Antibodies and antibody conjugates

Abstract

An object of the present application is to provide an anti-VEGF-A antibody and an antibody conjugate thereof.SOLUTION: Anti-VEGF-A antibodies and antibody conjugates thereof have been discovered, and it has been discovered that some embodiments of the antibodies can be conjugated to moieties such as HEMA-PC polymers, that some embodiments of the antibody conjugates can retain or enhance antibody activity, and that the antibodies and antibody conjugates can be particularly useful for the treatment of diabetic retinopathy. Furthermore, a method for conjugating polymers to proteins, e.g. antibodies such as IgG1, has been found.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Incorporation by reference of priority applications Any and all applications claiming foreign or domestic priority in an Application Data Sheet filed with this application are hereby incorporated by reference under 37 CFR 1.57.

[0002] Sequence Listing This application is submitted with an electronic Sequence Listing, which is provided as a file entitled KDIAK004.txt, created on December 28, 2016, and is 18,231 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0003] FIELD OF THE INVENTION The present invention relates to antibodies and antibody conjugates, and methods of using and making such antibodies, antibody conjugates, and other protein conjugates. [Background technology]

[0004] Diabetic retinopathy is the leading cause of blindness in people between the ages of approximately 20 and 64. Engelgau M, Geiss L, Saaddine J, Boyle J, et al. 2004. The Evolving Diabetes Burden in the United States. Ann of Int Med. 140 (11): 945-951. Diabetic retinopathy accounts for approximately 12% of new cases of blindness in the United States. In diabetic retinopathy, retinal blood vessels typically swell and leak fluid into the back of the eye. Hyperglycemia induces intramural thickening of the basement membrane, resulting in leaky or permeable blood vessels.

[0005] In diabetic retinopathy, changes in the blood vessels of the retina occur due to changes in blood sugar levels. Everyone with diabetes is at risk. The longer a person has diabetes, the higher the risk of developing some kind of eye problem. Between 40 and 45 percent of Americans diagnosed with diabetes have some stage of diabetic retinopathy. Causes and Risk Factors. Diabetic Retinopathy. United States National Library of Medicine. 15 September 2009.

[0006] Diabetic retinopathy first manifests as the development of microaneurysms within the retina. Microaneurysms occur when there is swelling of the capillaries (very small blood vessels) that supply the retina. The presence of relatively few microaneurysms usually does not cause vision problems. However, as retinopathy progresses to later stages, the potential for vision loss is significant. This early stage of retinopathy is called background diabetic retinopathy or nonproliferative diabetic retinopathy (NPDR). Patients with NPDR typically have no symptoms, but early detection of the disease is important because vision loss is highly likely once retinopathy progresses to later stages.

[0007] The next stage of diabetic retinopathy is when new blood vessels form at the back of the eye (proliferative diabetic retinopathy). These new blood vessels are leaky and can rupture and subsequently bleed, resulting in blurred or blurred vision. The lack of oxygen in the eye causes further new blood vessels to grow along the retina and into the vitreous humor. When these vessels rupture, further bleeding can occur, potentially severely damaging or destroying the retina. Leakage Fluid accumulation in the macula caused by diabetic blood vessels is called diabetic macular edema. Many patients with diabetic retinopathy develop diabetic macular edema.

[0008] There are three common treatment routes for patients with diabetic retinopathy: laser surgery, corticosteroid injections, and injections of anti-VEGF agents (e.g., Avastin® (bevacizumab), Lucentis® (ranibizumab), and Eylea® (aflibercept)). Laser surgery is generally effective in treating diabetic retinopathy, but laser-induced retinal damage is a frequent side effect. Steroid preparations, such as triamcinolone acetonide, have been administered via intravitreal injection for the treatment of diabetic retinopathy. However, frequent administration of the steroid solution is required to treat diabetic retinopathy. Intravitreal steroid therapy has also been associated with cataracts, steroid-induced glaucoma, and endophthalmitis.

[0009] Another method for treating diabetic retinopathy is intravitreal injection of anti-VEGF agents. In this regard, Lucentis® (ranibizumab) and Eylea® (aflibercept) have recently been approved for the treatment of diabetic retinopathy in patients with diabetic macular edema. VEGF-directed therapies are effective not only for diabetic retinopathy, but also for age-related macular degeneration (AMD), including neovascular (wet) AMD. Summary of the Invention

[0010] Provided herein is an antibody conjugate comprising an anti-VEGF-A antibody linked to a phosphorylcholine-containing polymer at a cysteine ​​outside the variable region of the antibody, wherein the cysteine ​​is added by recombinant DNA technology. Optionally, the anti-VEGF-A antibody has a light chain and a heavy chain, the heavy chain comprising an Fc region. Optionally, the anti-VEGF-A antibody is an immunoglobulin G (IgG). Optionally, the cysteine ​​is within the Fc region of the heavy chain. Optionally, the anti-VEGF-A heavy chain comprises a CDR H 1: GYDFTHYGMN (SEQ ID NO: 9), CDR H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H3: YPYYYGTSHWYFDV (SEQ ID NO: 11) and at position 221 (position via sequence counting found in SEQ ID NO: 3) is T, and the anti-VEGF-A light chain comprises CDR L 1: SASQDISNYLN (SEQ ID NO: 12), CDR L 2: FTSSLHS (SEQ ID NO: 13), and CDR L 3: QQYSTVPWT (SEQ ID NO: 14) and L at Kabat position 4. Optionally, the anti-VEGF-A heavy chain isotype is human IgG1.

[0011] Optionally, the heavy chain constant domain of the anti-VEGF-A antibody IgG1 has one or more mutations compared to the IgG1 constant domain to modulate effector function. Optionally, the mutations are at one or more of the following amino acid positions: E233X, L234X, L235X, G236X, G237X, A327X, A330X, and P331X (EU numbering), where X is any natural or unnatural amino acid. Optionally, the mutations are selected from the group consisting of E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S (EU numbering). Optionally, the mutations are L234A, L235A, and G237A (EU numbering). In some embodiments, the effector function is reduced. In some embodiments, CDC, ADCC, and / or ADCP are reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or more. In some embodiments, CDC is mediated by Fc binding to C1q, and ADCC and ADCP are mediated by Fc binding to various Fcγ receptors, and each of these binding interactions is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or more.

[0012] The cysteine ​​residue is optionally within the anti-VEGF-A heavy chain, and optionally Optionally, the anti-VEGF-A heavy chain is SEQ ID NO: 1 and the sequence of the anti-VEGF-A light chain is SEQ ID NO: 2. Optionally, the cysteine ​​is L443C (EU numbering).

[0013] Optionally, the phosphorylcholine-containing polymer comprises 2-(methacryloyloxyethyl)-2'-(trimethylammonium)ethyl phosphate (MPC) monomers shown below. [ka] The polymer comprises the following repeating unit: [ka] where n is an integer from 1 to 3000, and the wavy lines represent connection points between monomer units in the polymer.

[0014] Optionally, the polymer has three or more arms or is synthesized using an initiator having three or more polymer initiation sites. Optionally, the polymer has two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve arms or is synthesized using an initiator having two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve polymer initiation sites. Optionally, the polymer has two, three, six, or nine arms or is synthesized using an initiator having two, three, six, or nine polymer initiation sites. Optionally, the polymer has nine arms or is synthesized using an initiator having nine polymer initiation sites.

[0015] Optionally, the polymer has a molecular weight between about 300,000 and about 1,750,000 Da as measured by size exclusion chromatography-multi-angle light scattering (hereinafter "SEC-MALS"). Optionally, the polymer has a molecular weight between about 500,000 and about 1,000,000 Da. Optionally, the polymer has a molecular weight between about 600,000 and about 800,000 Da.

[0016] Optionally, the antibody conjugate is purified and the polymer is polydisperse. Optionally, the polymer has a polydispersity index (PDI) of less than 1.2. In some embodiments, any of the conjugate solutions provided herein can have a polydispersity index (PDI) of 1.8 or less, e.g., 1.7 or less, 1.6 or less, 1.5 or less, 1.4 or less, 1.3 or less, 1.2 or less, 1.1 or less, or 1 or less.

[0017] Optionally, an antibody conjugate comprising an anti-VEGF-A immunoglobulin G (IgG) conjugated to a polymer, wherein the polymer comprises an MPC monomer, the sequence of the anti-VEGF-A heavy chain is SEQ ID NO: 1, and the sequence of the anti-VEGF-A light chain is SEQ ID NO: 2, and the antibody is conjugated to the polymer only at C449 in SEQ ID NO: 1. Optionally, the polymer has nine arms, and the polymer has a molecular weight of between about 600,000 and about 800,000 Da.

[0018] Optionally, an antibody conjugate comprising an anti-VEGF-A immunoglobulin G (IgG) conjugated to a polymer, wherein the polymer comprises an MPC monomer, the sequence of the anti-VEGF-A heavy chain is SEQ ID NO: 1, and the sequence of the anti-VEGF-A light chain is SEQ ID NO: 2, and the antibody is conjugated to the polymer only at C443 (EU numbering), optionally the polymer has nine arms, and the polymer has a molecular weight of between about 600,000 and about 800,000 Da.

[0019] Optionally, the antibody conjugate has the structure: [ka] wherein each heavy chain of the anti-VEGF-A antibody is represented by the letter H and each light chain of the anti-VEGF-A antibody is represented by the letter L, the polymer is attached to the anti-VEGF-A antibody via the sulfhydryl of C449 in SEQ ID NO: 1, which attachment is shown on one of the heavy chains, and PC is [ka] wherein the wavy line represents a point of attachment to the remainder of the polymer portion, and X is a) OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halide, including Br; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±15%. Optionally, n1, n2, n3, n4, n5, n6, n7, n8, and n9 are independently integers from 0 to 3000. Optionally, n1, n2, n3, n4, n5, n6, n7, n8, and n9 are independently integers from 0 to 500. In some embodiments, X is OR, where R is a sugar, aminoalkyl, or one of the following residues: saturated C1-C 24 Alkyl, unsaturated C2-C 24 Alkenyl or C2-C 24 Alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and alkyl halides including polyhalogenated alkyls, -CO-O-R7, carbonyl -CCO-R7, -CO-NR8R9, -(CH2) n -COOR7, -CO-(CH) n -COOR7, -(CH2) n-NR8R9, ester, alkoxycarbonyl, aryloxycarbonyl, mono-substituted, multiply substituted, or unsubstituted variants, in which n is an integer from 1 to 6, and R7, R8, and R9 are hydrogen atoms, halogen atoms, the following residues: saturated C1-C 24 Alkyl, unsaturated C2-C 24 Alkenyl or C2-C 24 and alkyl halides, including polyhalogenated alkyls, 5-membered rings, and 6-membered rings, each independently selected from the group consisting of mono-, multiply-, or unsubstituted variants of alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and alkyl halides, including polyhalogenated alkyls, 5-membered rings, and 6-membered rings.

[0020] Optionally, the antibody conjugate has the structure: [ka] wherein each heavy chain of the anti-VEGF-A antibody is represented by the letter H and each light chain of the anti-VEGF-A antibody is represented by the letter L, the polymer is attached to the anti-VEGF-A antibody via the sulfhydryl of C443 (EU numbering), which attachment is shown on one of the heavy chains, and PC is [ka] wherein the wavy line represents a point of attachment to the remainder of the polymer portion, and X is a) OR, where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halide, including Br; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±15%. Optionally, n1, n2, n3, n4, n5, n6, n7, n8, and n9 are independently integers from 0 to 3000. Optionally, n1, n2, n3, n4, n5, n6, n7, n8, and n9 are independently integers from 0 to 500. In some embodiments, X is OR, wherein R is a sugar, aminoalkyl, or one of the following residues: saturated C1-C 24 Alkyl, unsaturated C2-C 24 Alkenyl or C2-C 24 Alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and alkyl halides including polyhalogenated alkyls, -CO-O-R7, carbonyl -CCO-R7, -CO-NR8R9, -(CH2) n -COOR7, -CO-(CH) n -COOR7, -(CH2) n -NR8R9, Ester, Alco and mono-, multiply, or unsubstituted variants of oxycarbonyl, aryloxycarbonyl, and aryloxycarbonyl, wherein n is an integer from 1 to 6, and R7, R8, and R9 are hydrogen atoms, halogen atoms, or the following residues: saturated C1-C 24 Alkyl, unsaturated C2-C 24 Alkenyl or C2-C 24and alkyl halides, including polyhalogenated alkyls, 5-membered rings, and 6-membered rings, each independently selected from the group consisting of mono-, multiply-, or unsubstituted variants of alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and alkyl halides, including polyhalogenated alkyls, 5-membered rings, and 6-membered rings.

[0021] In some embodiments, the antibody conjugates described above are provided in a liquid solution, optionally with a pharmaceutically acceptable carrier.

[0022] In some embodiments, an anti-VEGF-A antibody is provided having a heavy chain and a light chain. The heavy chain comprises a CDR H 1: GYDFTHYGMN (SEQ ID NO: 9), CDR H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H 3: YPYYYGTSHWYFDV (SEQ ID NO: 11) and at position 221 (the position via the sequence count found in SEQ ID NO: 3) is T, and the light chain contains CDR L 1: SASQDISNYLN (SEQ ID NO: 12), CDR L 2: FTSSLHS (SEQ ID NO: 13), and CDR L 3:QQYSTVPWT (SEQ ID NO: 14), and L at Kabat position 4. Optionally, the heavy chain isotype is IgG1, and the IgG1 constant domain comprises one or more of the following mutations to modulate effector function: E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S (EU numbering). Optionally, the antibody has L234A, L235A, and G237A (EU numbering). Optionally, the sequence of the anti-VEGF-A heavy chain is SEQ ID NO: 1, and the sequence of the anti-VEGF-A light chain is SEQ ID NO: 2.

[0023] In some embodiments, methods are provided for treating or preventing ocular diseases, comprising administering an antibody conjugate as described above or a pharmaceutical composition as described above. Optionally, the ocular disease is selected from the group consisting of diabetic retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic macular edema (DME), pathologic myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), central branch retinal vein occlusion (BRVO), corneal neovascularization, retinal neovascularization, retinopathy of prematurity (ROP), subconjunctival hemorrhage, and hypertensive retinopathy. Optionally, the disease is diabetic retinopathy.

[0024] In some embodiments, a method for making an antibody conjugate comprising an anti-VEGF-A antibody conjugated to a phosphorylcholine-containing polymer is provided, the method comprising the steps of conjugating an anti-VEGF-A antibody to a phosphorylcholine-containing polymer, the anti-VEGF-A antibody comprising a cysteine ​​residue added via recombinant DNA technology, the cysteine ​​being outside the variable region of the antibody, the phosphorylcholine-containing polymer comprising a sulfhydryl-specific reactive group selected from the group consisting of maleimide, vinyl sulfone, orthopyridyl disulfide, and iodoacetamide, and reacting the sulfhydryl-specific reactive group on the phosphorylcholine-containing polymer with the cysteine ​​residue on the anti-VEGF-A antibody to form the antibody conjugate.

[0025] Optionally, the anti-VEGF-A antibody is an immunoglobulin G (IgG), and the cysteine ​​is in the Fc region of the antibody. Optionally, the anti-VEGF-A antibody has a light chain and a heavy chain, and the anti-VEGF-A antibody heavy chain comprises a CDR H 1: GYDFTHYGMN (SEQ ID NO: 9), CDR H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H 3: YPYYYGTSHWYFDV (SEQ ID NO: 11) and at position 221 (position via sequence counting found in SEQ ID NO: 3) is T, and the anti-VEGF-A antibody light chain comprises CDRL 1: SASQDISNYLN (SEQ ID NO: 12), CDR L 2: FTSSLHS (SEQ ID NO: 13), and CDR L 3: QQYSTVPWT (SEQ ID NO: 14) and L at Kabat position 4. Optionally, the anti-VEGF-A antibody heavy chain isotype is IgG1.

[0026] Optionally, the IgG1 constant domain has one or more mutations compared to the IgG1 constant domain to modulate effector function. Optionally, the mutations are at one or more of the following amino acid positions: E233X, L234X, L235X, G236X, G237X, G236X, D270X, K322X, A327X, P329X, A330X, A330X, P331X, and P331X (EU numbering), where X is any natural or unnatural amino acid. Optionally, the mutations are selected from the group consisting of E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S (EU numbering). Optionally, the mutations are L234A, L235A, and G237A (EU numbering).

[0027] Optionally, the cysteine ​​residue added by recombinant DNA techniques is selected from the group consisting of Q347C (EU numbering) and L443C (EU numbering). Optionally, the cysteine ​​residue added by recombinant DNA techniques is L443C (EU numbering). Optionally, the sequence of the anti-VEGF-A heavy chain is SEQ ID NO: 1 and the sequence of the anti-VEGF-A light chain is SEQ ID NO: 2.

[0028] Optionally, the phosphorylcholine-containing polymer comprises 2-(methacryloyloxyethyl)-2'-(trimethylammonium)ethyl phosphate (MPC) monomers shown below. [ka] The polymer comprises the following repeating unit: [ka] where n is an integer from 1 to 3000, and the wavy lines represent connection points between monomer units in the polymer.

[0029] Optionally, the polymer has 3 or more arms. Optionally, the polymer has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 arms. Optionally, the polymer has 2, 3, 6, or 9 arms. Optionally, the polymer has 9 arms.

[0030] Optionally, the polymer has a molecular weight between about 300,000 and 1,750,000 Da. Optionally, the polymer has a molecular weight between about 500,000 and 1,000,000 Da. Optionally, the polymer has a molecular weight between about 600,000 and 800,000 Da.

[0031] Optionally, the method includes the additional step of contacting the anti-VEGF-A antibody with a thiol reducing agent under conditions to generate reduced cysteine ​​sulfhydryl groups, thereby generating a reduced anti-VEGF-A antibody in which all cysteine ​​residues are reduced. Optionally, the thiol reducing agent is selected from the group consisting of tris[2-carboxyethyl]phosphine hydrochloride (TCEP), dithiothreitol (DTT), dithioerythritol (DTE), sodium borohydride (NaBH), sodium cyanoborohydride (NaCNBH), β-mercaptoethanol (BME), cysteine ​​hydrochloride, and cysteine. Optionally, the thiol reducing agent is TCEP. Optionally, the thiol reducing agent is in between a 1-fold and a 100-fold molar excess relative to the concentration of the anti-VEGF-A antibody. Optionally, the thiol reducing agent is in between a 20-fold and a 50-fold molar excess relative to the concentration of the anti-VEGF-A antibody.

[0032] Optionally, the method further comprises removing the thiol reducing agent from the reduced anti-VEGF-A antibody and treating the reduced anti-VEGF-A antibody with an oxidizing agent, optionally the oxidizing agent being air, aqueous CuSO4, or dehydroascorbic acid (DHAA). Optionally, the method further comprises purifying the antibody complex.

[0033] Optionally, the antibody conjugate is purified using a technique selected from the group consisting of ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, affinity chromatography, and combinations thereof. Optionally, the purified antibody conjugate has at least 20% of the biological activity of the unconjugated anti-VEGF-A antibody. Optionally, the purified antibody conjugate retains at least 50% of the biological activity compared to the unconjugated anti-VEGF-A antibody. Optionally, the purified antibody conjugate retains at least 90% of the biological activity compared to the unconjugated anti-VEGF-A antibody. Optionally, the purified antibody conjugate has an increased half-life compared to the unconjugated anti-VEGF-A antibody. Optionally, the purified antibody conjugate has at least a 1.5-fold increased half-life compared to the unconjugated anti-VEGF-A antibody.

[0034] Optionally, the method further comprises polymerizing a free-radically polymerizable phosphorylcholine-containing monomer in a polymerization medium to provide said phosphorylcholine-containing polymer, said medium comprising said free-radically polymerizable phosphorylcholine-containing monomer, M t is a transition metal, q is the maximum oxidation state of the metal, and q-1 is the oxidation state of the metal, and the metal is a transition metal catalyst M t (q-1)+ and X' is a counter ion or group. t (q-1)+ X' (q-1) or the inactive metal salt M in its highest oxidation state together with a reducing agent capable of reducing the transition metal from an oxidatively inactive state to a reductively active state. tq+ X' q The transition metal catalyst, ligand, and initiator are provided in situ by providing

[0035] Optionally, the radical polymerizable phosphorylcholine-containing monomer is [ka] wherein R1 is H or C 1~6 R2, R3, and R4 are each methyl; and X and Y are each 2.

[0036] M if desired t is selected from the group consisting of Cu, Fe, Ru, Cr, Mo, W, Mn, Rh, Re, Co, V, Zn, Au, and Ag. Optionally, the metal catalyst is M t (q-1)+ X' (q-1) Optionally, M t (q-1)+ is Cu 1+ , Fe 2+ , Ru 2+ , Cr 2+ , Mo 2+ , W 2+ , Mn 3+ , Rh 3+ ,Re 2+ , Co + , V 2+ , Zn + , Au + , and Ag + and X' is selected from the group consisting of halogen, C 1~6 Alkoxy, (SO4) 1 / 2 , (PO4) 1 / 3 , (R7PO4) 1 / 2 , (R72PO4), triflate, hexafluorophosphate, methanesulfonate, arylsulfonate, CN, and R7CO2, wherein R7 is a linear or branched C alkyl group optionally substituted 1 to 5 times with H or halogen. 1~6 is an alkyl group. t(q-1)+ is Cu 1+ and X' is Br. Optionally, M t (q-1)+ is provided in situ. Optionally, M t q+ X q is CuBr2.

[0037] Optionally, the reducing agent is an inorganic compound, optionally selected from the group consisting of low oxidation level sulfur compounds, sodium bisulfite, sodium sulfite, inorganic salts containing metal ions, metals, hydrazine hydrate, and derivatives of such compounds.

[0038] Optionally, the reducing agent is a metal. Optionally, the reducing agent is Cu 0 is.

[0039] Optionally, the reducing agent is an organic compound selected from the group consisting of alkylthiols, mercaptoethanol, or carbonyl compounds that can be readily enolized, ascorbic acid, acetylacetonate, camphorsulfonic acid, hydroxyacetone, reducing sugars, monosaccharides, glucose, aldehydes, and derivatives of such organic compounds.

[0040] Optionally, the ligand is 2,2'-bipyridine, 4,4'-di-5-nonyl-2,2'-bipyridine, 4,4-dinonyl-2,2'-dipyridyl, 4,4',4''-tris(5-nonyl)-2,2':6',2''-terpyridine, N,N,N',N',N''-pentamethyldiethylenetriamine, 1,1,4,7,10,10-hexamethyltriethylenetetramine, tris(2-dimethylaminoethyl)amine, N,N-bis(2-pyridylmethyl)octadecyl amine, N,N,N',N'-tetra[(2-pyridal)methyl]ethylenediamine, tris[(2-pyridyl)methyl]amine, tris(2-aminoethyl)amine, tris(2-bis(3-butoxy-3-oxopropyl)aminoethyl)amine, tris(2-bis(3-(2-ethylhexoxy)-3-oxopropyl)aminoethyl)amine, and tris(2-bis(3-dodecoxy-3-oxopropyl)aminoethyl)amine. Optionally, the ligand is 2,2'-bipyridine.

[0041] Optionally, the initiator has the structure: [ka] wherein R1 is a nucleophilic reactive group, R2 comprises a linker, and R3 comprises a polymer synthesis initiator moiety having the structure: [ka] wherein R4 and R5 are the same or different and are selected from the group consisting of alkyl, substituted alkyl, alkylene, alkoxy, carboxyalkyl, haloalkyl, cycloalkyl, cyclic alkyl ether, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkylene, heterocycloalkyl, heterocycloalkylene, aryl, arylene, aryleneoxy, heteroaryl, amino, amido, and any combination thereof; Z is halogen or CN; and s is an integer between 1 and 20.

[0042] Optionally, Z is Br and R4 and R5 are each methyl. Optionally, R1 is selected from the group consisting of NH2-, OH-, and SH-. Optionally, R1 is NH2-. Optionally, R2 is alkyl, substituted alkyl, alkylene, alkoxy, carboxyalkyl, haloalkyl, cycloalkyl, cyclic alkyl ether, alkenyl, or aryl. Optionally, R2 is alkynylene, alkynyl, alkynylene, cycloalkylene, heterocycloalkyl, heterocycloalkylene, aryl, arylene, aryleneoxy, heteroaryl, amino, amido, and any combination thereof. [ka] wherein X and Y are the same or different and are integers from 1 to 20.

[0043] Optionally, X and Y are each 4. Optionally, R3 is [ka] wherein R6, R7, and R8 are the same or different, and [ka] [ka] and [ka] wherein Z is NCS, F, Cl, Br, or I. Thus, Z is Br. Optionally, R6, R7, and R8 are each [ka] is.

[0044] Optionally, the initiator has the structure: [ka] wherein A and B are the same or different and are integers from 2 to 12, and Z is any halide such as Br. Optionally, A and B are each 4.

[0045] Optionally, the method further comprises reacting the polymer with a maleimide reagent to provide a polymer having maleimide termini. Optionally, the maleimide compound is [ka] is.

[0046] In some embodiments, the options provided herein avoid one or more problems associated with other treatment methods or forms. For example, because intravitreal injections can be painful and require a clinical setting, these options allow for reduced administration frequency to less than one intravitreal injection per month. Furthermore, diabetic retinopathy patients with relatively unimpaired vision may be resistant to monthly intravitreal injections and may therefore be left untreated under other treatments. Therefore, there is a need for less frequently administered diabetic retinopathy treatments. In this context, treatments for the disease may be used early in the disease to prevent the progression of diabetic retinopathy and associated vision-threatening events.

[0047] In some embodiments, the antibody conjugate comprises (1) an anti-VEGF-A antibody and (2) a phosphorylcholine-containing polymer covalently attached to the antibody at a cysteine ​​outside the variable region of the antibody, the cysteine ​​being added by recombinant DNA techniques.

[0048] In some embodiments, the anti-VEGF-A antibody heavy chain comprises the CDR H 1: GYDFTHYGMN (SEQ ID NO: 9), CDR H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H 3:YPYYYGTSHWYFDV (SEQ ID NO: 11), wherein the anti-VEGF-A light chain comprises CDR L 1: SASQDISNYLN (SEQ ID NO: 12), CDR L 2: FTSSLHS (SEQ ID NO: 13), and CDR L 3: Contains QQYSTVPWT (SEQ ID NO: 14).

[0049] In some embodiments, the polymer conjugated to the antibody has a molecular weight between about 300,000 and about 1,750,000 Da as measured by size exclusion chromatography-multi-angle light scattering (hereinafter "SEC-MALS").

[0050] In some embodiments, the anti-VEGF-A antibody has a heavy chain and a light chain. The heavy chain has a CDR H 1: GYDFTHYGMN (SEQ ID NO: 9), CDR H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H 3: YPYYYGTSHWYFDV (SEQ ID NO: 11), wherein the light chain comprises CDR L 1: SASQDISNYLN (SEQ ID NO: 12), CDR L 2: FTSSLHS (SEQ ID NO: 13), and CDR L 3:QQYSTVPWT (SEQ ID NO: 14), wherein the heavy chain isotype is IgG1, and the IgG1 constant domain contains one or more of the following mutations to reduce effector function: E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S (EU numbering).

[0051] In some embodiments, any of the above methods includes providing an anti-VEGF-A antibody having a light chain and a heavy chain, wherein the anti-VEGF-A antibody heavy chain comprises a CDR H1: GYDFTHYGMN (SEQ ID NO: 9), CDR H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H 3: YPYYYGTSHWYFDV (SEQ ID NO: 11), wherein the anti-VEGF-A antibody light chain comprises CDR L 1: SASQDISNYLN (SEQ ID NO: 12), CDR L 2: FTSSLHS (SEQ ID NO: 13), and CDR L 3: The antibody comprising QQYSTVPWT (SEQ ID NO: 14) may be used.

[0052] In some embodiments, the antibody comprises a heavy chain amino acid variable region comprising SEQ ID NO:1 and a light chain amino acid variable region comprising SEQ ID NO:2.

[0053] In some embodiments, the antibody is a human IgG1 and the heavy chain constant domain is an immuno-mediated It contains one or more mutations that reduce endogenous effector function.

[0054] In some embodiments, the antibody is further conjugated to a polymer to further form a bioconjugate, wherein the bioconjugate has a molecular weight between about 450,000 and 1,900,000 daltons.

[0055] In some embodiments, the polydispersity index (PDI) is 1.5 or less.

[0056] In some embodiments, the antibody that binds to VEGF-A has a CDR within SEQ ID NO:1 H CDR 1 H 1. CDR in SEQ ID NO: 1 H CDR 2 H 2. CDR in SEQ ID NO: 1 H CDR 3 H 3. CDR in SEQ ID NO:2 L CDR 1 L 1. CDR in SEQ ID NO:2 L CDR 2 L 2. CDRs in SEQ ID NO:2 L CDR 3 L3. Contains at least one of the following mutations: L234A, L235A, and G237A (EU numbering), and at least one of the following mutations: Q347C (EU numbering) or L443C (EU numbering).

