased affinity for VEGF. Indeed, tyrosine sulfation of the Fab of therapeutic antibodies against other targets can result in the It has been shown that this dramatically increases the affinity and activity for antigens (see, for example, Loos et al., 2003). See, e.g., Bruno et al., 2015, PNAS 112: 12675-12680, and Choe et al., 2003, Cell 114: 161-170. Such post-translational modifications are mediated by ranibizumab, which inhibits the enzyme required for tyrosine sulfation. bevacizumab produced in CHO cells was not present in Escherichia coli (a host that does not have Unlike human retinal cells, CHO cells are not secretory cells. However, posttranslational tyrosine sulfation is limited (e.g., Mikkelsen and Ezban, 1999). 91, Biochemistry 30: 1533-1537, see especially the discussion on p. 1537).

[0073] For the above reasons, the production of HuPTMFabVEGFi, e.g. HuGlyFabVEGFi, is of great value in gene therapy - e.g. into the subretinal space within the eye(s) of a patient (human subject) diagnosed with nAMD, For example, a viral vector or other DNA expression construct encoding HuGlyFabVEGFi may be administered. The post-translationally modified retinal cells produced by the transduced retinal cells are continuously delivered to the eye. For example, a permanent data set that provides a fully human transgene product that is human glycosylated and sulfated can be used. This will be achieved by creating a “biologically superior” molecule for the treatment of nAMD. The cDNA construct for Fab VEGFi was used to transduce retinal cells. Proper co-translational and post-translational processing (glycosylation and protein sulfation) is ensured by The signal peptide used by retinal cells should be included to identify the signal peptide that is responsible for the transcription of the retinal Null arrays include, but are not limited to: [ka] (For examples of signal peptides that can be used, see, for example, See Stern et al., 2007, Trends Cell. Mol. Biol., 2:1-17, incorporated herein by reference. and Dalton and Barton, 2014, Protein Sci, 23: 517-525).

[0074] Alternatively, or as an additional treatment to gene therapy, HuPTMFabVEGFi products, e.g. HuG The glycoprotein of lyFabVEGFi is produced in a human cell line using recombinant DNA technology and injected intravitreally. The HuPTMFabVEGFi product, e.g., The glycoprotein can also be administered to patients with AMD or diabetic retinopathy. Some of the human cell lines that can be used for the production of recombinant glycoproteins include , including but 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 (e.g., HuPTMFabVEGFi products, e.g. Human cell lines that can be used for recombinant production of, for example, HuGlyFabVEGFi glycoproteins. For a review, see Dumont et al., 2015, Crit. Rev. Biotechnol. (Early online, published online on September 18, 2015, pp. 1-13) Human cell lines for pharmaceutical manufacturing: history, status, and future prospects iopharmaceutical manufacturing: history, status, and future perspectives). To ensure complete glycosylation, particularly sialic acid addition and tyrosine sulfation, The cell lines used for production were characterized as having α-2,6-sialyltransferase (or α-2,3- and α-2,6-sialyltransferase) and / or tyrosine-O- in retinal cells By engineering host cells to co-express the TPST-1 and TPST-2 enzymes that contribute to sulfation, It can be further enhanced.

[0075] HuPTFabVEGFi, e.g. HuGlyFabVEGFi, to the eye / retina along with delivery of other available therapeutic agents The combination of delivery of the additional treatment is encompassed by the methods provided herein. The gene therapy methods provided herein can be administered prior to, simultaneously with, or after the placement of the gene. Available treatments for nAMD that can be combined with include, but are not limited to, Levitra. laser photocoagulation, photodynamic therapy with verteporfin, and other similar therapeutic approaches. anti-VEGF agents, including pegaptanib, ranibizumab, aflibercept, or bevacizumab Additional treatments with anti-VEGF drugs, such as biologics, are This is sometimes called "rescue" treatment.

[0076] Unlike small molecule drugs, biologics usually have different efficacy, pharmacokinetic, and safety profiles. The gene includes a mixture of many variants with different modifications or forms having the same sequence. Or all molecules produced in a protein therapeutic approach are fully glycosylated. It is not essential that the glycoproteins be sulfated. Rather, the population of glycoproteins produced is , glycosylation containing sufficient 2,6-sialylation to show efficacy (about 1% to about 10% of the population The purpose of the gene therapy treatments provided herein is to treat retinal degeneration. Slow or stop the progression of vision loss with minimal intervention / invasive procedures The efficacy was evaluated by measuring BCVA (best corrected visual acuity), intraocular pressure, and stroke. Littlamp biomicroscopy, indirect ophthalmoscopy, SD-OCT (SD-optical coherence tomography), retina Can be monitored by electrogram (ERG). Vision loss, including retinal detachment Signs of infection, inflammation, and other safety events can also be monitored. Retinal thickness may be monitored to determine the effectiveness of the treatment provided herein. Without being bound by any particular theory, retinal thickness can be used as a clinical reading. Here, the greater the decrease in retinal thickness or the longer the period until retinal thickness increases, the better the treatment. The retinal thickness can be determined, for example, by SD-OCT, which uses low-coherence Interferometry is used to measure the echo time delay and amplitude of backscattered light reflected from an object of interest. OCT is a three-dimensional imaging technique that determines the axial resolution of tissue samples with an axial resolution of 3–15 μm. The ability to scan layers of material (e.g., the retina) makes SD-OCT more axially accurate than previous forms of technology. Improve lateral resolution and scanning speed (Schuman, 2008, Trans. Am. Opthamol Soc. 106:426-458). For example, retinal function can be determined by ERG. ERG is a method for determining the function of the retina. It is a non-invasive electrophysiological test of retinal function approved by the FDA for use in The light-sensitive cells of the eye (rods and cones) and the ganglion cells to which they connect, especially The researchers then examined their responses to flash stimuli.

[0077] 5.1 N-Glycosylation, Tyrosine Sulfation, and O-Glycosylation Anti-VEGF antibodies of HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, for use in the methods described herein. The amino acid sequence (primary sequence) of the antigen-binding fragment is N-glycosylated or tyrosine sulfation-free. In one embodiment, the amino acid sequence of the anti-VEGF antigen-binding fragment is The array contains at least one N-glycosylation site and at least one tyrosine sulfation site. Such moieties are described in more detail below. In one embodiment, the anti-VEGF antigen-binding fragment The amino acid sequence of the present invention contains at least one O-glycosylation site, which is This may be in addition to one or more N-glycosylation sites and / or tyrosine sulfation sites present in

[0078] 5.1.1 N-Glycosylation (reverse glycosylation site) A standard N-glycosylation sequence is known in the art as Asn-X-Ser (or Thr), where X is P It is known that the amino acid sequence of the ribozyme is α-cytosine (α-cytosine) and β-cytosine (β-cytosine) and can be any amino acid except ro. However, recently, The asparagine (Asn) residue of the antibody conforms to the reverse consensus motif Ser (or Thr)-X-Asn (herein It has been demonstrated that the nucleotide sequence of ... Valliere-Douglass et al., 2009, J. Biol. Chem. 284:32493-32506; and Valli See Ere-Douglass et al., 2010, J. Biol. Chem. 285:16012-16022. As disclosed herein, and contrary to current understanding, the method used in accordance with the present disclosure Anti-VEGF antigen-binding fragments for such purposes, such as ranibizumab, are Thus, the methods described herein include those that utilize the sequence Ser (or Thr)-X-Asn (where X can be any amino acid except Pro (also referred to herein as a "reverse N-glycosylation site"). The use of an anti-VEGF antigen-binding fragment comprising at least one N-glycosylation site comprising include.

[0079] In one embodiment, the methods described herein provide a method for the treatment of a mutated schizophrenia comprising the sequence Ser(or Thr)-X-Asn, X may be any amino acid except Pro) In one embodiment, the antibody comprises an anti-VEGF antigen-binding fragment that contains more than one N-glycosylation site. The methods described herein can be used to prepare a medicament comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 reverse N-glycosyltransferases. N-glycosylation sites and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 non-consensus N-glycosylation sites. This includes the use of anti-VEGF antigen-binding fragments that contain an sylation site (defined herein below).

[0080] In a specific embodiment, one or more reverse N-glycosyltransferases used in the methods described herein are The anti-VEGF antigen-binding fragments containing the cosylation site are ranibizumab, SEQ ID NO: 1 and SEQ ID NO: 2, respectively. In another specific embodiment, the one or more reverse transcriptases used in the method include two light and two heavy chains. The anti-VEGF antigen-binding fragments containing the N-glycosylation site include the Fab of bevacizumab, Includes light and heavy chains of SEQ ID NOs: 3 and 4.

[0081] (Non-consensus glycosylation site) In addition to reverse N-glycosylation sites, glutamine (Gln) residues in human antibodies are replaced with non-consensus It has recently been demonstrated that ribozymes can be glycosylated in the context of the motif Gln-Gly-Thr. See Valliere-Douglass et al., 2010, J. Biol. Chem. 285:16012-16022. Surprisingly, there is provided an anti-VEGF antigen-binding fragment for use according to the methods described herein, For example, ranibizumab contains several such non-consensus sequences. The methods described herein involve the use of a polypeptide having the sequence Gln-Gly-Thr (referred to herein as a "non-consensus N-glycosyltransferase"). an anti-VEGF antigen-binding domain that contains at least one N-glycosylation site (also referred to as an “N-glycosylation site”); Includes the use of fragments.

[0082] In some embodiments, the methods described herein provide for the production of 1, 2, 3, 4 or 5 nucleotide analogs comprising the sequence Gln-Gly-Thr. Use of anti-VEGF antigen-binding fragments containing 1, 5, 6, 7, 8, 9, 10, or more than 10 N-glycosylation sites Includes.

[0083] In specific embodiments, one or more non-conventional The anti-VEGF antigen-binding fragment containing the anti-VEGF N-glycosylation site is represented by ranibizumab (SEQ ID NO: 1, respectively). In another specific embodiment, the method comprises the steps of: The anti-VEGF antigen-binding fragment containing one or more non-consensus N-glycosylation sites used may be The Fab of ciprofloxacin (containing the light and heavy chains of SEQ ID NOs: 3 and 4, respectively) is included.

[0084] (Engineering of N-glycosylation sites) In one embodiment, the nucleic acid encoding the anti-VEGF antigen-binding fragment is (e.g., an N-group associated with an anti-VEGF antigen-binding fragment in its unmodified state) rather than (relative to the number of glycosylation sites) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more N-glycosylation sites (standard N-glycosylation consensus sequence, reverse N-glycosylation site , and non-consensus N-glycosylation sites). In embodiments, the introduction of a glycosylation site disrupts antigen binding to its antigen, VEGF. N-glycosylation at any position in the primary structure of the antigen-binding fragment is not permitted, as long as it does not affect the binding of the fragment. Glycosylation sites (standard N-glycosylation consensus sequences, reverse N-glycosylation sites, and non- This is achieved by inserting a consensus N-glycosylation site into the ribosome. The introduction creates an N-glycosylation site (i.e., an amino acid is introduced into the antigen-binding fragment / antibody). Instead of adding amino acids of the antigen-binding fragment / antibody to the selected N-glycosylation site, For example, to mutate to form an antigen-binding fragment or The addition of new amino acids to the primary structure of the antibody from which it is derived (i.e., glycosylation) or antigen-binding fragments or fragments thereof This is accomplished by mutating existing amino acids in the antibody from which the fragment is derived. Those skilled in the art can easily identify the amino acid sequence of a protein using techniques known in the art. The probe can be modified, for example, using recombinant approaches involving modification of a nucleic acid sequence encoding the protein. As such, those skilled in the art will recognize that such modifications can be readily made.

[0085] In a specific embodiment, the anti-VEGF antigen binding fragment used in the methods described herein The fragment is modified so that it can be hyperglycosylated when expressed in retinal cells. See Courtois et al., 2016, mAbs 8:99-112, which is incorporated herein by reference. In a specific embodiment, the anti-VEGF antigen-binding fragment is ranibizumab ( In another specific embodiment, the The anti-VEGF antigen-binding fragment comprises the Fab of bevacizumab (light chains and (including heavy chains).

[0086] (N-glycosylation of anti-VEGF antigen-binding fragment) Unlike small molecule drugs, biologics usually have different efficacy, pharmacokinetic, and safety profiles. The present invention includes a mixture of many variants having different modifications or forms. All molecules produced in protein therapeutic approaches are fully glycosylated and It is not essential that the glycoproteins be sulfated. Rather, the population of glycoproteins produced must be sulfated to be effective. should undergo sufficient glycosylation (including 2,6-sialylation) and sulfation to demonstrate The purpose of the gene therapy treatments provided herein is to slow or stop the progression of retinal degeneration. The objective of the study is to improve vision and slow down or prevent vision loss through minimally interventional / invasive procedures.

[0087] In a specific embodiment, the anti-VEGF antigen binding fragment used according to the methods described herein For example, ranibizumab has 10 of its N-glycosylation sites when expressed in retinal cells. However, those skilled in the art may be able to determine whether glycosylation is In order to obtain the benefit of It will be appreciated that glycosylation does not necessarily have to be performed. Rather, there may be benefits to glycosylation. If only a percentage of N-glycosylation sites are glycosylated and / or if a certain This can be achieved if only a small percentage of the expressed antigen-binding fragments are glycosylated. Thus, in one embodiment, the anti-VEGF antigen binding antibody used in accordance with the methods described herein The fragment, when expressed in retinal cells, utilizes 10% to 10% of its available N-glycosylation sites. 20%, 20%~30%, 30%~40%, 40%~50%, 50%~60%, 60%~70%, 70%~80%, 80 In one embodiment, the retinal cells are glycosylated to between 90% and 90%, or between 90% and 100%. When expressed in a 5'-terminal region of the VEGF antigen-binding fragment, 10 %~20%, 20%~30%, 30%~40%, 40%~50%, 50%~60%, 60%~70%, 70%~80% , 80%-90%, or 90%-100% of their available N-glycosylation sites At least one is glycosylated.

