RU2843347C1 - Antibodies and conjugates thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: there are presented anti-VEGF-A antibodies and pharmaceutical compositions containing them. Also disclosed is a method of treating or preventing an ophthalmic disease, comprising administering said antibodies or compositions to a subject. Besides, the invention refers to conjugates containing polymer-conjugated anti-VEGF-A antibodies. Antibody conjugates can maintain or enhance antibody activity.

EFFECT: antibodies and conjugates thereof can be used in treating ophthalmic diseases, especially diabetic retinopathy.

26 cl, 28 dwg, 7 tbl, 17 ex

Description

INCLUSION BY LINK OF ANY PRIORITY APPLICATIONS

Any and all applications for which foreign or domestic priority claims are indicated in an application data sheet filed with this application are hereby incorporated by reference in accordance with 37 CFR § 1.57.

SEQUENCE LIST

This application is filed together with a sequence listing in electronic format. The sequence listing is provided as file KDIAK004.txt, created on December 28, 2016, the size of which is 18231 bytes. The information of the sequence listing in electronic format is incorporated herein by reference in its entirety.

FIELD OF INVENTION

The present invention relates to antibodies and their conjugates and methods for using and producing said antibodies, their conjugates and other protein conjugates.

PRIOR ART

Diabetic retinopathy is the leading cause of blindness in people aged 20 to 64 years. 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. In the United States, diabetic retinopathy accounts for approximately 12% of new cases of blindness. Typically, in cases of diabetic retinopathy, the blood vessels in the retina become dilated and fluid leaks into the back of the eye. Hyperglycemia causes intramural and thickening of the basement membrane, leading to leakage or permeability of the blood vessels.

In diabetic retinopathy, changes in blood glucose levels cause changes in the blood vessels of the retina. All people with diabetes are at risk. The longer a person has diabetes, the higher the risk of developing some form of eye disease. Between 40 and 45 percent of Americans with diabetes have some stage of diabetic retinopathy. Causes and Risk Factors. Diabetic Retinopathy. United States National Library of Medicine. 15 September 2009.

Diabetic retinopathy first becomes apparent when a microaneurysm develops in the retina. Microaneurysms occur when the capillaries (very small blood vessels) that feed the retina become swollen. The presence of a relatively small number of microaneurysms usually does not cause vision problems. However, if retinopathy progresses to later stages, there is a significant risk of vision loss. This early stage of retinopathy is called background diabetic retinopathy or non-proliferative diabetic retinopathy (NPDR). Although patients with NPDR usually do not show symptoms, early detection of retinopathy is critical because if the disease progresses to later stages, significant vision loss is very likely.

In the next stage of diabetic retinopathy, neovascularization occurs in the back of the eye (proliferative diabetic retinopathy). The newly formed vessels leak, and the vessels may burst, which then bleed and cause blurred or cloudy vision. Due to the lack of oxygen in the eye, further neovascularization occurs. Blood vessels grow along the retina and into the vitreous. As these vessels burst, further bleeding occurs and the retina can be severely damaged or destroyed. The accumulation of fluid in the macula due to leaking blood vessels is called diabetic macular edema. Many patients with diabetic retinopathy develop diabetic macular edema.

There are three common treatment options for patients with diabetic retinopathy: laser surgery, corticosteroid injection, and injection of anti-VEGF agents (eg, AVASTIN® (bevacizumab), LUCENTIS® (ranibizumab), and Eylea® (aflibercept)). Although laser surgery is usually effective in treating diabetic retinopathy, laser-induced retinal damage is a common side effect. Steroid medications such as triamcinolone acetonide are administered by intravitreal injection to treat diabetic retinopathy. However, steroid solutions must be administered frequently to treat diabetic retinopathy. In addition, vitreous steroid treatment has been associated with the development of cataracts, steroid-induced glaucoma, and endophthalmitis.

Another treatment option for diabetic retinopathy is the 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-targeted therapy is effective not only in diabetic retinopathy but also in age-related macular degeneration (AMD), such as neovascular (wet) AMD.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to a conjugate of an antibody against VEGF-A linked via a cysteine outside the variable region of the antibody to a polymer containing phosphorylcholine, wherein said cysteine was added using recombinant DNA technology. Optionally, the antibody against VEGF-A comprises a light chain and a heavy chain, and the heavy chain comprises an Fc region. Optionally, the antibody against VEGF-A is immunoglobulin G (IgG). Optionally, the cysteine is in the Fc region of the heavy chain. Optionally, the heavy chain of the anti-VEGF-A antibody 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), and position 221 (by sequential counting as in SEQ ID NO: 3) is T, and the light chain of the anti-VEGF-A antibody 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 position 4 according to Kabat is L. Optionally, the heavy chain isotype of the anti-VEGF-A antibody is human IgG1.

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

The cysteine residue is optionally in the heavy chain of the anti-VEGF-A antibody and is optionally Q347C (EU numbering) or L443C (EU numbering). Optionally, the heavy chain of the anti-VEGF-A antibody is SEQ ID NO: 1 and the light chain sequence of the anti-VEGF-A antibody is SEQ ID NO: 2. Optionally, the cysteine is L443C (EU numbering).