[0057] In some embodiments, the antibody comprises all three of the following mutations: L234A, L235A, and G237A (EU numbering), and the antibody comprises L443C (EU numbering).

[0058] In some embodiments, a method for preparing a conjugated protein includes reducing one or more cysteines in a protein to form a decapped protein in solution, reoxidizing the decapped protein to reform at least one disulfide bond in the reduced protein while the engineered cysteine ​​residue in the protein remains in a free thiol state to form a reoxidized decapped protein in the solution, and adding at least one excipient to the solution to reduce polymer-induced protein precipitation. The method further includes adding a polymer to the solution and conjugating the polymer to the reoxidized decapped protein at the engineered cysteine ​​residue to form a conjugated protein. In some embodiments, the protein is an antibody, antibody-protein fusion, or binding fragment thereof. In some embodiments, the excipient is an acid or a base. In some embodiments, the excipient is selected from the group consisting of at least one of a detergent, a sugar, and a charged amino acid. In some embodiments, the reaction of the reduced protein with the polymer occurs under aqueous conditions between pH 6.0 and pH 8.5. In some embodiments, the amount of reduced protein is less than the amount of polymer. In some embodiments, the polymer is conjugated to the protein at 2-37 degrees Celsius. In some embodiments, the method further comprises contacting a solution containing the conjugated protein with an ion exchange medium, or a hydrophobic interaction or affinity chromatography medium. In some embodiments, the ion exchange medium, or the hydrophobic interaction or affinity chromatography medium separates the conjugated protein from the free polymer and the reoxidized decapped protein. In some embodiments, the polymer comprises a zwitterion. In some embodiments, the polymer comprises phosphorylcholine. In some embodiments, the polymer comprises a PEG linker linking the center of a polymer branch point to the maleimide functionality.

[0059] In some embodiments, an anti-VEGF antibody conjugate is provided that is capable of inhibiting at least 90% of the interaction between a VEGF ligand and a VEGF receptor.

[0060] In some embodiments, anti-VEGF antibodies are provided that inhibit at least 95% of the interaction between a VEGF ligand and a VEGF receptor.

[0061] In some embodiments, anti-VEGF antibodies are provided that inhibit at least 90% of the interaction between a VEGF ligand and a VEGF receptor. [Brief explanation of the drawings]

[0062] [Figure 1] FIG. 1 shows compound L. [Figure 2] FIG. 1 shows compound K. [Figure 3] FIG. 1 shows the synthesis of OG1802 from R3707. [Figure 4] FIG. 1 shows OG1786. [Figure 5] FIG. 1 shows the synthesis of OG1546 from OG1550. [Figure 6] FIG. 1 shows the synthesis of OG1784 from OG1546 and OG1563. [Figure 7] FIG. 1 shows the synthesis of OG1405 from OG1784. [Figure 8] FIG. 1 shows the synthesis of OG1785 from OG1405. [Figure 9] FIG. 1 shows the synthesis of OG1786 from OG1785. [Figure 10] FIG. 1 shows OG1802. [Figure 11] FIG. 1 shows compound E. [Figure 12] FIG. 1 depicts some embodiments of an anti-VEGF-A heavy chain with certain effector function mutations and L443C (position 449 in SEQ ID NO: 1 according to EU numbering). [Figure 13]FIG. 1 depicts some embodiments of an anti-VEGF-A light chain (SEQ ID NO: 2). [Figure 14] FIG. 1 depicts some embodiments of the bevacizumab heavy chain (SEQ ID NO: 3). [Figure 15] FIG. 1 depicts several embodiments of the bevacizumab light chain (SEQ ID NO: 4). [Figure 16] FIG. 1 depicts several embodiments of the ranibizumab heavy chain (SEQ ID NO: 5). [Figure 17] FIG. 1 depicts several embodiments of the ranibizumab light chain (SEQ ID NO: 6). [Figure 18] FIG. 1 illustrates some embodiments of methods for preparing antibody conjugates. [Figure 19] FIG. 1 shows ion exchanger analysis (A280 absorbance) for reactions A through G. [Figure 20] FIG. 1 shows the effect of various anti-VEGF molecules on the binding of biotin-VEGF to plate-bound VEGFR ECD-Fc proteins, and the IC50 values ​​of those effects. [Figure 21] FIG. 1 shows OG1950 binding affinity to VEGF measured by BIAcore single cycle kinetics. [Figure 22] FIG. 1 shows binding of OG1950 to Fcγ receptor I. [Figure 23] FIG. 1 shows the binding of OG1950 to Fcγ receptor IIIa. [Figure 24] FIG. 1 shows the binding of QG1950 to the human complement protein C1q. [Figure 25] FIG. 1 shows the results of proliferation assays (including IC50 values). [Figure 26] FIG. 1 shows the results of single cycle kinetics of VEGF binding to anti-VEGF agents. [Figure 27] FIG. 1 depicts some embodiments of nucleic acid sequences encoding heavy and light chain variable regions. [Figure 28]FIG. 1 shows the results of screening different samples (different excipients) after incubation in polymer solution (OG1802) at 2-8 degrees Celsius for 20 hours. DETAILED DESCRIPTION OF THE INVENTION

[0063] Anti-VEGF-A antibodies are provided herein. In some embodiments, these antibodies can be conjugated to a half-life extending moiety. In some embodiments, the conjugates can be used to treat certain conditions, such as diabetic retinopathy and / or age-related macular degeneration.

[0064] Further provided herein are methods for preparing antibody (any type of antibody) conjugate compositions, which in some embodiments allow for reduced aggregate formation of the desired antibody conjugate or increased efficiency in making the desired conjugate.

[0065] These and additional embodiments are provided below before a definitions section is provided.

[0066] definition An "angiogenic disorder" is a disorder or disease state characterized by altered, dysregulated, or unregulated angiogenesis. Examples of angiogenic disorders include neoplastic transformation (e.g., cancer) and ocular angiogenic disorders, including diabetic retinopathy and age-related macular degeneration.

[0067] An "ocular neovascular" disorder is a disorder characterized by altered, dysregulated, or non-regulated angiogenesis in a patient's eye. Such disorders include optic nerve head neovascularization, iris neovascularization, retinal neovascularization, choroidal neovascularization, corneal neovascularization, vitreous neovascularization, glaucoma, pannus, pterygium, macular edema, diabetic retinopathy, vascular retinopathy, retinal degeneration, uveitis, retinal inflammatory disease, and proliferative vitreoretinopathy.

[0068] The term "antibody" includes intact antibodies and their binding fragments. A binding fragment refers to a molecule other than an intact antibody that contains a portion of the intact antibody and binds to the antigen to which the intact antibody binds. Examples of binding fragments include Fv, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from multiple antibody fragments. scFv antibodies are described in Houston JS. 1991. Methods in Enzymol. 203:46-96. Furthermore, antibody fragments include single-chain polypeptides that have characteristics of a VH domain or a VL domain, i.e., that combine with a VL domain or a VH domain to form a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody.

[0069] The specific binding of an antibody to its target antigen is at least 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 M -1 Specific binding refers to an affinity for at least one target. Specific binding is detectable to a greater extent than and distinguishable from nonspecific binding that occurs to at least one unrelated target. Specific binding can be the result of bond formation between specific functional groups or a specific spatial fit (e.g., lock-and-key type), while nonspecific binding is usually the result of van der Waals forces. However, specific binding does not necessarily mean that an antibody or fusion protein binds to one and only one target.

[0070] The basic antibody structural unit is a tetramer of subunits. Each tetramer contains two identical pairs of polypeptide chains, each pair consisting of one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 amino acids or more, primarily responsible for antigen recognition. This variable region is first expressed linked to a cleavable signal peptide. A variable region without the signal peptide is sometimes referred to as a mature variable region. Thus, for example, a light chain mature variable region refers to a light chain variable region without the light chain signal peptide. However, reference to a variable region does not necessarily imply the presence of a signal sequence, and in fact, the signal sequence is cleaved upon expression and secretion of the antibody or fusion protein. The paired heavy and light chain variable regions define the binding region of the antibody. The carboxy-terminal portions of the light and heavy chains define the light chain constant region and heavy chain constant region, respectively. The heavy chain constant region is primarily responsible for effector function. In IgG antibodies, the heavy chain constant region is divided into CH1 region, hinge region, CH2 region, and CH3 region. The CH1 region is formed by disulfide bonds and non-covalent bonds. The hinge region provides flexibility between the binding and effector regions of the antibody and also provides a site for intermolecular disulfide bonding between the two heavy chain constant regions in the tetrameric subunit. The CH2 and CH3 regions are the primary sites for effector function and FcR binding.

[0071] Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" segment of about 12 or more amino acids, with the heavy chain also containing a "D" segment of about 10 or more amino acids (see Chapter 7 of Fundamental Immunology (Paul, W. et al., 2nd ed., Raven Press, New York, 1989) (incorporated by reference in its entirety for all purposes).

[0072] The mature variable regions of each light-heavy chain pair form the antibody binding site. Thus, an intact antibody has two binding sites, i.e., is bivalent. In natural antibodies, the binding sites are the same. However, bispecific antibodies can be generated, in which the two binding sites are different. (See, e.g., Songsivilai S, Lachmann PC. 1990. Bispecific antibody: a tool for diagnosis and treatment of disease. Clin Exp Immunol. 79:315-321; Kostelny SA, Cole MS, Tso (See JY. 1992. Formation of bispecific antibodies by the use of leucine zippers. J Immunol. 148: 1547-1553). All variable regions exhibit the same general structure, with three hypervariable regions, also called complementarity-determining regions or CDRs, connected to relatively conserved framework regions (FR). The CDRs of the two chains of each pair are aligned by the framework regions, enabling them to bind to a specific epitope. Both light and heavy chains contain, from N- to C-terminus, the following domains: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. For convenience, the variable heavy chain CDRs are referred to as CDRs. H 1. CDR H 2 and CDR H 3, and the variable light chain CDRs are CDRs L 1. CDR L 2 and CDR L3. The assignment of amino acids to each domain conforms to the definitions in Kabat EA et al., 1987 and 1991, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, USA) or Chothia C, Lesk AM. 1987. Canonical Structures for the Hypervariable Regions of Immunoglobulins. J Mol Biol 196:901-917; Chothia C, et al. 1989. Conformations of Immunoglobulin Hypervariable Regions. Nature 342:877-883. Kabat also provides a widely used numbering system (Kabat numbering) in which the same numbers are assigned to corresponding residues between different heavy chain variable regions or different light chain variable regions. While Kabat numbering can be used for antibody constant regions, EU numbering is more commonly used, as in the present application. Although specific sequences are given for the exemplary antibodies disclosed herein, it is understood that some or all of these molecules may have one to several amino acids at the amino or carboxy termini of the light and / or heavy chains missing or derivatized after expression of the protein chains, particularly the C-terminal lysine residue of the heavy chain.

[0073] The term "epitope" refers to the site on an antigen to which an antibody or extracellular capture fragment binds. Epitopes on a protein can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained upon exposure to denaturing solvents.

[0074] In contrast, epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three amino acids, and usually more, at least five or eight to ten amino acids, in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance spectroscopy. See, for example, "Epitope Mapping Protocols" in Methods in Molecular Biology, Vol. 66, edited by Glenn E. Morris (1996).

[0075] Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay showing the ability of one antibody to compete with another antibody for binding to a target antigen. The epitope of an antibody can also be identified by X-ray crystallography of the antibody (or Fab fragment) bound to the antigen to identify contact residues.

[0076] Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody.

[0077] Competition between antibodies is determined by an assay in which the specific binding of a reference antibody to a common antigen is inhibited by the antibody under test (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with a reference antibody if an excess of the test antibody (e.g., at least 2-fold, 5-fold, 10-fold, 20-fold, or 100-fold) inhibits binding of the reference antibody by at least 50%. In some embodiments, the test antibody inhibits binding of the reference antibody by 75%, 90%, or 99% as measured in a competitive binding assay. Antibodies identified by competition assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent epitopes sufficiently close to the epitope bound by the reference antibody that steric hindrance occurs.

[0078] The term "patient" includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0079] For the purposes of classifying amino acid substitutions as conservative or non-conservative, amino acids are grouped as follows: Group I (hydrophobic side chains): Met, Ala, Val, Leu, Ile; Group II (neutral hydrophilic side chains): Cys, Ser, Thr; Group III (acidic side chains): Asp, Glu; Group IV (basic side chains): Asn, Gin, His, Lys, Arg; Group V (residues that affect chain orientation): Gly, Pro; and Group VI (aromatic side chains): Trp, Tyr, Phe. Conservative substitutions involve substitutions between amino acids of the same class. Non-conservative substitutions involve exchanging a member of one of these classes for a member of another class.

[0080] Percentage sequence identity is determined using antibody sequences aligned to maximize Kabat numbering for variable regions or EU numbering for constant regions. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared to the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is calculated by dividing the number of positions occupied by the same amino acid in both the subject and reference antibody regions by the total number of aligned positions in the two regions, not counting gaps, and multiplying this result by 100 to convert it to a percentage. Sequence identity for other sequences can be determined by aligning the sequences using algorithms such as BESTFIT, FASTA, and TFASTA, with default gap parameters, as included in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wisconsin, or by visual inspection and best alignment (i.e., resulting in the highest percentage sequence similarity in the comparison window). The percentage of sequence identity is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where identical residues appear in both sequences, counting the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0081] A composition or method "comprising" one or more recited elements may include other elements not specifically recited, for example, a composition comprising an antibody may contain the antibody alone or in combination with other components.

[0082] The term "antibody-dependent cellular cytotoxicity" (ADCC) refers to a mechanism of cell death induction that depends on the interaction of immune cells with lytic activity (also called effector cells) with antibody-coated target cells (i.e., cells containing bound antibodies). Such effector cells include natural killer cells, monocytes / macrophages, and neutrophils. ADCC is triggered by the interaction between the Fc region of a cell-bound antibody and Fcy receptors, particularly FcγRI and FcγRIII, on immune effector cells such as neutrophils, macrophages, and natural killer cells. The target cells are eliminated by phagocytosis or lysis, depending on the type of intervening effector cell. Death of the antibody-coated target cells occurs as a result of effector cell activity.

[0083] The term opsonization, also known as "antibody-dependent cellular phagocytosis" or ADCP, refers to the process by which antibody-coated cells are wholly or partially internalized by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc region of immunoglobulins.

[0084] The term "complement-dependent cytotoxicity" or CDC refers to a cell death-inducing mechanism in which the Fc effector domain of a target-bound antibody activates a series of enzymatic reactions that ultimately result in the formation of holes in the target cell's membrane. Typically, antigen-antibody complexes, such as those on antibody-coated target cells, bind and activate the complement component Clq, which in turn activates the complement cascade leading to target cell death. Complement activation leads to the deposition of complement components on the target cell surface, which may also promote ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0085] Humanized antibodies are genetically engineered antibodies in which CDRs from a non-human "donor" antibody have been grafted onto human "acceptor" antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and 5,585,089; Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No. 6,407,213; Adair, U.S. Pat. Nos. 5,859,205 and 6,881,557; and Foote, U.S. Pat. No. 6,881,557). These acceptor antibody sequences can be, for example, mature human antibody sequences, composites of such sequences, consensus sequences of human antibody sequences, or germline region sequences. Thus, humanized antibodies are antibodies having some or all of the CDRs completely or substantially derived from the donor antibody, as well as variable region framework sequences and constant region sequences, if present, completely or substantially derived from human antibodies. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs completely or substantially derived from a donor antibody heavy chain, and a heavy chain variable region framework sequence and heavy chain constant region, if present, substantially derived from human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain has at least one, two, and usually all three CDRs completely or substantially derived from a donor antibody light chain, and a light chain variable region framework sequence and light chain constant region, if present, substantially derived from human light chain variable region framework and constant region sequences. In addition to nanobodies and dAbs, humanized antibodies have humanized heavy chains and humanized light chains. CDRs in a humanized antibody are substantially derived from corresponding CDRs in a non-human antibody when at least 85%, 90%, 95%, or 100% of corresponding residues (as defined by Kabat) are identical between the respective CDRs. An antibody chain variable region framework sequence or an antibody chain constant region is substantially derived from a human variable region framework sequence or human constant region when at least 85%, 90%, 95%, or 100% of the corresponding residues as defined by Kabat are identical.

[0086] Humanized antibodies often incorporate all six CDRs (which may be as defined by Kabat) from a murine antibody; however, humanized antibodies can also be made using fewer than all of the CDRs (e.g., at least three, four, or five CDRs from a murine antibody) (see, e.g., De Pascalis R, Iwahashi M, Tamura M, et al. 2002. Grafting “Abbreviated” Complementary-Determining Regions Containing Specificity-Determining Residues Essential for Ligand Contact to Engineer a Less Immunogenic Humanized Monoclonal Antibody. J Immunol. 169:3076-3084; Vajdos FF, Adams CW, Breece TN, Presta LG, de Vos AM, Sidhu, SS. 2002. Comprehensive functional maps of the antigen-binding site of an anti-ErbB2 antibody obtained with shotgun scanning mutagenesis. J Immunol. 169:3076-3084). Mol Biol. 320: 415-428, Iwahashi M, Milenic DE, Padlan EA, et al. 1999. CDR substitutions of a humanized monoclonal antibody (CC49): Contributions of individual CDRs to antigen binding and immunogenicity. Mol Immunol. 36:1079-1091, Tamura M, Milenic DE, Iwahashi M, et al. 2000. Structural correlates of an anticarcinoma antibody: Identification of specificity-determining regions (SDRs) and development of a minimally immunogenic antibody variant by retention of SDRs only. J Immunol. 164:1432-1441).

[0087] Chimeric antibodies are antibodies in which the mature light and heavy chain variable regions of a non-human (e.g., murine) antibody are combined with human light and heavy chain constant regions. Such antibodies substantially or completely retain the binding specificity of the murine antibody and contain approximately two-thirds human sequence.

[0088] A veneered antibody retains some, usually all, of the CDRs of the non-human antibody and some of the non-human variable region framework residues, but removes other variable region framework residues that may contribute to B-cell or T-cell epitopes, such as exposed residues (Padlan EA. 1991 A possible procedure for reducing the immunogenicity of antibody variable domains while preserving their ligand-binding properties. Mol Immunol. 28:489-98) is a type of humanized antibody in which residues from the CDRs of the non-human antibody are replaced with residues derived from the corresponding positions in a human antibody sequence. The result is an antibody in which the CDRs are completely or substantially derived from a non-human antibody and the variable region framework of the non-human antibody has been made more human-like by the substitutions. Human antibodies can be isolated from humans or can otherwise result from the expression of human immunoglobulin genes (e.g., in transgenic mice or in vitro by phage display).Methods for producing human antibodies include those described in Ostberg L, Pursch E. 1983. Human x (mouse x human) hybridomas stably producing human antibodies. Hybridoma 2:361-367, the trioma method described in U.S. Pat. No. 4,634,664 to Ostberg and U.S. Pat. No. 4,634,666 to Engleman et al., the use of transgenic mice containing human immunoglobulin genes (see, for example, Lonberg et al., International Publication WO93 / 12227 (1993), U.S. Pat. Nos. 5,877,397, 5,874,299, 5,814,318, 5,789,650, 5,770,429, 5,661,016, 5,633,425, 5,625,126, 5,569,825, 5,545,806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, International Publication No. WO91 / 10741 (1991)), and phage display methods (see, for example, Dower et al., International Publication No. WO91 / 17271 and McCafferty et al., International Publication No. WO92 / 01047, U.S. Pat. Nos. 5,877,218, 5,871,907, 5,858,657, 5,837,242, 5,733,743, and 5,565,332).

[0089] A "polymer" refers to a series of linked monomers. A polymer can be composed of multiple units of a single monomer (homopolymer) or different monomers (heteromonomer). High molecular weight polymers can be prepared from monomers including, but not limited to, acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinylpyridines, vinylpyrrolidones, and vinyl esters such as vinyl acetate. Additional monomers are useful for high molecular weight polymers. When two different monomers are used, the two monomers are called "comonomers," meaning that the different monomers copolymerize to form a single polymer. The polymer can be linear or branched. When the polymer is branched, each polymer chain is called a "polymer arm." The end of the polymer arm attached to the initiator moiety is the proximal end, and the growing chain end of the polymer arm is the distal end. At the growing chain end of the polymer arm, the polymer arm end group can be a radical scavenger or another group.

[0090] "Initiator" refers to a compound capable of initiating polymerization using a monomer or comonomer. The polymerization can be conventional free radical polymerization or controlled / "living" radical polymerization, such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation-termination (RAFT) polymerization, or nitroxide-mediated polymerization (NMP). The polymerization can also be "quasi" controlled polymerization, such as degenerate transfer. When an initiator is suitable for ATRP, it contains a labile bond I that can homolytically cleave to form an initiator fragment, a radical capable of initiating radical polymerization, and a radical scavenger I' that reacts with a radical on a growing polymer chain to reversibly terminate polymerization. The radical scavenger I' is typically a halogen but can also be an organic moiety such as a nitrile. In some embodiments, the initiator contains one or more 2-bromoisobutyrate groups as sites for polymerization by ATRP.

[0091] A "chemical linker" refers to a chemical moiety that joins two groups together, such as a half-life extending moiety and a protein. The linker may be cleavable or non-cleavable. A cleavable linker can be, among others, a hydrolyzable linker, an enzyme-cleavable linker, a pH-sensitive linker, a photolabile linker, or a disulfide linker. Other linkers include homobifunctional and heterobifunctional linkers. A "linking group" is a functional group capable of forming a covalent bond consisting of one or more bonds to a bioactive agent. Non-limiting examples include those shown in Table 1 of International Publication WO2013059137 (incorporated by reference).

[0092] The term "reactive group" refers to a group capable of reacting with another chemical group to form a covalent bond, i.e., a group that is covalently reactive under appropriate reaction conditions, and generally represents a point of attachment to another substance. The reactive group is a moiety such as a maleimide or succinimide ester that is capable of chemically reacting with a functional group on a different moiety to form a covalent bond. Reactive groups generally include nucleophiles, electrophiles, and photoactivatable groups.

[0093] "Phosphorylcholine" is also written as "PC" and refers to the following: [ka] where * indicates a connection point. The phosphorylcholine is a zwitterionic group, including its salts (such as inner salts) and its protonated and deprotonated forms.

[0094] A "phosphorylcholine-containing polymer" is a polymer that contains phosphorylcholine. A "zwitterion-containing polymer" refers to a polymer that contains a zwitterion.

[0095] Poly(acryloyloxyethyl phosphorylcholine)-containing polymer refers to a polymer containing 2-(acryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate (HEA-PC shown in Example 6 below) as a monomer.

[0096] Poly(methacryloyloxyethyl phosphorylcholine)-containing polymers refer to polymers that contain 2-(methacryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate (HEMA-PC or MPC) as a monomer (see below). [ka]

[0097] As used herein, "MPC" and "HEMA-PC" are interchangeable.

[0098] "Molecular weight" in the context of the polymer may be expressed as either the number average molecular weight, the weight average molecular weight, or the peak molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to the peak molecular weight. These molecular weight determinations, i.e., the number average molecular weight (Mn), the weight average molecular weight (Mw), and the peak molecular weight (Mp), may be measured using size exclusion chromatography or other liquid chromatography techniques. Other methods for measuring molecular weight values ​​may also be used, such as using end-group analysis to determine number average molecular weight or measuring colligative properties (e.g., freezing point depression, boiling point elevation, or osmotic pressure), or using light scattering techniques, ultracentrifugation, or viscometry to determine weight average molecular weight. In some embodiments, molecular weight is measured by SEC-MALS (size exclusion chromatography-multi-angle light scattering). In some embodiments, the polymeric reagent is typically polydisperse (i.e., the number average molecular weight and weight average molecular weight of the polymer are not equal) and may have a low polydispersity, e.g., less than about 1.5, as determined, for example, by a PDI value derived from SEC-MALS measurements. In some embodiments, the polydispersity index (PDI) is in the range of about 1.4 to about 1.2. In some embodiments, the PDI is less than about 1.15, less than 1.10, less than 1.05, or less than 1.03.

[0099] The phrase "a" or "an" entity refers to one or more of that entity, for example, a compound refers to one or more compounds or at least one compound. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0100] "About" refers to possible variations in measurements taken among different instruments, samples, and sample preparations.

[0101] "Protected," "protected form," "protecting group," and "protecting group" refer to the presence of a group (i.e., a protecting group) that prevents or inhibits reaction of a particular chemically reactive functional group in a molecule under certain reaction conditions. Protecting groups vary depending on the type of chemically reactive group being protected, as well as the reaction conditions used and the presence of additional reactive or protecting groups, if any, in the molecule. Suitable protecting groups include those found in Greene et al., "Protective Groups in Organic Synthesis," 3rd Edition, John Wiley and Sons, New York, 1999.

[0102] "Alkyl" refers to a straight-chain or branched saturated aliphatic radical having the indicated number of carbon atoms. For example, C1-C6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Other alkyl groups include, but are not limited to, heptyl, octyl, nonyl, decyl, etc. Alkyl can contain any number of carbons, e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6, and 5-6 carbons. Alkyl groups are typically monovalent, but can be divalent, for example, by joining two moieties together.

[0103] The term "lower" as referred to above and hereinafter in connection with organic radicals or compounds defines compounds or radicals that may be branched or unbranched, containing up to 7 or 4 carbon atoms, respectively, and (when unbranched) 1 or 2 carbon atoms.

[0104] "Alkylene" refers to an alkyl group, as defined above, that links at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkylene can be attached to the same atom or different atoms of the alkylene. For example, a straight chain alkylene is -(CH2) where n is 1, 2, 3, 4, 5, or 6. n Alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene, and hexylene.

[0105] Substituents for alkyl and heteroalkyl radicals (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) may be -OR', ═O, ═NR', ═N-OR', -NR'R'', -SR', -halogen, in a number ranging from zero to (2m'+1), where m' is the total number of carbon atoms in such radical. , -SiR'R''R''', -OC(O)R', -C(O)R', -CO2R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)2R', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R'', -CN, and -NO2. R', R'' and R''' each independently represent hydrogen, unsubstituted (C1-C8) alkyl and heteroalkyl groups, unsubstituted aryl groups, aryl groups substituted with 1-3 halogens, unsubstituted alkyl, alkoxy, or thioalkoxy groups, or aryl-(C1-C4)alkyl groups. When R' and R" are attached to the same nitrogen atom, they can combine with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include 1-pyrrolidinyl and 4-morpholinyl. The term "alkyl" includes groups such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CHOCH3, etc.). In some embodiments, substituted alkyl and heteroalkyl groups have 1 to 4 substituents. In some embodiments, substituted alkyl and heteroalkyl groups have 1, 2, or 3 substituents. An exception is perhaloalkyl groups (e.g., pentafluoroethyl, etc.).

[0106] Substitution of alkyl and heteroalkyl radicals (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) The groups may be one or more of a variety of groups selected from, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —COR′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)R′, —S(O)NR′R″, —NRSOR′, —CN, and —NO, in a number ranging from zero to (2m′+1), where m′ is the total number of carbon atoms in such radical. R', R", R'", and R"" each independently represent hydrogen, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, e.g., an aryl group substituted with 1 to 3 halogens, a substituted or unsubstituted alkyl group, an alkoxy or thioalkoxy group, or an arylalkyl group. When a compound contains more than one R group, for example, each of the R groups is independently selected, as is the case for each of the R', R", R'", and R"" groups when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can combine with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups that contain carbon atoms bonded to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CHOCH3, etc.).

[0107] "Alkoxy" refers to an alkyl group having an oxygen atom attached to either the connection point or two carbon atoms of the alkoxy group. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 2-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, and the like. These alkoxy groups can be further substituted with various substituents as described. For example, these alkoxy groups can be further substituted with halogens to form "haloalkoxy" groups.

[0108] The term "carboxyalkyl" refers to an alkyl group (as defined herein) substituted with a carboxy group. The term "carboxycycloalkyl" refers to a cycloalkyl group (as defined herein) substituted with a carboxy group. The term alkoxyalkyl refers to an alkyl group (as defined herein) substituted with an alkoxy group. As used herein, the term "carboxy" refers to carboxylic acids and esters thereof.

[0109] "Haloalkyl" refers to an alkyl, as defined above, in which some or all of the hydrogen atoms have been replaced with halogen atoms. Halogen (halo) refers to chloro or fluoro, but may also be bromo or iodo. For example, haloalkyl includes trifluoromethyl, fluoromethyl, 1,2,3,4,5-pentafluorophenyl, and the like. The term "perfluoro" defines a compound or radical in which all available hydrogens have been replaced with fluorine. For example, perfluorophenyl refers to 1,2,3,4,5-pentafluorophenyl, perfluoromethyl refers to 1,1,1-trifluoromethyl, and perfluoromethoxy refers to 1,1,1-trifluoromethoxy.