[0088] In a specific embodiment, the anti-VEGF antigen binding fragment used according to the methods described herein At least 10%, 20%, 30%, 40%, 50%, 6% or more of the N-glycosylation sites present in the fragment 0%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the retinal cells were treated with anti-VEGF antigen-binding fragments. When expressed in That is, the N-glycosylated portion of the resulting HuGlyFabVEGFi is At least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the positions are glycosyl It will be made into a rule.

[0089] In another specific embodiment, the anti-VEGF antigen binding agent used according to the methods described herein At least 10%, 20%, 30%, 40%, 50%, 60% of the N-glycosylation sites present in the fusion fragment , 70%, 75%, 80%, 85%, 90%, 95%, or 99% of retinal cells have anti-VEGF antigen binding. When the fragment is expressed, the Asn residue (or other relevant residue) present at the N-glycosylation site is ) is glycosylated with the same linking glycan. At least 50%, 60%, 70%, 75%, 80% of the N-glycosylation sites of the resulting HuGlyFabVEGFi , 85%, 90%, 95%, or 99% are identical linked glycans.

[0090] Anti-VEGF antigen-binding fragments for use in accordance with the methods described herein, such as ranibizumab, When expressed in membranous cells, the N-glycosylation sites of the antigen-binding fragments are expressed in a variety of different groups. The N-glycans of the antigen-binding fragment can be glycosylated with any of the N-glycans known in the art. For example, Bondt et al., 2014, Mol. & Cell. Proteomics 13.11 3029-3039 (herein incorporated by reference in its entirety for its disclosure of Fabs and associated N-glycans). (incorporated herein) characterized the glycans associated with Fab and determined the Fab and Fc The moieties contain different glycosylation patterns, with the Fab glycans being galactosylated, sialic, and Bisection (e.g., bisecting GlcNac glycans) but lower in fucosylation compared to Fc glycans As in Bondt, Huang et al., 2006, Anal. Biochem. 349:1 97-207, which is incorporated herein by reference in its entirety for its disclosure of Fab and associated N-glycans. In the case of the Fab (in which the Fab is integrated), most of the glycans were found to be sialylated. However, the Fab fragment of the antibody examined by Huang (produced in a mouse cell background) In the above, the sialic residues identified are N-acetylglucosamine (NAc), ... N-glycolylneuraminic acid (Neu5Gc or Neu5Ac, the predominant human sialic acid) The nucleotide sequence was NeuGc (not natural to humans). 014, Anal. Chem. 86:5661-5666 (in its entirety for disclosure of N-glycans associated with its Fabs). (which is incorporated herein by reference) describes the lysing of N-glycans associated with commercially available antibodies. The braille is listed.

[0091] Importantly, the anti-VEGF antigen-binding fragments used in accordance with the methods described herein, such as laminin, When nibizumab is expressed in human retinal cells, it can be expressed in prokaryotic host cells (e.g., E. coli) or The need for in vitro production in eukaryotic host cells (eg, CHO cells) is bypassed. Alternatively, the methods described herein (e.g., the use of a fusion protein for expressing an anti-hVEGF antigen-binding fragment) can be used. As a result of the use of membrane cells, the N-glycosylation sites of the anti-VEGF antigen-binding fragments are advantageously The glycans are decorated with glycans that are relevant and beneficial to the treatment of the disease. Significant advantages can be achieved when utilizing CHO cells or E. coli for the production of antibodies / antigen-binding fragments. For example, CHO cells (1) do not express 2,6 sialyltransferase; , cannot add 2,6-sialic acid during N-glycosylation, and (2) cannot use Neu5Ac as the sialic acid. and E. coli does not naturally possess the necessary structures for N-glycosylation. Therefore, in one embodiment, the present invention is directed to a method for the expression of the present invention in a retinal cell. The anti-VEGF antigen-binding fragments that give rise to the HuGlyFabVEGFi used in the method of treatment described herein are The protein is glycosylated in a manner that is N-glycosylated in human retinal cells, e.g., retinal pigment cells. is glycosylated in the manner in which proteins are glycosylated in CHO cells. In another embodiment, the retinal cells are not expressed in the therapeutic methods described herein. The anti-VEGF antigen-binding fragment, resulting in HuGlyFabVEGFi, used in this study was designed to detect the protein binding to human retinal cells. For example, the glycosylated retinal pigment cells are N-glycosylated in the manner in which they are N-glycosylated, Such glycosylation is not possible using prokaryotic host cells, e.g., E. coli. Of course this is not possible.

[0092] In one embodiment, the HuGlyFabVEGFi used in accordance with the methods described herein, e.g. Baranibizumab is a compound that binds one, two, three, four, five, or more different N-groups associated with the Fab of a human antibody. In a specific embodiment, the N-glycan associated with the Fab of a human antibody comprises a B Ondt 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. 86:5661-5 In one embodiment, the N-glycan is selected from the group consisting of N-glycans as set forth in 666. The HuGlyFabVEGFi used, for example ranibizumab, does not contain NeuGc.

[0093] In a specific embodiment, the HuGlyFabVEGFi used in accordance with the methods described herein, For example, ranibizumab is glycosylated with glycans that contain predominantly 2,6-linked sialic acid. In one embodiment, the HuGlyFabVEGFi contains 2,6-linked sialic acid. In some embodiments, the HuGly is sialic acid-added, i.e., contains two or more sialic acids. Each N-glycosylation site of FabVEGFi contains a glycan containing 2,6-linked sialic acid. 100% of the N-glycosylation sites of the HuGlyFabVEGFi are glycans containing 2,6-linked sialic acid. In another specific embodiment, the HuGlyF used in accordance with the methods described herein At least 20%, 30%, 40%, 50%, 60%, 70%, or 75% of the N-glycosylation sites of abVEGFi , 80%, 85%, 90%, 95%, or 99% of the glycans contain 2,6-linked sialic acid. In another specific embodiment, the HuGl used in accordance with the methods described herein is At least 10%-20%, 20%-30%, 30%-40%, 40%-50% of the N-glycosylation sites of yFabVEGFi %~50%, 50%~60%, 60%~70%, 70%~80%, 80%~90%, or 90%~99% is 2,6-bonded In another specific embodiment, the polypeptide is glycosylated with a glycan containing a conjugated sialic acid. The antigen-binding fragments expressed in retinal cells according to the methods described herein (i.e., HuGl At least 20%, 30%, 40% of the yFabVEGFi (e.g., antigen-binding fragment resulting from ranibizumab) , 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% are 2,6-linked sialic acids In another specific embodiment, the glycosylated glycan comprises the glycan described herein. The antigen-binding fragments expressed in retinal cells by the methods described (i.e., HuGlyFabVEGFi, For example, at least 10%-20%, 20%-30%, 30%-40%, 4% of the Fab that produces ranibizumab 0%~50%, 50%~60%, 60%~70%, 70%~80%, 80%~90%, or 90%~99% is 2,6 In another specific embodiment, the glycosylated glycan contains a sialic acid-linked glycan. In one embodiment, the sialic acid is Neu5Ac. Only a certain percentage of glycosylation sites are 2,6-sialylated or polysialylated. The remaining N-glycosylation may contain different N-glycans or no N-glycans at all. The glycosylated nucleotides do not remain glycosylated (i.e., remain unglycosylated).

[0094] When HuGlyFabVEGFi is 2,6-polysialylated, it contains multiple sialic acid residues, e.g. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 sialic acid residues. , when HuGlyFabVEGFi is polysialylated, it is 2-5, 5-10, 10-20, 20-30, In one embodiment, the HuGlyFabVEGFi comprises 30 to 40, or 40 to 50 sialic acid residues. When sialylated, it is 2,6-linked (sialic acid) n (where n is a number between 1 and 100) can be any number).

[0095] In a specific embodiment, the HuGlyFabVEGFi used in accordance with the methods described herein, For example, ranibizumab is glycosylated predominantly with glycans containing bisecting GlcNAc. In one embodiment, each N-glycosylation site of the HuGlyFabVEGFi is bisected. The N-glycosylation site of the HuGlyFabVEGFi contains a glycan containing a linking GlcNAc. 100% of the glycans contain a bisecting GlcNAc. In order to reduce the number of N-glycosylation sites of HuGlyFabVEGFi used according to the methods described herein, At least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% is glycosylated with glycans containing a bisecting GlcNAc. In an embodiment, the N-glycosylation of HuGlyFabVEGFi for use in accordance with the methods described herein At least 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60% %~70%, 70%~80%, 80%~90%, or 90%~99% contains bisecting GlcNAc In another specific embodiment, the glycan is glycosylated with the glycan described herein. The antigen-binding fragment expressed in the retinal cells according to the method (i.e., HuGlyFabVEGFi, e.g. At least 20%, 30%, 40%, 50%, 60%, 70% of the antigen-binding fragments that produce ranibizumab , 75%, 80%, 85%, 90%, 95%, or 99% of the glycans contain a bisecting GlcNAc. In another specific embodiment, the polypeptide is glycosylated by the methods described herein. Antigen-binding fragments (i.e., HuGlyFabVEGFi, e.g., ranibizumab) expressed in retinal cells At least 10%-20%, 20%-30%, 30%-40%, 40%- 50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-99% are bisexual It is glycosylated with glycans containing glycan-binding GlcNAc.

[0096] In one embodiment, the HuGlyFabVEGFi used in accordance with the methods described herein, e.g. Baranibizumab is hyperglycosylated, i.e., it does not contain any naturally occurring N-glycosylation sites. In addition to the N-glycosylation that occurs from HuGlyFabVEGFi, the HuGlyFabVEGFi also exhibits antigen binding that occurs in HuGlyFabVEGFi. Contains glycans at N-glycosylation sites engineered to be present in the amino acid sequence of the fragment In one embodiment, the HuGlyFabVEGFi used in accordance with the methods described herein, e.g. Baranibizumab is hyperglycosylated but does not contain NeuGc.

[0097] 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, incubation with hydrazine allows the polysaccharide to bind to its associated protein. (Ludger Free Hydrazinolysis Glycan Release Kit, Oxfordshire, UK) The nucleophile hydrazine acts as a bridge between the polysaccharide and the carrier protein. It attacks the glycosidic bond, releasing the attached glycan. During this process, the N-acetyl group Glycans are lost and must be reconstituted by N-acetylation. and enzymes such as PNGase F and Endo H. These enzymes can be used to completely remove hydrazine with fewer side reactions than hydrazine. The released glycans are purified on a carbon column and subsequently labeled with the fluorescent substrate 2-aminobenzamide. The reducing end of the polysaccharide can be labeled with dimethylformamide. The labeled polysaccharide was then subjected to the same procedure as described in Royle et al., Anal Biochem 2003. Separation was performed on a GlycoSep-N column (GL Sciences) according to the HPLC protocol in 2010, 304(1):70-90. The resulting fluorescence chromatograms can be used to determine the length and repeating units of the polysaccharide. Structural information was obtained by collecting individual peaks and subsequent MS / MS analysis. This allows the composition of monosaccharides and the sequence of repeating units to be ascertained. The homogeneity of the polysaccharide composition can be further specified. The peaks can be analyzed by MALDI-MS / MS, and the results confirm the sequence of the glycans. Each peak in the chromatogram represents a polymer, e.g., a glycan. Each cDNA corresponds to a specific number of repeating units and fragments thereof, such as sugar residues. The chromatogram allows the measurement of the length distribution of polymers, e.g., glycans. The spacing is a measure of the polymer length, while the fluorescence intensity is a measure of the molar ratio of each polymer, e.g., glycan. Other methods for assessing the glycans associated with antigen-binding fragments include: Bondt et al., 2014, Mol. & Cell. Proteomics 13.11:3029-3039, Huang et al., 200 6, Anal. Biochem. 349:197-207, and / or Song et al., 2014, Anal. Chem. 86:5661- There is a method described in 5666.

[0098] The homogeneity or heterogeneity of the glycan patterns associated with antibodies (including antigen-binding fragments) can be determined by: Both the length or size of the glycan and the number of glycans present across a glycosylation site As such, methods known in the art can be used to determine the length or size of the glycan, and This can be assessed using methods that measure hydrodynamic radius, such as HPLC, size exclusion, etc. Hydrodynamic semi-analyses were performed by normal-phase, reverse-phase, and anion-exchange HPLC, as well as capillary electrophoresis. If the number of glycosylation sites in a protein is large, the size of the glycosylation site can be measured. The variation in hydrodynamic radius is larger compared to carriers with smaller particle sizes. However, However, when single glycan chains were analyzed, these chains appeared to be more uniform due to their more controlled length. Glycan length can be determined by hydrazinolysis, SDS-PAGE, and capillary gel electrophoresis. Furthermore, homogeneity can be measured by the use of specific glycosylation sites. These factors may mean that the pattern changes to a broader or narrower range. The amount of the peptide can be measured by LC-MS / MS.