Optionally, the phosphorylcholine-containing polymer comprises 2-(methacryloyloxyethyl)-2'-(trimethylammonium)ethyl phosphate (MPC) monomers as follows:

.

So, the polymer includes the following repeating units:

where n is an integer from 1 to 3000 and the wavy lines indicate the attachment points between monomer units in the polymer.

The polymer optionally has three or more arms, or is synthesized with an initiator comprising 3 or more polymer initiation sites. Optionally, the polymer has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 arms, or is synthesized with an initiator comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 polymer initiation sites. Optionally, the polymer has 2, 3, 6 or 9 arms, or is synthesized with an initiator comprising 2, 3, 6 or 9 polymer initiation sites. Optionally, the polymer has 9 arms or is synthesized with an initiator comprising 9 polymer initiation sites.

Optionally, the polymer has a molecular weight of about 300,000 to about 1,750,000 Da, as determined by size exclusion chromatography (SEC) with multi-angle light scattering (MALS) (hereinafter "SEC-MALS"). Optionally, the polymer has a molecular weight of about 500,000 to about 1,000,000 Da. Optionally, the polymer has a molecular weight of about 600,000 to about 800,000 Da.

Optionally, the antibody conjugate is purified and the polymer is polydisperse. Optionally, the polymer has a polydispersity index (PI) of less than 1.2. In some embodiments, any of the conjugate solutions provided herein may have a polydispersity index (PI) of equal to or less than 1.8, such as less than or equal to 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.

Optionally, an antibody conjugate comprising immunoglobulin G (IgG) against VEGF-A is linked to a polymer comprising MPC monomers, wherein the heavy chain sequence of the anti-VEGF-A antibody is SEQ ID NO: 1 and the light chain sequence of the anti-VEGF-A antibody is SEQ ID NO: 2, and wherein the antibody is linked only at position C449 of SEQ ID NO: 1 to the polymer. Optionally, the polymer has 9 arms; and the polymer has a molecular weight of from about 600,000 to about 800,000 Da.

Optionally, an antibody conjugate comprising immunoglobulin G (IgG) against VEGF-A is linked to a polymer comprising MPC monomers, wherein the heavy chain sequence of the anti-VEGF-A antibody is SEQ ID NO: 1 and the light chain sequence of the anti-VEGF-A antibody is SEQ ID NO: 2, and wherein the antibody is linked only at C443 (EU numbering) to the polymer. Optionally, the polymer has 9 arms; and the polymer has a molecular weight of from about 600,000 to about 800,000 Da.

Optionally, the antibody conjugate has the following structure:

,

wherein each heavy chain of the anti-VEGF-A antibody is designated by the letter H and each light chain of the anti-VEGF-A antibody is designated by the letter L; the polymer is linked to the anti-VEGF-A antibody via the sulfhydryl group C449 of SEQ ID NO: 1, wherein this linkage is depicted on one of the heavy chains; PC is , where the wavy line indicates the point of attachment to the rest of the polymer; where 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 plus or minus 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, a monosubstituted, polysubstituted, or unsubstituted version of the following moieties: saturated C ₁ -C ₂₄ alkyl, unsaturated C ₂ -C ₂₄ alkenyl, or C ₂ -C ₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano and halogenated alkyl including polyhalogenated alkyl, -CO-OR ₇ , carbonyl -CCO-R ₇ , -CO-NR ₈ R ₉ , -(CH ₂ ) _n -COOR ₇ , -CO-(CH) _n -COOR ₇ , -(CH ₂ ) _n -NR ₈ R ₉ , ester, alkoxycarbonyl, aryloxycarbonyl, where n is an integer from 1 to 6, wherein each of R ₇ , R ₈ and R ₉ is individually selected from the group consisting of a hydrogen atom, a halogen atom, a monosubstituted, polysubstituted or unsubstituted version of the following residues: saturated C ₁ -C ₂₄ alkyl, unsaturated C _{2 -C24} alkenyl or _C2 - _C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboxyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano and halogenated alkyl including polyhalogenated alkyl, 5-membered ring and 6-membered ring.

Optionally, the antibody conjugate has the following structure:

where each heavy chain of the anti-VEGF-A antibody is designated by the letter H, and each light chain of the anti-VEGF-A antibody is designated by the letter L; the polymer is linked to the anti-VEGF-A antibody via the sulfhydryl group C443 (EU numbering), this linkage is depicted on one of the heavy chains; PC is , where the wavy line indicates the point of attachment to the rest of the polymer; where 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 plus or minus 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, a monosubstituted, polysubstituted, or unsubstituted version of the following moieties: saturated C ₁ -C ₂₄ alkyl, unsaturated C ₂ -C ₂₄ alkenyl, or C ₂ -C ₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarbonyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano and halogenated alkyl including polyhalogenated alkyl, -CO-OR ₇ , carbonyl -CCO-R ₇ , -CO-NR ₈ R ₉ , -(CH ₂ ) _n -COOR ₇ , -CO-(CH) _n -COOR ₇ , -(CH ₂ ) _n -NR ₈ R ₉ , ester, alkoxycarbonyl, aryloxycarbonyl, where n is an integer from 1 to 6, wherein each of R ₇ , R ₈ and R ₉ is individually selected from the group consisting of a hydrogen atom, a halogen atom, a monosubstituted, polysubstituted or unsubstituted version of the following residues: saturated C ₁ -C ₂₄ alkyl, unsaturated C _{2 -C24} alkenyl or _C2 - _C24 alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboxyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano and halogenated alkyl including polyhalogenated alkyl, 5-membered ring and 6-membered ring.