[0110] "Fluoro-substituted alkyl" refers to an alkyl group in which one, some, or all of the hydrogen atoms have been replaced by fluorine.

[0111] A "cytokine" is a member of a group of protein signaling molecules that can be involved in intercellular communication in immune and inflammatory responses. Cytokines are typically found in approximately 8-3 It is a small, water-soluble glycoprotein with a mass of 5 kDa.

[0112] "Cycloalkyl" refers to a cyclic hydrocarbon group containing from about 3 to 12, 3 to 10, or 3 to 7 ring carbon atoms. Cycloalkyl groups include fused, bridged, and spiro ring structures.

[0113] "Intracyclic" refers to an atom or group of atoms that form part of a ring structure.

[0114] "Exocyclic" refers to an atom or group of atoms attached to the ring structure but which does not define the ring structure.

[0115] The term "cyclic alkyl ether" refers to a 4- or 5-membered cyclic alkyl group having 3 or 4 ring carbon atoms and 1 ring oxygen or sulfur atom (e.g., oxetane, thietane, tetrahydrofuran, tetrahydrothiophene), or a 6- to 7-membered cyclic alkyl group having 1 or 2 ring oxygen or sulfur atoms (e.g., tetrahydropyran, 1,3-dioxane, 1,4-dioxane, tetrahydrothiopyran, 1,3-dithiane, 1,4-dithiane, 1,4-oxathiane).

[0116] "Alkenyl" refers to either a straight-chain or branched hydrocarbon having 2 to 6 carbon atoms and at least one double bond. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl. Alkenyl groups can have 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6, and even 5 to 6 carbons. Alkenyl groups are typically monovalent, but can be divalent, for example, by linking two moieties together.

[0117] "Alkenylene" refers to an alkenyl group, as defined above, that is, a divalent hydrocarbon radical, that is linked to at least two other groups. The two moieties linked to the alkenylene can be linked to the same atom or different atoms of the alkenylene. Alkenylene groups include, but are not limited to, ethenylene, propenylene, isopropenylene, butenylene, isobutenylene, sec-butenylene, pentenylene, and hexenylene.

[0118] "Alkynyl" refers to either a straight-chain or branched hydrocarbon having 2 to 6 carbon atoms and at least one triple bond. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadinyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadinyl, 1,4-pentadinyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadinyl, 1,4-hexadinyl, 1,5-hexadinyl, 2,4-hexadinyl, or 1,3,5-hexatriyl. Alkynyl groups can have 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6, and even 5 to 6 carbons. Alkynyl groups are typically monovalent, but can be divalent, for example, by linking two moieties together.

[0119] "Alkynylene" refers to an alkynyl group, as defined above, that is, a divalent hydrocarbon radical, that is linked to at least two other groups. The two moieties linked to the alkynylene can be linked to the same atom or different atoms of the alkynylene. Alkynylene groups include, but are not limited to, ethynylene, propynylene, butynylene, sec-butynylene, pentynylene, and hexynylene.

[0120] "Cycloalkyl" refers to a saturated or partially unsaturated monocyclic, fused bicyclic, or bridged polycyclic ring containing from 3 to 12 ring atoms, or the number of atoms indicated. Monocyclic rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Bicyclic and polycyclic rings include, for example, norbornane, decahydronaphthalene, and adamantane. For example, C 3~8 Cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbornane.

[0121] "Cycloalkylene" refers to a cycloalkyl group, as defined above, that is, a divalent hydrocarbon radical, that is attached to at least two other groups. The two moieties attached to the cycloalkylene can be attached to the same atom or different atoms of the cycloalkylene. Cycloalkylene groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cyclooctylene.

[0122] "Heterocycloalkyl" refers to a ring system having from 3 to about 20 ring members and from 1 to about 5 heteroatoms, such as N, O, and S. Other heteroatoms may also be useful, including, but not limited to, B, Al, Si, and P. The heteroatoms may be oxidized, such as, but not limited to, -S(O)- and -S(O)-. For example, heterocycles include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, morpholino, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, piperidinyl, indolinyl, quinuclidinyl, and 1,4-dioxa-8-azaspiro[4.5]dec-8-yl.

[0123] "Heterocycloalkylene" refers to a heterocycloalkyl group, as defined above, that is linked to at least two other groups. The two moieties linked to the heterocycloalkylene can be linked to the same atom or different atoms of the heterocycloalkylene.

[0124] "Aryl" refers to a monocyclic, fused bicyclic, tricyclic or higher aromatic ring containing 6 to 16 ring carbon atoms. For example, aryl can be phenyl, benzyl, or naphthyl. "Arylene" refers to a divalent radical derived from an aryl group. The aryl group may be mono-, di-, or tri-substituted by one, two, or three radicals selected from alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, aminoalkyl, trifluoromethyl, alkylenedioxy, and oxy-C2-C3-alkylene, all of which may be optionally further substituted, e.g., as defined above, or 1-naphthyl or 2-naphthyl, or 1-phenanthrenyl or 2-phenanthrenyl. Alkylenedioxy is a divalent substituent attached to two adjacent carbon atoms of phenyl, e.g., methylenedioxy or ethylenedioxy. Oxy-C2-C3-alkylene is also a divalent substituent attached to two adjacent carbon atoms of phenyl, e.g., oxyethylene or oxypropylene. An example of oxy-C2-C3-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

[0125] In some embodiments, aryl is naphthyl, phenyl, or phenyl mono- or di-substituted with alkoxy, phenyl, halogen, alkyl, or trifluoromethyl, particularly phenyl, or phenyl mono- or di-substituted with alkoxy, halogen, or trifluoromethyl, especially phenyl.

[0126] Examples of substituted phenyl groups as R include, for example, 4-chlorophenyl, optionally substituted with a heterocycle. Phen-1-yl, 3,4-dichlorophen-1-yl, 4-methoxyphen-1-yl, 4-methylphen-1-yl, 4-aminomethylphen-1-yl, 4-methoxyethylaminomethylphen-1-yl, 4-hydroxyethylaminomethylphen-1-yl, 4-hydroxyethyl-(methyl)-aminomethylphen-1-yl, 3-aminomethylphen-1-yl, 4-N-acetylaminomethylphen-1-yl, 4-aminophen-1-yl, 3-aminophen-1-yl, 2-aminophen-1-yl, 4-phenyl-phen-1-yl, 4-(imidazole) 4-(2-methoxyethylaminomethyl)-phen-1-yl and 4-(pyrrolidin-1-ylmethyl)-phen-1-yl, 4-(thiophenyl)-phen-1-yl, 4-(3-thiophenyl)-phen-1-yl, 4-(4-methylpiperazin-1-yl)-phen-1-yl, and 4-(piperidinyl)-phenyl, and 4-(pyridinyl)-phenyl.

[0127] "Arylene" refers to an aryl group, as defined above, that links at least two other groups. The two moieties linked to the arylene can be linked to the same atom or different atoms of the arylene. Arylene groups include, but are not limited to, phenylene.

[0128] "Aryleneoxy" refers to an arylene group, as defined above, in which one of the moieties bonded to the arylene is bonded through an oxygen atom. Aryleneoxy groups include, but are not limited to, phenyleneoxy.

[0129] Similarly, the substituents on the aryl and heteroaryl groups vary and include, in numbers ranging from zero to the total number of open valences on the aromatic ring system, -halogen, -OR', -OC(O)R', -NR'R'', -SR', -R', -CN, -NO2, -CO2R', -CONR'R'', -C(O)R', -OC(O)NR'R'', -NR''C(O)R', -NR''C(O)2R', -NR'-C(O)NR''R''', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(N H2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R'', -N3, -CH(Ph)2, perfluoro(C1-C4)alkoxy, and perfluoro(C1-C4)alkyl, where R', R'', and R''' are independently selected from hydrogen, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4)alkyl, and (unsubstituted aryl)oxy-(C1-C4)alkyl.

[0130] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring are represented by the formula -TC(O)-(CH), where T and U are independently -NH-, -O-, -CH-, or a single bond, and q is an integer from 0 to 2. q Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be of the formula -A-(CH) ( ... r and optionally replaced with a substituent -B-. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may have the formula -(CH) where s and t are independently integers from 0 to 3 and X is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. s -X-(CH2) tThe substituent R' in -NR'- and -S(O)2NR'- is selected from hydrogen or unsubstituted (C1-C6) alkyl.

[0131] "Heteroaryl" refers to a monocyclic or fused bicyclic or tricyclic ring containing 5 to 16 ring atoms, with 1 to 4 of the ring atoms being N, O, or S heteroatoms, respectively. For example, heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any other radical substituted, particularly mono- or di-substituted, with, for example, alkyl, nitro, or halogen. Pyridyl refers to 2-pyridyl, 3-pyridyl, or 4-pyridyl, preferably 2-pyridyl or 3-pyridyl. Thienyl refers to 2-thienyl or 3-thienyl. In some embodiments, quinolinyl represents 2-quinolinyl, 3-quinolinyl, or 4-quinolinyl. In some embodiments, isoquinolinyl represents 1-isoquinolinyl, 3-isoquinolinyl, or 4-isoquinolinyl. In some embodiments, benzopyranyl and benzothiopyranyl can represent 3-benzopyranyl or 3-benzothiopyranyl, respectively. In some embodiments, thiazolyl can represent 2-thiazolyl or 4-thiazolyl. In some embodiments, triazolyl can be 1-triazolyl, 2-triazolyl, or 5-(1,2,4-triazolyl). In some embodiments, tetrazolyl can be 5-tetrazolyl.

[0132] In some embodiments, heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothienyl, oxazolyl, indazolyl, or any of the substituted, particularly mono- or di-substituted, radicals thereof.

[0133] The term "heteroalkyl" refers to an alkyl group having from one to three heteroatoms, such as N, O, and S. Other heteroatoms may also be useful, including, but not limited to, B, Al, Si, and P. The heteroatoms may be oxidized, such as, but not limited to, -S(O)- and -S(O)2-. For example, heteroalkyls may include ethers, thioethers, alkylamines, and alkylthiols.

[0134] The term "heteroalkylene" refers to a heteroalkyl group, as defined above, that is linked to at least two other groups. The two moieties linked to the heteroalkylene can be linked to the same atom or different atoms of the heteroalkylene.

[0135] "Electrophile" refers to an ion or atom or collection of atoms, which may be ionic, that has an electrophilic center, i.e., a center that is electrophilic, and that can react with a nucleophile. An electrophile (or electrophile reagent) is a reagent that forms a bond with its reaction partner (nucleophile) by accepting both bonding electrons from the reaction partner.

[0136] "Nucleophile" refers to an ion or atom or collection of atoms, which may be ionic, that has a nucleophilic center, i.e., a center seeking an electrophilic center, or that can react with an electrophile. A nucleophile (or nucleophile reagent) is a reagent that forms a bond with its reaction partner (electrophile) by donating both bonding electrons. A "nucleophilic group" refers to a nucleophile after it has reacted with a reactive group. Non-limiting examples include amino, hydroxyl, alkoxy, haloalkoxy, etc.

[0137] "Maleimide" refers to a pyrrole-2,5-dione-1-yl group having the structure: [ka]

[0138] It reacts with sulfhydryls (e.g., thioalkyls) to form an -S-maleimide group having the structure: [ka] where "·" represents the point of attachment of the maleimide group and the wavy line represents the point of attachment of the thiolic sulfur atom to the remainder of the original sulfhydryl-bearing group.

[0139] For purposes of this disclosure, "naturally occurring amino acids" found in proteins and polypeptides are L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamine, L-glutamic acid, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and / or L-valine. "Unnaturally occurring amino acids" found in proteins are any amino acids other than those listed as naturally occurring amino acids. Unnatural amino acids include, but are not limited to, D-isomers of the naturally occurring amino acids and mixtures of the D and L-isomers of the naturally occurring amino acids. Other amino acids, such as N-α-methyl amino acids (e.g., sarcosine), 4-hydroxyproline, desmosine, isodesmosine, 5-hydroxylysine, ε-N-methyllysine, and 3-methylhistidine, are found in naturally occurring proteins, but are generally introduced by methods other than ribosomal translation of mRNA and are therefore considered to be unnatural amino acids found in proteins for the purposes of this disclosure.

[0140] "Linear" in reference to the shape, configuration, or overall structure of a polymer refers to a polymer having a single polymer arm.

[0141] "Branched," in reference to the shape, configuration, or overall structure of a polymer, refers to a polymer having two or more polymer "arms" extending from a core structure contained within an initiator. The initiator may be used in atom transfer radical polymerization (ATRP) reactions. A branched polymer may have two polymer chains (arms), three polymer arms, four polymer arms, five polymer arms, six polymer arms, seven polymer arms, eight polymer arms, nine polymer arms, or more. Each polymer arm extends from a polymer initiation site. Each polymer initiation site may be a site for polymer chain growth by the addition of a monomer. For example, but not limited to, in ATRP, the polymer initiation site on the initiator may be a transition metal, such as a copper halide. Typically, the halide is an organic halide that undergoes a reversible oxidation-reduction process catalyzed by a metal compound. In some embodiments, the halide is bromine.

[0142] A "pharmaceutically acceptable excipient" refers to an excipient that can be included in a composition, that does not cause significant adverse toxicological effects to a patient, and that has been or can be approved by the FDA for therapeutic use, particularly in humans. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solution, lactated Ringer's solution, normal sucrose, normal glucose, etc.

[0143] Therapeutic proteins are administered in an effective regimen, meaning a dosage, route of administration, and frequency of administration that delays the onset of the disorder, reduces the severity of the disorder, prevents further deterioration of the disorder, and / or ameliorates at least one sign or symptom of the disorder. If the patient already has the disorder, the regimen can be referred to as a therapeutically effective regimen. If the patient is at increased risk for the disorder compared to the general population but has not yet shown symptoms, the regimen can be referred to as a prophylactically effective regimen. In some instances, therapeutic or prophylactic efficacy can be observed in an individual patient compared to historical controls or past experience with the same patient. In other instances, therapeutic or prophylactic efficacy can be demonstrated in preclinical or clinical trials of a treated patient population compared to a control population of untreated patients.

[0144] The "biological half-life" of a substance is a pharmacokinetic parameter that specifies the time required for half of the substance to be eliminated from a tissue or organism after its introduction.

[0145] "OG1786" is a nine-arm initiator used in polymer synthesis having the structure shown in Figure 30, which shows OG1786 in the form of a salt with trifluoroacetic acid. OG1786 may be used as other salts or as the free base.

[0146] "OG1801" is a polymer of approximately (±15%) 750 kDa (depending on Mn or Mp) made using OG1786 as the initiator for ATRP synthesis using the monomer HEMA-PC.

[0147] "OG1802" is maleimide-functionalized OG1801 and is shown in Figure 36, in which n1, n2, n3, n4, n5, n6, n7, n8, and n9 are each a (positive) integer (from 0 up to about 3000) such that the total molecular weight (Mw) of the polymer is 750,000 ± 15% Daltons.

[0148] Multi-angle light scattering (MALS) is a technique for analyzing macromolecules by impinging a laser beam on the molecule and inducing an oscillating dipole in the molecule due to the light's oscillating electric field. This oscillating dipole re-emits light, which can be measured using a MALS detector such as the Wyatt miniDawn TREOS. The intensity of the emitted light depends on the magnitude of the induced dipole in the macromolecule, which is proportional to the polarizability of the macromolecule; the larger the induced dipole, the greater the intensity of the scattered light. Therefore, to analyze scattering from solutions of such macromolecules, it is necessary to know their polarizability relative to the surrounding medium (e.g., solvent). This can be determined by measuring the change in refractive index of the solution, Δn, related to the change in molecular concentration, Δc, by measuring the dn / dc (= Δn / Δc) value using a Wyatt Optilab T-rEX differential refractive index detector. The two molecular weight parameters used in MALS measurements are the number average molecular weight (Mn) and the weight average molecular weight (Mw), where polydispersity index (PDI) is equal to Mw divided by Mn. SEC also allows the determination of another average molecular weight, the peak molecular weight Mp, which is defined as the molecular weight of the highest peak in the SEC.

[0149] PDI is used as a measure of the broadness of the molecular weight distribution of polymers and bioconjugates resulting from the conjugation of individual proteins (e.g., OG1950) to polydisperse biopolymers (e.g., OG1802). For protein samples, the polydispersity is close to 1.0 because the protein sample is a translation product in which all protein molecules in solution are expected to have approximately the same length and molecular weight. In contrast, biopolymers are polydisperse when polymer chains of various lengths are synthesized during the polymerization process; therefore, determining the PDI of a sample as one of its quality characteristics is crucial for a narrow molecular weight distribution.

[0150] Size exclusion chromatography (SEC) is a chromatographic technique that separates molecules in a solution by size. Typically, the sample is run through a column packed with resins of various pore sizes, with the resin expected to be inert to the analytes as they pass through the column, separating them from one another based on their specific size and the pore size characteristic of the selected column.

[0151] In contrast to relying on a set of SEC calibration standards, the coupling of SEC with MALS, or SEC / MALS, provides an accurate distribution of molecular weights and sizes (root-mean-square radii). This type of arrangement has many advantages over traditional column calibration methods. Because light scattering and concentration are measured for each elution fraction, molecular weight and size can be determined independently of elution position. This is particularly relevant for species that are non-spherical macromolecules, such as biopolymers (OG1802) or biocomplexes (OG1953), which typically do not elute with behavior that a set of column calibration standards could describe.

[0152] In some embodiments, the SEC / MALS analysis involves a Waters HPLC system including an Alliance 2695 solvent delivery module equipped with a Shodex SEC-HPLC column (7.8 x 300 mm) and a Waters 2996 photodiode array detector. An Optilab T-rEX refractive index detector is connected online. Waters Empower software is used to control the Waters HPLC system, and Wyatt ASTRA V6.1.7.16 software is used to acquire MALS data from the Wyatt miniDawn TREOS, dn / dc data from the T-rEX detector, and mass recovery data using the A280 absorbance signal from a Waters 2996 photodiode array detector. SEC is performed in 1x PBS, pH 7.4, at a flow rate of 1 ml / min. Upon sample injection, the MALS and RI signals are analyzed by the ASTRA software to determine absolute molecular weights (Mp, Mw, Mn) and polydispersity index (PDI). The calculations also involve input dn / dc values ​​of 0.142 and 0.183 for the polymer and protein, respectively. Regarding the dn / dc value of the OG1953 bioconjugate, the dn / dc is calculated to be about 0.148 based on the measured MW of the polymer and the protein using the following formula: dn / dc of the complex = 0.142 × [polymer MW / (polymer MW + protein MW)] + 0.183 × [protein MW / (polymer MW + protein MW)]

[0153] where the polymer MW of OG1802 is 800 kDa and the protein MW of OG1950 is 146 kDa.

[0154] overview Anti-VEGF antibodies and antibody conjugates are provided herein. In some embodiments, the antibodies themselves are distinct from other anti-VEGF agents and provide superior results over other anti-VEGF agents. In some embodiments, the anti-VEGF antibody conjugates are distinct from other antibodies and provide superior results over other anti-VEGF agents. and / or other antibody conjugates exhibit surprising superiority over the expected activity of other antibody conjugates.

[0155] Historically, conjugation of a molecule to a protein often reduces the protein's binding interaction with its intended target. In some embodiments of the present disclosure, the same level of reduction as expected is not always observed when conjugating to a location outside the active site. Evidence provided herein demonstrates the opposite effect. In some embodiments, without intending to be limited by theory, the conjugate may be superior to an antibody alone. For example, the interaction between a ligand and its specific receptor is often driven by stereospecific interactions between the ligand and its receptor, directed by the interaction of hydrophilic amino acids on the ligand with hydrophilic amino acids on the receptor, with water molecules front and center in these interactions. At the same time, this hydrophilic stereospecificity is further enhanced by de-emphasizing and / or suppressing nonspecific hydrophobic interactions that may be largely mediated / formed by hydrophobic-to-hydrophobic amino acids.

[0156] In some embodiments, anti-VEGF antibody conjugates are provided that are capable of inhibiting at least 90% of the interaction between a VEGF ligand ("VEGFL") and a VEGF receptor ("VEGFR"). For example, the antibody conjugates may inhibit at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or virtually all of the interaction between a VEGFR and a VEGFL. In some embodiments, the noted inhibition occurs at saturating concentrations. In some embodiments, anti-VEGF antibody conjugates are provided that inhibit at least 95% of the interaction between a VEGF ligand and a VEGF receptor. As an example of such superiority, see Figure 20 for the ability of OG1953 (and the antibody conjugates provided herein) to inhibit to a greater extent than Lucentis® (ranibizumab) or Avastin® (bevacizumab), or even the antibody OG1950 (unconjugated). Indeed, while it is possible to expect that the addition of a polymer to an antibody (to form an antibody conjugate) may or may not have some detrimental effect on the binding / activity of the antibody, this result was unexpected since it was not expected that the addition would actually improve the inhibitory capacity of the antibody in this way.

[0157] In some embodiments, the antibody or antibody complex inhibits at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity and / or interaction between VEGFR and VEGFL. In some embodiments, the IC50 value can be 0.1 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 100 nM, or less than any one or more of the foregoing values. In some embodiments, the KD is 2×10 -13 M, 1 x 10 -13 M, 1 x 10 -12 M, 1 x 10 -11 M, 1 x 10 -10M, or any one of the foregoing values. In some embodiments, the IC50 value can be 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, or any one of the foregoing values.

[0158] In some embodiments, anti-VEGF antibodies are provided that inhibit at least 90% of the interaction between a VEGF ligand and a VEGF-receptor. For example, the antibody can inhibit at least 91, 92, 93, 94, 95, 96, 97, 98, 99, or virtually all of the interaction between a VEGFR and a VEGFL. An example of such superiority is OG1950 (and the antibodies provided herein), which inhibits to a greater extent than Lucentis® (ranibizumab) or Avastin® (bevacizumab). Please refer to Figure 20 for the capabilities of the body.

[0159] In some embodiments, other antibodies, such as Lucentis® (ranibizumab) or Avastin® (bevacizumab), can be conjugated to one or more of the polymers described herein by one or more of the processes described herein. In some embodiments, any antibody or antibody fragment can be conjugated to one or more of the polymers described herein by one or more of the processes described herein.

[0160] In some embodiments, the antibody comprises a heavy chain amino acid variable region comprising SEQ ID NO: 1 and a light chain amino acid variable region comprising SEQ ID NO: 2. In some embodiments, the antibody is conjugated to one or more of the polymers provided herein. In some embodiments, the conjugated antibody is at least 90% identical to SEQ ID NO: 1 and / or 2. In some embodiments, the antibody comprises six CDRs in SEQ ID NO: 1 and SEQ ID NO: 2, and a point mutation of L443C (EU numbering, or 449C in SEQ ID NO: 1). In some embodiments, the conjugated antibody is at least 90% identical to SEQ ID NO: 1 and / or 2 and comprises the following mutations: L234A, L235A, and G237A (EU numbering), and at least one of the following mutations: Q347C (EU numbering) or L443C (EU numbering).

[0161] In some embodiments, an antibody that binds to VEGF-A is provided, the antibody comprising a CDR within SEQ ID NO: 1. H CDR 1 H 1. CDR in SEQ ID NO: 1 H CDR 2 H 2. CDR in SEQ ID NO: 1 H CDR 3 H 3. CDR in SEQ ID NO:2 L CDR 1 L 1. CDR in SEQ ID NO:2 L CDR 2 L 2. CDRs in SEQ ID NO:2 L CDR 3 L 3. Contains at least one of the following mutations: L234A, L235A, and G237A (EU numbering), and at least one of the following mutations: Q347C (EU numbering) or L443C (EU numbering).

[0162] As will be understood by one of skill in the art, with reference to the present specification, any of the antibodies provided herein can be conjugated to any of the polymers provided herein, and / or any of the antibodies provided herein can have a cysteine ​​added to allow for site-specific conjugation to a polymer.

[0163] "VEGF" or "vascular endothelial growth factor" refers to a human vascular endothelial growth factor that affects the process of angiogenesis or neovascularization. In particular, the term VEGF refers to any member of a class of growth factors that (i) bind to a VEGF receptor, such as VEGFR-1 (Flt-1), VEGFR-2 (KDR / Flk-1), or VEGFR-3 (FLT-4), (ii) activate the tyrosine kinase activity associated with the VEGF receptor, and (iii) thereby affect the process of angiogenesis or neovascularization.

[0164] The VEGF factor family consists of five closely related glycoproteins: VEGF-A (also known as VPE), -B, -C, -D, and PGF (placental growth factor). Of these, VEGF-A is the most well-studied and is the target of antiangiogenic therapy. Ferrara et al. (2003) Nat. Med. 9:669-676. VEGF-A is produced by both alternative splicing and proteolysis, resulting in a number of different isotypes, namely VEGF-A 206 , VEGF-A 189 , VEGF-A 165 , and VEGF-A 121 These isoforms differ in their ability to bind to heparin and non-signaling binding proteins called neuropilins. All of these isoforms are biologically active as dimers.

[0165] The various actions of VEGF are mediated by the binding of VEGF, e.g., VEGF-A (P15692), -B (P49766), -C (P49767), and -D (Q43915), to receptor tyrosine kinases (RTKs). VEGF family receptors belong to class V RTKs, each of which contains seven Ig-like domains in its extracellular domain (ECD). In humans, VEGF binds to three RTKs: VEGFR-1 (Flt-1) (P17948), VEGFR-2 (KDR, Flk-1) (P935968), and VEGFR-3 (Flt-4) (P35916). Unless otherwise clear from the context, a reference to VEGF refers to any of VEGF-A, -B, -C, -D, and PGF, which are either naturally occurring isoforms or naturally occurring or artificial variants having at least 90%, 95%, 98%, 99%, or 100% sequence identity to the naturally occurring sequence. In some embodiments, such VEGF is human VEGF. Similarly, a reference to VEGFR refers to any of VEGR-1, R-2, or R-3, including any naturally occurring isoform or naturally occurring or artificial variant having at least 90%, 95%, 98%, 99%, or 100% sequence identity to the naturally occurring sequence.

[0166] VEGF antagonist therapy has been approved for the treatment of certain cancers and wet AMD. Bevacizumab (Avastin, Genentech / Roche) is a humanized murine monoclonal antibody that binds to and neutralizes human VEGF, particularly all isoforms of VEGF-A and biologically active proteolytic fragments of VEGF-A. See, e.g., Ferrara N, Hillan KJ, Gerber HP, Novotny W. 2004. Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov. 3(5):391-400. Bevacizumab has been approved for the treatment of certain cancers. The protein sequences of the heavy and light chains of bevacizumab (DrugBank DB00112) are shown in SEQ ID NO:3 (heavy chain) and SEQ ID NO:4 (light chain).

[0167] Bevacizumab variable light chain CDRs are CDRs L 1: SASQDISNYLN (SEQ ID NO: 12), CDR L 2: FTSSLHS (SEQ ID NO: 13) and CDR L 3:QQYSTVPWT (SEQ ID NO: 14). Bevacizumab variable heavy chain CDRs are CDRs H 1:GYTFTNYGMN, CDR H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H 3:YPHYYGSSHWYFDV. The CDRs are defined by Kabat, with the exception that the combined Kabat / Chothia definition is used for CDRH1. In some embodiments, cysteines can be added to the bevacizumab sequence, and the antibody (and / or variants containing the six CDRs of bevacizumab) can be conjugated to any one or more of the polymers provided herein.

[0168] Another anti-VEGF molecule, ranibizumab (Lucentis® (ranibizumab), Genentech / Roche), derived from the same murine monoclonal antibody as bevacizumab, is approved for the treatment of wet AMD. Ranibizumab is an antibody fragment or Fab. Ranibizumab was generated by affinity maturation of the variable heavy and variable light chains of bevacizumab. The sequences of the ranibizumab heavy and light chains (published by Novartis) are set forth in SEQ ID NOs: 5 and 6, respectively. In some embodiments, cysteines can be added to the ranibizumab sequence, and the antibody (and / or variants comprising the six CDRs of ranibizumab) can be conjugated to any one or more of the polymers provided herein.

[0169] The ranibizumab CDRs are the same as bevacizumab except that they have been refined after affinity maturation. The ranibizumab variable light chain CDRs are CDRs L 1: SASQDISNYLN (SEQ ID NO: 12), CDR L 2: FTSSLHS (SEQ ID NO: 13) and CDR L 3:QQYS TVPWT (SEQ ID NO: 14). Ranibizumab variable heavy chain CDRs are CDRs H 1: GYDFTHYGMN (SEQ ID NO: 9), CDR H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H 3: YPYYYGTSHWYFDV (SEQ ID NO: 11).