[0099] (Advantages of N-glycosylation) N-glycosylation provides a significant advantage to the HuGlyFabVEGFi used in the methods described herein. Such an advantage is due to the fact that E. coli does not naturally possess the components necessary for N-glycosylation. This cannot be achieved by production of antigen-binding fragments in E. coli because the Some advantages cannot be achieved through antibody production in, for example, CHO cells, because The structures required for the addition of certain glycans (e.g., 2,6-sialic acid and bisecting GlcNAc) The CHO cells lack the structural components and can synthesize glycans that are not typical for humans, such as Neu5Gc See, for example, Song et al., 2014, Anal. Chem. 86:5661-5666. Therefore, the anti-VEGF antigen-binding fragments described herein, e.g., ranibizumab, are Non-target N-glycosylation sites (including both reverse and non-consensus glycosylation sites) By virtue of the discovery that the anti-VEGF antigen-binding fragments contain glyceryl esters, such The present invention provides a method for expressing antigen-binding fragments in a manner that provides improved therapeutic benefits (and thus improved advantages associated with antigen-binding fragments). In particular, expression of anti-VEGF antigen-binding fragments in human retinal cells was shown to be consistent with otherwise antigen-binding HuGlyFabVEGFi( For example, ranibizumab).

[0100] Non-canonical glycosylation sites are typically associated with low levels of glycosylation in antibody populations (e.g., 1 ~5%), whereas functional benefits are more pronounced in immune-privileged organs, such as the eye. (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, For this purpose, any technique known to those skilled in the art can be used, such as enzyme-linked immunosorbent assay (ELISA) or a label. Surface plasmon resonance (SPR) can be used to determine the effect of Fab glycosylation on antibody half-life. To determine the effect, for example, blood or By measuring the level of radioactivity in an organ (e.g., the eye), any of the methods known to those skilled in the art can be used to determine the level of radioactivity. The following techniques can be used to determine the stability of antibodies, e.g., aggregation or protein unfolding. To determine the effect of Fab glycosylation on the level of glycosylation, any method known to those skilled in the art can be used. Techniques such as differential scanning calorimetry (DSC), high performance liquid chromatography (HPLC), e.g. Size-exclusion high-performance liquid chromatography (SEC-HPLC), capillary electrophoresis, mass spectrometry , or turbidity measurements can be used. , 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more glycosylated antigen binding In one embodiment, the antibody is an antibody that binds to the HuGlyFabVEGFi gene. 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% from the population of original binding fragments % or more of the antigen-binding fragments are glycosylated at non-canonical sites. In the standard range of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or more In some embodiments, these non-canonical sites are glycosylated. Glycosylation of antigen-binding fragments at the β-terminal position was not observed in antigen-binding fragments produced in HEK293 cells. The amount of glycosylation at these non-canonical sites was increased by 25%, 50%, 100%, 200%, 300%, or 400%. %, 400%, 500% or more larger.

[0101] The presence of sialic acid on HuGlyFabVEGFi used in the methods described herein is May affect the clearance rate of bVEGFi, e.g., from the vitreous humor Therefore, the sialic acid pattern of HuGlyFabVEGFi was used to determine the optimal clearance rate. Methods for assessing the clearance rate of antigen-binding fragments include , are known in the art. See, for example, Huang et al., 2006, Anal. Biochem. 349: Please see 197-207.

[0102] In another specific embodiment, the advantage conferred by N-glycosylation is reduced aggregation. Occupied N-glycosylation sites mask aggregation-prone amino acid residues, preventing aggregation. Such N-glycosylation sites can reduce the aggregation of the antibody as used herein. Either inherent in the original binding fragment or capable of being engineered into an antigen-binding fragment as used herein and is less likely to aggregate when expressed, e.g., in retinal cells. Methods for assessing antibody aggregation are known in the art. See, e.g., Courtois et al., 2016, mAbs, which is incorporated by reference in its entirety. Please see 8:99-112.

[0103] In another specific embodiment, the advantage conferred by N-glycosylation is reduced immunogenicity. Such N-glycosylation sites are conjugated to the antigen-binding fragments used herein. can be expressed or engineered into an antigen-binding fragment as used herein, The HuGlyFab expressed is less immunogenic when expressed, for example, in retinal cells. Bring on the VEGFi.

[0104] In another specific embodiment, the advantage conferred by N-glycosylation is Stability. N-glycosylation of a protein is known to confer stability to the protein. It is known in the art that methods for evaluating protein stability due to N-glycosylation are It is well known in the art. See, for example, Sola and Griebenow, 2009, J Pharm Sci., 98( 4): 1223-1245.

[0105] In another specific embodiment, the advantage conferred by N-glycosylation is increased binding affinity. The presence of N-glycosylation sites in the variable domain of an antibody changes its affinity to its antigen. It is known in the art that the affinity of antibodies can be increased. See, e.g., Wang et al., 2016, J. Immunol. 196:1435-1441. Measuring Antibody Binding Affinity Assays for determining the degree of cytotoxicity are known in the art. See, e.g., Wright et al., 1991. , EMBO J. 10:2717-2723; and Leibiger et al., 1999, Biochem. J. 338:529-538. I want to be illuminated.

[0106] 5.1.2 Tyrosine Sulfation Tyrosine sulfation occurs when glutamic acid (E) or aspartic acid (As) is attached within +5 to -5 of the tyrosine (Y) position. It has a paragic acid (D) and the -1 position of Y is a neutral or acidic charged amino acid, but is not sulfated. Neutralizing basic amino acids, such as arginine (R), lysine (K), or histidine (H) Surprisingly, for use according to the methods described herein, Anti-VEGF antigen-binding fragments for the treatment of VEGF-related diseases, such as ranibizumab, contain tyrosine sulfation sites (see Figure 1). Thus, the methods described herein include methods for detecting at least one tyrosine sulfation site. and the use of an anti-VEGF antigen-binding fragment, such as HuPTMFabVEGFi, The binding fragment may be tyrosine sulfated when expressed in a retinal cell.

[0107] Importantly, tyrosine sulfated antigen-binding fragments, such as ranibizumab, are not naturally occurring. It cannot be produced in E. coli, which does not have the enzyme required for tyrosine sulfation. O cells are deficient in tyrosine sulfation—they are not secretory cells and do not produce posttranslational tyrosine sulfation. They have limited capacity for oxidation. See, e.g., Mikkelsen and Ezban, 1991, Biochem. Mistry 30: 1533-1537. Advantageously, the methods provided herein include Anti-VEGF antigen binding in retinal cells that are secretory and do possess the capacity for tyrosine sulfation It is necessary to express a fragment such as HuPTMFabVEGFi, e.g. ranibizumab. Kanan et al., 2, reported the production of secreted tyrosine sulfated glycoproteins by cells. 009, Exp. Eye Res. 89: 559-567 and Kanan and Al-Ubaidi, 2015, Exp. Eye Res. . See 133:126-131.

[0108] Tyrosine sulfation is advantageous for several reasons, including increasing the affinity of therapeutic antibodies to their targets. Tyrosine sulfation of the original binding fragment can dramatically increase affinity and avidity for the antigen. See, for example, Loos et al., 2015, PNAS 112: 12675-12680, and Choe et al. See, e.g., 2003, Cell 114: 161-170. Assays for detecting tyrosine sulfation The method is known in the art. See, for example, Yang et al., 2015, Molecules 20:213. Please see 8-2164.

[0109] 5.1.3 O-Glycosylation O-glycosylation is the process by which enzymatic N-acetylgalactosylation occurs at serine or threonine residues. The amino acid residues present in the hinge region of the antibody can be O-glycosylated. In one embodiment, the antibodies used in accordance with the methods described herein are A VEGF antigen-binding fragment, such as ranibizumab, contains all or part of the hinge region and thus It can be O-glycosylated when expressed in human retinal cells. The affinity of the HuPTMFab fragments provided herein is compared to, for example, antigen-binding fragments produced in E. coli. This gives VEGFi, for example HuGlyFabVEGFi, another advantage because E. coli Naturally, they do not contain a mechanism equivalent to that used in human O-glycosylation. (Instead, O-glycosylation in E. coli is thought to be due to the fact that the bacterium contains a specific O-glycosylation machinery. For example, see Faridmoayer et al., 2007, J. Bacteriol. 189:8088-8098.) To have glycans, N-glycosylated HuPTMFabVEGFi, for example HuGlyFabVEGFi, has the same advantageous properties as N-glycosylated HuGlyFabVEGFi. It has certain characteristics (as discussed above).

[0110] 5.2 Constructs and Formulations For use in the methods, the term anti-VEGF antigen-binding fragment or anti-VEGF antigen-binding fragment is used herein. Viral vectors or other DNA expression constructs encoding hyperglycosylated derivatives of the fragments Viral vectors and other DNA expression constructs provided herein Any method suitable for delivery of a transgene to a target cell (e.g., a retinal pigment epithelial cell) can be used. Vehicles for delivery of transgenes include viral vectors, liposomes, and other lipid-containing complexes. bodies, other macromolecular complexes, synthetically modified mRNAs, unmodified mRNAs, small molecules, non-biologically active molecules (e.g. gold particles), polymeric molecules (e.g., dendrimers), naked DNA, plasmids, phages, In some embodiments, the nucleic acid sequence may be a gene, a transposon, a cosmid, or an episome. The vector is a targeted vector, e.g., a vector targeted to retinal pigment epithelial cells. .

[0111] In some embodiments, the disclosure provides a nucleic acid for use, wherein the nucleic acid comprises: CMV promoter, Rous sarcoma virus (RSV) promoter, MM T promoter, EF-1α promoter, UB6 promoter, chicken β-actin promoter a CAG promoter, an RPE65 promoter, and an opsin promoter. A HuPTMFabVEGFi, e.g., HuGlyFabVEGFi, operably linked to a promoter of choice. Code.

[0112] In certain embodiments, the term "nucleic acid" refers to one or more nucleic acids (e.g., polynucleotides) that are The nucleic acid may comprise DNA, RNA, or a combination of DNA and RNA. In one embodiment, the DNA comprises a promoter sequence, a sequence of a gene of interest (transgene), (e.g., an anti-VEGF antigen-binding fragment), a non-translated region, and a termination sequence. In some embodiments, the viral vectors provided herein comprise the following sequences: The promoter is operably linked to an elephant gene.

[0113] In certain embodiments, the nucleic acids (e.g., polynucleotides) and nucleic acids disclosed herein are The nucleic acid sequence may be codon optimized, for example, by any codon optimization technique known to those of skill in the art. (See, for example, the review by Quax et al., 2015, Mol Cell 59:149-161).

[0114] In specific embodiments, the constructs described herein comprise the following components: ) AAV2 inverted terminal repeats flanking the expression cassette; (2) a) CMV enhancer / chicken β-activator a) the CB7 promoter containing the chicken β-actin intron, and c) (3) a control element including a rabbit β-globin polyA signal; and (4) heavy and light chain polypeptides. separated by a self-cleaving Furin(F) / F2A linker that ensures equal expression of peptides , comprising nucleic acid sequences encoding the heavy and light chains of the anti-VEGF antigen-binding fragment.

[0115] (5.2.1 mRNA) In one embodiment, the vectors provided herein comprise a modified nucleotide sequence encoding a gene of interest. mRNA (e.g., a transgene, e.g., an anti-VEGF antigen-binding fragment portion). Synthesis of modified and unmodified mRNA for delivery of transgenes to cells is described, for example, in its entirety. Hansson et al., J. Biol. Chem., 2015, 290(9):5661, incorporated herein by reference. In certain embodiments, the anti-VEGF antigen-binding fragments described herein are The present invention provides modified mRNA encoding the polypeptide.

[0116] 5.2.2 Viral Vectors Viral vectors include adenoviruses, adeno-associated viruses (AAV, e.g., AAV8), Lentivirus, helper-dependent adenovirus, herpes simplex virus, poxvirus rus, hemagglutinin virus of Japan (HVJ), alfa These include vaccinia virus, rabies virus, and retrovirus vectors. Vectors include murine leukemia virus (MLV) and human immunodeficiency virus (HIV)-based vectors. Alphavirus vectors include Semliki Forest virus (SFV) and In one embodiment, the viruses provided herein include Sindbis virus (SIN). In one embodiment, the vector provided herein is a recombinant viral vector. The viral vector is engineered to be replication-deficient in humans. In the present study, the viral vector is a hybrid vector, e.g., a "helpless" adenovirus vector. In one embodiment, the AAV vector is disposed in a human endovirus vector. The document describes a viral capsid from a first virus and a viral endonucleaser from a second virus. In a specific embodiment, a viral vector comprising an envelope protein is provided. In a more specific embodiment, the second virus is vesicular stomatitis virus (VSV). The envelope protein is the VSV-G protein.

[0117] In certain embodiments, the viral vectors provided herein are HIV-based viruses. In one embodiment, the HIV-based vectors provided herein are , comprising at least two polynucleotides, wherein the gag and pol genes are derived from the HIV genome. However, the env gene is derived from another virus.

[0118] In some embodiments, the viral vectors provided herein are herpes simplex viruses. In one embodiment, the simple vector provided herein is a viral vector based on Rupesvirus-based vectors do not contain one or more immediate early (IE) genes and are therefore non- It is modified to be cytotoxic.

[0119] In certain embodiments, the viral vectors provided herein are MLV-based viruses. In one embodiment, the MLV-based vectors provided herein are , containing up to 8 kb of heterologous DNA in place of viral genes.

[0120] In some embodiments, the viral vectors provided herein are lentiviral-based. In one embodiment, the lentivirus provided herein is a viral vector. In one embodiment, the vector is derived from a human lentivirus. The lentiviral vector is derived from a non-human lentivirus. Thus, the lentiviral vectors provided herein are packaged in a lentiviral capsid. In one embodiment, the lentiviral vectors provided herein are caged. The nucleic acid sequence may contain one or more of the following elements: a long terminal repeat, a primer binding site, a polypurine sequence, It contains a polypurine tract, an att site, and an encapsidation site.

[0121] In some embodiments, the viral vectors provided herein are alphavirus vectors. In one embodiment, the alphavirus vectors provided herein are The virus vector is a recombinant replication-defective alphavirus. The alphavirus replicon in the alphavirus vector provided herein is Targeting to specific cell types by displaying functional heterologous ligands on the surface of the lysosomes do.

[0122] In some embodiments, the viral vectors provided herein are AAV-based viruses. In a preferred embodiment, the viral vector provided herein is is an AAV8-based viral vector. In one embodiment, AAV8-based viral vectors retain tropism for retinal cells. In one embodiment Therefore, the AAV-based vectors provided herein contain the AAV rep gene (required for replication). and / or encodes the AAV cap gene (required for synthesis of capsid protein). Several AAV serotypes have been identified. In one embodiment, the AAV-based vectors provided herein In one embodiment, the vector comprises components derived from one or more serotypes of AAV. The AAV-based vectors provided in this document are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV23, AAV24, AAV25 Capsid components derived from one or more of AV8, AAV9, AAV10, AAV11, or AAVrh10 In a preferred embodiment, the AAV-based vectors provided herein include AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV17, AAV18, AAV19, AAV20, AAV21, AAV22, AAV30, AAV40, AAV50, AAV60, AAV70, AAV81, AAV90, AAV10 It contains components derived from one or more of the AV9, AAV10, AAV11, or AAVrh10 serotypes.

[0123] In certain embodiments, the AAV used in the methods described herein is Anc80 or Anc80L. 65, which is incorporated by reference in its entirety, see Zinn et al., 2015, Cell Rep. 12(6): 1056-1068. In one embodiment, the method of claim 1, further comprising administering to said subject the subject the composition as described herein. The AAV used in the method comprises one of the following amino acid insertions: LGETTRP or LALGETTRP. No. 9,193, 919, 920, 930, 940, 950, 960, 970, 980, 990, 991, 992, 993, 994, 995, 996, 997, 998, 9 ... Nos. 956; 9458517; and 9,587,282, and U.S. Patent Application Publication No. 2016 / 0376323. As described herein. In one embodiment, the method for use in the method described herein The AAV is AAV.7m8, each of which is incorporated by reference in its entirety. Nos. 9,193,956; 9,458,517; and 9,587,282, and U.S. Patent Application Publication No. 2 In one embodiment, the method described herein is as described in US Pat. The AAV used in the present invention can be any AAV disclosed in U.S. Pat. No. 9,585,971, such as AAV-PHP. In some embodiments, the AAV used in the methods described herein is The following patents and patent applications are incorporated herein by reference in their entirety: U.S. Pat. No. 7,906,111; No. 8,524,446; No. 8,999,678; No. 8,628,966; No. 8,927,514; No. 8,734 ,809; US No. 9,284,357; No. 9,409,953; No. 9,169,299; No. 9,193,956; No. 9458517 Nos. 2015 / 0374803; 2015 / 0126588; and 9,587,282. 7 / 0067908; 2013 / 0224836; 2016 / 0215024; 2017 / 0051257; and International Patents. The AAV is disclosed in any of applications PCT / US2015 / 034799; PCT / EP2015 / 053335.

[0124] AAV8-based viral vectors are used in some of the methods described herein. Nucleic acid sequences of AAV-based viral vectors and methods for producing recombinant AAV and AAV capsids Methods for this purpose are described, for example, in U.S. Pat. Nos. 7, 28, 311, 321 and 322, each of which is incorporated herein by reference in its entirety. 2,199 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. As used herein, AAV (e.g., avian vein immunoglobulin (AVV)) encoding a transgene (e.g., an anti-VEGF antigen-binding fragment) is In a specific embodiment, a viral vector based on the present invention is provided. The present invention provides an AAV8-based viral vector encoding an anti-VEGF antigen-binding fragment. In a specific embodiment, the AAV8-based virus encoding ranibizumab is described herein. Provides the Russ Vector.

[0125] In some embodiments, single-stranded AAV (ssAAV) can be used as described above. In some embodiments, a self-complementary vector, such as a scAAV, can be used (e.g., See, for example, Wu, 2007, Human Genetics, each of which is incorporated by reference in its entirety. Therapy, 18(2):171-82, McCarty et al., 2001, Gene Therapy, Vol 8, Number 16, P ages 1248-1254; and U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683. Please refer to.

[0126] In one embodiment, the viral vector used in the methods described herein comprises: Adenovirus-based viral vectors. Recombinant adenovirus vectors are The recombinant adenovirus can be used to introduce VEGF antigen-binding fragments. It may or may not have a deletion of E3, and an expression cassette may be inserted in either deleted region. The recombinant adenovirus can be a first generation vector into which E2 and The second generation vector may contain a complete or partial deletion of the E3 and E4 regions. -dependent adenoviruses are expressed by the adenovirus inverted terminal repeat and packaging signal ( φ) The transgene is inserted between the packaging signal and the 3' ITR. It contains additional sequences to maintain the genome close to the wild-type size of approximately 36 kb. An exemplary protocol for producing an adenoviral vector includes: Alba et al., 2005, “Empty A Adenoviruses: the latest generation of adenoviruses for gene therapy (Gutless adenovirus: las "Genetic generation of adenovirus for gene therapy," Gene Therapy 12:S18-S27 This can be done.

[0127] In one embodiment, the viral vector used in the methods described herein comprises: The recombinant lentiviral vector is a lentivirus-based vector that delivers an anti-VEGF antigen. To create the construct, four processes were performed: Plasmid: A plasmid containing the Gag / pol sequence, a plasmid containing the Rev sequence, an envelope protein A plasmid containing the VSV protein (i.e., VSV-G) and packaging elements and an anti-V A Cis plasmid containing an EGF antigen-binding fragment gene is used.

[0128] For the production of lentiviral vectors, the four plasmids were transfected into cells (i.e., HEK293 The transfection agent is then co-transfected into the target cell line (the base cell line). In particular, polyethyleneimine or calcium phosphate can be used. Harvest lentivirus (lentiviruses need to bud from cells to be active). (Therefore, there is no need and should not be any cell harvesting.) The supernatant is filtered (0.45 μm), followed by the addition of magnesium chloride and benzonase. Can be varied, with the most GMP-compliant using TFF and column chromatography Other processes use ultracentrifugation and column chromatography. Exemplary processes for the production of lentiviral vectors include The protocol is described in Lesch et al., both of which are incorporated herein by reference in their entireties. , 2011, "Lentivirus generated in 293T suspension cells containing baculovirus vectors" Production and purification of lentiviral vectors erated in 293T suspension cells with baculoviral vectors), Gene Therapy 18:53 1-538, and Ausubel et al., 2012, "Production of CGMP-Grade Lentiviral Vectors ( Production of CGMP-Grade Lentiviral Vectors)”, Bioprocess Int. 10(2):32-43. It can be put out.

[0129] In a specific embodiment, the vectors used in the methods described herein are When introducing the vector into cells (e.g., retinal cells in vivo or in vitro) , glycosylated and / or tyrosine sulfated by the cell, An anti-VEGF antigen-binding fragment (e.g., ranibizumab) is expressed such that a variant of the fragment is expressed. In a specific embodiment, the expressed anti-VEGF antigen-binding fragment is a vector encoding the contains the glycosylation and / or tyrosine sulfation patterns described above in Section 5.1.

[0130] 5.2.3 Promoters and Modifiers of Gene Expression In some embodiments, the vectors provided herein are capable of mediating gene delivery or gene expression. In one embodiment, the gene includes a regulatory component (e.g., an "expression control element"). The vectors provided herein contain components that regulate gene expression. In the present invention, the vectors provided herein include components that affect binding or targeting to cells. In some embodiments, the vectors provided herein are capable of being integrated into cells. Contains components that affect the localization of a polynucleotide (e.g., a transgene) within a cell In certain embodiments, the vectors provided herein are capable of transporting, e.g., a polynucleotide. Use as a detectable or selectable marker to detect or select transfected cells The present invention includes a component that can

[0131] In certain embodiments, the viral vectors provided herein comprise one or more promoters In some embodiments, the promoter is a constitutive promoter. In embodiments, the promoter is an inducible promoter. The promoter is a hypoxia-inducible promoter. In one embodiment, the promoter In one embodiment, the promoter comprises a hypoxia inducible factor (HIF) binding site. In one embodiment, the promoter comprises a HIF-1α binding site. In one embodiment, the HIF binding site comprises an RCGTG motif. For details regarding the location and sequence, see, for example, See Schodel et al., Blood, 2011, 117(23):e207-e217. In these cases, the promoter is involved in the binding of hypoxia-inducible transcription factors other than HIF transcription factors. In some embodiments, the viral vectors provided herein are capable of inducing transcriptional activity in response to hypoxia. The gene contains one or more IRES sites that are preferentially translated in the presence of hypoxia. The disclosure and factors involved therein are set forth in, for example, US Pat. See Kenneth and Rocha, Biochem J., 2008, 414:19-29.

[0132] In one embodiment, the promoter is the CB7 promoter (see, e.g., U.S. Pat. No. 6,213,945, which is incorporated by reference in its entirety). Dinculescu et al., 2005, Hum Gene Ther 16:649-66, incorporated herein by reference. In some embodiments, the CB7 promoter is expressed by a vector. It contains other expression control elements that enhance expression of the transgene being driven. In the present specification, other expression control elements include chicken β-actin intron and / or rabbit In one embodiment, the promoter comprises a TATA block. In some embodiments, the promoter comprises one or more elements. In embodiments, one or more promoter elements are in inverted orientation relative to each other In some embodiments, the promoter elements may be arranged to function in a coordinated manner. In one embodiment, the elements of the promoter are arranged to function independently. In one embodiment, the viral vector provided herein is arranged to Immediate early gene promoter, SV40 early promoter, Rous sarcoma virus (RS) long tail repeat sequence, and one or more promoters selected from the group consisting of rat insulin promoter In certain embodiments, the vectors provided herein include vectors selected from AAV, MLV, MMTV, , SV40, RSV, HIV-1, and HIV-2 LTRs. In one embodiment, the vectors provided herein comprise a 1 Including the above tissue-specific promoters (e.g., retinal pigment epithelial cell-specific promoters) In some embodiments, the viral vectors provided herein are In certain embodiments, the vectors provided herein comprise a VMD2 promoter. nothing.

[0133] In some embodiments, the viral vectors provided herein include a nucleic acid sequence other than a promoter. In one embodiment, the viral vectors provided herein comprise one or more regulatory elements. In some embodiments, the viral vectors provided herein include enhancers. In some embodiments, the viral vector provided herein comprises a repressor. In some embodiments, the vector comprises an intron or a chimeric intron. The provided viral vectors contain polyadenylation sequences.

[0134] 5.2.4 Signal peptides In certain embodiments, the vectors provided herein regulate the delivery of proteins. In certain embodiments, the viral vectors provided herein comprise one or more The signal peptide includes the above signal peptide. Also, the signal peptide is referred to herein as a "leader peptide." In some embodiments, the signal peptide may be referred to as a "leader peptide." The peptide allows the transgene product (e.g., an anti-VEGF antigen-binding fragment portion) to undergo proper pathway expression in the cell. In one embodiment, it is possible to achieve packaging (e.g., glycosylation). In such a case, the signal peptide controls the expression of the transgene product (e.g., the anti-VEGF antigen-binding fragment portion). In one embodiment, the signal peptide can be used to achieve proper localization in cells. The tide allows the transgene product (e.g., the anti-VEGF antigen-binding fragment portion) to be selected from the cells. The vectors and transgenes provided herein can be used in conjunction with Examples of signal peptides that can be used can be found in Table 1. Table 1. Signal peptides for use with the vectors provided herein [Table 1]

[0135] 5.2.5 Polycistronic Messages - IRES and F2A Linkers Internal ribosome entry site. A single construct can be bound by a cleavable linker or an IRES. The transduced cells are engineered to encode both heavy and light chains separated by a More separate heavy and light chain polypeptides can be expressed. In embodiments, the viral vectors provided herein are polycistronic (e.g., For example, viral constructs can deliver internal ribosomal (isistronic) messages. It can encode heavy and light chains separated by an IRES element (IRES For examples of the use of elements to generate bicistronic vectors, see, e.g. Gurtu et al., 1996, Biochem. Biop, which is incorporated herein by reference in its entirety. Hys. Res. Comm. 229(1):295-8). IRES elements are The use of an IRES in AAVs allows the entire Furling et al., 2001, Gene Ther 8(11): 854-858, which is incorporated herein by reference. In one embodiment, the bicistronic message is The polynucleotide(s) contained within the viral vector are limited in size. In one embodiment, the bicistronic message is delivered by an AAV virus-based vector. The vector can be contained within a vector (e.g., an AAV8-based vector).

[0136] Furin-F2A linker. In another embodiment, the viral vector provided herein is a cleavable linker, such as a self-cleaving Furin / F2A (F / F2A) linker (each in its entirety) Fang et al., 2005, Nature Biotechnology 2, which is incorporated herein by reference. 3: 584-590, and Fang, 2007, Mol Ther 15: 1153-9). The ribosome encodes the primary and light chains.