[0170] In some embodiments, an antibody conjugate is provided having an anti-VEGF-A antibody linked to a phosphorylcholine-containing polymer at a cysteine ​​outside the variable region of the antibody, where the cysteine ​​has been added by recombinant DNA technology. In some embodiments, the polymer is linked to a single cysteine. In some embodiments, "added by recombinant DNA technology" refers to the replacement of the cysteine ​​residue with a non-cysteine ​​amino acid present at the same position in a known or existing antibody sequence or consensus antibody sequence. Thus, for example, if the antibody is an IgG1 and its heavy chain has a leucine at EU position 443, the leucine is replaced with a cysteine ​​by recombinant DNA technology (L443C, EU numbering, or 449C in SEQ ID NO: 1). Correspondingly, the native IgG1 sequence at EU position 347 is Q (glutamine), and the Q is replaced with a cysteine ​​by recombinant DNA technology to yield Q347C.

[0171] In some embodiments, the anti-VEGF-A antibody has a light chain and a heavy chain, and the heavy chain has an Fc region. In some embodiments, the cysteine ​​is within the Fc region, and the anti-VEGF-A antibody is an immunoglobulin G (IgG). In some embodiments, the anti-VEGF-A heavy chain has a CDR H 1: GYDFTHYGMN (SEQ ID NO: 9), CDR H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H 3: YPYYYGTSHWYFDV (SEQ ID NO: 11) and at position 221 (position via sequence counting found in SEQ ID NO: 3) is T, and the anti-VEGF-A light chain has CDR L 1: SASQDISNYLN (SEQ ID NO: 12), CDR L 2: FTSSLHS (SEQ ID NO: 13), and CDR L 3: QQYSTVPWT (SEQ ID NO: 14) and L at Kabat position 4.

[0172] In some embodiments, the anti-VEGF-A heavy chain isotype is IgG1. In some embodiments, the IgG1 constant domain has one or more mutations compared to an IgG1 constant domain (e.g., the constant region of SEQ ID NO: 3) to modulate effector function. In some embodiments, the effector function mutations are one or more of the following: E233X, L234X, L235X, G236X, G237X, A327X, A330X, and P331X (EU numbering), where X is any natural or unnatural amino acid. In some embodiments, the mutations are selected from the group consisting of E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S (EU numbering). In some embodiments, the antibody complex has the following mutations: L234A, L235A, and G237A (EU numbering).

[0173] In some embodiments, the cysteine ​​residue is in the anti-VEGF-A heavy chain and is Q347C (EU numbering) or L443C (EU numbering). In some embodiments, the cysteine ​​residue is L443C (EU numbering, or 449C in SEQ ID NO: 1). In some embodiments, the sequence of the anti-VEGF-A heavy chain is SEQ ID NO: 1 and the sequence of the anti-VEGF-A light chain is SEQ ID NO: 2.

[0174] In some embodiments, the phosphorylcholine-containing polymer comprises 2-(methacryloyloxyethyl)-2'-(trimethylammonium)ethyl phosphate (MPC) monomers, as shown below. [ka]

[0175] The polymer comprises the following repeating unit: [ka] where n is an integer from 1 to 3000, and the wavy lines represent connection points between monomer units in the polymer.

[0176] In some embodiments, the polymer has three or more arms or is synthesized using an initiator having three or more polymer initiation sites. In some embodiments, the polymer has two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve arms or is synthesized using an initiator having two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve polymer initiation sites. More preferably, the polymer has three, six, or nine arms or is synthesized using an initiator having three, six, or nine polymer initiation sites. In some embodiments, the polymer has nine arms or is synthesized using an initiator having nine polymer initiation sites.

[0177] In some embodiments, the attached polymer has a molecular weight (SEC-MALs) between about 300,000 and about 1,750,000 Da. In some embodiments, the polymer has a molecular weight between about 500,000 and about 1,000,000 Da. In some embodiments, the polymer has a molecular weight between about 600,000 and about 900,000 Da. In some embodiments, the polymer has a molecular weight between about 750,000 and about 850,000 Da. In some embodiments, the polymer has a molecular weight between about 800,000 and about 850,000 Da. In some embodiments, the polymer has a molecular weight between about 750,000 and about 800,000 Da.

[0178] In some embodiments, any of the antibodies described herein can be further conjugated to a polymer to form a bioconjugate having a molecular weight (total, SEC-MALs) between about 350,000 and 2,000,000 daltons, e.g., between about 450,000 and 1,900,000 daltons, between about 550,000 and 1,800,000 daltons, between about 650,000 and 1,700,000 daltons, between about 750,000 and 1,600,000 daltons, between about 850,000 and 1,500,000 daltons, between about 900,000 and 1,600,000 daltons, between about 1000,000 and 1,800,000 daltons, between about 1500,000 and 1,900,000 daltons, between about 1600,000 and 1,600,000 daltons, between about 1700,000 and 1,800,000 daltons, between about 1800,000 and 1,900,000 daltons, between about 2000,000 and 2000,000 daltons, between about 2500,000 and 2500,000 daltons, between about 3000,000 and 3000,000 daltons, between about 3500,000 and 2,000,000 daltons, between about 4500,000 and 1,900,000 daltons, between about 5500,000 and 1, The amino acid sequence may be between about 950,000 and 1,400,000 daltons, between about 950,000 and 1,300,000 daltons, between about 900,000 and 1,000,000 daltons, between about 1,000,000 and 1,300,000 daltons, between about 850,000 and 1,300,000 daltons, between about 850,000 and 1,000,000 daltons, and between about 1,000,000 and 1,200,000 daltons.

[0179] In some embodiments, the antibody conjugate is purified. In some embodiments, the polymer is polydisperse, i.e., the PDI of the polymer is not 1.0. In some embodiments, the PDI is less than 1.5. In some embodiments, the PDI is less than 1.4. In some embodiments, the PDI is less than 1.3. In some embodiments, the PDI is less than 1.2. In some embodiments, the PDI is less than 1.1.

[0180] In some embodiments, the antibody conjugate comprises an anti-VEGF-A immunoglobulin G (IgG) conjugated to a polymer, the polymer comprising an MPC monomer, the anti-VEGF-A heavy chain having the sequence SEQ ID NO: 1, the anti-VEGF-A light chain having the sequence SEQ ID NO: 2, and the antibody being conjugated to the polymer only at C449 in SEQ ID NO: 1. In some embodiments, the polymer has nine arms and a molecular weight between 600,000 and 1,000,000 Da.

[0181] In some embodiments, the antibody conjugate comprises an anti-VEGF-A immunoglobulin G (IgG) conjugated to a polymer, the polymer comprising an MPC monomer, the anti-VEGF-A heavy chain sequence is SEQ ID NO: 1, the anti-VEGF-A light chain sequence is SEQ ID NO: 2, and the antibody is conjugated to the polymer only at C443 (EU numbering, or 449C in SEQ ID NO: 1). In some embodiments, the polymer has 9 arms and a molecular weight between 600,000 and 1,000,000 Da.

[0182] In some embodiments, the antibody conjugate has the structure: [ka] wherein each heavy chain of the anti-VEGF-A antibody is represented by the letter H, and each light chain of the anti-VEGF-A antibody is represented by the letter L; the polymer is attached to the anti-VEGF-A antibody via the sulfhydryl at C449 of SEQ ID NO: 1, the attachment being shown on one of the heavy chains; and PC [ka] wherein the wavy line represents the point of attachment to the remainder of the polymer, and X is a) OR where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halide, including Br; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±10%. In certain embodiments, n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000. In certain embodiments, n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 500. In some embodiments, X is OR, wherein R is a sugar, aminoalkyl, or one of the following residues: saturated C1-C 24 Alkyl, unsaturated C2-C 24Alkenyl or C2-C 24 Alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and alkyl halides including polyhalogenated alkyls, -CO-O-R7, carbonyl -CCO-R7, -CO-NR8R9, -(CH2) n -COOR7, -CO-(CH) n -COOR7, -(CH2) n -NR8R9, ester, alkoxycarbonyl, and mono-, multiply, or unsubstituted variants of aryloxycarbonyl, wherein n is an integer from 1 to 6, and R7, R8, and R9 are hydrogen atoms, halogen atoms, or the following residues: saturated C1-C 24 Alkyl, unsaturated C2-C 24 Alkenyl or C2-C 24 and alkyl halides, including polyhalogenated alkyls, 5-membered rings, and 6-membered rings, each independently selected from the group consisting of mono-, multiply-, or unsubstituted variants of alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and alkyl halides, including polyhalogenated alkyls, 5-membered rings, and 6-membered rings.

[0183] In some embodiments, the antibody conjugate has the structure: [ka] wherein each heavy chain of the anti-VEGF-A antibody is represented by the letter H, and each light chain of the anti-VEGF-A antibody is represented by the letter L; the polymer is conjugated to the anti-VEGF-A antibody via the sulfhydryl of C443 (EU numbering, or 449C in SEQ ID NO: 1), the linkage being shown on one of the heavy chains; and PC [ka] wherein the wavy line represents the point of attachment to the remainder of the polymer, and X is a) OR where R is H, methyl, ethyl, propyl, or isopropyl; b) H; or c) any halide, including Br; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±10%. In certain embodiments, n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000. In certain embodiments, n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 500. In some embodiments, X is OR, wherein R is a sugar, aminoalkyl, or one of the following residues: saturated C1-C 24 Alkyl, unsaturated C2-C 24 Alkenyl or C2-C 24 Alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and alkyl halides including polyhalogenated alkyls, -CO-O-R7, carbonyl -CCO-R7, -CO-NR8R9, -(CH2) n -COOR7, -CO-(CH) n -COOR7, -(CH2)n -NR8R9, ester, alkoxycarbonyl, aryloxycarbonyl, mono-substituted, multiply substituted, or unsubstituted variants, in which n is an integer from 1 to 6, and R7, R8, and R9 are hydrogen atoms, halogen atoms, the following residues: saturated C1-C 24 Alkyl, unsaturated C2-C 24 Alkenyl or C2-C 24 and alkyl halides, including polyhalogenated alkyls, 5-membered rings, and 6-membered rings, each independently selected from the group consisting of mono-, multiply-, or unsubstituted variants of alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and alkyl halides, including polyhalogenated alkyls, 5-membered rings, and 6-membered rings.

[0184] In some embodiments, the antibody conjugate is in a liquid formulation. In some embodiments, the antibody conjugate is combined with a pharmaceutically acceptable carrier.

[0185] In some embodiments, an anti-VEGF-A antibody is provided. The anti-VEGF-A antibody heavy chain comprises at least the following CDR sequences: H 1: GYDFTHYGMN (SEQ ID NO: 9), CDR H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H 3:YPYYYGTSHWYFDV (SEQ ID NO: 11). In some embodiments, the anti-VEGF-A heavy chain has these CDRs and additionally has a threonine (T) at position 221 (the position via the sequence count found in SEQ ID NO: 3). In some embodiments, the anti-VEGF-A light chain has at least the following CDRs: CDR L 1: SASQDISNYLN (SEQ ID NO: 12), CDR L 2: FTSSLHS (SEQ ID NO: 13) and CDRL 3:QQYSTVPWT (SEQ ID NO: 14). In some embodiments, the anti-VEGF-A antibody has these CDRs and additionally has a leucine (L) at Kabat position 4. In some embodiments, the anti-VEGF-A antibody heavy chain isotype is IgG1 and has a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In some embodiments, the light chain isotype is kappa.

[0186] In some embodiments, the IgG1 domain of the anti-VEGF-A antibody comprises one or more mutations that modulate effector functions such as ADCC, ADCP, and CDC. In some embodiments, the IgG1 mutations reduce effector function. In some embodiments, the amino acids used for effector function mutations include E233X, L234X, L235X, G236X, G237X, G236X, D270X, K322X, A327X, P329X, A330X, where X is any natural or unnatural amino acid. , A330X, P331X, and P331X (EU numbering). In some embodiments, the mutations include one or more of the following: E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S (EU numbering). In some embodiments, the anti-VEGF-A heavy chain has the following mutations: L234A, L235A, and G237A (EU numbering). In some embodiments, the number of effector function mutations relative to the native human IgG1 sequence is fewer than 10. In some embodiments, the number of effector function mutations relative to the native human IgG1 sequence is fewer than 5, fewer than 4, fewer than 3, fewer than 2, or fewer than 1. In some embodiments, the antibody has reduced Fcγ binding and / or complement C1q binding such that the antibody's ability to elicit effector function is reduced. This is particularly advantageous for ophthalmic indications / disorders.

[0187] In some embodiments, the anti-VEGF-A antibody comprises one or more of the following amino acid mutations: L234A, L235A, G237A (EU numbering), and L443C (EU numbering, or 449C in SEQ ID NO: 1).

[0188] In some embodiments, the anti-VEGF-A antibody is human immunoglobulin G (IgG1) or a portion thereof.

[0189] In some embodiments, the VEGF-A antibody has a heavy chain constant domain comprising one or more mutations that reduce immune-mediated effector function.

[0190] In some embodiments, an anti-VEGF-A antibody is provided, the anti-VEGF antibody comprising a CDR comprising the sequence GYDFTHYGMN (SEQ ID NO: 9). H 1. CDR containing the sequence WINTYTGEPTYAADFKR (SEQ ID NO: 10) H 2. CDR containing the sequence YPYYYGTSHWYFDV (SEQ ID NO: 11) H 3. CDR containing the sequence SASQDISNYLN (SEQ ID NO: 12) L 1. CDR containing the sequence FTSSLHS (SEQ ID NO: 13) L 2, and sequence QQYSTVPWT (SEQ ID NO: 14) CDR L 3.

[0191] Alternatively, the IgG domain may be IgG2, IgG3, or IgG4, or may be a composite in which the constant region is formed from more than one of these isotypes (e.g., the CH1 region of IgG2 or IgG4 and the hinge, CH2, and CH3 regions of IgG1). Such domains may contain mutations at one or more of the EU positions mentioned for IgG1 to reduce and / or alter effector function. Human IgG2 and IgG4 have reduced effector function compared to human IgG1 and IgG3.

[0192] The anti-VEGF-A heavy chain has a cysteine ​​residue added by recombinant DNA technology as a mutation that can be used to conjugate a half-life extending moiety. In some embodiments, the mutation is (EU numbering) Q347C (EU numbering) and / or L443C (EU numbering, or 449C in SEQ ID NO: 1). In some embodiments, the mutation is L443C (EU numbering, or 449C in SEQ ID NO: 1). In some embodiments, the stoichiometry of antibody to polymer is 1:1, i.e., the conjugate has one molecule of antibody conjugated to one molecule of polymer.

[0193] The half-life of the anti-VEGF-A antibody can be extended by the attachment of a "half-life ("half-life") extending moiety" or "half-life ("half-life") extending group." Half-life extending moieties include peptides and proteins that can be expressed in-frame with (or optionally chemically conjugated to) the biologic of interest, as well as various polymers that can be attached or conjugated to one or more amino acid side chain or terminal functional groups, such as -SH, -OH, -COOH, -CONH, -NH, or one or more N- and / or O-glycan structures. Half-life extending moieties generally act to increase the in vivo circulatory half-life of a biologic.

[0194] Examples of peptide / protein half-life extending moieties include Fc fusions (Capon DJ, Chamow SM, Mordenti J, et al. Designing CD4 immunoadhesions for AIDS therapy. Nature. 1989. 337:525-31), human serum albumin (HAS) fusion (Yeh P, Landais D, Lemaitre M, et al. Design of yeast-secreted albumin derivatives for human therapy: biological and antiviral properties of a serum albumin-CD4 genetic conjugate. Proc Natl Acad Sci USA. 1992. 89:1904-08 ), carboxy-terminal peptide (CTP) fusion (Fares FA, Suganuma N, Nishimori K, et al. Design of a long-acting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin beta subunit to the follitropin beta subunit. Proc Natl Acad Sci USA. 1992. 89:4304-08), genetic fusion of non-exact repeat peptide sequences (XTEN) fusions (Schellenberger V, Wang CW, Geething NC, et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat Biotechnol. 2009. 27:1186-90), elastin-like peptide (ELP) (MCpherson DT, Morrow C, Minehan DS, et al. Production and purification of a recombinant elastomeric polypeptide, G(VPGVG19-VPGV, from Escherichia coli. Biotechnol Prog. 1992). 8:347-52), human transferrin fusion proteins (Prior CP, Lai CH, Sadehghi H et al. Modified transferrin fusion proteins. International Patent Publication WO2004 / 020405, 2004), proline-alanine-serine (PAS) (Skerra A, Theobald I, Schlapsky M. Biological active proteins having increased in vivo and / or vitro stability. International Patent Publication WO2008 / 155134(A1) 2008), homoamino acid polymers (HAP) (Schlapschy M, Theobald I, Mack H, et al.Fusion of a recombinant antibody fragment with a homo-amino acid polymer: effects on biophysical properties and prolonged plasma half-life. Protein Eng Des Sel. 2007. 20:273-84) and gelatin-like protein (GLK) fusion (Huang YS, Wen XF, Zaro JL, et al. Engineering a pharmacologically superior form of granulocyte-colony-stimulating factor by fusion with gelatin-like protein polymer. Eur J. Pharm Biopharm. 2010. 72:435-41).

[0195] Examples of polymeric half-life extending moieties include polyethylene glycol (PEG), branched PEG, PolyPEG® (Warwick Effect Polymers; Coventry, UK), polysialic acid (PSA), starch, hydroxyethyl starch (HES), hydroxyalkyl starch (HAS), carbohydrates, polysaccharides, pullulan, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, dextran, carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene-maleic anhydride copolymer, polystyrene-maleic anhydride copolymer, poly(1-hydroxymethylethylene hydroxymethyl formal) (PHF), zwitterionic polymers, phosphorylcholine-containing polymers and MPC-containing polymers, poly(Gly x -Ser y), hyaluronic acid (HA), heparosan polymer (HEP), fleximer, dextran, and polysialic acid (PSA).

[0196] In one embodiment, half-life extending moieties can be conjugated to antibodies via the free amino groups of the protein using N-hydroxysuccinimide (NHS) esters. Reagents targeted for conjugation to amine groups can react randomly with the ε-amine groups of lysines, the α-amine group of the N-terminal amino acid, and the δ-amine group of histidines.

[0197] However, the anti-VEGF-A antibodies disclosed herein have numerous amine groups available for polymer conjugation, and therefore, polymer conjugation to free amino groups may negatively affect the antibody protein's ability to bind to VEGF.

[0198] In some embodiments, half-life extending moieties are conjugated to one or more free SH groups using any suitable thiol-reactive chemistry, including but not limited to maleimide chemistry, or preoxidation followed by attachment of a polymeric hydrazide or polymeric amine to the carbohydrate moiety of the antibody. In some embodiments, maleimide conjugation is used. In some embodiments, conjugation occurs at a naturally occurring or engineered cysteine.

[0199] In some embodiments, the polymer is covalently attached to cysteine ​​residues introduced into the anti-VEGF-A antibody by site-directed mutagenesis. In some embodiments, these cysteine ​​residues are used in the Fc portion of the antibody. In some embodiments, sites for introducing cysteine ​​residues into the Fc region are provided in International Publication No. WO 2013 / 093809, U.S. Patent No. 7,521,541, International Publication No. WO 2008 / 020827, U.S. Patent No. 8,008,453, U.S. Patent No. 8,455,622, and U.S. Patent Application Publication No. 2012 / 0213705, which are incorporated herein by reference for all purposes. In some embodiments, the cysteine ​​mutations are Q347C (EU numbering) and L443C, which refer to human IgG heavy chains according to EU numbering.

[0200] In some embodiments, conjugates are provided of an antibody and a high molecular weight polymer that functions as a half-life extender. In some embodiments, conjugates are provided that include an antibody conjugated to a polymer formed from one or more monomeric units, at least one of the monomeric units being bound to a zwitterionic polymer bearing a zwitterionic group. In some embodiments, the zwitterionic group is phosphorylcholine.

[0201] In some embodiments, one of the monomer units is HEMA-PC. In some embodiments, the polymer is synthesized from a single monomer that is HEMA-PC.

[0202] In some embodiments, the antibody conjugates comprise two monomers, each of which is HEMA-PC. In some embodiments, the conjugate has 3, 6, or 9 arms. In some embodiments, the conjugate has 9 arms. In some embodiments, the conjugate has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 polymer arms, where the monomer is HEMA-PC. In some embodiments, the conjugate has 3, 6, or 9 arms. In some embodiments, the conjugate has 9 arms.

[0203] In some embodiments, the polymer-antibody conjugate has a polymer portion having a molecular weight between 100,000 and 1,500,000 Da. In some embodiments, the conjugate has a polymer portion having a molecular weight between 500,000 and 1,000,000 Da. In some embodiments, the conjugate has a polymer portion having a molecular weight between 600,000 and 800,000 Da. In some embodiments, the conjugate has a polymer portion having a molecular weight between 600,000 and 850,000 Da and has nine arms. When a molecular weight is given for an antibody conjugated to a polymer, the molecular weight is the molecular weight of the protein, including any carbohydrate moieties associated with the protein, plus the molecular weight of the polymer.

[0204] In some embodiments, anti-VEGF-A antibodies are provided comprising a HEMA-PC polymer having a molecular weight, as measured by Mw, of between about 100 kDa and 1650 kDa. In some embodiments, the molecular weight, as measured by Mw, of the polymer is between about 500 kDa and 1000 kDa. In some embodiments, the molecular weight, as measured by Mw, of the polymer is between about 600 kDa and about 900 kDa. In some embodiments, the molecular weight, as measured by Mw, of the polymer is 750 kDa ± 15%.

[0205] In some embodiments, the polymer is prepared from an initiator suitable for ATRP having one or more polymer initiation sites. In some embodiments, the polymer initiation site comprises a 2-bromoisobutyrate site. In some embodiments, the initiator has three or more polymer initiation sites. In some embodiments, the initiator has three, four, five, six, seven, eight, nine, ten, eleven, or twelve polymer initiation sites. In some embodiments, the initiator has three, six, or nine polymer initiation sites. In some embodiments, the initiator has nine polymer initiation sites. In some embodiments, the initiator is OG1786.

[0206] The anti-VEGF-A antibody may be produced by recombinant expression, including (i) producing recombinant DNA by genetic engineering, (ii) introducing the recombinant DNA into prokaryotic or eukaryotic cells, for example, but not limited to, by transfection, electroporation, or microinjection, (iii) culturing the transformed cells, (iv) expressing the antibody, for example, continuously or upon induction, and (v) isolating the antibody, for example, from the culture medium or by recovering the transformed cells, to (vi) obtain purified antibody.

[0207] The anti-VEGF-A antibody can be produced by expression in a suitable prokaryotic or eukaryotic host system capable of producing a pharmacologically acceptable antibody molecule. Examples of eukaryotic cells include mammalian cells such as CHO, COS, HEK293, BHK, SK-Hip, and HepG2. Other suitable expression systems include prokaryotes (e.g., E. coli, including the pET / BL21 expression system), yeast (Saccharomyces cerevisiae and / or Pichia pastoris), and insect cells.

[0208] A wide variety of vectors can be used to prepare the antibodies disclosed herein and are selected from eukaryotic and prokaryotic expression vectors. Examples of prokaryotic expression vectors include, but are not limited to, plasmids such as pRESET, pET, and pAD, and promoters used in prokaryotic expression vectors include, but are not limited to, one or more of lac, trc, trp, recA, or araBAD. Examples of vectors for karyotic expression include (i) for expression in yeast, vectors such as, but not limited to, pAO, pPIC, pYES, or pMET, which use promoters such as, but not limited to, AOX1, GAP, GAL1, or AUG1; (ii) for expression in insect cells, vectors such as, but not limited to, pMT, pAc5, pIB, pMIB, or pBAC, which use promoters such as, but not limited to, PH, p10, MT, Ac5, OpIE2, gp64, or polh; and (iii) for expression in mammalian cells, vectors such as, but not limited to, pSVL, pCMV, pRc / RSV, pcDNA3, or pBPV, which use promoters such as, but not limited to, CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and β-actin, and in one embodiment, vectors derived from viral systems such as, but not limited to, vaccinia virus, adeno-associated virus, herpes virus, or retrovirus.

[0209] Methods for conjugating proteins to polymers In some embodiments, a method for preparing a therapeutic protein half-life extending moiety conjugate is provided, the method comprising conjugating a therapeutic protein having a cysteine ​​residue added by recombinant DNA techniques to a half-life extending moiety having a sulfhydryl-specific reactive group selected from the group consisting of maleimide, vinyl sulfone, orthopyridyl disulfide, and iodoacetamide to provide the therapeutic protein half-life extending moiety conjugate.

[0210] In some embodiments, a method for preparing an OG1950 antibody conjugate from OG1950 is provided. As shown in FIG. 18, the method involves reducing the OG1950 protein with a 50-fold molar excess of TCEP reducing agent (FIG. 18). After reduction, the antibody is oxidized to form a decapped OG1950 antibody in which the naturally occurring inter- and intrachain disulfide bonds between the light and heavy chains are formed, but the engineered cysteine ​​at position L443C (EU numbering, or 449C in SEQ ID NO: 1) of the heavy chain remains uncapped (FIG. 18). The OG1950 is then conjugated by adding excipients and a 5- to 10-fold molar excess of a maleimide biopolymer (FIG. 18). The biopolymer is attached to the OG1950 antibody via a covalent thiol ether bond (FIG. 18). After conjugation, the OG1950 antibody conjugate is purified to remove both unconjugated antibody and polymer (FIG. 18).

[0211] The proteins and processes described above can also be modified. Accordingly, in some embodiments, a process for preparing a conjugated protein (not necessarily an antibody or an anti-VEGF antibody) is provided. The process includes reducing one or more cysteines in a protein to form a decapped protein in solution. After reduction of the one or more cysteines, the decapped protein is reoxidized to re-establish at least one disulfide bond in the reduced protein, while ensuring that the engineered cysteine ​​residue in the protein remains in a free thiol form to form a reoxidized decapped protein in solution. At least one excipient is then added to the solution, which excipient reduces polymer-induced protein precipitation. After addition of the excipient, a polymer is added to the solution, and the polymer is conjugated to the reoxidized decapped protein at the engineered cysteine ​​residue to form a conjugated protein.

[0212] In some embodiments, the molar excess of the reducing agent can vary to any functional amount. In some embodiments, a 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, or 90x molar excess of the reducing agent (not necessarily TCEP in all embodiments) can be used. In some embodiments, any antibody (therapeutic or otherwise) can be used. In some embodiments, a 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, or 15x molar excess of maleimide biopolymer can be used. In some embodiments, there is an excess of decapped protein relative to polymer. In some embodiments, the amount of reduced protein is less than the amount of polymer. In some embodiments, the amount of reduced protein is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the amount of polymer. In some embodiments, 10-15 times more polymer than protein is used. In some embodiments, the amount of the reduced antibody is greater than the amount of the polymer. In some embodiments, the amount of the polymer is greater than the amount of the reduced antibody.

[0213] In some embodiments, the purification step is optional.

[0214] In some embodiments, the method of making an antibody conjugate comprises conjugating an anti-VEGF-A antibody to a phosphorylcholine-containing polymer. In some embodiments, the method comprises conjugating an anti-VEGF-A antibody to a phosphorylcholine-containing polymer. The anti-VEGF-A antibody comprises an amino acid residue added by recombinant DNA techniques. In some embodiments, the added amino acid residue is a cysteine ​​residue. In some embodiments, the cysteine ​​residue is added outside the variable region of the antibody. The cysteine ​​residue can be added to either the heavy or light chain of the antibody.

[0215] In some embodiments, the polymer comprises or consists of a phosphorylcholine-containing polymer. In some embodiments, the phosphorylcholine-containing polymer comprises a sulfhydryl-specific reactive group selected from the group consisting of maleimide, vinyl sulfone, orthopyridyl disulfide, and iodoacetamide. In some embodiments, the sulfhydryl-specific reactive group on the phosphorylcholine-containing polymer reacts with the cysteine ​​residue on the anti-VEGF-A antibody to form the antibody conjugate.

[0216] In some embodiments, the conjugated protein may be an antibody, antibody-protein fusion, or binding fragment thereof. In some embodiments, the protein is not an antibody but an enzyme, ligand, receptor, or other protein, or a mutant or variant thereof. In some embodiments, the native protein contains at least one disulfide bond and at least one non-native cysteine.

[0217] In some embodiments, the excipient can be an acid or a base. In some embodiments, the excipient is a surfactant, a sugar, or a charged amino acid. In some embodiments, the excipient helps keep the protein in solution during conjugation to the polymer. In some embodiments, the excipient is added to the solution containing the protein before adding the polymer to the solution containing the protein.

[0218] In some embodiments, the reaction occurs under aqueous conditions between about pH 5 and about pH 9. In some embodiments, the reaction occurs between 6.0 and 8.5, between 6.5 and 8.0, or between 7.0 and 7.5.