[0137] For example, a Furin-F2A linker is included in the expression cassette separating the heavy and light chain coding sequences. Incorporating structure: leader-heavy chain-furin site-F2A site-leader-light chain-polyA This can result in a construct having the following structure:

[0138] Amino acid sequence [ka] The F2A site containing the Additional linkers that can be used include, but are not limited to: [ka] There is.

[0139] When the ribosome encounters the F2A sequence in an open reading frame, peptide bond formation occurs. This leads to either the termination of translation or to the continued translation of the downstream sequence (light chain). The self-processing sequence of provides an additional chain of amino acids at the C-terminal end of the heavy chain. However, such additional amino acids are subsequently converted to the F2A site by the host cell's furin. It is cleaved at the furin site located immediately before and after the heavy chain sequence and is converted to carboxypeptidase The resulting heavy chain has the sequence of the Furin linker used. and 1, 2, 3, or 4 at the C-terminus, depending on the carboxypeptidase that cleaves the linker in vivo. or more additional amino acid sequences, or such additional amino acids may be included. (See, for example, Fang et al., 17 April 2005, Nature Biotechnol. Advance Online Fang et al., 2007, Molecular Therapy 15(6):1153-1159; Luke, 2012, (See, for example, J. Am. Chem. Soc., 1999, 144: 161-186.) The linker is a stretch of four basic amino acids, e.g., RKRR, RRRR, RRKR, or RKKR. Once this linker is cleaved by carboxypeptidase, an additional 0, 1, 2, 3, or 4 amino acids, e.g., R, RR, RK, RKR, RRR, RRK, RKK, RKRR, RRRR, RRKR, Alternatively, additional amino acids may remain such that RKKR may remain on the C-terminus of the heavy chain. In embodiments, once the linker is cleaved by the carboxypeptidase, additional amines are In some embodiments, the amino acid is not present for use in the methods described herein. 5%, 10%, 15%, or Approximately 20% of the population has 1, 2, 3, or 4 amino acids remaining on the C-terminus of the heavy chain after cleavage. In one embodiment, the Furin linker has the sequence RXK / RR, such that The additional amino acids are R, RX, RXK, RXR, RXKR, or RXRR, where X is any amino acid. In some embodiments, the additional amino acid is at the C-terminus of the heavy chain, e.g., alanine (A). It cannot remain.

[0140] In one embodiment, the expression cassettes described herein include the polynucleotides The viral vector is contained within a viral vector that is constrained with respect to size. In the present invention, the expression cassette is an AAV virus-based vector (e.g., an AAV8-based vector). -) can be included.

[0141] (5.2.6 Untranslated Regions) In certain embodiments, the viral vectors provided herein contain one or more untranslated regions. In some embodiments, the UTRs are involved in protein expression. In one embodiment, the UTRs are optimized for a desired level of expression. In one embodiment, the UTRs are optimized for the half-life of the transgene mRNA. In one embodiment, the UTRs are optimized for the secondary structure of the mRNA of the transgene. It is optimized for

[0142] 5.2.7 Inverted terminal repeats In some embodiments, the viral vectors provided herein contain one or more inverted terminal repeats. The ITR sequence is what allows the virion of the viral vector to contain the recombinant gene expression cassette. In one embodiment, the ITRs can be used to package the AAV For example, derived from AAV8 or AAV2 (e.g., each of which is incorporated by reference in its entirety herein). See 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).

[0143] 5.2.8 Transgene The HuPTMFabVEGFi encoded by the transgene, e.g., HuGlyFabVEGFi, is not limited to these. Although not limited to, antigen-binding fragments of antibodies that bind to VEGF, e.g., bevacizumab; anti-VEGF Fab or the Fab fragment of such bevacizumab or ranibizumab. The Fab portion may be engineered to contain additional glycosylation sites on the (e.g., a derivative of bevacizumab with hyperglycosylation on the Fab domain of the full-length antibody) For a description, see Courtois et al., which is incorporated herein by reference in its entirety. See 2016, mAbs 8: 99-112.

[0144] In one embodiment, the vector provided herein comprises an anti-VEGF antigen-binding fragment transgene. In a specific embodiment, the anti-VEGF antigen-binding fragment transgene encodes a retinal cell. In one embodiment, the expression control elements are suitable for expression in cells. In the present study, the anti-VEGF antigen-binding fragment transgene was synthesized by the bevacizumab light and heavy chain cDNA sequences ( In one embodiment, the anti-VEGF antigen-binding fragment derivative comprises a Fab portion of SEQ ID NO: 10 and SEQ ID NO: 11. The transgene contains the ranibizumab light and heavy chain cDNA sequences (SEQ ID NOs: 12 and 13, respectively). In one embodiment, the anti-VEGF antigen binding fragment transgene comprises the light chains of SEQ ID NOs: 3 and 4, respectively. In one embodiment, the antibody encodes an anti-VEGF antigen binding domain, and the antibody encodes a bevacizumab Fab comprising a heavy chain and a low chain. The fusion fragment transgene has at least 85%, 86%, 87%, 88% identical sequence to the sequence set forth in SEQ ID NO:3. , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical In one embodiment, the anti-VEGF antibody encodes an antigen-binding fragment comprising a light chain comprising the amino acid sequence The GF antigen-binding fragment transgene has at least 85%, 86%, 87%, or 100% identical sequence to the sequence set forth in SEQ ID NO:4. %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% In one embodiment, the antigen-binding fragment encodes an antigen-binding fragment comprising a heavy chain comprising an amino acid sequence identical to that of the heavy chain. The anti-VEGF antigen-binding fragment transgene has at least 85% and 85% identical sequence to the sequence set forth in SEQ ID NO:3. 6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or a light chain comprising an amino acid sequence that is 99% identical to the sequence set forth in SEQ ID NO:4, and Also 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, The antigen-binding fragments each encode a heavy chain that contains an amino acid sequence that is 98% or 99% identical to the antigen-binding fragments. In certain embodiments, the anti-VEGF antigen binding fragment transgene comprises the light chain of SEQ ID NOs: 1 and 2, respectively. and a heavy chain comprising hyperglycosylated ranibizumab. The anti-VEGF antigen-binding fragment transgene has at least 85% and 85% identical sequence to the sequence set forth in SEQ ID NO:1. 6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or In one embodiment, the antigen-binding fragment comprises a light chain that comprises an amino acid sequence that is 99% identical to the antigen-binding fragment of the present invention. In the present invention, the anti-VEGF antigen-binding fragment transgene has at least the sequence set forth in SEQ ID NO:2. Also 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, The antigen-binding fragments each encode a heavy chain that contains an amino acid sequence that is 98% or 99% identical to the antigen-binding fragments. In one embodiment, the anti-VEGF antigen binding fragment transgene has the sequence set forth in SEQ ID NO:1. At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98% or 99% identical amino acid sequence to that set forth in SEQ ID NO:2. Sequences with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, or 99% identical to the heavy chain of any one of the antigen-binding fragments of the present invention. Code the following:

[0145] In one embodiment, the anti-VEGF antigen binding fragment transgene comprises the light chain and heavy chain, and one or more of the following mutations: L118N (heavy chain), E195N (light chain), or Q160N or It encodes a hyperglycosylated bevacizumab Fab with Q160S (light chain). In an embodiment, the anti-VEGF antigen binding fragment transgene comprises the light and heavy chains of SEQ ID NOs: 1 and 2. and one or more of the following mutations: L118N (heavy chain), E195N (light chain), or Q160N or Q160S. (light chain) encoding hyperglycosylated ranibizumab. Antigen-binding fragment introduction The cDNA sequences of the genes can be found, for example, in Table 2. In certain embodiments, the antigen binding The cDNA sequence of the fusion fragment transgene is the signal sequence of SEQ ID NO: 10 and 11 or SEQ ID NO: 12 and 13. with one or more of the signal sequences listed in Table 1.

[0146] In one embodiment, the anti-VEGF antigen binding fragment transgene encodes an antigen binding fragment, and the nucleotide sequences of the six bevacizumab CDRs. The original binding fragment transgene encodes an antigen-binding fragment and contains the nucleotides of the six ranibizumab CDRs. In one embodiment, the anti-VEGF antigen-binding fragment transgene comprises a ranibizumab sequence. An antigen-binding fragment comprising a heavy chain variable region including the heavy chain CDRs 1 to 3 (SEQ ID NOs: 20, 18, and 21) of the mab In one embodiment, the anti-VEGF antigen binding fragment transgene encodes ranibizumab. The antigen-binding fragment includes a light chain variable region including light chain CDR1 to 3 (SEQ ID NOs: 14 to 16) of In one embodiment, the anti-VEGF antigen-binding fragment transgene comprises the heavy chain CDR1-CDR2-CDR3 of bevacizumab. 3 (SEQ ID NOs: 17 to 19). In the above, the anti-VEGF antigen-binding fragment transgene is a light chain CDR1-3 of bevacizumab (SEQ ID NO: 1 In one embodiment, the antigen-binding fragment encodes an antigen-binding fragment comprising a light chain variable region comprising: The anti-VEGF antigen-binding fragment transgene is composed of the heavy chain CDRs 1 to 3 of ranibizumab (SEQ ID NOs: 20, 18, and 2 1), and a light chain comprising the light chain CDRs 1 to 3 of ranibizumab (SEQ ID NOs: 14 to 16). In one embodiment, the anti-VEGF antigen-binding fragment encodes an antigen-binding fragment comprising a variable region. The transgene comprises a heavy chain variable region including heavy chain CDRs 1 to 3 (SEQ ID NOs: 17 to 19) of bevacizumab, and and an antigen-binding fragment comprising a light chain variable region including the light chain CDRs 1 to 3 (SEQ ID NOs: 14 to 16) of bevacizumab. Code a piece. Table 2. Exemplary transgene sequences [Table 2] TIFF2025000642000008.tif221170TIFF2025000642000009.tif215170TIFF2025000642000010.tif216170TIFF2025000642000011.tif92170

[0147] (5.2.9 Constructs) In one embodiment, the viral vector provided herein comprises the following components in the following order: The element: a) a constitutive or hypoxia-inducible promoter sequence, and b) encoding a transgene. In some embodiments, the transgene comprises a sequence that binds to the VEGF antigen (e.g., an anti-VEGF antigen-binding fragment portion). The sequence encoding the gene comprises multiple ORFs separated by IRES elements. In one embodiment, the ORF encodes the heavy and light chain domains of an anti-VEGF antigen-binding fragment. In the present invention, the transgene coding sequence is contained in one ORF separated by F / F2A sequences. In one embodiment, the sequence comprising the transgene is the F / F2A sequence. The heavy and light chain domains of the anti-VEGF antigen-binding fragment are separated by a sequence. In embodiments, the viral vectors provided herein comprise the following elements in the following order: a) a constitutive or hypoxia-inducible promoter sequence, and b) a sequence encoding a transgene. (e.g., an anti-VEGF antigen-binding fragment portion), where the transgene contains a signal peptide of VEGF. The transgene comprises a light and heavy chain sequence separated by an IRES element (SEQ ID NO:5). In one embodiment, the viral vectors provided herein encode the following sequences: The following elements, in this order: a) a constitutive or hypoxia-inducible promoter sequence, and b) a vector. a sequence encoding a transgene (e.g., an anti-VEGF antigen-binding fragment portion), The vector contains the VEGF signal peptide (SEQ ID NO: 5) and the transgene is bound to a cleavable F / F2A sequence. Thus, they encode separated light and heavy chain sequences.

[0148] In one embodiment, the viral vector provided herein comprises the following components in the following order: Elements: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or hypoxia-inducible promoter. a motor sequence, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence; h) a sequence encoding a transgene (e.g., an anti-VEGF antigen-binding fragment portion); i) j) a fourth linker sequence; k) a polyA sequence; l) a fifth linker sequence; and m) Contains the second ITR sequence.

[0149] In one embodiment, the viral vector provided herein comprises the following components in the following order: Elements: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or hypoxia-inducible promoter. a motor sequence, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence; h) a sequence encoding a transgene (e.g., an anti-VEGF antigen-binding fragment portion); i) j) a fourth linker sequence; k) a polyA sequence; l) a fifth linker sequence; and m) A second ITR sequence, wherein the transgene contains the signal peptide of VEGF (SEQ ID NO:5). The transgene encodes light and heavy chain sequences separated by a cleavable F / F2A sequence. .

[0150] 5.2.10 Vector Production and Testing The viral vectors provided herein can be produced using host cells. The provided viral vectors can be expressed in mammalian host cells, such as A549, WEHI, 10T1 / 2, BHK, MDC, K, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1 080, HepG2, primary fibroblasts, hepatocytes, and myoblasts can be used. The viral vectors provided herein can be used in humans, monkeys, mice, rats, rabbits, or rabbits. It can be produced using host cells derived from Mus ster.

[0151] The host cell is transformed with the transgene and associated elements (i.e., the vector genome), as well as means for producing the virus in a host cell, e.g., replication and capsid genes (e.g. For example, the AAV rep and cap genes are used to stably transform the AAV8 capsid. Methods for producing recombinant AAV vectors having the peptides are described herein, which are hereby incorporated by reference in their entirety. See Section IV of the Detailed Description of U.S. Patent No. 7,282,199 B2, which is incorporated herein by reference. The genome copy titer of the vector can be determined, for example, by TAQMAN® analysis. Virions can be harvested, for example, by CsCl2 precipitation.