[0219] In some embodiments, the polymer is conjugated to the protein at 2-37 degrees Celsius. In some embodiments, the conjugation occurs at 0-40 degrees Celsius, 5-35 degrees Celsius, 10-30 degrees Celsius, and 15-25 degrees Celsius.

[0220] In some embodiments, the complexed proteins described herein can be contacted with an ion exchange medium, or a hydrophobic interaction or affinity chromatography medium for purification (to separate the complexed from the uncomplexed). In some embodiments, the ion exchange medium, hydrophobic interaction chromatography medium, and / or affinity chromatography medium can be used to separate the complexed proteins from the uncomplexed proteins. The complexed protein is separated from the depolymerized protein and from the reoxidized decapped protein.

[0221] In some embodiments, the methods described herein and summarized in FIG. 18 involve excipients that can promote and / or maintain solubility. In some embodiments, the methods allow the solution to maintain the solubility, i.e., interaction, of the two constituent components. This can include the solubility of the protein and the polymer, as well as the solubility of the final complex. In some embodiments, without the excipient approach, the protein may be soluble, but when the biopolymer is added, the solubility of the solution (e.g., protein) may decrease, resulting in the protein settling / precipitation from solution. Of course, if the protein precipitates, it will not be available to efficiently conjugate with the biopolymer. Therefore, excipients can be used to maintain the solubility of the protein in the presence of the biopolymer, allowing the two to combine to form the protein complex (or antibody complex, as shown in FIG. 18). This also allows the complex to maintain solubility.

[0222] In some embodiments, the polymers disclosed herein can include one or more of the following: zwitterions, phosphorylcholine, or PEG linkers bridging the central polymer branch point to the maleimide functional group. In some embodiments, any of the polymers provided herein can be attached to proteins by the methods provided herein.

[0223] In some embodiments, any of the proteins provided herein can be conjugated to any of the polymers provided herein by one or more of the methods provided herein.

[0224] In some embodiments, the processes provided herein allow for large-scale processing for producing and purifying protein and / or antibody conjugates. In some embodiments, the working volume is at least 1 liter, e.g., 1 liter, 10 liters, 100 liters, 1,000 liters, 5,000 liters, 10,000 liters, or more. In some embodiments, the amount of the antibody conjugate produced and / or purified can be 0.1 gram, 1 gram, 10 grams, 100 grams, 1000 grams, or more.

[0225] In some embodiments, the therapeutic protein can be any of the anti-VEGF-A antibodies described herein having a cysteine ​​residue added by recombinant DNA techniques. In some embodiments, the anti-VEGF antibody heavy chain has the following CDRs: H 1: GYDFTHYGMN (SEQ ID NO: 9), CDR H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H 3:YPYYYGTSHWYFDV (SEQ ID NO: 11). The heavy chain may also have a threonine (T) at position 221 (the position via the sequence count found in SEQ ID NO: 3). In some embodiments, the anti-VEGF light chain has the following CDRs: CDR L 1: SASQDISNYLN (SEQ ID NO: 12), CDR L 2: FTSSLHS (SEQ ID NO: 13), and CDR L 3: QQYSTVPWT (SEQ ID NO: 14). The anti-VEGF-A light chain may also have a leucine (L) at Kabat position 4.

[0226] In some embodiments, the anti-VEGF-A antibody is an IgG1. In some embodiments, the heavy chain comprises one or more mutations to modulate effector function. In some embodiments, the mutations are at one or more of the following amino acid positions: E233, L234, L235, G236, G237, A327, A330, and P331 (EU numbering). In some embodiments, the mutations are selected from the group consisting of E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S (EU numbering). In some embodiments, the mutations are L234A, L235A, and G237A (EU numbering).

[0227] In some embodiments, the cysteine ​​residues added to the therapeutic protein by recombinant DNA technology must not participate in Cys-Cys disulfide bonds. In this regard, the therapeutic protein may be a dimer. For example, a complete anti-VEGF-A antibody has two light chains and two heavy chains. If a Cys residue is introduced into the heavy chain, for example, the complete antibody will have two such introduced cysteines at the same position, and these cysteine ​​residues may potentially form intrachain disulfide bonds. If the introduced cysteine ​​residues tend to form or overlap Cys-Cys disulfide bonds, the introduced Cys residues will not be useful for conjugation. Methods to avoid positions within the heavy and light chains that result in intrachain disulfide bonds are known in the art. See, for example, U.S. Patent Application Publication No. 2015 / 0158952.

[0228] In some embodiments, the cysteine ​​residue introduced via recombinant DNA technology is selected from the group consisting of Q347C and L443C (EU numbering). In some embodiments, the cysteine ​​residue is L443C (EU numbering, or 449C in SEQ ID NO: 1). In some embodiments, the antibody heavy chain has the amino acid sequence set forth in SEQ ID NO: 1 and the light chain has the amino acid sequence of SEQ ID NO: 2.

[0229] In some embodiments, the sulfhydryl-specific reactive group is a maleimide.

[0230] In some embodiments, the half-life extending moiety is polyethylene glycol (PEG), branched PEG, PolyPEG® (Warwick Effect Polymers Ltd.; Coventry, UK), polysialic acid (PSA), starch, hydroxyethyl starch (HES), hydroxyalkyl starch (HAS), carbohydrates, polysaccharides, pullulan, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, dextran, carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene-maleic anhydride copolymer, polystyrene-maleic anhydride copolymer, poly(1-hydroxymethylethylene hydroxymethyl formal) (PHF), zwitterionic polymers, a phosphorylcholine-containing polymers, and 2-methacryloyloxy-2'-ethyltrimethylammonium phosphate (MPC)-containing polymers.

[0231] In some embodiments, the half-life extending moiety is a zwitterionic polymer. In some embodiments, the zwitterion is phosphorylcholine, i.e., a phosphorylcholine-containing polymer. In some embodiments, the polymer is composed of MPC units.

[0232] In some embodiments, the MPC polymer has three or more arms. In some embodiments, the MPC polymer has two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve arms. In some embodiments, the MPC polymer has three, six, or nine arms. In some embodiments, the MPC polymer has nine arms. In some embodiments, the polymer is synthesized using an initiator having two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more polymer initiation sites.

[0233] In some embodiments, the MPC polymer has a molecular weight between about 300,000 and 1,750,000 Da. In some embodiments, the MPC polymer has a molecular weight between about 500,000 and 1,000,000 Da, or between about 600,000 and 900,000 Da.

[0234] In some embodiments, the method for preparing a therapeutic protein half-life extending moiety conjugate includes the additional step of contacting the therapeutic protein with a thiol reducing agent under conditions that generate reduced cysteine ​​sulfhydryl groups. As discussed above, it is preferred that the cysteine ​​residues added by recombinant DNA technology be unpaired, i.e., not involved in Cys-Cys intrachain disulfide bonds, or not substantially involved in such bonds. However, it is known that Cys residues that are not involved in such Cys-Cys disulfide bonds and that are free for conjugation can react with free cysteines in the medium to form disulfide adducts. See, for example, International Publication WO 2009 / 052249. Such derivatized cysteines are no longer available for conjugation. To prevent the newly added cysteines from forming disulfide adducts, the purified protein is treated with a reducing agent, e.g., dithiothreitol. However, such treatment with a reducing agent will reduce all of the cysteine ​​residues in the therapeutic protein, including many of the unique cysteines involved in inter- and intra-chain Cys-Cys disulfide bonds. These unique Cys-Cys disulfides are generally important for protein stability and activity, and they must be reformed. In some embodiments, all unique (e.g., inter- and intra-chain) Cys-Cys disulfides are reformed.

[0235] After reduction to remove the cysteine ​​disulfide adducts, the therapeutic protein is exposed to oxidizing conditions and / or an oxidizing agent for a period of time, e.g., overnight, to reform the inherent inter- and intrachain disulfide residues. In some embodiments, overnight exposure to air can be used to achieve the reformation of the inherent disulfide bonds. In some embodiments, an oxidizing agent is used to rebuild the inherent disulfides. In some embodiments, the oxidizing agent is selected from the group consisting of aqueous CuSO4 and dehydroascorbic acid (DHAA). In some embodiments, the oxidizing agent is DHAA. In some embodiments, the range of DHAA used is between 5 and 30 equivalents. In some embodiments, the range is between 10 and 20 equivalents. In some embodiments, the range is 15 equivalents.

[0236] In some embodiments, the thiol reducing agent is selected from the group consisting of tris[2-carboxyethyl]phosphine hydrochloride (TCEP), dithiothreitol (DTT), dithioerythritol (DTE), sodium borohydride (NaBH), sodium cyanoborohydride (NaCNBH), β-mercaptoethanol (BME), cysteine ​​hydrochloride, and cysteine. In some embodiments, the thiol reducing agent is TCEP.

[0237] In some embodiments, the concentration of the thiol reducing agent is between 1 and 100-fold molar excess relative to the concentration of the therapeutic protein. In some embodiments, the concentration of the thiol reducing agent is between 20 and 50-fold molar excess relative to the concentration of the therapeutic protein. In some embodiments, the thiol reducing agent is removed after incubation with the therapeutic protein and prior to oxidation of the therapeutic protein.

[0238] In some embodiments, the method for conjugating a therapeutic protein to a half-life extending moiety further comprises purifying the therapeutic protein conjugate after conjugation, in some embodiments, the therapeutic protein conjugate is purified using a technique selected from the group consisting of ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and affinity chromatography, or a combination thereof.

[0239] In some embodiments, the therapeutic protein conjugate retains at least 20% of the biological activity compared to the unconjugated therapeutic protein. In some embodiments, the therapeutic protein conjugate retains at least 50% of the biological activity compared to the unconjugated therapeutic protein. In some embodiments, the therapeutic protein conjugate retains at least 90% of the biological activity compared to the native therapeutic protein.

[0240] In some embodiments, the therapeutic protein conjugate has an increased half-life relative to the unconjugated therapeutic protein. In some embodiments, the therapeutic protein conjugate has a half-life that is increased by at least 1.5-fold relative to the unconjugated therapeutic protein. In some embodiments, the therapeutic protein conjugate has a half-life that is increased by at least 5-fold relative to the unconjugated therapeutic protein.

[0241] In some embodiments, the zwitterionic polymer of the method of conjugating a therapeutic protein to a half-life extending moiety is a radically polymerizable monomer having a zwitterionic group, and the method comprises the additional step of polymerizing the free-radically polymerizable zwitterionic monomer in a polymerization medium to provide a polymer, the medium comprising the radically polymerizable zwitterionic monomer, M t is a transition metal, q is a higher oxidation state of the metal, and q-1 is a lower oxidation state of the metal; t (q-1)+ and X' is a counter ion or group. t (q-1)+ X' (q-1)or the inactive metal salt M in a higher oxidation state together with a reducing agent capable of reducing the transition metal from an oxidatively inactive state to a reduced, active state. t q+ X' q The transition metal catalyst, ligand, and initiator are provided in situ by providing

[0242] To function as an ATRP transition metal catalyst, the transition metal must have at least two readily accessible oxidation states separated by one electron, a higher oxidation state and a lower oxidation state. In ATRP, a reversible redox reaction cycles the transition metal catalyst between its higher and lower oxidation states, causing the polymer chain to cycle between having a growing chain end and a dormant chain end. See, e.g., U.S. Pat. No. 7,893,173.

[0243] In some embodiments, the radically polymerizable zwitterionic monomer is [ka] wherein R1 is H or C 1~6 is alkyl, ZW is a zwitterion, and n is an integer from 1 to 6.

[0244] In some embodiments, the radical polymerizable monomer is [ka] wherein R1 is H or C 1~6 alkyl, R2, R3, and R4 are the same or different, and H or C 1~4 alkyl, and X and Y are the same or different and are integers from 1 to 6. In some embodiments, R1, R2, R3, and R4 are each methyl, and X and Y are each 2.

[0245] In some embodiments, the radical polymerizable monomer is [ka] where R1 is H or C 1~6 alkyl, R2 and R3 are the same or different, and H or C 1~4 R is alkyl, R is PO, SO, or CO, and X and Y are the same or different and are integers from 1 to 6. In some embodiments, R, R, and R are methyl, R is PO, and X and Y are each 2.

[0246] In some embodiments, the monomer is [ka] where R1 is H or C 1~6 alkyl, R2, R3, and R4 are the same or different, and H or C 1~4 R is alkyl, R is PO, SO, or CO, and X and Y are the same or different and are integers from 1 to 6. In some embodiments, R, R, R, and R are methyl, R is PO, and X and Y are 2.

[0247] In some embodiments, the transition metal M t is selected from the group consisting of Cu, Fe, Ru, Cr, Mo, W, Mn, Rh, Re, Co, V, Zn, Au, and Ag. In some embodiments, the metal catalyst is M t (q-1)+ X' (q-1) It is supplied as a salt in the form M t (q-1)+ is Cu 1+ , Fe 2+ , Ru 2+ , Cr 2+ , Mo 2+ , W 2+ , Mn 3+ , Rh 3+,Re 2+ , Co + , V 2+ , Zn + , Au + , and Ag + X' is selected from the group consisting of halogen, C 1~6 Alkoxy, (SO4) 1 / 2 , (PO4) 1 / 3 , (R7PO4) 1 / 2 , (R72PO4), triflate, hexafluorophosphate, methanesulfonate, arylsulfonate, CN, and R7CO2, wherein R7 is a linear or branched C alkyl group optionally substituted 1 to 5 times with H or halogen. 1~6 In some embodiments, M t (q-1)+ is Cu 1+ and X' is Br.

[0248] In some embodiments, M t (q-1)+ is provided in situ. In some embodiments, M t q+ X q is CuBr2. In some embodiments, the reducing agent is an inorganic compound. In some embodiments, the reducing agent is selected from the group consisting of low oxidation level sulfur compounds, sodium bisulfite, inorganic salts containing metal ions, metals, hydrazine hydrate, and derivatives of such compounds. In some embodiments, the reducing agent is a metal. In some embodiments, the reducing agent is Cu 0 is.

[0249] In some embodiments, the reducing agent is an organic compound selected from the group consisting of alkylthiols, mercaptoethanol, or readily enolizable carbonyl compounds, ascorbic acid, acetylacetonate, camphorsulfonic acid, hydroxyacetone, reducing sugars, monosaccharides, glucose, aldehydes, and derivatives of such organic compounds.

[0250] In some embodiments, the ligand is 2,2'-bipyridine, 4,4'-di-5-nonyl-2,2'-bipyridine, 4,4-dinonyl-2,2'-dipyridyl, 4,4',4''-tris(5-nonyl)-2,2':6',2''-terpyridine, N,N,N',N',N''-pentamethyldiethylenetriamine, 1,1,4,7,10,10-hexamethyltriethylenetetramine, tris(2-dimethylaminoethyl)amine, N,N-bis(2-pyridylmethyl)octyl ... In some embodiments, the ligand is selected from the group consisting of tadecylamine, N,N,N',N'-tetra[(2-pyridal)methyl]ethylenediamine, tris[(2-pyridyl)methyl]amine, tris(2-aminoethyl)amine, tris(2-bis(3-butoxy-3-oxopropyl)aminoethyl)amine, tris(2-bis(3-(2-ethylhexoxy)-3-oxopropyl)aminoethyl)amine, and tris(2-bis(3-dodecoxy-3-oxopropyl)aminoethyl)amine. In some embodiments, the ligand is 2,2'-bipyridine.

[0251] In some embodiments, the initiator has the structure: [ka] wherein R1 is a nucleophilic reactive group, R2 comprises a linker, and R3 comprises a polymer synthesis initiator moiety having the structure: [ka] wherein R4 and R5 are the same or different and are selected from the group consisting of alkyl, substituted alkyl, alkylene, alkoxy, carboxyalkyl, haloalkyl, cycloalkyl, cyclic alkyl ether, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkylene, heterocycloalkyl, heterocycloalkylene, aryl, arylene, aryleneoxy, heteroaryl, amino, amido, or any combination thereof; Z is halogen or CN; and s is an integer between 1 and 20.

[0252] In some embodiments, Z is Br and R4 and R5 are each methyl. In some embodiments, R1 is selected from the group consisting of NH2-, OH-, and SH-.

[0253] In some embodiments, R2 is alkyl, substituted alkyl, alkylene, alkoxy, carboxyalkyl, haloalkyl, cycloalkyl, cyclic alkyl ether, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkylene, heterocycloalkyl, heterocycloalkylene, aryl, arylene, aryleneoxy, heteroaryl, amino, amido, or any combination thereof. In some embodiments, R2 is [ka] wherein X and Y are the same or different and are integers from 1 to 20. In some embodiments, X and Y are each 4.

[0254] In some embodiments, R3 is [ka] wherein R6, R7, and R8 are the same or different, and [ka] [ka] , and [ka] wherein Z is NCS, F, Cl, Br, or I. In some embodiments, Z is Br and R6, R7, and R8 are each selected from the group consisting of [ka] is.

[0255] In some embodiments, the initiator has the structure: [ka] wherein A and B are the same or different and are integers from 2 to 12, and Z is any halide, for example, Br. In some embodiments, A and B are each 4.

[0256] In some embodiments, the method further comprises reacting the polymer with a maleimide reagent to provide a polymer having maleimide termini. In some embodiments, the maleimide compound is [ka] is.

[0257] treatment method In some embodiments, methods are provided for treating or preventing ocular diseases, the methods comprising administering a therapeutic protein selected from the group consisting of anti-VEGF-A antibodies (and antibody conjugates). In some embodiments, any one or more of the antibodies or antibody conjugates provided herein can be used as a method for treating and / or preventing ocular diseases. The method comprises administering to a subject any one or more of the antibodies or antibody conjugates provided herein.

[0258] In some embodiments, methods are provided for treating or preventing ocular diseases, comprising administering to a subject in need thereof any of the antibodies and / or antibody conjugates described herein. In some embodiments, the disease can be age-related macular degeneration (AMD) or diabetic macular edema (DME). In some embodiments, the disease can be wet AMD.

[0259] In some embodiments, the ocular disease is selected from one or more of the group consisting of diabetic retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic macular edema (DME), pathologic myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), central branch retinal vein occlusion (BRVO), corneal neovascularization, retinal neovascularization, retinopathy of prematurity (ROP), subconjunctival hemorrhage, and hypertensive retinopathy. In some embodiments, the ocular disease is diabetic retinopathy.

[0260] In some embodiments, the antibody or antibody conjugate is administered no more frequently than once a month. In some embodiments, the antibody or antibody conjugate is administered twice a month or weekly. In some embodiments, the antibody or antibody conjugate is administered every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, or every twelve months.

[0261] In some embodiments, one or more of the compositions provided herein can reduce the high treatment burden associated with intravitreal injection of anti-VEGF agents for the treatment of wet (proliferative) age-related macular degeneration (AMD). Real-world results for patients with wet AMD lag behind the clinical outcomes demonstrated in Phase 3 clinical trials, such as the MARINA and ANCHOR trials for Lucentis (ranibizumab) and the VIEW 1 and VIEW 2 trials for Eylea (aflibercept). Anti-VEGF therapeutic agents with longer intraocular residence times, which can be administered less frequently and therefore with a more tolerable profile, can provide more patients with real-world results closer to Phase 3 clinical outcomes.

[0262] In some embodiments, compounds, including antibody conjugates and anti-VEGF-A antibodies, described herein are used to treat patients with underlying diabetic retinopathy or non-proliferative diabetic retinopathy, but with little or no vision loss. In some embodiments, such patients are dosed by intravitreal injection less frequently than once per month. In some embodiments, such patients are dosed six times per year. In some embodiments, such patients are dosed no more than four times per year. In some embodiments, the patient is dosed no more than three times per year. In some embodiments, the patient is dosed no more than twice per year. In some embodiments, the patient is dosed no more than once per year. In some embodiments, the subject receives the antibody or antibody conjugate by intravitreal injection.

[0263] The therapeutic proteins described herein (eg, both antibodies and antibody conjugates) can be used by in vivo expression of such polypeptides in a patient, ie, by gene therapy.

[0264] There are two major approaches to getting the nucleic acid (optionally contained in a vector) into a patient's cells: in vivo and ex vivo. For in vivo delivery, the nucleic acid is injected directly into the patient, usually at the site where the therapeutic protein is needed, i.e., where the biological activity of the therapeutic protein is required. For ex vivo therapy, the patient's cells are removed, the nucleic acid is introduced into these isolated cells, and the modified cells are administered to the patient directly or, for example, encapsulated in a porous membrane that is implanted into the patient (see, e.g., U.S. Patent No. 5,629,994). (See US Pat. Nos. 4,892,538 and 5,283,187). A variety of techniques are available for introducing nucleic acids into living cells. These techniques vary depending on whether the nucleic acid is transferred into cultured cells in vitro or into the cells of the intended host in vivo. Suitable techniques for transferring nucleic acids into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, transduction, cell fusion, DEAE-dextran, calcium phosphate precipitation, and the like. Transduction involves contacting a replication-defective recombinant viral (including retroviral) particle with a cellular receptor, followed by the transfer of the nucleic acid contained in the particle into that cell. A commonly used vector for ex vivo gene delivery is the retrovirus.

[0265] In some embodiments, the in vivo nucleic acid transfer techniques include transfection using viral or non-viral vectors (such as adenovirus, lentivirus, herpes simplex virus I, or adeno-associated virus (AAV)) and lipid-based systems (useful lipids for lipid-mediated gene transfer include DOTMA, DOPE, and DC-Chol; see, e.g., Tonkinison et al., Cancer Investigation, 14(1): 54-65 (1996)). In some embodiments, the vector used in gene therapy is a virus, including adenovirus, AAV, lentivirus, or retrovirus. Viral vectors, such as retroviral vectors, contain at least one transcriptional promoter / enhancer or locus-directing element, or other elements that control gene expression by other means, such as alternative splicing, nuclear RNA transport, or post-translational modification of messengers. Furthermore, viral vectors, such as retroviral vectors, contain a nucleic acid molecule that, when transcribed in the presence of a gene encoding the therapeutic protein, is operably linked to the gene and acts as a translation initiation sequence. Such vector constructs also contain a packaging signal appropriate for the virus used, a long terminal repeat (LTR) or portion thereof, and positive and negative strand primer binding sites (if not already present in the viral vector). Furthermore, such vectors typically contain a signal sequence for secretion of the PRO polypeptide from a host cell containing the vector. In some embodiments, the signal sequence for this purpose is a mammalian signal sequence. In some embodiments, the signal is a signal sequence native to the therapeutic protein. Optionally, the vector construct may also contain a signal directing polyadenylation, as well as one or more restriction sites and a translation termination sequence. By way of example, such vectors typically contain a 5' LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3' LTR or portion thereof.Other vectors that are non-viral, such as cationic lipids, polylysine, and dendrimers, are available.

[0266] In some situations, it is desirable to provide a nucleic acid source with a target cell targeting agent, such as a cell surface membrane protein or an antibody specific to those target cells, a ligand for a receptor on those target cells, etc. When using liposomes, for example, capsid proteins or fragments thereof that target specific cell types, antibodies against proteins that are internalized during the cell cycle, and proteins that bind to cell surface membrane proteins associated with endocytosis to target proteins to intracellular localization and extend their intracellular half-life, and / or to promote uptake, may be used. Receptor-mediated endocytosis techniques are described, for example, by Wu et al., J. Biol. Chem., 262: 4429-4432 (1987) and Wagner et al., Proc. Natl. Acad. Sci. USA, 87: 3410-3414 (1990). For a review of currently known gene marking and gene therapy protocols, see Anderson et al., Science, 256: 808-813 (1992). See also International Publication WO 93 / 25673 and the references cited therein.

[0267] Suitable gene therapy and methods for producing retroviral particles and structural proteins can be found, for example, in US Pat. No. 5,681,746.

[0268] In some embodiments, methods are provided for treating or preventing eye diseases in a mammal, wherein a nucleic acid molecule encoding a therapeutic protein selected from the group consisting of anti-VEGF-A antibodies is administered, and in some embodiments, the nucleic acid is depicted in Figure 27.

[0269] In some embodiments, the heavy chain is set forth in SEQ ID NO: 1 and the light chain is set forth in SEQ ID NO: 2. In some embodiments, the nucleic acid molecule is administered via ex vivo gene therapy.

[0270] Therapeutic proteins can be incorporated into pharmaceutical compositions with pharmaceutically acceptable excipients. Pharmaceutical compositions adapted for oral administration can be provided as discrete units such as capsules, solutions, syrups, or suspensions (in aqueous or non-aqueous liquids, or as edible foams or whips, or as emulsions). Suitable excipients for tablets or hard gelatin capsules include lactose, corn starch or derivatives thereof, stearic acid or salts thereof. Suitable excipients for use with soft gelatin capsules include, for example, vegetable oils, waxes, fats, semisolid polyols, or liquid polyols. For the preparation of solutions and syrups, excipients that can be used include, for example, water, polyols, and sugars. For the preparation of suspensions, oils (e.g., vegetable oils) can be used to provide oil-in-water or water-in-oil suspensions.

[0271] Pharmaceutical compositions can be adapted for nasal administration in which the excipient is a solid, including a coarse powder, e.g., having a particle size of 20-500 microns, that is administered by sniffing, i.e., rapid inhalation through the nasal passages from a container of the powder held close to the nose. Suitable compositions in which the excipient is a liquid for administration as a nasal spray or nasal drops include aqueous or oil solutions of the active ingredient. Pharmaceutical compositions adapted for administration by inhalation include the fine particle aerosols that can be generated by various types of metered-dose pressurized aerosols, nebulizers, or insufflators.

[0272] Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the osmotic pressure of the formulation substantially isotonic with that of the intended recipient's blood, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. Examples of excipients that may be used for injection solutions include water, alcohols, polyols, glycerin, and vegetable oils. The compositions may be provided in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition, requiring the addition of a sterile liquid carrier, such as water for injection, immediately before use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Pharmaceutical compositions may be substantially isotonic, meaning an osmotic pressure of about 250 to 400 mOsm / kg of water.

[0273] The pharmaceutical composition may include preservatives, solubilizers, stabilizers, humectants, emulsifiers, sweeteners, colorants, odorants, salts (substances may be provided in the form of pharmaceutically acceptable salts), buffers, coating agents, or antioxidants. The pharmaceutical composition may also include a therapeutically active agent in addition to the substance. The pharmaceutical composition may be used in combination with one or more pharmaceutically acceptable excipients. Such excipients may include, but are not limited to, saline, buffered saline (phosphate buffered saline), glucose, liposomes, water, glycerol, ethanol, and combinations thereof.

[0274] The antibodies and pharmaceutical compositions containing the antibodies can be administered in a regimen effective for treating or preventing disease in a patient, including, for example, oral, intravitreal, intravenous, subcutaneous, intramuscular, intraosseous, intranasal, topical, intraperitoneal, and intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration or routes, among others. The active agent for treatment or prophylaxis can be administered to an individual as an injectable composition, for example, as a sterile aqueous dispersion. In some embodiments, the agent is isotonic or substantially isotonic.

[0275] For administration to mammals, and particularly humans, the dosage of the active agent is from 0.01 mg / kg body weight, typically around 1 mg / kg. A physician can determine the actual dosage most appropriate for an individual, which will depend on factors such as the individual's age, weight, sex, and response, the disease or disorder being treated, and the age and condition of the individual being treated. The dosages listed above are representative of the average case. Of course, there may be instances where higher or lower dosages are merited. In some embodiments, the dosage may be 0.5 to 20 mg / eye, e.g., 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, or 19 mg.

[0276] This dosage can be repeated as frequently as needed (e.g., once a week, once every two weeks, once a month, once every two months, four times a year, twice a year, once a year). If side effects occur, the amount and / or frequency of the dosage can be reduced in accordance with normal clinical practice. In one embodiment, the pharmaceutical composition can be administered at a frequency of once a day to once every 30 days. In one embodiment, the pharmaceutical composition can be administered at a frequency of twice a 30 day period. In one embodiment, the pharmaceutical composition can be administered at a frequency of once a week.

[0277] The antibodies and pharmaceutical compositions may be used alone or in combination with other therapeutic compounds or molecules, such as anti-inflammatory drugs, analgesics, or antibiotics. Such administration with other compounds may be simultaneous, separate, or sequential. The components may be provided in the form of a kit, which may optionally include instructions.

[0278] The antibodies and pharmaceutical compositions disclosed herein can be used for the treatment or prevention of diseases, particularly the ocular diseases or conditions described herein.

[0279] When so used, the conjugates are typically formulated and administered in the form of eye drops and / or ointments for ocular, intraocular, and / or intravitreal injection, and / or juxtascleral injection, and / or subretinal injection, and / or sub-Tenon injection, and / or suprachoroidal injection, and / or subconjunctival administration, and / or topical administration. Such antibodies and compositions can be delivered intravitreally in a variety of ways, for example, as devices and / or depots that allow for sustained release of the compound into the vitreous, including those described in references such as "Intraocular Drug Delivery," Jaffe, Ashton, and Pearson, editors, Taylor & Francis (March 2006). In one example, the device can be in the form of a minipump and / or matrix and / or passive diffusion system and / or encapsulated cells that release the compound over an extended period of time ("Intraocular Drug Delivery," Jaffe, Ashton, and Pearson, editors, Taylor & Francis (March 2006)).

[0280] Formulations for ocular, intraocular, or intravitreal administration can be prepared by methods known in the art and using ingredients known in the art. A key requirement for effective treatment is adequate penetration into the eye. Unlike diseases of the anterior segment of the eye, where drugs can be delivered topically, retinal diseases require a more site-specific approach. Eye drops and ointments rarely penetrate deep into the eye, and the blood-ocular barrier prevents systemically administered drugs from penetrating into ocular tissues. Therefore, the drug delivery method of choice for treating retinal diseases such as AMD and CNV is usually direct intravitreal injection. Intravitreal injections are usually repeated at intervals that depend on the patient's condition and the properties and half-life of the drug being delivered.

[0281] Therapeutic antibodies and related conjugates are generally placed into a container having a sterile access port, for example, an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. Such compositions may also be supplied in the form of pre-filled syringes.

[0282] A "stable" formulation is one in which the protein therein or the protein conjugated to a polymer having a half-life extending moiety essentially retains its physical stability and / or chemical stability and / or biological activity upon storage. The term "stable" refers to a formulation that shows little or no signs of instability, including aggregation and / or deamidation. For example, the formulation provided may remain stable for at least two years when stored at a temperature of 5-8°C as indicated.

[0283] A variety of analytical techniques for measuring protein stability are available in the art, see, for example, Peptide and Protein Drug Delivery, 247-301 (Vincent Lee, ed., New York, NY, 1991) and Jones, 1993 Adv. Drug Delivery Rev. 10:29-90. Stability can be measured at a selected temperature for a selected period of time. In some embodiments, the formulations are stable upon storage for at least 6 months, 12 months, 12-18 months, or 2 years or more.

[0284] A protein, such as an antibody or fragment thereof, "retains its physical stability" when, in a pharmaceutical formulation, it shows no signs of aggregation, precipitation, deamidation, and / or denaturation when examined visually for color and clarity or as measured by UV light scattering or size exclusion chromatography.

[0285] A protein "retains its chemical stability" if, in a pharmaceutical formulation, there exists a chemical stability at a given time such that the protein is still considered to retain its biological activity. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Chemical alterations can include changes in size (e.g., clipping), which can be assessed using, for example, size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOFMS). Other types of chemical alterations include changes in charge (e.g., resulting from deamidation), which can be assessed by, for example, ion-exchange chromatography. An antibody "retains its biological activity" in a pharmaceutical formulation if, for example, the biological activity of the antibody at a given time, as determined by an antigen-binding assay, is within about 10% (within assay error) of the biological activity exhibited at the time the pharmaceutical formulation was prepared.

[0286] Protein polymer conjugates are conjugates in which the chemical bonds between the protein and the polymer are intact. A complex "retains its chemical stability" if, for example, its chemical bonds have not been hydrolyzed or otherwise broken. The protein portion of the complex maintains its chemical stability as described above.

[0287] The term "isotonic" means that the formulation of interest has essentially the same osmotic pressure as human blood or vitreous humor for intravitreal injection. Isotonic formulations generally have an osmotic pressure of up to about 250-400 mOsm. Isotonicity can be measured, for example, using a vapor pressure osmometer or a freezing point osmometer.

[0288] As used herein, "buffer" refers to a buffer solution that resists pH changes due to the action of its acid-base complex components. In some embodiments, the buffer has a pH of about 3.0 to about 8.0, e.g., about 4.5 to 8, or about pH 6 to about 7.5, or about 6.0 to about 7.0, or about 6.5 to 7.0, or about pH 7.0 to about 7.5, or about 7.1 to about 7.4. pHs at any point between the above ranges are also contemplated.

[0289] In some embodiments, "PBS" phosphate buffered saline, Tris-based buffers and histidine-based buffers are used.

[0290] In some embodiments, the PBS buffer is composed of at least NaHPO, KHPO, and NaCl, adjusted to provide an appropriate pH. In some embodiments, the buffer may contain other pharmaceutical excipients, such as KCl and other salts, surfactants, and / or preservatives, to provide a stable storage solution.

[0291] "Preservatives" are compounds that can be included in formulations to essentially inhibit bacterial activity within them, thus, for example, facilitating the production of multi-use formulations. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl alcohol, and benzyl alcohol, alkylparabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

[0292] In some embodiments, a formulation that is safe for human use or animal testing must have a sufficiently low level of endotoxin. "Endotoxin" is lipopolysaccharide (LPS) derived from the cell membrane of gram-negative bacteria. Endotoxin consists of a hydrophilic polysaccharide moiety covalently bound to a hydrophobic lipid moiety (lipid A). Raetz CR, Ulevitch RJ, Wright SD, Sibley CH, Ding A, Nathan CF. 1991. Gram-negative endotoxin: an extraordinary lipid with profound effects on eukaryotic signal transduction. FASEB J. 5(12):2652-2660. Lipid A is responsible for most of the biological activity of endotoxins, i.e., their toxicity. Endotoxins are released in large amounts when bacterial cells die, as well as during growth and division. Endotoxins are highly heat-stable and are not destroyed by standard sterilization conditions. Extreme heat or pH, e.g., 180-250°C and treatment with acids or bases greater than 0.1 M, must be used (Petsch D, Anspach F. 2000. Endotoxin removal from protein solutions. J Biotechnol. 76: 97-119). Needless to say, such conditions are extremely harmful to biological products.

[0293] In the biotechnology and pharmaceutical industries, endotoxins can be found in both product manufacturing processes and final products. Because bacteria can grow in nutrient-poor media, including water, saline, and buffers, endotoxins are ubiquitous unless precautions are taken. Injection of endotoxins into animals or humans can cause a wide variety of pathophysiological effects, including endotoxic shock, tissue damage, and even death. Ogikubo Y, Ogikubo Y, Norimatsu M, Noda K, Takahashi J, Inotsume M, Tsuchiya M, Tamura Y. 2004. Evaluation of the bacterial endotoxin test for quantifications of endotoxin contamination of porcine vaccines. Biologics 32:88-93.

[0294] Fever and shock are induced in mammals upon intravenous injection of low concentrations (1 ng / mL) of endotoxin (Fiske JM, Ross A, VanDerMeid RK, McMichael JC, Arumugham. 2001. Method for reducing endotoxin in Moraxella catarrhalis UspA2 protein preparations. J Chrom B. 753:269-278). The maximum endotoxin level for intravenous administration of pharmaceutical biologics is set by all pharmacopeias at 5 endotoxin units (EU) per kg of body weight per hour (Daneshiam M, Guenther A, Wendel A, Hartung T, Von Aulock S. 2006. In vitro pyrogen test for toxic or immunomodulatory drugs. J Immunol Method 313:169-175). EU is a measure of endotoxin bioactivity. For example, 100 pg of the standard endotoxin EC-5 and 120 pg of endotoxin from E. coli O111:B4 have an activity of 1 EU (Hirayama C, Sakata M. 2002. Chromatographic removal of endotoxin from protein solutions by polymer particles. J Chrom B 781:419-432). Meeting this threshold level has always been difficult in biological research and the pharmaceutical industry (Berthold W, Walter J. 1994. Protein Purification: Aspects of Processes for Pharmaceutical Products. Biologicals 22:135-150; Petsch D, Anspach FB. 2000. Endotoxin removal from protein solutions. J Biotech 76:97-119).

[0295] The presence of endotoxins in drugs delivered by intravitreal injection is of particular concern. Intravitreal injection of a drug (penicillin) was first performed by Rycroft in 1945. Rycroft BW. 1945. Penicillin and the control of deep intraocular infection. British J Ophthalmol 29 (2): 57-87. The vitreous is a chamber capable of introducing and maintaining high levels of drugs for relatively long periods of time. Drug concentrations achievable by intravitreal injection far exceed those that can be produced by local or systemic administration (e.g., intravenous administration).

[0296] One of the most dangerous complications that can occur from intravitreal injections is endophthalmitis. Endophthalmitis is divided into two categories: infectious endophthalmitis and sterile endophthalmitis. Infectious endophthalmitis is generally caused by bacteria, fungi, or parasites. Symptoms of infectious endophthalmitis include severe Symptoms include pain, loss of vision, and redness of the conjunctiva and underlying episclera. Infectious endophthalmitis requires urgent diagnosis and treatment. Possible treatments include intravitreal antibiotic injections and, in some cases, pars plana vitrectomy. Removal of the blind and painful eye is sometimes called enucleation. See, e.g., Christy NE, Sommer A. 1979. Antibiotic prophylaxis of postoperative endophthalmitis. Ann Ophthalmol 11 (8): 1261-1265.

[0297] Sterile endophthalmitis, in contrast, does not involve an infectious agent and can be defined as acute intraocular inflammation of the vitreous cavity that resolves without the need for intravitreal antibiotics and / or vitreoretinal surgery. If vitreous microbiological testing is performed, it must be proven negative to maintain the diagnosis of sterile endophthalmitis. Marticorena J, Romano V, Gomez-Ulla F. 2012 "Sterile Endophthalmitis after Intravitreal Injections" Med Inflam. 928123.

[0298] It has been observed that intravitreal injection of endotoxin-contaminated biologics can cause sterile endophthalmitis. Marticorena, et al. Bevacizumab (Avastin) is approved by the U.S. Food and Drug Administration for the treatment of glioblastoma, as well as metastatic colorectal cancer, advanced nonsquamous non-small cell lung cancer, and metastatic renal cancer. Bevacizumab is also widely used off-label for the treatment of wet AMD. Bevacizumab is supplied by the manufacturer as a 100 mg / 4 ml solution. This solution cannot be used directly for intravitreal injection; it must be compounded by a pharmacist. Clusters of cases of sterile endophthalmitis have been observed and have been explained as resulting from inadvertent endotoxin contamination of bevacizumab by compounding pharmacists.

[0299] Given the devastating clinical consequences of intravitreal endotoxin injection, the total amount of endotoxin that can be administered to a patient via intravitreal administration is very limited. In some embodiments, a solution is provided having an antibody or antibody conjugate with an endotoxin level not exceeding 5.0 EU / ml. In some embodiments, the endotoxin level does not exceed 1.0 EU / ml. In some embodiments, the endotoxin level does not exceed 0.5 EU / ml. In some embodiments, the endotoxin level does not exceed 0.2 EU / ml. In some embodiments, the endotoxin level does not exceed 2 EU / ml, 1 EU / ml, 0.5 EU / ml, 0.2 EU / ml, 0.1 EU / ml, 0.09 EU / ml, 0.08 EU / ml, 0.07 EU / ml, 0.06 EU / ml, 0.05 EU / ml, 0.04 EU / ml, 0.03 EU / ml, 0.02 EU / ml, or 0.01 EU / ml.

[0300] Two commonly used FDA-approved tests for the presence of endotoxin are the rabbit pyrogen test and the Limulus amebocyte extract (LAL) assay (Hoffman S, et al. 2005. International validation of novel pyrogen tests based on human monocytoid cells J. Immunol. Methods 298:161-173, Ding JL, Ho BA. 2001. New era in pyrogen testing. Biotech. 19:277-281). The rabbit pyrogen test was developed in the 1920s and involves monitoring the temperature rise in rabbits injected with a test solution. However, its use has declined significantly over the years due to its high cost and long turnaround time. Much more common is the LAL test. LAL is derived from the blood of the American horseshoe crab and clots when exposed to endotoxin.

[0301] One of the simplest LAL assays is the LAL gel clot assay. Essentially, the LAL clotting assay is combined with serial dilutions of the sample in question. Gel formation is proportional to the amount of endotoxin in the sample. Serial dilutions are prepared from the sample, and each dilution is assayed for its ability to form an LAL gel. A negative reaction is included at some point. The amount of endotoxin in the original sample can be estimated from the dilution assay.

[0302] Other LAL tests have also been developed, including the turbidimetric LAL assay (Ong KG, Lelan JM, Zeng KF, Barrett G, Aourob M, These include the turbidimetric and chromogenic LAL assays (Haishima Y, Hasegawa C, Yagami T, Tsuchiya T, Matsuda R, Hayashi Y. 2003. Estimation of uncertainty in kinetic-colorimetric assay of bacterial endotoxins. J Pharm Biomed Analysis. 32:495-503). Turbidimetric and chromogenic assays are much more sensitive and quantitative than simple gel clot dilution assays.

[0303] In some embodiments, provided herein are methods for reducing the amount of endotoxin in a composition comprising an antibody disclosed herein, the method comprising contacting the composition with an affinity chromatography resin that binds the antibody, eluting the antibody from the affinity chromatography resin to form an affinity chromatography eluate having the antagonist, contacting the affinity chromatography eluate with an ion exchange resin that binds the antibody, and eluting the antibody from the ion exchange resin, wherein the antibody eluted from the ion exchange resin is substantially free of endotoxin.

[0304] The above-described method for reducing endotoxin levels, or other methods or processes recited herein, can be performed in the order of the steps described above, or the steps can be performed in a different order as desired, or one or more of the steps can be repeated. In one embodiment, the method for reducing the amount of endotoxin in a composition is performed in the order of the steps described. In some embodiments, the contacting step with the affinity chromatography resin, washing step, and elution step are repeated more than once in the same order before contacting the affinity chromatography eluate with the ion exchange resin. The method can also include a filtration step, for example, using a 0.1 micron, 0.22 micron, or 0.44 micron filter, which can be performed on any one or more of the eluates removed after each resin binding step.

[0305] In certain instances, the steps of contacting the composition with an affinity chromatography resin, washing, and eluting the antibody from the affinity chromatography resin can be repeated more than once before contacting the initial eluate with an ion exchange resin. In one embodiment, the affinity chromatography resin comprises recombinant Protein A.

[0306] ("rProteinA") resin. One example of a suitable recombinant Protein A resin is rProteinA Sepharose FF® resin (Amersham, Piscataway, NJ). In another embodiment, a suitable affinity chromatograph is In one embodiment, the affinity chromatography resin will comprise a Protein G chromatography resin. In another embodiment, a suitable affinity chromatography resin comprises a Protein A / Protein G mixed resin. In another embodiment, a suitable affinity chromatography resin comprises a hydrophobic charge-inducing resin containing a 4-mercaptoethylpyridine ligand, such as MEP HyperCel® resin (BioSepra, Cergy-Saint-Christophe, France).

[0307] In some embodiments, the ion exchange resin comprises an anion exchange resin. As those skilled in the art know, ion exchangers can be based on a variety of materials, both in terms of the matrix and the attached charged groups. For example, the following matrices, in which the materials mentioned are generally soluble, may be used: MacroCap Q (GE Healthcare Biosciences, Piscataway, NJ), agarose-based matrices (such as Sepharose CL-6B®, Sepharose Fast Flow®, and Sepharose High Performance®), cellulose-based matrices (such as DEAE Sephacel®), dextran-based matrices (such as Sephadex®), silica-based matrices, and synthetic polymer-based matrices. For the anion exchange resin, the charged groups covalently attached to the matrix may be, for example, diethylaminoethyl, quaternary aminoethyl, and / or quaternary ammonium. In some embodiments, the anion exchange resin comprises quaternary amine groups. An exemplary anion exchange resin having quaternary amine groups for binding the anti-M-CSF antibody is Q Sepharose® resin (Amersham, Piscataway, NJ).

[0308] In other aspects, if endotoxin levels are higher than desired after subjecting the composition to the aforementioned anion exchange chromatography step, the composition may alternatively be subjected to a cation exchange resin. In some embodiments, any endotoxin in the composition should have a binding to the ion exchange resin that is unique to the protein, allowing for purification of the protein of interest from the endotoxin. In this regard, endotoxins are negatively charged and generally bind to anion exchange resins. If both the protein and the endotoxin bind to anion exchange resins, purification of one from the other can be achieved by using a salt gradient to elute the two in different fractions. The relative binding of the protein to a particular resin can also be achieved by altering the pH of the buffer relative to the pI of the protein. In some embodiments, cation exchange chromatography is the only ion exchange chromatography employed.

[0309] In some embodiments, if endotoxin levels are too high after the anion exchange resin, the composition can be further subjected to a second ion exchange step, for example, by contacting the composition with a cation exchange resin, followed by a washing step, and then elution from the ion exchange resin. In some embodiments, the cation exchange resin contains sulfonic groups for binding. Exemplary cation exchange resins are SP Sepharose® resin FF (Amersham, Piscataway, NJ) and Poros XS (CEX) (Life Technologies, Grand Island, NY).

[0310] In some embodiments, after producing the antibody protein solution with a specified level of endotoxin, there are multiple steps before final formulation of the protein. In some embodiments, a half-life extending moiety is conjugated to the protein. The conjugate is then formulated into the final formulation that is injected into the patient. In some embodiments, the conjugate is purified again with an ion exchange resin, which may be a cation exchange resin. In other embodiments, the protein is formulated In all cases, routine testing should be employed to prevent endotoxin contamination of the protein sample or the protein polymer complex. [Example]

[0311] Example 1. Route 1: Synthesis of OG1802 The first route for the synthesis of OG1802 is as follows: First, a TFA / amine salt initiator (compound L) having the structure shown in Figure 1 was synthesized as follows.

[0312] First, compound K, having the structure shown in Figure 2, was synthesized as follows: Compound J (OG1563) (1.9 g, 2.67 mmol, 3.3 equiv.) was placed in a 200 mL round-bottom flask under nitrogen. [ka] and compound E (0.525 g, 0.81 mmol, 1.0 equiv) (see Figure 11), followed by dimethylformamide (10 mL), followed by diisopropylethylamine (2.5 mL, 14.6 mmol, 18 equiv). The flask was cooled to 0°C using an ice bath. To this was added propylphosphonic anhydride solution (50 wt% in ethyl acetate, 2.5 mL, 4.04 mmol, 5 equiv) over approximately 6 minutes.

[0313] The reaction was allowed to warm to room temperature and stirred for 15 minutes. The reaction was quenched by the addition of water (20 mL), saturated aqueous sodium bicarbonate (20 mL), and ethyl acetate (100 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (75 mL). The combined organic layers were washed with saturated aqueous sodium bicarbonate (30 mL), 0.5 M aqueous citric acid (40 mL), water (25 mL), and saturated aqueous sodium chloride (40 mL), then dried (sodium sulfate), filtered, and concentrated in vacuo. The residue yielded 2.0 g (0.80 mmol, 99%) of compound K, which was used without further purification. 1H NMR (400 MHz DMSO-d6): δ = 1.36 (s, 9H, OC CH3 ), 1.90 (s, 54H, CC (CH3)2 Br), 2.31 (t, J = 7.2 Hz, 6H, C CH2 CH2NH), 2.98 (d, J = 5.6 Hz, 6H, C CH2 NH), 3.04 (q, J = 6.0 Hz, 2H, OCH2 CH2 NH), 3.18 (s, 2H, O CH2 C), 3.3-3.37 (m, 8H, CH2 ), 3.47-3.55 (m, 12H, CH2 ), 3.58 (s, 6H, O CH2 C), 3.87 (s, 6H, O=C CH2 O), 4.27 (s, 18H, C CH2 OC=O), 6.74 (br t, 1H, CH2 NH C=O), 7.69 (t, J = 6.8 Hz, 3H, CH2 NH C=O), 7.84 (t, J = 6.0 Hz, 3H, CH2 NH C=O). LC-MS (ES, m / z): calculated for [(M+2H-boc) / 2]+ (C84H136Br9N7O33+2H-Boc) / 2: 1196.6; found: 1196.6.

[0314] Compound L (Figure 1) was then synthesized as follows. To a 100 mL round-bottom flask under nitrogen was added compound K (2.0 g, 0.8 mmol), dichloromethane (10 mL), followed by trifluoroacetic acid (5 mL). The reaction was stirred at room temperature for 30 minutes. The reaction was concentrated under vacuum. Dichloromethane (10 mL) was used to dilute the reaction, which was then concentrated under vacuum. The residue was dissolved using acetonitrile (10 mL), filtered through a syringe filter (Acrodisc CR25, PN 4225T), loaded onto a preparative HPLC column, and eluted with 60% aqueous acetonitrile (containing 0.1% trifluoroacetic acid) up to 98% aqueous acetonitrile (containing 0.1% trifluoroacetic acid). Tubes containing the product were pooled, concentrated under vacuum, frozen, and placed on a lyophilizer. This yielded 990 mg (0.4 mmol, 50% over two steps) of compound L as a white powder. 1H NMR (400 MHz DMSO-d6): δ = 1.90 (s, 54H, CC (CH3)2 Br), 2.31 (t, J = 7.2 Hz, 6H, C CH2 CH2NH), 2.97-3.0 (m, 8H, C CH2 NH and OCH2 CH2 NH), 3.17 (s, 2H, O CH2 C), 3.3 (q, 6H, CH2 CH2 NHC=O), 3.4-3.59 (m, 20H, CH2 ), 3.87 (s, 6H, O=C CH2 O), 4.27 (s, 18H, C CH2 OC=O), 7.69-7.84 (m, 9H, CH2 NH C=O and NH3 + both). LC-MS (ES, m / z): calculated for [(M+2H) / 2]+ (C84H136Br9N7O33+2H) / 2: = 1196.6; found: 1197.4.

[0315] Next, MPC polymer was synthesized using compound L as the initiator. The initiator is typically prepared as a stock solution of approximately 100 mg / mL in DMF. The initiator and ligand (2,2'-bipyridyl) were placed in a Schlenk tube. The resulting solution was cooled to -78 °C using a dry ice / acetone mixture and degassed under vacuum for 10 minutes. The tube was refilled under argon, and the catalyst (CuBr unless otherwise noted) kept under argon was placed in the Schlenk tube (the molar ratio of initiator / catalyst (CuBr) / bromine atoms on the ligand was maintained at 1 / 1 / 2). The solution immediately turned dark brown. The Schlenk tube was sealed and quickly purged by applying short vacuum / argon cycles. A HEMA-PC solution was prepared by mixing a predetermined amount of monomer, prepared in a glovebox under nitrogen, with 200-proof degassed ethanol. The monomer solution was added dropwise (via cannula) to the Schlenk tube (and homogenized by gentle stirring). The temperature was maintained at -78°C. Full vacuum was applied to the reaction mixture for at least 10-15 minutes until bubbling from the solution ceased. The tube was then backfilled with argon and allowed to warm to room temperature. The solution was stirred, and as polymerization proceeded, the solution became viscous. After 3-8 hours, or simply overnight, the reaction was quenched by direct exposure to air to oxidize Cu(I) to Cu(II), turning the mixture blue-green in color, and the mixture was passed through a silica column to remove the copper catalyst. The collected solution was concentrated by rotary evaporation, and the resulting mixture was either precipitated with tetrahydrofuran or dialyzed against water, followed by lyophilization to yield a free-flowing white powder. Table 1.1 below shows the polymer data for polymers using Compound L as the initiator. [Table 1]

[0316] Next, the maleimide Mal-PEG4-PFP ester was conjugated to the 750 kDa polymer mentioned above (as shown in Figure 29) to provide OG1802. Polymer R3707 (515 mg of the 750 kDa polymer prepared using L as the initiator) was placed in a 20 mL vial and dissolved in ethanol (4.0 mL) after stirring for 40 minutes. To this was added 22 μL of a 1% 4-methylmorpholine solution in acetonitrile. In a separate vial, Mal-PEG4-PFP (1.97 mg) was dissolved in acetonitrile (1.0 mL), and this solution was added to the polymer solution at room temperature over approximately 2 minutes. The resulting solution was stirred overnight. The reaction mixture was diluted with 2 mL of 0.1% aqueous trifluoroacetic acid (pH ∼5), followed by ∼12 mL of water, filtered through a syringe filter (Acrodisc Supor, PN4612), and evenly distributed across three Amicon centrifugal membrane dialysis tubes (30,000 mwco). The tubes were diluted and mixed with ∼5 mL of water each and centrifuged at 3200 rpm for 25 minutes. The filtrate was removed for analysis, while the retentate was diluted and mixed with ∼10 mL of water per tube. The centrifugation procedure was repeated five more times, after which the retentate was removed and placed in a vial. The Amicon membrane tubes were washed with water (2 x ∼2 mL per tube), which was then mixed with the retentate. The retentate solution was filtered through a syringe filter (Acrodisc Supor, PN4612), frozen, and placed on a freeze dryer. This yielded 485 mg of a white powder.

[0317] Example 2. Synthesis of initiator OG1786 OG1786 is a nine-arm initiator for polymer synthesis used as a precursor for the synthesis of OG1802. Each arm is capped with 2-bromoisobutyrate, which can initiate polymerization by ATRP. OG1786 is a trifluoroacetic acid (TFA) salt, as shown in Figure 30. OG1786 is prepared as follows: First, OG1550 is reacted with TFA (trifluoroacetic acid) to produce OG1546, as shown in Figure 31.

[0318] Into a 1 L round-bottom flask equipped with a magnetic stir bar and dropping funnel was added OG1550 (14.8 g), methyl tert-butyl ether (MTBE) (350 ml), and water (30 ml). The mixture was stirred to dissolve the OG1550 and then cooled in an ice bath. To this mixture was added a solution of trifluoroacetic acid (4.9 ml) in water (90 ml) dropwise over 90 minutes. After the addition was complete, the mixture was stirred for an additional 15 minutes, then removed from the ice bath and allowed to warm to room temperature. The mixture was stirred for an additional 4-5 hours (after removal from the ice bath) until TLC indicated that approximately 5% starting material remained and the pH of the aqueous solution was between 3 and 4 (pH paper).

[0319] The mixture was partitioned. The MTBE layer was washed with water (30 ml). After combining the aqueous layers, the aqueous layers were extracted with MTBE (150 ml). This second MTBE phase was washed with water (30 ml). The combined aqueous layers were washed a third time with MTBE (100 ml). This third The MBTE phase was washed with water (25 ml) and the aqueous layers were mixed again (approximately 250 ml, pH approximately 4 according to pH paper).

[0320] The product was collected by lyophilization. 11.5 g of a white solid was obtained. This material is extremely hygroscopic and is best handled under nitrogen. The product was confirmed by LCMS.

[0321] The prepared OG1546 was then reacted with OG1563 (as shown in Figure 32) to give OG1784.

[0322] In a 250 ml flask equipped with a stir bar and under nitrogen, OG1546 (hygroscopic, 9.0 g) was added, followed by N,N-dimethylformamide (110 ml). The mixture was stirred at room temperature until all of OG1546 was dissolved (approximately 15 min). OG1563 (29.9 g) was then added, and the mixture was stirred for an additional 3 min until the OG1563 was also dissolved. The resulting solution was cooled in an ice bath, and N,N-diisopropylethylamine (37.6 ml) was added over 3 min, followed by the dropwise addition of 50% propylphosphonic anhydride (T3P) in ethyl acetate (34.5 ml) over 5 min (the addition of T3P is exothermic). After the T3P addition was complete, the flask was removed from the cooling bath and allowed to reach room temperature. Samples were then taken at 5 min intervals for LCMS analysis. The reaction exhibited a very light yellow / tan color.

[0323] After 20 minutes, the reaction was cooled again in an ice bath, and 5 ml of water was added. The mixture was then removed from the cooling bath, and an additional 50 ml of water was added, followed by 50 ml of 0.5 M citric acid and then isopropyl acetate (300 ml). The mixture was partitioned. The aqueous phase (approximately 300 ml) was extracted with additional isopropyl acetate (150 mL). The aqueous phase was identified as AQ1 in the HPLC assay. The combined organics were washed with aqueous citric acid (115 ml, 65 mM, which was a mixture of 15 ml of 0.5 M citric acid and 100 ml of water). The aqueous phase was identified as AQ2 (pH approximately 3). The organic phase was washed with water / saturated sodium chloride (100 ml / 25 ml). The aqueous phase was identified as AQ3 (pH approximately 3). The organic phase was finally washed with saturated sodium chloride (100 ml). The aqueous phase was identified as AQ4. None of the AQ fractions contained any product (data not shown). LCMS showed product in the organic phase. The product was dried over sodium sulfate (80 g), filtered, washed with isopropyl acetate (75 mL), and concentrated on a rotary evaporator to a tan oil (33.2 g). The crude product was stored under nitrogen overnight.

[0324] The next day, the crude product was allowed to reach room temperature and then dissolved in acetonitrile / water (46 ml / 12 ml) and filtered using an HPLC filter disk (Cole-Parmer PTFE 0.2 μm, product number 02915-20). The filtrate was divided into three equal portions and purified in three runs.