[0152] In vitro assays, such as cell culture assays, can be used to detect the vectors described herein. Expression of the transgene from the vector can be measured, thus indicating, for example, the efficacy of the vector. For example, the PER.C6® cell line (Lonza), which is a cell line derived from human fetal retinal cells; or retinal pigment epithelial cells, such as the retinal pigment epithelial cell line hTERT RPE-1 (available from ATCC®) Once expressed, expression can be assessed using Glycosylation and tyrosine sulfation associated with the expressed product (i.e., HuGlyFabVEGFi) The HuGlyFabVEGFi can be characterized, including determining the pattern of glycosyl The patterns of tyrosine sulfation and how they are determined are discussed in Section 5.1.1. The sequences and methods for their determination are discussed in Section 5.1.2. The benefits of glycosylation / sulfation of EGFi can be determined by assays known in the art, such as the fifth step. This may be determined using the methods described in Sections 5.1.1 and 5.1.2.

[0153] (5.2.11 Composition) A composition comprising a vector encoding a transgene as described herein and a suitable carrier is described. Suitable carriers (e.g., for subretinal and / or intraretinal administration) can be readily determined by one of skill in the art. will be selected.

[0154] (5.3 Gene Therapy) Introduction of a therapeutically effective amount of the compound to a human subject having an eye disease caused by increased angiogenesis. Methods for administering the transgene construct are described. More specifically, For administering a therapeutically effective amount of a transgene construct to a patient, particularly a subretinal and / or Methods for intraretinal administration are described. In certain embodiments, such therapeutically effective amounts Preventing exudation using methods for subretinal and / or intraretinal administration of transgene constructs Patients with AMD or diabetic retinopathy can be treated.

[0155] Introduction of a therapeutically effective amount to patients diagnosed with eye diseases caused by increased angiogenesis Methods for subretinal and / or intraretinal administration of genetic constructs are described. In embodiments, such a therapeutically effective amount of the transgene construct is administered subretinal and / or The method for intraretinal administration can be used to treat AMD; and in particular, wet AMD (neovascular AMD) or glycosuria. Patients diagnosed with diabetic retinopathy can be treated.

[0156] Also provided herein are subretinal and / or intraretinal administration of a therapeutically effective amount of a transgene construct. Methods for administration of a therapeutically effective amount of a transgene construct to the retinal pigment epithelium Provide a method for providing.

[0157] 5.3.1 Target Patient Population In one embodiment, the methods provided herein involve treating cancer caused by increased angiogenesis. The drug is intended for administration to patients diagnosed with an eye disease that is

[0158] In certain embodiments, the methods provided herein include administering to a patient diagnosed with severe AMD. In certain embodiments, the methods provided herein are for administration of reduced It is intended for administration to patients diagnosed with AMD.

[0159] In certain embodiments, the methods provided herein are directed to a patient diagnosed with severe wet AMD. In some embodiments, the methods provided herein are for administration to a subject. The drug is intended for administration to patients diagnosed with wet AMD.

[0160] In certain embodiments, the methods provided herein are directed to a patient diagnosed with severe diabetic retinopathy. For administration to a patient. In certain embodiments, the methods provided herein include: It is intended for administration to patients diagnosed with reduced diabetic retinopathy.

[0161] In certain embodiments, the methods provided herein relate to a patient who is responsive to treatment with an anti-VEGF antibody. The study is intended for administration to patients diagnosed with AMD who have been identified as having AMD.

[0162] In certain embodiments, the methods provided herein involve treatment with an anti-VEGF antigen-binding fragment. It is intended for administration to patients diagnosed with AMD who are identified as likely to respond.

[0163] In some embodiments, the methods provided herein include administering a vitreous sample prior to gene therapy treatment. Patients diagnosed with AMD who have been identified as responding to treatment with intracellularly injected anti-VEGF antigen-binding fragments It is intended for administration to patients.

[0164] In some embodiments, the methods provided herein are administered with LUCENTIS® (Ranibiz mab), EYLEA® (aflibercept), and / or AVASTIN® (vestib For administration to patients diagnosed with AMD who have been identified as responsive to treatment with vasodilator therapy (vacizumab) It is of the following.

[0165] 5.3.2 Dosage and Mode of Administration The therapeutically effective dose of the recombinant vector is in the range of ≧0.1 mL to ≦0.5 mL, preferably 0.1 to 0.30 Injection volumes in the range of mL (100-300 μl), and most preferably 0.25 mL (250 μl) It should be delivered subretinal at a concentration of at least 0.330 μg / mL in the vitreous humor or 0.400 μg / mL in the aqueous humor (the ocular 0.110 μg / mL C minThe dose required to maintain transgene product concentration at 100 mg / kg / day for 3 months was then, intravitreal C of the transgene product in the range of 1.70–6.60 μg / mL min Concentration, and / or or in the range of 0.567 to 2.20 μg / mL in the ocular chamber C min The concentration should be maintained. The transgene product is produced continuously (either under the control of a constitutive promoter or in the presence of hypoxia). If an inducible promoter is used, it is induced by hypoxic conditions) and maintained at a lower concentration Vitreous humor concentration is the patient's concentration of the vitreous humor or fluid collected from the anterior chamber. This can be measured directly in the sample or by measuring the patient's serum concentration of the transgene product. The effect of the transgene product on the overall systemic exposure can be estimated and / or monitored using a The ratio of 1:90,000 to total body exposure is approximately 1:90,000 (see, for example, US Pat. No. 6,313,943, incorporated herein by reference in its entirety). Xu L et al., 2013, Invest. Opthal. Vis. Sci. 54: 1616-1624, p. 16 See Table 5 on p. 21 and p. 1623 for the vitreous and serum concentrations of ranibizumab. I want to be illuminated.

[0166] In certain embodiments, the dosage is administered to the patient's eye (e.g., subretinal and / or In one embodiment, 1×10 9 ~1×10 11 In a specific embodiment, 1×10 genome copies are administered. 9 ~5×10 9 Geno In a specific embodiment, 6×10 9 ~3×10 10 Administering genome copies In a specific embodiment, 4×10 10 ~1×10 11 A genome copy is administered.

[0167] 5.3.3 Sampling and Efficacy Monitoring The effect of the treatment methods provided herein on visual impairment was measured using a BCVA (best corrected visual acuity), intraocular pressure, and sclerosing eyelids. Can be measured by lit-lamp biomicroscopy and / or indirect ophthalmoscopy do.

[0168] The effect of the therapeutic methods provided herein on physical changes to the eye / retina can be measured using SD-OCT (SD-optical coherence tomography). It can be measured by computed tomography.

[0169] Efficacy can be monitored as measured by electroretinography (ERG).

[0170] The effects of the methods of treatment provided herein include vision loss, infection, inflammation, and retinal detachment. Other safety events can be monitored by measuring indicia.

[0171] Retinal thickness can be monitored to determine the effectiveness of the treatments provided herein. Without being bound to any particular theory, the use of retinal thickness as a clinical reading The greater the decrease in retinal thickness, or the longer the period until the retinal thickness increases, Treatment becomes more effective. For example, retinal function can be determined by ERG. ERG is a It is a non-invasive electrophysiological test of retinal function approved by the FDA for use in The light-sensitive cells of the eye (rods and cones) and the ganglion cells to which they connect, especially The retinal thickness can be determined, for example, by SD-OCT. Low-coherence interferometry is used to measure echo time delays and their reflections from the object of interest. OCT is a three-dimensional imaging technique that determines the amplitude of axially scattered light. It is possible to scan layers of tissue samples (e.g., retina) that are as small as 1 μm, making SD-OCT a step beyond anything seen in the past. This improves axial resolution and scan speed over conventional techniques (Schuman, 2008, Trans. ns. Am. Opthamol. Soc. 106:426-458).

[0172] 5.4 Combination Therapy The methods of treatment provided herein can be used in combination with one or more additional therapies. In some embodiments, the methods of treatment provided herein are administered in conjunction with laser photocoagulation. In the present specification, the method of treatment is provided in combination with photodynamic therapy with verteporfin. Carry out.

[0173] In one aspect, the methods of treatment provided herein include, but are not limited to, HuPTMF abVEGFi, e.g., HuGlyFabVEGFi produced in human cell lines (Dumont et al., 2015, supra), or other anti-VEGF agents, such as pegaptanib, ranibizumab, aflibercept, or with intravitreal (IVT) injection of anti-VEGF drugs, including bevacizumab.

[0174] Additional treatments can be administered prior to, concurrently with, or subsequent to the gene therapy treatment. .

[0175] The efficacy of gene therapy treatments can be evaluated using standard therapies, such as, but not limited to, HuPTMFabVE GFi, e.g., HuGlyFabVEGFi produced in a human cell line, or other anti-VEGF agents, e.g. Anti-VEGF agents, including pegaptanib, ranibizumab, aflibercept, or bevacizumab Elimination or reduction of the frequency of rescue treatment using intravitreal (IVT) injections of drugs It can be shown. Table 3. Sequence Listing [Table 3] TIFF2025000642000013.tif228170TIFF2025000642000014.tif225170TIFF20250006420 00015.tif228170TIFF2025000642000016.tif226170TIFF2025000642000017.tif114170

[0176] 6. Working Examples 6.1 Example 1: Bevacizumab Fab cDNA-Based Vectors Derivatives containing the Fab portions of the bevacizumab light and heavy chain cDNA sequences (SEQ ID NOs: 10 and 11, respectively) A bevacizumab Fab cDNA-based vector containing the transgene was constructed. comprises a nucleic acid comprising a signal peptide selected from the group listed in Table 1. Light and Heavy Chains The nucleotide sequence encoding the IRES element is used to create a bicistronic vector. Optionally, the vector further comprises a hypoxia-inducible The promoter is

[0177] 6.2 Example 2: Ranibizumab cDNA-Based Vectors Ranibizumab Fab light and heavy chain cDNA (part of SEQ ID NOs: 12 and 13, respectively, signal peptide The ranibizumab Fab cDNA-based vector contained a transgene containing the Fab gene (which does not encode the Fab gene). The transgene is constructed by integrating a signal peptide selected from the group listed in Table 1. The nucleotide sequences encoding the light and heavy chains are bicistronic. Separated by an IRES element or 2A cleavage site to create a vector. Optional Additionally, the vector further comprises a hypoxia-inducible promoter.

[0178] 6.3 Example 3: Hyperglycosylated Bevacizumab Fab cDNA-Based Vectors One or more of the following mutations: L118N (heavy chain), E195N (light chain), or Q160N or Q160S ( Bevacizumab light and heavy chain cDNA sequences with mutations to the sequences encoding the light chain A hyperglycosylated bevacizumab containing a transgene containing the Fab portion of SEQ ID NOs: 10 and 11, respectively. A mab Fab cDNA-based vector was constructed, and the transgene was selected from the group listed in Table 1. The nucleic acid encoding the light and heavy chains includes a nucleic acid containing a signal peptide of choice. The sequence may be modified by an IRES element or a 2A cleavage site to create a bicistronic vector. Optionally, the vector further comprises a hypoxia inducible promoter.

[0179] 6.4 Example 4: Hyperglycosylated Ranibizumab cDNA-Based Vectors One or more of the following mutations: L118N (heavy chain), E195N (light chain), or Q160N or Q160S ( Ranibizumab Fab light and heavy chain cDNAs (which A transgene containing a portion of SEQ ID NO: 12 and SEQ ID NO: 13, respectively, which does not encode a signal peptide A hyperglycosylated ranibizumab Fab cDNA-based vector containing the The transgene comprises a nucleic acid comprising a signal peptide selected from the group listed in Table 1. Light Chain The nucleotide sequence encoding the heavy chain is Optionally, the vector further comprises an IRES element or a 2A cleavage site. Contains an oxygen inducible promoter.

[0180] 6.5 Example 5: Ranibizumab-Based HuGlyFabVEGFi Ranibizumab Fab cDNA-based vectors (see Example 2) were engineered in an AAV8 background. The resulting product, Lani, was expressed in the PER.C6® cell line (Lonza). Determine that the visumab-based HuGlyFabVEGFi is stably produced. The N-glycosylation of is confirmed by hydrazinolysis and MS / MS analysis. See reference Mol. & Cell. Proteomics 13.11:3029-3039. We found that HuGlyFabVEGFi is N-glycosylated, with 2,6 sialic acid being the predominant modification. The advantageous properties of N-glycosylated HuGlyFabVEGFi are confirmed by methods known in the art. The HuGlyFabVEGFi was found to have high stability and high affinity to its antigen (VEGF). The stability of the compound can be evaluated by the Sola and Gr See Iebenow, 2009, J Pharm Sci., 98(4): 1223-1245 for methods to assess affinity. See Wright et al., 1991, EMBO J. 10:2717-2723 and Leibiger et al., 1999, Biochem. See Hem. J. 338:529-538.

[0181] 6.6 Example 6: Treatment of Wet AMD with Ranibizumab-Based HuGlyFabVEGFi To determine the advantageous properties of ranibizumab-based HuGlyFabVEGFi (see Example 5) Based on these results, the ranibizumab Fab cDNA-based vector, when expressed as a transgene, It is believed to be useful in the treatment of wet AMD. at a dose sufficient to achieve a concentration of transgene product of at least 0.330 μg / mL Cmin in the culture solution. AAV8 encoding ranibizumab Fab is administered. Following treatment, subjects are shown to demonstrate improvement in wet AMD symptoms. Evaluate the good.