[0325] The filtrate was loaded onto a RediSep Rf Gold C18 column (275 g, SN69-2203-339, lot number 24126-611Y) equilibrated with 50% acetonitrile / water. The material was eluted at 100 ml / min using the following gradient (solvent A: water, solvent B: acetonitrile). All appropriate fractions were checked by HPLC. Fractions deemed sufficiently pure (from all three runs) were pooled and concentrated on a rotary evaporator (water bath temperature maintained at approximately 20°C), followed by partitioning between dichloromethane (100 ml) and water (5 ml) / saturated sodium chloride (25 ml). The aqueous phase was extracted twice more with dichloromethane (2 x 30 ml). The combined organics were dried over sodium sulfate (35 g), filtered, washed with DCM (30 ml), and concentrated. Product identity and purity were confirmed by LCMS. The isolated yield and purity of the R5172 and R5228 lots are shown in Table 2.1. [Table 2]

[0326] Next, OG1405 was prepared from OG1784 as shown in Figure 33. In a 500 ml round-bottom flask equipped with a magnetic stir bar, OG1784 (20.9 g) was added, followed by dichloromethane (50 ml) and then trifluoroacetic acid (20 ml). The mixture was stirred at room temperature, and complete deprotection was indicated by HPLC analysis within 23 minutes. The mixture was concentrated on a rotary evaporator, redissolved in dichloromethane (25 ml), reconcentrated, then redissolved in acetonitrile (25 ml), and reconcentrated. The product was confirmed by LCMS. The above material (OG1405, 34.5 g, assuming a quantitative yield of 21.0 g) was used as a crude oil in the next step. No purification was necessary.

[0327] Next, OG1405 was reacted with OG1402 to prepare OG1785, as shown in Figure 34. OG1402 (5.5 g) was placed in a 500 ml flask under nitrogen and equipped with a stir bar, followed by acetonitrile (70 ml), followed by N,N-diisopropylethylamine (26.3 ml) and T3P solution (see above) (7.9 ml). The solution was stirred at room temperature for 30 minutes, then cooled in an ice-water bath, and a solution of OG1405 (the crude oil from above, 34.5 g) in acetonitrile (70 ml) was added. The mixture was allowed to warm to room temperature. After 20 minutes, the reaction was cooled in an ice-water bath and quenched with water (5 ml). The mixture was then concentrated to half its volume under vacuum using a rotary evaporator. A sample was taken for LCMS.

[0328] Further water (50 mL) was added, followed by 0.5 M citric acid (75 mL) and isopropyl acetate (175 mL). The mixture was partitioned over 5 minutes. The aqueous phase was extracted with additional isopropyl acetate (50 mL). The combined organics were washed with aqueous citric acid (0.13 M, 30 mL, consisting of 10 mL of 0.5 M citric acid and 20 mL of water). The organics were then washed with a mixture of saturated sodium chloride (25 mL) and water (25 mL), followed by a final wash with saturated sodium chloride (25 mL). The organics were then dried over sodium sulfate (124 g), filtered, washed with isopropyl acetate (30 mL), and concentrated on a rotary evaporator to a tan oil (27.3 g). A sample was taken for LCMS analysis.

[0329] The oil was dissolved in acetonitrile / water (3:1, 15 ml / 5 ml), filtered through HPLC filter discs (Cole-Parmer PTFE membrane 0.2 μm, product number 02915-20), and divided into three equal portions, each of which was individually purified as follows.

[0330] The aliquot was loaded onto a Redi-Sep Gold C18 column (275 g, SN-69-2203-339, lot 241234-611W) equilibrated with 50% solvent B (acetonitrile) / 50% solvent A (water). The material was then purified by reverse-phase HPLC using a gradient of solvent A:water / solvent B:acetonitrile. Appropriate fractions were pooled. The residue was partitioned between dichloromethane (150 mL) and water (5 mL) / saturated sodium chloride (25 mL). The aqueous phase was extracted twice with dichloromethane (2 x 50 mL). The combined organics were dried over sodium sulfate (60 g), filtered, washed with dichloromethane (40 mL), and concentrated. Various analytical methods, including LCMS, confirmed the structure and purity. OG1785 was isolated as a foaming solid (R5329, 19.0 g, 83% yield, 95.1% purity (absorbance 210 nm) and stored under nitrogen at 4 °C.

[0331] The tert-butyloxycarbonyl protecting group on OG1785 was then removed using trifluoroacetic acid (TFA) to generate OG1786, as shown in FIG.

[0332] Example 3. Synthesis of polymer OG1801 Polymer OG1801 is first prepared from initiator OG1786. OG1801 is an amine functional group, which is more stable (than maleimide) during polymer synthesis. To synthesize polymer OG1801, a modified ATRP was used in which copper species (Cu(I)) are generated in situ by adding metallic copper to Cu(II). The starting materials and reagents required for the reaction are calculated based on the batch input of monomer (HEMA-PC) OG47 and the target molecular weight (MW).

[0333] In a glovebox, 50 g of monomer OG47 was weighed and 200 mL of degassed EtOH was added to dissolve the monomer at room temperature. A sample was then taken for monomer concentration testing. Cu(II), Bpy, and Cu(0) were weighed into a 500 mL flask and purged with argon. The monomer solution was then added to the flask, the flask was sealed with a stopper, and vacuum was applied for 25 minutes until all bubbles disappeared. The reaction gradually changed color from light green to dark green, and then to light brown. In a glovebox, approximately 200 mg of initiator OG1786 was weighed and dissolved in approximately 2000 μL of DMF at room temperature to make a 100 mg / mL stock solution. A sample was taken for initiator concentration and purity testing, and the initiator solution was added to the flask under argon. The reaction solution turned dark brown and became viscous over time. The system was sealed and allowed to react for two days.

[0334] OG1801 was then prepared for the addition of the maleimide and the catalyst (copper) was removed as follows: A pre-packed RediSep® Rf normal phase silica column was used to remove the catalyst. The column size was selected based on the amount of copper in the reaction mixture. For example, a 330 g column (catalog number 69-2203-330, column size 330 g, CV=443 mL) was used for a 50 g batch of OG1801. Teflon tubing was used for all connections because EtOH was the elution solvent.

[0335] After copper removal, all fractions were transferred to a round-bottom flask in each batch, and the EtOH was evaporated to dryness on a rotary evaporator at 45-50°C under reduced pressure. The volume of EtOH collected during this step was monitored to ensure that the EtOH was removed by more than 90%. The polymer was dissolved in 250 mL of WFI and filtered using a 0.2 μm filter, resulting in a clear to light yellow polymer solution at a concentration of approximately 150 mg / mL. The solution could be stored at 2-8°C for up to 3 months before use.

[0336] Example 4. Synthesis of polymer OG1802 The starting materials and reagents required for the reaction are calculated based on the batch input of OG1801. The linker is 3-maleimidopropionic acid NHS ester. 30 ml of 0.5 M sodium phosphate (in WFI, pH 8) was added to 50 g of polymer solution (approximately 150 mg / mL). Stirring was performed for 1 minute. The pH was measured by pH paper and found to be 8.0. 204.8 mg of linker was weighed and dissolved in 4.1 mL of DMF to create a 50 mg / mL stock solution. The linker solution was added dropwise at 815 μL per minute to the polymer solution with vigorous stirring. 4095 μL of linker solution was added over 5 minutes. The reaction was allowed to proceed at room temperature for 30 minutes. The reaction was quenched with 20 mL of 5% acetic acid to achieve a final pH of 5. The solution was filtered using a 1 L vacuum filter (0.2 μm).

[0337] OG1802 (see Figure 36) was then purified as follows. A Millipore crossflow cassette was used for aqueous polymer purification. The polymer solution was concentrated to 250 mL (approximately 200 mg / mL). Fresh WFI was added from the reservoir, and the flow rate of the fresh WFI was adjusted to the same as the permeate (approximately 2 mL / min). UF / DF was performed overnight at 2-8°C. Typically, 2.5 L of WFI was used (a 10-fold volume ratio relative to the polymer solution). A retentate sample was collected for purity testing. The target purity was >98%. The polymer solution was filtered through a 1 L 0.2 μm filter bottle. The polymer solution could be stored at 2-8°C for up to 3 months before conjugation.

[0338] Example 5. Additional phosphorylcholine polymers HEA-PC polymer was synthesized as described below. HEA-PC (2-(acryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate) is an acrylate, in contrast to the methacrylate HEMA-PC described above, and has the following structure: [ka]

[0339] HEA-PC was polymerized with the initiator shown in Example 1 as Compound L. [Table 3]

[0340] A 200 mg / mL initiator stock solution was prepared by dissolving 2.2 mg of initiator in 11 μL of dry DMF, and a 200 mg / mL ligand solution was prepared by dissolving 4.6 mg of Me6TREN in 23 μL of dry DMF. 8.25 μL of the initiator stock solution and 13.6 μL of the ligand were dispensed into a tube. The tube was degassed at -78°C for 5 minutes, refilled with argon, and 1.2 mg of CuBr was added. The tube was degassed and refilled with argon. A stock solution of HEA-PC in methanol (0.461 g of HEA-PC was weighed and dissolved in 0.5 mL of methanol) was added to the reaction vessel at -78°C. The solution was added to the vial. The vial was rinsed with 200 μL of methanol, and the solution in the -78°C reaction vessel was added, followed by 0.5 mL of distilled water, followed by another 200 μL of water. The reaction was thoroughly degassed until no more bubbling was observed and all heterogeneity had disappeared (solid particles had dissolved or disappeared). The reaction was backfilled with 4 psi of argon and allowed to proceed at room temperature for 1 hour. The reaction was already viscous. The reaction was allowed to proceed for approximately 1 hour. A solution of bipyridine in methanol (5 mg in 0.5 μL) was added. Another 2-3 mL of methanol was added, and the catalyst was allowed to oxidize overnight at 4°C. The conversion, as determined by 1H NMR, was estimated to be 94%.

[0341] The next day, the polymer was dialyzed and subjected to SEC / MALS analysis using a Shodex SB806M_HQ column (7.8 × 300 mm) in 1 × PBS (pH 7.4) at a flow rate of 1 ml / min, yielding a PDI of 1.157, Mn of 723.5 kDa, Mp of 820.4 kDa, and Mw of 837.2 kDa (before dialysis, the PDI was 1.12, Mn was 695 kDa, and Mp was 778 kDa). Maleimide functional groups were then added to the polymer to enable it to be conjugated to proteins.

[0342] Next, a maleimide Mal-PEG4-PFP ester (see Example 1 above) was conjugated to the HEA-PC polymer as described in Example 1. The resulting maleimide-functionalized HEA-PC polymer can then be conjugated to sulfhydryl groups as discussed herein for HEMA-PC polymers.

[0343] Acrylamide-PC polymers were also prepared using the monomer 2-(acrylamyl)ethyl-2-(trimethylammonium)ethyl phosphate (Am-PC) having the structure: [ka]

[0344] The Am-PC was used in polymerizations using a three-arm initiator (TFA salt) having the following structure: [ka]

[0345] The synthesis of Am-PC polymer was carried out as follows. [Table 4]

[0346] A 200 mg / mL stock solution of the ligand was prepared by dissolving 9 mg of Me6TREN in 45 μL of dry DMF. Add 19.7 μL of the stock solution to the reaction vessel. A 200 mg / mL stock solution of the initiator was prepared by dissolving 6.5 mg of material in 32.5 μL of DMF. Add 11 μL of the initiator stock solution to the ligand. Degas for 5 minutes. Add 1 mg of CuBr. A 200 mg / mL stock solution of CuBr2 was prepared by dissolving 4 mg of CuBr2 in 20 μL of DMF. Add 0.5 g of the monomer (AmPC) to 1 mL of methanol (slow dissolution / viscous solution), followed by 1 μL of the CuBr2 stock solution. Add the monomer solution dropwise to the reaction mixture. Wash with 1 mL of water. The reaction mixture is thoroughly degassed (freeze-thawed) and the reaction is allowed to proceed for 24 hours.

[0347] The Am-PC polymer may then be dialyzed. The molecular weight of the polymer was determined by SEC / MALS: Mn 215 kDa, Mp 250 kDa, and PDI 1.17. Conversion was estimated to be 94% by 1H NMR. Maleimide functional groups can be added to the Am-PC polymer as discussed above for HEMA-PC and HEA-PC. The maleimide-functionalized Am-PC polymer can be conjugated to proteins as described above.

[0348] Example 6. Reverse Ellman assay to calculate free maleimide in compounds After addition of maleimide functional groups to polymer OG1801 to form OG1802 (see above), the amount of functional (i.e., conjugable) maleimide in the sample was determined using an Ellman's assay. Thiols convert Ellman's reagent (DTNB) to TNB and then to TNB2 in water at neutral and alkaline pH, which produces a yellow color (measured at 412 nm). A calibration curve was established using cysteine. Because maleimides react with thiols, this assay actually measures the remaining thiol (cysteine). The inhibition rate was calculated as (thiol remaining after initial thiol-maleimide polymer addition) / (initial thiol) and is expressed as a percentage.

[0349] Reagents used in the assay: A standard curve was generated using cysteine ​​ranging from 62.5 μM to 2 μM. Polymer stock solutions were prepared by dissolving the powder in 1×PBS (reaction buffer) at pH 7.4 and mixing thoroughly. Equimolar polymer and cysteine ​​solutions were mixed and reacted for 30 minutes at 27°C. 150 μM DTNB solution was added to the cysteine ​​standards and polymer / cysteine ​​reactions and color was developed for 5 minutes at 27°C. OD was read at 412 nm on a Spectramax plate reader and the Soft Percent inhibition was calculated using max Pro software and the cysteine ​​standard curve.

[0350] Example 7. Protein sequence of an antibody (OG1950) comprising an anti-VEGF-A antibody heavy chain and an anti-VEGFA antibody light chain with an L443C (EU numbering, or 449C in SEQ ID NO: 1) mutation An anti-VEGF-A antibody containing the L443C (EU numbering) mutation was cloned having the sequence shown below in SEQ ID NO:1 (Figure 12) (heavy chain).An anti-VEGF-A antibody light chain was cloned having the sequence shown below in SEQ ID NO:2 (Figure 13).

[0351] Example 8a. Purification and decap of OG1950 The heavy and light chains of OG1950 may be cloned into expression plasmids and transfected into CHO cells. The cells may be cultured in an appropriate medium and harvested. OG1950 may be purified using the techniques described above. The cysteine ​​residue at position 443 of OG1950 (L443C (EU numbering)) is typically "capped" or oxidized by chemicals in the cell culture medium and is unavailable for conjugation. In this regard, purified OG1950 may be subjected to a decap (i.e., reduction) procedure to remove the cap, allowing the free (i.e., not involved in a Cys-Cys disulfide bond) cysteine ​​residue to be conjugated to a maleimide functional group on a polymer. Decap (decap) may be performed by mixing purified OG1950 with a 30-fold molar excess of the reducing agent TCEP (3,3',3''-phosphantriyltripropanoic acid) for 1 hour at 25°C. The reduction reaction using TCEP can be monitored by SDS-PAGE. After denaturation, the OG1950 protein can be washed by UFdF using a Pellion XL ultrafiltration cassette and a buffer consisting of 20 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM TCEP to remove the cap. The TCEP reagent can then be removed in the same UFdF setup containing 20 mM Tris pH 7.5, 150 mM NaCl. The reduced OG1950 can then be refolded using air (ambient oxygen), again followed by SDS-PAGE as an assay.

[0352] Example 8b. Purification and decap of OG1950 The heavy and light chains of OG1950 may be cloned into expression plasmids and transfected into CHO cells. The cells may be cultured in an appropriate medium and harvested. OG1950 may be purified using the techniques described above. The cysteine ​​residue at position 443 of OG1950 (L443C (EU numbering)) is typically "capped" or oxidized by chemicals in the cell culture medium and is unavailable for conjugation. In this regard, purified OG1950 may be subjected to a decap (i.e., reduction) procedure to remove the cap, allowing the free (i.e., not involved in a Cys-Cys disulfide bond) cysteine ​​residue to be conjugated to a maleimide functional group on a polymer. Decap (decap) may be performed by mixing purified OG1950 with a 30-fold molar excess of the reducing agent TCEP (3,3',3''-phosphantriyltripropanoic acid) for 1 hour at 25°C. The reduction reaction using TCEP can be monitored by SDS-PAGE. After reduction, the OG1950 protein can be washed using an ultrafiltration / diafiltration (UF / DF) system using a Millipore Pellicon XL ultrafiltration cassette with a 30 kDa MWCO membrane and a buffer consisting of 20 mM Tris pH 7.5, 150 mM NaCl, and 0.5 mM TCEP to remove the cap and excess TCEP. The remaining TCEP reagent can then be removed in the same UF / DF setup containing 20 mM Tris pH 7.5, 150 mM NaCl. Reduced OG1950 can then be reoxidized using dHAA for 1 hour at ambient temperature, followed by UF / DF to remove dHAA, forming decapped OG1950. The decapped state is monitored by SDS-PAGE assay.

[0353] Example 9. Conjugation of OG1950 to MPC polymer Decapped OG1950 may be conjugated to polymeric OG1802. An excess of OG1802 is used (10-20 fold molar excess). Conjugation can be monitored by SDS-PAGE, and the reaction can be run to near complete conjugation. The OG1950 conjugate can be purified by cation exchange chromatography and buffer exchanged into the formulation buffer by UF / DF. The polymeric OG1950 conjugate can be purified by chromatography as described above.

[0354] Example 10. SPR binding kinetics of OG1950 This example illustrates the binding of OG1950 to VEGF-165 in a single-cycle kinetic BIAcore™ experiment.

[0355] SPR interaction analysis of OG1950 with human VEGF-165 was performed on a BIAcore™ T200 system (GE Healthcare) equipped with a Protein A chip (GE Healthcare). Single-cycle kinetics was performed. Antibody capture was performed using HBS-EP. + The experiments were performed at a concentration of 25 μg / mL in buffer (0.01 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 0.15 M sodium chloride, and 0.05% polysorbate 20) with a 25-second pulse and a flow rate of 10 μl / min. Various concentrations of recombinant human VEGF-165 (R&D Systems) were then added with a 60-second pulse, a flow rate of 30 μl / min, and a final dissociation time of 1000 seconds. The experiments were performed at 25°C. The surface was regenerated with 10 mM glycine pH 1.7 for 1 minute at a flow rate of 50 μL / min. Binding kinetics analysis was performed using Biacore T200 evaluation software for responses fitted to a global 1:1 interaction. The results are summarized in Table 10.1 and Figure 21. [Table 5] OG1950 exhibits a KD of less than 1 pM (above instrument sensitivity) in a 1:1 binding fit model.

[0356] Example 11. Conjugation of OG1953 and purification using cation exchange chromatography OG1950 Protein Expression and Protein Preparation: OG1950 protein was expressed in a mammalian GSCHOK1 expression system and subsequently purified using a Protein A affinity column. The purity of the Protein A column-purified OG1950 exceeded 90% based on size-exclusion chromatography and SDS-PAGE. The engineered specific cysteine ​​residue in OG1950 was available for thiol conjugation to the OG1802 biopolymer. The thiol-reactive chemical group in OG1802 reacted to form a stable covalent bond to form the OG1953 bioconjugate. To accomplish this, 1 mg of OG1950 was completely reduced with tris(2-carboxyethyl)phosphine hydrochloride (TCEP) reducing agent, followed by removal of TCEP by buffer exchange using a 30 KDa centrifugal concentrator. The fully reduced OG1950 protein was then reoxidized to ensure that all expected protein disulfide bonds were formed except for the engineered cysteine ​​residue, which remained in a reduced state for conjugation to the biopolymer via thiol-specific conjugation chemistry (uncapped OG1950).

[0357] Conjugation Reaction: The conjugation reaction was performed by mixing 100 μg of decapped OG1950 with a 15-fold molar excess of OG1802 biopolymer to a final protein concentration of 2 mg / mL in Tris buffer (20 mM Tris, 100 mM NaCl, pH 7.5). Various additives were evaluated in different conjugation reactions, as shown in Table 11.1, to compare their effect on conjugation efficiency. All reactions were made at a fixed volume and incubated at 4°C for 20 hours. Table 11.1 and Figure 19 show the ion exchanger analysis (A280 absorbance) and fractionation of conjugation reactions A–G. Reaction A contained buffer only; reaction B contained OG1950 antibody only; reaction C contained OG1950 antibody and OG1802 polymer; and reaction D contained OG1950 antibody, OG1802 polymer, and sucrose. Reaction E contained OG1950 antibody, OG1802 polymer, and trehalose; reaction F contained OG1950 antibody, OG1802 polymer, and glutamic acid; and reaction G contained OG1950 antibody, OG1802 polymer, and aspartic acid. Reactions B through G were initiated with an input of 100 μg of OG1950 protein for a fixed total reaction volume. At reaction completion, equal volumes of reactions B through G were injected for IEX analysis. A comparison of conjugation efficiencies is shown in Figure 19. Peak 1 (P1) represents the excess biopolymer (OG1802) in each reaction that was not conjugated to the protein, failed to bind to the ion exchange column, and thus eluted as the unbound fraction. Peak 2 (P2) represents the antibody-polymer bioconjugate in each reaction. Peak 3 (P3) represents the free antibody that was not conjugated to the polymer in each reaction. [Table 6]

[0358] As can be seen from the above results, various excipients enabled significantly higher conjugation efficiencies. Without wishing to be limited by theory, such excipients aid in maintaining the solubility of the components, which aids in conjugation efficiency.

[0359] Cation exchange analysis of OG1953 conjugation reactions: Upon completion of the conjugation reactions, 5 μl of each reaction mixture was diluted 3-fold with column equilibration buffer (20 mM sodium acetate, pH 5.5) for cation exchange chromatography (IEX) analysis and separation using a Shodex SP-825 HPLC column. IEX was performed in a bind-and-elute mode, where the reaction mixture was first diluted to a lower salt concentration to allow both the conjugate and unreacted protein to bind to the column, followed by a salt gradient elution with increasing NaCl concentration to elute the conjugate (OG1953) and uncomplexed free protein (OG1950) at different retention times due to differential charge variation. As shown in Figure 19, the IEX analysis results demonstrate excellent separation of the OG1953 conjugate (peak P2) from the uncomplexed free polymer (OG1802) represented by peak P1 and the uncomplexed free protein (OG1950) represented by peak P3.

[0360] Large-scale purification of OG1953 conjugate for activity analysis: Conjugation reactions D and E were pooled and further separated by IEX, and the conjugate peak (P2) elution fractions were collected and concentrated for activity analysis.

[0361] Example 12. Effect of anti-VEGF molecules on biotin-VEGF on VEGFR using ELISA The ability of OG1950, OG1953, and other anti-VEGF molecules to inhibit the binding of biotin-VEGF-165 to VEGFR was examined in an ELISA assay. 96-well ELISA plates were filled with 1 μg / mL of recombinant VEGF R1-Fc protein (R&D The plates were coated with a blocking buffer (R&D systems, part number 321-FL-050). The plates were incubated at room temperature overnight. The plates were then washed and blocked with blocking buffer (1% BSA in 1x PBS, pH 7.4) for at least 90 minutes with gentle shaking. A 3-fold dilution series of the test samples was prepared. Starting at 400 nM, the samples were mixed with biotin-VEGF (R&D systems, part number custom06). The final concentration of biotin-VEGF was 4 ng / mL (100 pM). The highest concentration of the sample dilution series was 85 μg / mL. After dilutions were prepared, the samples were incubated at room temperature for at least 30 minutes and then washed three times. After washing, 100 μL of the sample / biotin-VEGF mixture was transferred to each well. The plates were incubated at room temperature for at least 90 minutes with gentle shaking. After incubation, 100 μl of 1:1500 diluted SA-HRP (R&D systems, product number A7906) was added to each well. The incubated plate was protected from light for approximately 20 minutes. The plate was then washed three times. After washing, 100 μl of TMB substrate (R&D systems, product number DY998) was added. The plate was incubated and protected from light for approximately 30 minutes. Color development was monitored. Color development was stopped by adding 50 μl of stop solution. The plate was read at 450 nm. The results are shown in Figure 20.

[0362] These results demonstrate that the OG1953 antibody / conjugate achieved a higher maximum inhibition of biotin-VEGF binding to VEGFR compared with OG1950 (unconjugated antibody) and the commercially available VEGF inhibitors Lucentis® (ranibizumab) and Avastin® (bevacizumab). In a typical VEGF ligand-VEGF receptor binding assay, addition of OG1953 inhibited 97-98% of VEGF ligand binding to the VEGF receptor. This compares with 75-87% inhibition of VEGF ligand / receptor binding with OG1950, and only 68-78% inhibition of VEGF ligand / receptor binding with Lucentis® (ranibizumab) or Avastin® (bevacizumab), respectively. Furthermore, previous studies have shown that the addition of a concentration of 400 nM of either OG1950, Lucentis® (ranibizumab), or Avastin® (bevacizumab) does not result in 100% inhibition of VEGF ligand binding to the VEGF receptor.

[0363] Figure 20 also shows that, compared to the antibody alone (anti-VEGF), another antibody conjugate, the anti-VEGF bioconjugate (anti-VEGF BC), achieved a higher maximum inhibition rate of VEGF binding to the VEGF receptor. Collectively, these results suggest that (i) compared to currently available VEGF inhibitors, the OG1953 antibody conjugate significantly inhibits VEGF binding to the VEGF receptor. These results indicate that (ii) VEGF antibodies are more effective at inhibiting VEGF ligand binding to the VEGF receptor, and (iii) that conjugation of VEGF antibodies to sites outside the active site region can increase inhibition of VEGF ligand binding to the VEGF receptor.

[0364] In some embodiments, provided herein are anti-VEGF antibody conjugates that exhibit an increased level of inhibitory potency compared to (or at least the same level of) the anti-VEGF antibody alone.

[0365] Example 13. Method for measuring binding of OG1950 to Fcγ receptors I and IIIa Binding kinetic experiments were performed at 25°C using a BIAcore T200. Anti-His antibodies were immobilized on a CM5 chip. Histidine-tagged FcγRI and FcγRIIIa antibodies at 0.5 μg / mL in HBS-EP buffer (0.01 M HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, 0.005% Tween-20) were injected individually over 60 seconds using a flow rate of 5 μL / min only through the active flow cell. Next, antibody candidates and Avastin® (bevacizumab), used as a positive control, were injected into the reference and active flow cells using a 60-second injection at 30 μL / mL using a single-cycle kinetics method. Antibody concentrations ranging from 0.48 to 300 nM were used for FcγRI, and antibody concentrations ranging from 7.8 nM to 2000 nM were used for FcγRIIIa. The flow cell was regenerated after each measurement with a 60-second injection of 10 mM glycine pH 1.7 using a flow rate of 50 μg / mL. Data were double-referenced using subtraction of both the reference flow cell and a blank cycle. Analysis was performed using BIA evaluation software. Results are shown in Figures 22 and 23. The results indicate that OG1950 did not exhibit significant binding to either Fcγ receptor I or IIIa in this assay.

[0366] Example 14. Method for measuring binding of OG1950 to human complement protein C1q Complement binding properties were assessed by C1q binding ELISA. The antibody panel was titrated in 1x PBS at a 1:2 ratio from a highest concentration of 10 μg / mL for overnight coating at 4°C. Plates were then blocked after a 2-hour blocking step in 1% BSA. Purified human C1q was then applied at a concentration of 5 μg / mL in 1% BSA for 2 hours at room temperature, followed by detection with HRP-conjugated anti-human C1q antibody and TMB development. The results are shown in Figure 24. The results indicate that C1q has a higher binding affinity for Avastin® (bevacizumab) compared to OG1950 at antibody concentrations between 10 μg / mL and 0.625 μg / mL.

[0367] In some embodiments, OG1950 has less than 10% of the binding of Avastin. In some embodiments, OG1950 has less binding to C1q than Avastin® (bevacizumab).

[0368] Example 15. Effect of anti-VEGF agents on VEGF-stimulated HRMVEC proliferation Human retinal microvascular endothelial cell (HuRMVEC) proliferation assays were performed as follows. Cells were maintained in complete CSC medium (Cell Systems, Part Number 4Zo-500) supplemented with 2% CultureBoost (Cell Systems, Part Number 4CB-500) and 0.2% Bac-off (Cell Systems, Part Number 4ZO-643) and seeded at a density of 10,000 cells per well in 96-well plates in assay medium (serum-free medium (Cell Systems, Part Number 4Z3-500-S) and 5% FBS). VEGF inhibitors were first added to each well at the indicated concentrations. Thirty minutes later, VEGF165 (R&D Systems, Part Number 293-VE-500 / CF) was added to a final concentration of 1.3 nM. After 3 days, cells were incubated with WST-1 cell proliferation assay reagent and measured at OD450nm.

[0369] The results showed that two independent preparations of OG1953 (OG1953A and OG1953B) both exhibited potent inhibition of HuRNVEC proliferation. In this assay, both the maximal inhibition rate and IC50 of OG1953 are comparable to that of the antibody alone, OG1950. The maximal inhibition rate of OG1953 is also significantly better than that of Avastin® (bevacizumab) and Eylea® (aflibercept). The results, including IC50 values ​​and a comparison of these values ​​with Lucentis® (ranibizumab), Eylea® (aflibercept), and Avastin® (bevacizumab), are shown in Figure 25.