[0182] 6.7 Example 7: Single-dose subretinal administration induces VEGF-binding protein 1 (VEGF) expression in transgenic Rho / VEGF mice (Retinal neovascularization is reduced) This study uses the juvenile transluminal retina, a model of neovascular changes in the human retina with nAMD. Single dose in transgenic Rho / VEGF mice (Tobe, 1998, IOVS 39(1):180-188) This study demonstrates the in vivo efficacy of the HuPTMFabVEGFi vector described in Section 5.2 of Rho / VEGF mice. The rhodopsin promoter constitutively regulates the expression of human VEGF165 in photoreceptor cells from postnatal day 10. The retina begins to drive the retinal wall, and new blood vessels branch out from the deep capillary bed of the retina and grow into the subretinal space. The production of VEGF is sustained, and thus new blood vessels grow. The retinal vascular lesions continue to grow and expand within the subretinal space and are seen in people with neovascular age-related macular degeneration. They form vast webs similar to those that exist in the human body (Tobe, 1998, op. cit.).

[0183] The vector used in this study (referred to herein as "vector 1") contains the AAV2 inverted terminal We have developed a humanized mAb antigen-binding fragment that binds to and inhibits human VEGF adjacent to the intracellular repeats (ITRs). The heavy chain and Expression of the β-actin and light chains was driven by the CB7 promoter, which consists of the chicken β-actin promoter and CMV enhancer. The vector also contains the chicken β-actin intron and Contains rabbit β-globin polyA signal. In vector 1, the heavy and light chains of anti-VEGF Fab are The nucleic acid sequences encoding the strands are separated by a self-cleaving furin (F) / F2A linker. Rho / VEGF mice were subretinal injected with vector 1 or control (n = 10-17 per group), and the mice were then cultured for 1 week. The amount of retinal neovascularization was quantified after a period of time.

[0184] In Rho / VEGF mice given vector 1, phosphate-buffered saline (PBS) or Compared to mice receiving a null AAV8 vector, the total area of ​​retinal neovascularization was reduced in a dose-dependent manner. The efficacy criteria were a statistically significant reduction in the area of ​​retinal neovascularization. This criterion was set as a 1×10 7 The lowest dose of GC / eye vector 1 was Retinal neovascularization in a mouse transgenic Rho / VEGF model of nAMD in rat subjects It was determined that this was effective in reducing the

[0185] 6.8 Example 8: Bi-transgenic Tet / Opsin / VEGF expression by subretinal administration of a single dose Reduces retinal detachment in mice This study is a human study in which inducible expression of VEGF leads to severe retinopathy and retinal detachment. A transgenic mouse model of ocular neovascular disease in elephants - Tet / opsin / VEGF mouse (Ohno-Matsui, 2002 Am. J. Pathol. 160(2):711-719) This study demonstrates the in vivo efficacy of a single dose of Vector 1 to prevent VEGF in Tet / opsin / VEGF mice. Human VEGF in photoreceptor cells 165 Transgenic mice with doxycycline-inducible expression of These transgenic mice were given doxycycline in their drinking water. Doxycycline induces very high levels of VEGF in photoreceptor cells. The expression of IL-17 was induced, resulting in massive vascular leakage, eventually reaching 80–90% within 4 days of induction. 0% of mice develop complete exudative retinal detachment.

[0186] Tet / opsin / VEGF mice (10 per group) were injected subretinally with Vector 1 or control. Ten days after injection, doxycycline was added to the drinking water to induce VEGF expression. , image each fundus and perform a complete retinal scan of each individual by a person blinded to the treatment group. , partial detachment, or complete detachment.

[0187] These data (shown in Figure 5) demonstrate that treatment with Vector 1 results in increased expression of Tet / opsin / VEGF mice - Low incidence and severity of retinal detachment in animal models of ocular neovascular disease in human subjects Prove that you have done so.

[0188] 6.9 Example 9: AAV8 gene therapy expressing anti-VEGF protein in a mouse model (Strongly inhibits subretinal neovascularization and vascular leakage) In this example, the methods, results, and conclusions of the experiments described in Examples 7 and 8 are summarized.

[0189] Methods. VEGF expression in photoreceptor cells is driven by the rhodopsin promoter. 165 The expression of Transgenic mice (rho / VEGF mice) were injected with 3 × 10 6 ~1×10 10 Vector 1, in doses ranging from genomic copies (GC) 1 × 10 10 GC null vector, Subretinal injections of either 0.01% or 0.1% PBS were administered (n=10 per group). Subretinal neovascularization (S The area of ​​VEGF in photoreceptor cells was measured. 165 Doxycycline (DOX)-induced expression of The double transgenic mice (Tet / opsin / VEGF mice) were injected with 1 × 10 8 ~1×10 10 A subretinal injection of GC vector 1 was administered to the other eye, with no injection in the other eye, or 1 x 10 in each eye 10 The other eye received a subretinal injection of GC null vector and the other eye received PBS. Ten days after injection, 2 mg / ml DOX was added to the drinking water, and 4 days later, the bottom photograph was taken to show complete retinal detachment (R D) The presence or absence of partial retinal detachment was graded. Vector 1 x 10 8 ~1×10 10 One week after subretinal injection of GC, the transgene of Vector 1 was Product levels were measured by ELISA analysis of eye homogenates.

[0190] Results: Compared to eyes of rho / VEGF mice injected with null vector, ≥ 1 × 10 7 GC's Bek Mice injected with Tur1 experienced a significant reduction in the mean area of ​​SNV, ≤3 × 10 7 was injected with In the eye, there is a moderate decrease, ≥ 1 × 10 8 GC-injected eyes experienced a >50% decrease. 3 ×10 9 Or 1×10 10 GC-injected eyes experienced almost complete clearance of SNVs. Tet / opsin In / VEGF mice, ≥ 3 × 10 8 Eyes injected with GC vector 1 showed a significant reduction in exudative RD, with a 3 × 10 9 Or 1×10 10 GC The complete detachment rate was reduced by 70-80% in irradiated eyes. 9 of eyes injected with GC vector 1 The majority had protein levels below the limit of detection, but 3 × 10 9 Or 1×10 10 GC All irradiated eyes had detectable levels, with average levels per eye of 342.7 ng and 286. The amount was 2 ng.

[0191] Conclusion: Subretinal injection of vector 1 was used as a gene therapy to correct SNVs in rho / VEGF mice. Dose-dependent inhibition was observed, with 3 × 10 9 Or 1×10 10 These were almost completely suppressed by the GC dose. The same dose demonstrated robust protein product expression and complete expression in Tet / opsin / VEGF mice. There was a significant reduction in all exudative RD.

[0192] 6.10 Example 10: Gene Therapy for Neovascular AMD: In Subjects with Neovascular AMD (nAMD) A dose escalation study to evaluate the safety and tolerability of vector-based gene therapy using Brief summary of the study: Excess vascular endothelial growth factor (VEGF) is a factor in the progression of neovascular (wet) age-related macular degeneration. It plays an important role in promoting angiogenesis and edema in neovascular AMD (nAMD). Visumab (LUCENTIS®, Genentech) and aflibercept (EYLEA®) VEGF inhibitors (anti-VEGF), including VEGF inhibitors (Tregon, Regeneron), have been shown to be safe and effective for the treatment of nAMD. Anti-VEGF treatments have been shown to improve vision. It is usually administered by intravitreal injection, which can be a significant burden for patients. Recombinant adeno-associated virus (AAV) gene carrying the coding sequence of a soluble anti-VEGF protein This therapeutic vector is a long-term stable delivery of therapeutic proteins, which is a promising strategy to develop a gene therapy for nAMD. Following treatment, the current favorable benefit-risk profile preserves vision while The therapeutic burden of available treatments can be reduced.

[0193] Detailed description of the study. This dose escalation study is a vector-based study in previously treated subjects with nAMD. -1 Designed to evaluate the safety and tolerability of gene therapy. Three doses will be administered to approximately 18 patients. Subjects who meet the inclusion / exclusion criteria and experience an anatomic reaction to their first anti-VEGF injection will be included in the study. Subjects receiving the treatment are given a single dose of Vector 1 administered by subretinal delivery. The gene encoding the monoclonal antibody fragment that binds to and neutralizes the activity of VEGF is included. The AAV8 vector used in this study will be a 24-week randomized controlled trial. Safety will be evaluated during the first 24 weeks after administration of vector 1 (first study period). After completion of the first study period, subjects were followed up at 104 weeks post-treatment with Vector 1. Evaluate up to.

[0194] Dosage: 3 doses: 3×10 9 GC vector 1, 1 x 10 10 GC vector 1, and 6 × 10 10 G.C. Use Vector 1.

[0195] Outcome Measures. The primary outcome measure was safety over a 26-week time frame - ocular and extraocular sites. - Incidence of adverse events (AEs) and serious adverse events (SAEs).

[0196] Secondary outcome measures included:

[0197] Safety over a 106-week time frame - incidence of ocular and non-ocular AEs and SAEs.

[0198] - Change in best corrected visual acuity (BCVA) over a 106-week time frame.

[0199] Over a 106-week time frame - Change in central retinal thickness (CRT) as measured by SD-OCT.

[0200] Rescue injections over a 106-week time frame - Mean number of rescue injections.

[0201] Over a 106-week time frame - Choroidal neovascularization (CNV) measured by fluorescein angiography (FA) NV), and changes in the size of the lesion, as well as changes in the leakage area CNV

[0202] Eligibility Criteria. The following eligibility criteria:

[0203] Minimum age: 50 years old

[0204] Maximum age: (none)

[0205] Gender:All

[0206] Gender-based: None

[0207] Acceptance of healthy volunteers: No, will be applied to the study.

[0208] Selection criteria: Patients ≥ 50 years of age with a diagnosis of subfoveal CNV secondary to AMD in the study eye and a history of He was receiving intravitreal anti-VEGF therapy. For the first patient in each cohort, BCVA was ≦20 / 100 to ≧20 / 400 (Early treatment of diabetic retinopathy) study [ETDRS] letter ≤ 65 and ≥ 35), and then BCVA ≤ 2 for the remainder of the cohort 0 / 63 to ≥20 / 400 (ETDRS letter is ≤75 and ≥35). History of needing and responding to anti-VEGF therapy. Responded to anti-VEGF at study entry (as assessed by SD-OCT at week 1). The test subject's eye must be pseudophakic (post-cataract surgery). Aspartate aminotransferase / alanine aminotransferase (AST / A LT) < 2.5 × upper limit of normal (ULN); total bilirubin (TB) < 1.5 × ULN; prothrombin time ( PT) < 1.5 × ULN; hemoglobin (Hb) > 10 g / dL (men) and > 9 g / dL (women); platelets >100×10 3 / μL; Estimated glomerular filtration rate (eGFR)>30 mL / min / 1.73 m 2 It is. Willing and unable to provide written, signed informed consent It won't be.

[0209] Exclusion criteria The study eye has CNV or macular edema secondary to any cause other than AMD. Any condition that would prevent improvement of vision in the test subject's eye, such as fibrosis, atrophy, or There is a central retinal epithelial break in the fovea. -Current or history of retinal detachment in the test eye. -Has advanced glaucoma in the test eye. - Within 6 months prior to screening, the study subject had received intravitreal therapy other than anti-VEGF therapy, e.g. History of receiving intrauterine steroid injections or investigational products. The presence of an implant (excluding intraocular lenses) in the study subject's eye at screening. - Myocardial infarction, cerebrovascular accident, or transient ischemic attack within the past 6 months. Uncontrolled hypertension (systolic blood pressure [BP] > 180 mmHg despite maximal medical treatment) Hg, diastolic BP>100 mmHg).

[0210] 6.11 Example 11: Protocol for Treating Human Subjects The present example relates to gene therapy treatment of patients with neovascular (wet) age-related macular degeneration (nAMD). This example is an updated version of Example 10. In this example, Vector 1, a soluble Replication-deficient adeno-associated vaccinia carrying the coding sequence for the anti-VEGF Fab protein (described in Example 7) The aim of the gene therapy treatment is to administer AAV8 to patients with nAMD. Slow or stop the progression of membrane degeneration and prevent vision loss with minimal intervention / invasive procedures The objective of the present invention is to slow down or prevent the

[0211] Dosage and route of administration. A volume of 250 μL of Vector 1 was injected subretinal into the eye of the subject requiring treatment. The subjects will be administered a single dose via delivery. 9 GC / eye, 1×10 10 GC / eye or 6x 10 10 Give a dose of GC / eye.

[0212] Subretinal delivery is performed in subjects under local anesthesia by a retinal surgeon. The procedure includes a central vitreous incision and Standard three-port pars plana vitrectomy with removal of the vitreous followed by subretinal cannulation ( 38 The subretinal delivery of Vector 1 to the subretinal space is automated and performed via a vitreous catheter. Deliver 250 µL into the subretinal space via the corpectomy machine.

[0213] Gene therapy can be administered in combination with one or more therapies for the treatment of wet AMD. For example, gene therapy is a method for treating cancer using laser photocoagulation, photodynamic therapy using verteporfin, and other methods. including, but not limited to, pegaptanib, ranibizumab, aflibercept, or bevacizumab. It is administered in combination with intravitreal anti-VEGF agents, including mabs.

[0214] Starting approximately 4 weeks after administration of Vector 1, patients were administered intravitreal ranibizumab in the affected eye. Breathe treatment can be administered.