[0370] Example 16. Single cycle kinetics (SCK) of VEGF binding to anti-VEGF agents captured on a Protein A chip at 25°C Binding kinetics was performed on Avastin® (bevacizumab), OG1950, and OG1953 using a BIAcore T200 at 25°C. Briefly, anti-VEGF agents were captured on a Protein A chip (GE). 1 μg / ml OG1950 and Avastin® (bevacizumab) were flowed at 25 μl / min for 2 minutes. 10 μg / ml OG1953 was flowed at 10 μl / min for 10 minutes. For single-cycle kinetics, VEGF (recombinant; R&D Systems) at 0.56 nM, 1.67 nM, 5 nM, 15 nM, and 45 nM, respectively, was flowed over the captured antibody for a contact time of 240 seconds and allowed to dissociate for 30 minutes. Analysis was performed using BiaEvaluation software (GE). All sensorgrams were double-based subtracted and fitted 1:1 using the Langmuir binding model. The dissociation rates of these anti-VEGF agents may be underestimated in this experiment due to dissociation between the anti-VEGF agents and the Protein A capture complex.

[0371] The results, including the calculated KD, ka, and kd values, are presented in FIG.

[0372] Example 17. Excipient screening experiment for prevention of OG1802 polymer-induced IgG1 precipitation Using the standard conjugation reaction process setup, the OG1950 conjugation reaction mixture was found to become cloudy with a precipitate immediately upon mixing. Further investigation revealed that the precipitate was the protein itself, and that the low conjugation efficiency observed by SDS-PAGE or ion exchange analysis was due to the protein being lost to precipitation instead of participating in the conjugation reaction.

[0373] Initial troubleshooting experiments performed revealed that conditions that did not result in a clear reaction solution included: (1) decreasing the polymer molar excess ratio from greater than 10 to less than 5; (2) pre-adjusting the pH of the reaction solution from the standard neutral pH range (e.g., pH 6.5-7.5) to a more acidic (e.g., pH 5) or basic (e.g., pH 8.5); (3) testing other IgG1 protein samples with similar or different isoelectric points (pI) than OG1950; (4) buffer-exchanging the sample storage buffer to 1x PBS, pH 7.4, or 20 mM Tris buffer, 100 mM NaCl, pH 7.4; and (5) pre-adjusting the OG1802 solution in 20 mM Tris buffer, pH 7.4.

[0374] Proteins are known to have a net surface charge in aqueous solutions that aids in the solubility of proteins. These amino acids are called hydrophilic amino acids and include arginine, lysine, aspartic acid, and glutamic acid. At a neutral pH of 7, the side chains of these amino acids are positively charged for arginine and lysine, and negatively charged for aspartic acid and glutamic acid. Changes in the solution pH can alter the intrinsic protein solubility and Therefore, this approach has been applied in some of the troubleshooting experiments mentioned above, such as (2). Theoretically, the solubility of proteins in aqueous solutions varies depending on the level of hydrophobicity or hydrophilicity of the surface. Proteins with more hydrophobic surfaces will precipitate more easily. The addition of ions (e.g., NaCl or other salts) neutralizes some of the interaction between water particles and the protein, resulting in an electronic shielding effect that causes the proteins to bind to each other and begin to aggregate, reducing solubility. It is currently hypothesized that the biopolymers directly or indirectly adjust the protein surface charge and / or exposed surface to promote intermolecular hydrophobic interactions that cause protein precipitation.

[0375] Excipients that have been determined to modify protein solubility include the following categories: (i) detergents, including neutral detergents (e.g., 0.1-1% polysorbate 20 or Tween 20) or charged detergents (e.g., 0.1-1% sodium dodecyl sulfate (SDS)), (ii) sugars (e.g., 6% trehalose or 6% sucrose), (iii) negatively charged amino acids (e.g., 0.03-1 mM glutamic acid or 0.03-1 mM aspartic acid) or positively charged amino acids (e.g., 1-100 mM lysine or arginine), (iv) chaotropic agents or denaturants (e.g., 1-100 mM urea, guanidine hydrochloride analogs, or 1-100 mM arginine), (v) polyethylene glycols (e.g., 0.03-1 mM PEG 8000), and (vi) organic solvents (e.g., 20% ethanol).

[0376] In additional design of experiments (DOE) studies, various excipients from each of the above categories were selected based on their suitability for formulation processing suitable for human injectable use. Furthermore, extreme acidic pH of 4 and extreme basic pH of 9 were included in such evaluations. A standard IgG1 protein sample without engineered cysteines was selected for such evaluations to minimize the possibility of interference from unpaired cysteine ​​residues complicating precipitation observations. Table 17.1 illustrates such a matrix. Shaded codes / rows represent conditions that result in a clear solution, while unshaded rows represent conditions that result in varying degrees of turbidity or precipitation. [Table 7]

[0377] The resulting precipitated (or non-precipitated) solution is shown in FIG.

[0378] All patent applications, websites, other publications, accession numbers, etc., cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be incorporated by reference. Where different versions of a sequence are associated with an accession number at different times, the version associated with that accession number as of the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or, if applicable, the filing date of the prior application referencing that accession number. Similarly, where different versions of a publication, website, or the like are published at different times, the most recently published version as of the effective filing date of this application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect disclosed herein can be used in combination with any other, unless specifically indicated otherwise. While some embodiments have been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be understood that certain changes and modifications may be practiced that are within the scope of the appended claims. Other embodiments of the present invention include the following. [1] An antibody conjugate comprising (1) an anti-VEGF-A antibody and (2) a phosphorylcholine-containing polymer, the polymer being covalently attached to the antibody at a cysteine ​​outside the variable region of the antibody. wherein the cysteine ​​has been added by recombinant DNA technology. [2] The antibody complex according to [1], wherein the anti-VEGF-A antibody has a light chain and a heavy chain, and the heavy chain comprises an Fc region. [3] The antibody conjugate according to [2], wherein the cysteine ​​is within the Fc region of the heavy chain. [4] The antibody conjugate according to [3], wherein the anti-VEGF-A antibody is immunoglobulin G (IgG). [5] The anti-VEGF-A antibody heavy chain comprises CDR H1: GYDFTHYGMN (SEQ ID NO: 9), CDR H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H 3: YPYYYGTSHWYFDV (SEQ ID NO: 11), wherein the anti-VEGF-A light chain comprises CDR L 1: SASQDISNYLN (SEQ ID NO: 12), CDR L 2: FTSSLHS (SEQ ID NO: 13), and CDR L 3: The antibody complex according to [3] or [4], which comprises QQYSTVPWT (sequence number 14). [6] The antibody conjugate according to [4] or [5], wherein the anti-VEGF-A heavy chain isotype is human IgG1. [7] The antibody conjugate according to [6], wherein the heavy chain constant domain of the anti-VEGF-A antibody has one or more mutations compared to the constant domain of human IgG1, thereby regulating effector function.[8] 8. The antibody conjugate of [7], wherein the mutations are at one or more of the following amino acid positions: E233X, L234X, L235X, G236X, G237X, A327X, A330X, and P331X (EU numbering), where X is any natural or unnatural amino acid. [9] The antibody conjugate according to [8], wherein the mutation is selected from the group consisting of E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S (EU numbering).

[10] The antibody conjugate of [9], comprising the following mutations: L234A, L235A, and G237A (EU numbering).

[11] The antibody conjugate according to

[10] , wherein the cysteine ​​is in the heavy chain of the anti-VEGF-A antibody and is Q347C (EU numbering) or L443C (EU numbering).

[12] The antibody conjugate according to

[11] , wherein the sequence of the anti-VEGF-A antibody heavy chain is SEQ ID NO: 1 and the sequence of the anti-VEGF-A antibody light chain is SEQ ID NO: 2.

[13] The antibody conjugate according to

[11] , wherein the cysteine ​​is L443C (EU numbering).

[14] The antibody conjugate according to any one of [1] to

[13] , wherein the phosphorylcholine-containing polymer comprises a 2-(methacryloyloxyethyl)-2'-(trimethylammonium)ethyl phosphate (MPC) monomer shown below. [ka]

[15] The antibody conjugate according to

[14] , wherein the polymer has three or more arms or is synthesized using an initiator having three or more polymerization initiation sites.

[16] The antibody conjugate of

[15] , wherein the polymer has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 arms, or is synthesized using an initiator having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 polymerization initiation sites.

[17] The antibody conjugate of

[16] , wherein the polymer has 2, 3, 6, or 9 arms, or is synthesized using an initiator having 2, 3, 6, or 9 polymerization initiation sites.

[18] The antibody conjugate according to

[17] , wherein the polymer has nine arms or is synthesized using an initiator having nine polymerization initiation sites.

[19] The antibody conjugate described in

[18] , wherein the polymer has a molecular weight of between about 300,000 and about 1,750,000 Da as measured by size exclusion chromatography-multi-angle light scattering (hereinafter "SEC-MALS").

[20] The antibody conjugate according to

[19] , wherein the polymer has a molecular weight between about 500,000 and about 1,000,000 Da. [twenty one] The antibody conjugate described in

[20] , wherein the polymer has a molecular weight of between about 750,000 and about 850,000 Da. [twenty two] A purified antibody conjugate according to any one of [1] to

[21] . [twenty three] The antibody conjugate according to

[22] , wherein the polymer is a polydisperse system. [twenty four] The antibody conjugate according to

[23] , wherein the polymer has a polydispersity index (PDI) of less than about 1.2. [twenty five] An antibody conjugate comprising an anti-VEGF-A immunoglobulin G (IgG) conjugated to a polymer, wherein the polymer comprises an MPC monomer, the sequence of the anti-VEGF-A antibody heavy chain is SEQ ID NO: 1, the sequence of the anti-VEGF-A antibody light chain is SEQ ID NO: 2, and the antibody is SEQ ID NO: 1. The antibody conjugate is bound to the polymer at C449 in the

[26] the polymer has nine arms, and The polymer has a molecular weight between about 600,000 and about 900,000 Da.

[25] The antibody conjugate according to

[26] .

[27] having the following structure: [ka] During the ceremony, each heavy chain of the anti-VEGF-A antibody is represented by the letter H and each light chain of the anti-VEGF-A antibody is represented by the letter L; the polymer is attached to the anti-VEGF-A antibody via the sulfhydryl of C443 (EU numbering), which attachment is shown on one of the heavy chains; PC [ka] wherein the wavy line represents the point of attachment to the rest of the polymer portion, and X is a) OR where R is H, methyl, ethyl, propyl, isopropyl, b) H, or c) any halide, including Br; and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±15%;

[25] The antibody conjugate according to

[26] .

[28] A pharmaceutical composition comprising the antibody conjugate according to any one of [1] to

[27] in the form of a liquid solution.

[29] A pharmaceutical composition comprising the antibody conjugate according to any one of [1] to

[27] and a pharmaceutically acceptable carrier.

[30] An anti-VEGF-A antibody having a heavy chain and a light chain, The heavy chain comprises CDR H 1: GYDFTHYGMN (SEQ ID NO: 9), CDR H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H 3: YPYYYGTSHWYFDV (SEQ ID NO: 11), wherein the light chain comprises CDR L 1: SASQDISNYLN (SEQ ID NO: 12), CDR L 2: FTSSLHS (SEQ ID NO: 13), and CDR L 3: QQYSTVPWT (SEQ ID NO: 14), and The heavy chain isotype is IgG1, and the IgG1 constant domain contains one or more of the following mutations to modulate effector function: E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S (EU numbering). The anti-VEGF-A antibody.

[31] The antibody of

[30] , comprising the following mutations: L234A, L235A, and G237A (EU numbering).

[32] The antibody according to

[30] , wherein the sequence of the anti-VEGF-A heavy chain is SEQ ID NO: 1 and the sequence of the anti-VEGF-A light chain is SEQ ID NO: 2.

[33] The antibody according to any one of

[30] to

[32] , which is an IgG.

[34] A method for treating or preventing an eye disease, comprising administering the antibody conjugate according to any one of [1] to

[27] , or the pharmaceutical composition according to

[28] or

[29] .

[35]

[34] The method for treatment or prevention according to

[34] , wherein the ocular disease is selected from the group consisting of diabetic retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic macular edema (DME), pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, central retinal vein occlusion (CRVO), central branch retinal vein occlusion (BRVO), corneal neovascularization, retinal neovascularization, retinopathy of prematurity (ROP), subconjunctival hemorrhage, and hypertensive retinopathy.

[36] The method for treatment or prevention according to

[34] , wherein the disease is diabetic retinopathy.

[37] 1. A method for preparing an antibody conjugate comprising an anti-VEGF-A antibody conjugated to a phosphorylcholine-containing polymer, the method comprising: conjugating an anti-VEGF-A antibody to a phosphorylcholine-containing polymer; the anti-VEGF-A antibody comprises a cysteine ​​residue added via recombinant DNA techniques, the cysteine ​​being outside the variable region of the antibody; the phosphorylcholine-containing polymer comprises a sulfhydryl-specific reactive group selected from the group consisting of maleimide, vinyl sulfone, orthopyridyl disulfide, and iodoacetamide; and The sulfhydryl-specific reactive group on the phosphorylcholine-containing polymer reacts with the cysteine ​​residue on the anti-VEGF-A antibody to form the antibody conjugate. The method.

[38] The method according to

[37] , wherein the anti-VEGF-A antibody is immunoglobulin G (IgG) and the cysteine ​​is present in the Fc region of the antibody.

[39] The anti-VEGF-A antibody has a light chain and a heavy chain, and the anti-VEGF-A antibody heavy chain has a CDR H 1: GYDFTHYGMN (SEQ ID NO: 9), CDR H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H 3: YPYYYGTSHWYFDV (SEQ ID NO: 11), wherein the anti-VEGF-A antibody light chain comprises CDR L 1: SASQDISNYLN (SEQ ID NO: 12), CDR L 2: FTSSLHS (SEQ ID NO: 13), and CDR L 3: The method according to

[37] or

[38] , comprising QQYSTVPWT (sequence number 14).

[40] The method according to

[38] or

[39] , wherein the anti-VEGF-A antibody heavy chain isotype is IgG1.

[41] The method according to

[40] , wherein the effector function is regulated by the anti-VEGF-A antibody constant domain having one or more mutations compared to the IgG1 constant domain.

[42] 41. The method of claim 41, wherein the mutation is at one or more of the following amino acid positions: E233X, L234X, L235X, G236X, G237X, G236X, D270X, K322X, A327X, P329X, A330X, A330X, P331X, and P331X (EU numbering), where X is any natural or unnatural amino acid.

[43] The method of

[42] , wherein the mutation is selected from the group consisting of E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S (EU numbering).

[44] The method according to

[43] , wherein the mutations are L234A, L235A, and G237A (EU numbering).

[45] The method according to

[44] , wherein the cysteine ​​residue added by recombinant DNA technology is selected from the group consisting of Q347C (EU numbering) and L443C (EU numbering).

[46] The method according to

[45] , wherein the cysteine ​​residue added by recombinant DNA technology is L443C (EU numbering).

[47] The method according to

[45] or

[46] , wherein the sequence of the anti-VEGF-A antibody heavy chain is SEQ ID NO: 1 and the sequence of the anti-VEGF-A antibody light chain is SEQ ID NO: 2.

[48] The method according to

[37] , wherein the sulfhydryl-specific reactive group is maleimide.

[49] The method according to

[48] , wherein the phosphorylcholine-containing polymer comprises 2-(methacryloyloxyethyl)-2'-(trimethylammonium)ethyl phosphate (MPC) monomer as shown below. [ka]

[50]

[49] The method according to

[49] , wherein the polymer has three or more arms or is synthesized using an initiator having three polymer initiation sites.

[51] 50. The method of claim 50, wherein the polymer has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 arms or is synthesized using an initiator having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 polymer initiation sites.

[52] 51. The method of claim 51, wherein the polymer has 2, 3, 6, or 9 arms, or is synthesized using an initiator having 2, 3, 6, or 9 polymer initiation sites.

[53] The method of

[52] , wherein the polymer is synthesized using an initiator having nine arms or nine polymer initiation sites.

[54] 53. The method of claim 53, wherein the polymer has a molecular weight between about 300,000 and 1,750,000 Da.

[55] 54. The method of claim 54, wherein the polymer has a molecular weight between about 600,000 and 1,000,000 Da.

[56]

[55] The method of

[55] , wherein the polymer has a molecular weight of between about 600,000 and 850,000 Da.

[57] 37. The method of claim 37, further comprising contacting the anti-VEGF-A antibody with a thiol reducing agent under conditions to generate reduced cysteine ​​sulfhydryl groups to generate a reduced anti-VEGF-A antibody in which all cysteine ​​residues are reduced.

[58] 57. The method of claim 57, wherein the thiol reducing agent is selected from the group consisting of tris[2-carboxyethyl]phosphine hydrochloride (TCEP), dithiothreitol (DTT), dithioerythritol (DTE), sodium borohydride (NaBH), sodium cyanoborohydride (NaCNBH), β-mercaptoethanol (BME), cysteine ​​hydrochloride, and cysteine.

[59]

[58] The method according to

[58] , wherein the thiol reducing agent is TCEP.

[60] 59. The method of claim 59, wherein the concentration of the thiol reducing agent is between 1 and 100-fold molar excess relative to the concentration of the anti-VEGF-A antibody.

[61] 60. The method of claim 60, wherein the concentration of the thiol reducing agent is between 20 and 50 times molar excess relative to the concentration of the anti-VEGF-A antibody.

[62] 60. The method of claim 60, further comprising the steps of removing the thiol reducing agent from the reduced anti-VEGF-A antibody and treating the reduced anti-VEGF-A antibody with an oxidizing agent.

[63] The method according to

[61] , wherein the oxidizing agent is air, an aqueous CuSO4 solution, or dehydroascorbic acid (DHAA).

[64] The method according to

[37] , further comprising the step of purifying the antibody complex.

[65] The method of

[64] , wherein the antibody complex is purified using a technique selected from the group consisting of ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, affinity chromatography, and combinations thereof.

[66]

[65] The method of

[65] , wherein the purified antibody conjugate retains at least 20% of the biological activity compared to the unconjugated anti-VEGF-A antibody.

[67]

[66] The method of

[66] , wherein the purified antibody conjugate retains at least 50% of the biological activity compared to the unconjugated anti-VEGF-A antibody.

[68] 67. The method of claim 67, wherein the purified antibody conjugate retains at least 90% of the biological activity of the unconjugated anti-VEGF-A antibody.

[69] 66. The method of claim 66, wherein the purified antibody conjugate has an increased half-life compared to an unconjugated anti-VEGF-A antibody.

[70] 69. The method of claim 69, wherein the purified antibody conjugate has a half-life that is increased by at least 1.5-fold compared to an unconjugated anti-VEGF-A antibody.

[71] The method further comprises polymerizing free-radically polymerizable phosphorylcholine-containing monomers in a polymerization medium to provide the phosphorylcholine-containing polymer, the medium comprising the radical polymerizable phosphorylcholine-containing monomer, M t is a transition metal, q is the maximum oxidation state of the metal, and q-1 is the oxidation state of the metal, and the metal is a transition metal catalyst M t (q-1)+ and X' is a counter ion or group. t (q-1)+ X' (q-1)or the inactive metal salt M in its highest oxidation state together with a reducing agent capable of reducing the transition metal from an oxidatively inactive state to a reductively active state. t q+ X' q the transition metal catalyst being provided in situ by providing ligand, and initiator The method according to

[37] , comprising:

[72] The radical polymerizable phosphorylcholine-containing monomer [ka] wherein R1 is H or C 1~6 is alkyl, R2, R3, and R4 are each methyl; and X and Y are each 2 The method described in

[71] .

[73] M t is selected from the group consisting of Cu, Fe, Ru, Cr, Mo, W, Mn, Rh, Re, Co, V, Zn, Au, and Ag.

[74] The metal catalyst is M t (q-1)+ X' (q-1) The method according to

[73] , wherein the compound is provided as a salt in the form

[75] M t (q-1)+ is Cu 1+ , Fe 2+ , Ru 2+ , Cr 2+ , Mo 2+ , W 2+ , Mn 3+ , Rh 3+ ,Re 2+ , Co + , V 2+ , Zn + , Au + , and Ag +and X' is selected from the group consisting of halogen, C 1~6 Alkoxy, (SO4) 1 / 2 , (PO4) 1 / 3 , (R7PO4) 1 / 2 , (R72PO4), triflate, hexafluorophosphate, methanesulfonate, arylsulfonate, CN, and R7CO2, wherein R7 is a linear or branched C alkyl group optionally substituted 1 to 5 times with H or halogen. 1~6 The method according to

[74] , wherein the alkyl group is an alkyl group.

[76] M t (q-1)+ is Cu 1+ and X' is Br.

[77] M t (q-1)+ is provided in situ.

[78] M t q+ X q

[77] The method according to

[77] , wherein is CuBr2.

[79]

[72] The method according to

[72] , wherein the reducing agent is an inorganic compound.

[80] 79. The method of claim 79, wherein the inorganic compound is selected from the group consisting of low oxidation level sulfur compounds, sodium bisulfite, inorganic salts containing metal ions, metals, hydrazine hydrate, and derivatives of such compounds.

[81]

[80] The method according to

[80] , wherein the reducing agent is a metal.

[82] The reducing agent is Cu 0 The method according to

[81] ,

[83]

[72] The method according to

[72] , wherein the reducing agent is an organic compound.

[84]

[83] The method according to

[83] , wherein the organic compound is selected from the group consisting of alkylthiols, mercaptoethanol, or carbonyl compounds that can be easily enolized, ascorbic acid, acetylacetonate, camphorsulfonic acid, hydroxyacetone, reducing sugars, monosaccharides, glucose, aldehydes, and derivatives of such organic compounds.

[85] The ligand may be 2,2'-bipyridine, 4,4'-di-5-nonyl-2,2'-bipyridine, 4,4-dinonyl-2,2'-dipyridyl, 4,4',4''-tris(5-nonyl)-2,2':6',2''-terpyridine, N,N,N',N',N''-pentamethyldiethylenetriamine, 1,1,4,7,10,10-hexamethyltriethylenetetramine, tris(2-dimethylaminoethyl)amine, N,N-bis(2-pyridylmethyl)octadecylamine, N,N ,N',N'-tetra[(2-pyridal)methyl]ethylenediamine, tris[(2-pyridyl)methyl]amine, tris(2-aminoethyl)amine, tris(2-bis(3-butoxy-3-oxopropyl)aminoethyl)amine, tris(2-bis(3-(2-ethylhexoxy)-3-oxopropyl)aminoethyl)amine, and tris(2-bis(3-dodecoxy-3-oxopropyl)aminoethyl)amine.

[86] The method according to

[85] , wherein the ligand is 2,2'-bipyridine.

[87] The initiator has the structure: [ka] wherein R1 is a nucleophilic reactive group, R2 comprises a linker, and R3 comprises a polymer synthesis initiator moiety having the structure: [ka] wherein R4 and R5 are the same or different and are selected from the group consisting of alkyl, substituted alkyl, alkylene, alkoxy, carboxyalkyl, haloalkyl, cycloalkyl, cyclic alkyl ether, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkylene, heterocycloalkyl, heterocycloalkylene, aryl, arylene, aryleneoxy, heteroaryl, amino, amido, and any combination thereof; Z is halogen or CN and s is an integer between 1 and 20; The method described in

[72] .

[88] The method according to

[87] , wherein Z is Br, and R4 and R5 are each methyl.

[89] The method according to

[88] , wherein R1 is selected from the group consisting of NH2-, OH-, and SH-.

[90] The method according to

[89] , wherein R1 is NH2-.

[91] 90. The method of claim 90, wherein R2 is alkyl, substituted alkyl, alkylene, alkoxy, carboxyalkyl, haloalkyl, cycloalkyl, cyclic alkyl ether, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkylene, heterocycloalkyl, heterocycloalkylene, aryl, arylene, aryleneoxy, heteroaryl, amino, amido, and any combination thereof.

[92] R2 [ka]

[91] The method according to

[91] , wherein X and Y are the same or different and are integers from 1 to 20.

[93] The method according to

[92] , wherein X and Y are each 4.

[94] R3 [ka] wherein R6, R7, and R8 are the same or different, and [ka] [ka] and [ka] wherein Z is NCS, F, Cl, Br, or I; The method described in

[93] .

[95] The method according to

[94] , wherein Z is Br.

[96] R6, R7, and R8 are [ka] The method according to

[94] ,

[97] The initiator has the following structure: [ka]

[96] . The method of claim 96, wherein A and B are the same or different and are integers from 2 to 12, and Z is Br.

[98] The method according to

[97] , wherein A and B are each 4.

[99] 98. The method of claim 98, further comprising reacting the polymer with a maleimide reagent to provide a maleimide-terminated polymer.

[0100] The maleimide compound [ka] The method according to

[99] .

[0101] a heavy chain amino acid variable region comprising SEQ ID NO: 1, and Light chain amino acid variable region comprising SEQ ID NO:2 An antibody having

[0102] The antibody described in

[0101] , wherein the antibody is human IgG1 and the heavy chain constant domain contains one or more mutations that reduce immune-mediated effector function.

[0103] The antibody is further conjugated to a polymer to form a bioconjugate, and the bioconjugate An antibody conjugate described in

[0101] having a molecular weight between approximately 350,000 and 1,900,000 daltons.

[0104] An antibody conjugate according to

[0103] , having a polydispersity index (PDI) of 1.5 or less.

[0105] An antibody that binds to VEGF-A, CDRs in SEQ ID NO: 1 H CDR 1 H 1. CDRs in SEQ ID NO: 1 H CDR 2 H 2. CDRs in SEQ ID NO: 1 H CDR 3 H 3. CDRs in SEQ ID NO:2 L CDR 1 L 1. CDRs in SEQ ID NO:2 L CDR 2 L 2. CDRs in SEQ ID NO:2 L CDR 3 L 3. At least one of the following mutations: L234A, L235A, and G237A (EU numbering), and At least one of the following mutations (EU numbering): Q347C or L443C The antibody comprising:

[0106] The antibody described in

[0105] , wherein the antibody contains all three of the following mutations: L234A, L235A, and G237A (EU numbering), and also contains L443C (EU numbering).

[0107] 1. A process for preparing a conjugated protein, comprising: reducing one or more cysteines in the protein to form a decapped protein in solution; reoxidizing the decapped protein to re-establish at least one disulfide bond in the reduced protein, while the engineered cysteine ​​residue in the protein remains in a free thiol state, thereby forming a reoxidized decapped protein in the solution; adding at least one excipient to said solution, said excipient reducing polymer-induced protein precipitation; adding a polymer to the solution; and conjugating the polymer to the reoxidized decapped protein at the engineered cysteine ​​residue to form a conjugated protein. The method comprising the steps of:

[0108] The method described in

[0107] , wherein the protein is an antibody, an antibody-protein fusion, or a binding fragment thereof.

[0109] The manufacturing method described in

[0108] , wherein the excipient is an acid or a base.

[0110] The manufacturing method described in

[0109] , wherein the excipient is selected from the group consisting of at least one of a surfactant, a sugar, and a charged amino acid.

[0111] The method described in

[0107] , wherein the reaction between the reduced protein and the polymer occurs under aqueous conditions between pH 6.0 and pH 8.5.

[0112] The method according to claim 0107, wherein the amount of the reduced protein is less than the amount of the polymer. The polymer is conjugated to the protein at a temperature of 2 to 37 degrees Celsius. The manufacturing method described.

[0114] The method according to

[0107] further comprises contacting a solution containing the complexed protein with an ion exchange medium, or a hydrophobic interaction chromatography or affinity chromatography medium.

[0115] The method of claim 0114, wherein the complexed protein is separated from the free polymer and the reoxidized, uncapped protein by the ion exchange medium, or by hydrophobic interaction chromatography or affinity chromatography medium.

[0116] The method described in

[0107] , wherein the polymer contains zwitterions.

[0117] The method described in

[0107] , wherein the polymer contains phosphorylcholine.

[0118] The method described in

[0107] , wherein the polymer contains a PEG linker that links the center of the polymer branch point to the maleimide functional group.

[0119] An anti-VEGF antibody conjugate capable of inhibiting at least 90% of the interaction between a VEGF ligand and a VEGF receptor.

[0120] An anti-VEGF antibody conjugate that inhibits at least 95% of the interaction between a VEGF ligand and a VEGF receptor.

[0121] An anti-VEGF antibody conjugate that inhibits at least 90% of the interaction between a VEGF ligand and a VEGF receptor.

Claims

[Claim 1] An antibody conjugate comprising (1) an anti-VEGF-A antibody and (2) a phosphorylcholine-containing polymer, wherein the polymer is covalently attached to the antibody at a cysteine ​​outside the variable region of the antibody, the cysteine ​​being added by recombinant DNA technology.

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