[0215] Patient Subpopulations. Suitable patients include: · Have been diagnosed with nAMD; · Respond to anti-VEGF treatment; · Requiring frequent injections of anti-VEGF treatment; ·Be male or female aged 50 years or older; Have a BCVA of ≤20 / 100 and ≥20 / 400 (ETDRS letters ≤65 and ≥35) in the affected eye r; · Have a BCVA of ≤20 / 63 and ≥20 / 400 (ETDRS letter ≤75 and ≥35); ·Has a documented diagnosis of subfoveal CNV secondary to AMD in the affected eye; CNV lesion characteristics: Lesion size <10 disc areas (typical disc The surface area is 2.54 mm 2), with blood and / or scarring of lesion size <50%; - Less than 8 months (or less) prior to treatment with an anti-VEGF agent for the treatment of nAMD in affected eyes Both received four intravitreal injections and had their anatomical responses documented with SD-OCT; and / or Subretinal or intraretinal fluid present in the affected eye, as evidenced by SD-OCT , It may include a patient.

[0216] Prior to treatment, patients were screened for one or more of the following criteria: ·Having CNV or macular edema secondary to any cause other than AMD in the affected eye; Blood in affected eyes occupying ≥ 50% of the AMD lesion and > 1.0 mm below the fovea 2 The blood exist; Any condition that prevents improvement of VA in the affected eye, such as fibrosis, atrophy, or foveal with a central retinal epithelial break; ·Has ongoing or a history of retinal detachment in the affected eye; ·Having advanced glaucoma in the affected eye; Any affected eye condition that may increase the risk to the subject of vision loss, preventing or treating and requesting medical or surgical intervention that interferes with the test procedure or evaluation; History of intraocular surgery in the affected eye within 12 weeks prior to screening Lithium-aluminum-garnet capsulotomy was performed >10 weeks prior to the screening visit is acceptable if Intravitreal therapy other than anti-VEGF therapy in the affected eye within 6 months prior to screening, e.g. For example, a history of receiving intravitreal steroid injections or investigational products; ·Presence of an implant (excluding intraocular lens) in the affected eye at screening; History of malignancy requiring chemotherapy and / or radiation within 5 years prior to screening ( Localized basal cell carcinoma is acceptable); - May cause retinal toxicity and affect known treatments or vision. or any drug with known retinal toxicity, such as chloroquine or hydroxychloroquine. history of concurrent treatment with Having an ocular or periocular infection that may interfere with the surgical procedure in the affected eye; - Myocardial infarction, cerebrovascular accident, or transient ischemic attack within the past 6 months of treatment; Uncontrolled hypertension (systolic blood pressure [BP] > 180 mmHg despite maximal medical treatment) Hg, diastolic BP >100 mmHg); · undergoing any concomitant treatment that may interfere with the ocular surgical procedure or recovery process; Known hypersensitivity to ranibizumab or any of its components or to any of the vectors have a history of hypersensitivity to a particular drug; which, in the opinion of the investigator, would compromise the subject's safety or success in participating in the study Any serious or unstable medical or psychological condition. Aspartate aminotransferase (AST) / alanine aminotransferase (ALT) > 2.5 × upper limit of normal (ULN) Total bilirubin > 1.5 × ULN. However, if the subject has previously had Gilbert's syndrome and total bilirubin In those without a known history of fractionated bilirubin where <35% of the bins are conjugated bilirubin Limited to Prothrombin time (PT) > 1.5 x ULN Hemoglobin <10 g / dL for male subjects and <9 g / dL for female subjects Platelets <100×10 3 / μL Estimated glomerular filtration rate (GFR) < 30 mL / min / 1.73 m 2 is may indicate that this treatment is not suitable for the patient.

[0217] 1 or more of the following rescue criteria: Associated with retinal fluid accumulation on spectral domain optical coherence tomography (SD-OCT) Loss of 5 letters of vision (according to best corrected visual acuity [BCVA]) New or persistent subretinal or intraretinal lesions on SD-OCT associated with choroidal neovascularization (CNV) Increased fluid levels New eye bleeding If this is the case, patients should be given a period of treatment to monitor disease activity, starting approximately 4 weeks after gene therapy. Intravitreal ranibizumab rescue therapy in the affected eye may be administered. Set Insights: Visual acuity of 20 / 20 or better and central retinal thickness of "normal" as assessed by SD-OCT Yes, or Visual acuity and SD-OCT were stable after two consecutive injections If one of the following occurs, further rescue injections should be withheld at the discretion of the treating physician. It can be placed.

[0218] If injections are withheld, restart injections if visual acuity or SD-OCT worsens according to the above criteria. do.

[0219] Measures of clinical goals. The primary clinical goals include slowing or halting the progression of retinal degeneration. and slowing or preventing vision loss. Clinical goals include: These include, but are not limited to, pegaptanib, ranibizumab, aflibercept, or bevacizumab. Elimination or replacement of rescue therapy using intravitreal injections of anti-VEGF drugs, including ciprofloxacin The clinical goal is to reduce or prevent vision loss and / or or by reduction or prevention of retinal detachment.

[0220] The clinical objectives were to measure BCVA (best corrected visual acuity), intraocular pressure, slit lamp biomicroscopy, and This is determined by direct ophthalmoscopy and / or SD-OCT (SD-optical coherence tomography). The goal was to measure the mean change from baseline in BCVA over time, according to baseline BCVA. Measurement of gain or loss of ≥ 15 letters compared with baseline, based on CRT measured by SD-OCT over time Measure of mean change from baseline, mean number of ranibizumab rescue injections over time time to first rescue ranibizumab injection, and CNV and lesion size over time FA-based measures of mean change from baseline in leak area over time Measurement of mean change from baseline in ocular VEGF protein, serum and urinary vector excretion performing an immunogenicity analysis and / or measuring the immunogenicity of vector 1, i.e., measuring Nabs to AAV; Determined by measuring binding antibodies to AAV, measuring antibodies to VEGF, and / or performing ELISpot do.

[0221] The clinical goal is to assess the baseline area of ​​geographic atrophy (GAA) over time by fundus autofluorescence (FAF). Measure the mean change from baseline, measure the occurrence of new areas of geographic atrophy by FAF (baseline in subjects without geographic atrophy in the line), compared with baseline by BCVA, Measurement of the proportion of subjects gaining or losing ≥5 and ≥10 letters, respectively, compared to the previous year - Determination of the proportion of subjects with a 50% decrease in injection, and no fluid on SD-OCT It is determined by measuring the ratio of

[0222] Improvement / efficacy with administration of Vector 1 was observed at approximately 4 weeks, 12 weeks, 6 months, and 12 months after baseline , 24 months, 36 months, or any other desired time point. Treatment with Vector 1 can improve visual acuity by 5%, 10%, 15%, or 20% from baseline. , 30%, 40%, 50% or more. The improvement / effectiveness is spectrum Central retinal thickness (CRT) measured by SD-OCT at 4 weeks, 12 weeks, The mean change from baseline at 6, 12, 24, and 36 months was assessed. Treatment with Vector 1 reduced central retinal thickness by 5%, 10%, 15%, or 20% from baseline. %, 30%, 40%, 50% or more.

[0223] (Equivalent) Although the invention has been described in detail with reference to specific embodiments thereof, functionally equivalent variations are contemplated. It will be understood that any form of the invention is within the scope of the present invention. In addition to those described above, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description and accompanying drawings. It will be apparent that such modifications are intended to fall within the scope of the appended claims. Those of skill in the art will appreciate that many of the specific embodiments of the invention described herein are illustrative and not restrictive. Equivalents should be recognized or ascertainable using no more than routine experimentation. Such equivalents are intended to be encompassed by the scope of the following claims.

[0224] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety. Each such patent or patent application is specifically and individually incorporated by reference in its entirety. Each of the above-mentioned references is incorporated herein by reference to the same extent as if the references were set forth herein. It is included.

Claims

1. 1. Use of a recombinant viral vector in the manufacture of a medicament for treating neovascular age-related macular degeneration (nAMD) in a human subject, comprising: the vector comprises an AAV8 capsid and an expression cassette, the expression cassette comprises nucleotide sequences encoding the heavy and light chains of an anti-hVEGF antigen-binding fragment separated by a self-cleaving furin (F) / F2A linker; the anti-hVEGF antigen-binding fragment is (A) a light chain CDR1 comprising the amino acid sequence of SASQDISNYLN (SEQ ID NO: 14); a light chain CDR2 comprising the amino acid sequence of FTSSLHS (SEQ ID NO: 15); and a light chain CDR3 comprising the amino acid sequence of QQYSTVPWT (SEQ ID NO: 16); and Heavy chain CDR1 comprising the amino acid sequence of GYDFTHYGMN (SEQ ID NO: 20); a heavy chain CDR2 comprising the amino acid sequence of WINTYTGEPTYAADFKR (SEQ ID NO: 18); and a heavy chain CDR3 comprising the amino acid sequence of YPYYYGTSHWYFDV (SEQ ID NO: 21); or (B) a heavy chain variable region comprising amino acids 1 to 123 of SEQ ID NO:2, and a light chain variable region comprising amino acids 1 to 107 of SEQ ID NO:1; or (C) a heavy chain comprising the amino acid sequence of SEQ ID NO:2, and a light chain comprising the amino acid sequence of SEQ ID NO:

1. and The above uses, wherein the treatment comprises administering the viral vector to the eye of the human subject, such that the anti-hVEGF antigen-binding fragment is expressed by cells of the eye of the human subject.

2. the expression cassette is flanked by AAV2 inverted terminal repeats (ITRs); and The expression cassette comprises: (i) CB7 promoter consisting of chicken β-actin promoter and CMV enhancer; (ii) chicken β-actin intron; (iii) (a) one or more IL-2 signal peptides; and (b) a heavy chain CDR1 comprising the amino acid sequence of GYDFTHYGMN (SEQ ID NO: 20), a heavy chain CDR2 comprising the amino acid sequence of WINTYTGEPTYAADFKR (SEQ ID NO: 18), and a heavy chain CDR3 comprising the amino acid sequence of YPYYYGTSHWYFDV (SEQ ID NO: 21); or A heavy chain variable region comprising amino acids 1-123 of SEQ ID NO:2; or A heavy chain comprising the amino acid sequence of SEQ ID NO:2; and (c) a self-cleaving furin (F) / F2A linker; and (d) a light chain CDR1 comprising the amino acid sequence of SASQDISNYLN (SEQ ID NO: 14), a light chain CDR2 comprising the amino acid sequence of FTSSLHS (SEQ ID NO: 15), and a light chain CDR3 comprising the amino acid sequence of QQYSTVPWT (SEQ ID NO: 16); or A light chain variable region comprising amino acids 1-107 of SEQ ID NO:1; or A light chain comprising the amino acid sequence of SEQ ID NO:1 A nucleotide sequence encoding (iv) Rabbit β-globin poly(A) signal The use according to claim 1, comprising:

3. the antigen-binding fragment comprises a light chain and a heavy chain; the heavy chain comprises a VH CDR1 of SEQ ID NO: 20, a VH CDR2 of SEQ ID NO: 18, and a VH CDR3 of SEQ ID NO: 21; and The use according to claim 1 or 2, wherein the light chain comprises a VL CDR1 of SEQ ID NO: 14, a VL CDR2 of SEQ ID NO: 15, and a VL CDR3 of SEQ ID NO:

16.

4. 3. The use according to claim 1 or 2, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:2 and the light chain comprises the amino acid sequence of SEQ ID NO:

1.

5. The use according to claim 1 or 2, wherein the anti-hVEGF antigen-binding fragment comprises a heavy chain variable region comprising amino acids 1 to 123 of SEQ ID NO:2 and a light chain variable region comprising amino acids 1 to 107 of SEQ ID NO:

1.

6. The use according to any one of claims 1 to 5, wherein the antigen-binding fragment is a Fab, a F(ab')2, or a single chain variable domain (scFv).

7. The use according to any one of claims 1 to 6, wherein the expressed antigen-binding fragment is glycosylated.

8. The use according to claim 7 , wherein the expressed antigen-binding fragment comprises α2,6-sialylated glycans.

9. The use according to claim 7 or 8, wherein the expressed antigen-binding fragment does not contain NeuGc.

10. The use according to any one of claims 1 to 9, wherein the expressed antigen-binding fragment comprises tyrosine sulfation.

11. The use according to any one of claims 1 to 10, wherein said treatment comprises delivery of said vector by subretinal administration.

12. The use according to any one of claims 1 to 11, wherein said treatment comprises expression of said anti-hVEGF antigen-binding fragment in retinal cells.

13. The use according to any one of claims 1 to 12, wherein the treatment comprises expression of the anti-hVEGF antigen-binding fragment in photoreceptor cells.

14. The use according to any one of claims 1 to 13, wherein the treatment comprises expression of the anti-hVEGF antigen-binding fragment in retinal pigment epithelial cells.

15. The use according to any one of claims 1 to 14, wherein the treatment comprises expression of the anti-hVEGF antigen-binding fragment in rod cells, cone cells, horizontal cells, bipolar cells, amacrine cells, ganglion cells and / or Müller cells.

16. 16. The use of any one of claims 1 to 15, wherein the treatment comprises administering the viral vector at a dosage sufficient to maintain a concentration of the anti-hVEGF antigen-binding fragment in the vitreous humor of at least 0.330 μg / mL or in the aqueous humor of at least 0.110 μg / mL for three months.

17. 17. The use according to any one of claims 1 to 16, wherein said treatment comprises administering said viral vector in a dose sufficient to slow or stop the progression of retinal degeneration and slow or prevent vision loss with minimal intervention or invasive procedures.

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