In some embodiments, the antibody conjugate is provided as described above in a liquid solution. Optionally, the liquid solution comprises a pharmaceutically acceptable carrier.

In some embodiments, an anti-VEGF-A antibody is provided that comprises 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) and position 221 (by sequential counting as in SEQ ID NO: 3) is T and 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 position 4 according to Kabat is L. Optionally, the heavy chain isotype is IgG1, wherein the IgG1 constant domain has one or more of the following mutations to modulate effector function (EU numbering): E233P, L234V, L234A, L235A, G237A, A327G, A330S and P331S. Optionally, the antibody has (EU numbering) L234A, L235A and G237A. Optionally, the heavy chain sequence of the anti-VEGF-A antibody is SEQ ID NO: 1 and the light chain sequence of the anti-VEGF-A antibody is SEQ ID NO: 2.

In some embodiments, a method of treating or preventing an ophthalmological disease is provided. The method comprises administering an antibody conjugate as described above or a pharmaceutical composition as described above. Optionally, the ophthalmological 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), branch retinal vein occlusion (BRVO), corneal neovascularization, retinal neovascularization, retinopathy of prematurity (ROP), subconjunctival hemorrhage and hypertensive retinopathy. Optionally, the disease is diabetic retinopathy.

In some embodiments, a method is provided for producing an antibody conjugate comprising an anti-VEGF-A antibody conjugated to a polymer comprising phosphorylcholine. The method comprises the step of: conjugating an anti-VEGF-A antibody to a polymer comprising phosphorylcholine; wherein the anti-VEGF-A antibody comprises a cysteine residue added using recombinant DNA technology, and wherein the cysteine is outside the variable region of the antibody; wherein the polymer comprising phosphorylcholine comprises a sulfhydryl-specific reactive group selected from the group consisting of maleimide, vinyl sulfone, orthopyridyl disulfide, and iodoacetamide; and wherein the sulfhydryl-specific reactive group on the polymer comprising phosphorylcholine reacts with the cysteine residue on the anti-VEGF-A antibody to produce the antibody conjugate.

Optionally, the anti-VEGF-A antibody is immunoglobulin G (IgG) and the cysteine is in the Fc region of the antibody. Optionally, the anti-VEGF-A antibody comprises a light chain and a heavy chain, wherein the heavy chain of the anti-VEGF-A antibody 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), and position 221 (by sequential counting as in SEQ ID NO: 3) is T, and the light chain of the anti-VEGF-A antibody 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 position 4 according to Kabat is L. Optionally, the heavy chain isotype of the antibody against VEGF-A is IgG1.

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

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

Optionally, the phosphorylcholine-containing polymer comprises 2-(methacryloyloxyethyl)-2'-(trimethylammonium)ethyl phosphate (MPC) monomers as shown below:

,

So, the polymer includes the following repeating units:

where n is an integer from 1 to 3000 and the wavy lines indicate the attachment points between monomer units in the polymer.

Optionally, the polymer has three 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.

Optionally, the polymer has a molecular weight of from about 300,000 to 1,750,000 Da. Optionally, the polymer has a molecular weight of from about 500,000 to 1,000,000 Da. Optionally, the polymer has a molecular weight of from about 600,000 to 800,000 Da.

Optionally, the method comprises an additional step comprising the step of reacting the anti-VEGF-A antibody with a thiol reducing agent under conditions that produce a reduced sulfhydryl group of cysteine to form 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 ( _NaBH4 ), sodium cyanoborohydride ( _NaCNBH3 ), β-mercaptoethanol (BME), cysteine hydrochloride, and cysteine. Optionally, the thiol reducing agent is TCEP. Optionally, the thiol reducing agent is in 1-fold to 100-fold molar excess relative to the concentration of the anti-VEGF-A antibody. Optionally, the thiol reducing agent is in 20-fold to 50-fold molar excess relative to the concentration of the anti-VEGF-A antibody.

Optionally, the method further comprises 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. Optionally, the oxidizing agent is air, aqueous _CuSO4 , or dehydroascorbic acid (DHA). Optionally, the method further comprises the step of purifying the antibody conjugate.

Optionally, the antibody conjugate is purified by a method selected from the group consisting of ion exchange chromatography, hydrophobic chromatography, size exclusion chromatography, affinity chromatography, and combinations thereof. Optionally, the purified antibody conjugate retains at least 20% of the biological activity relative to an unconjugated anti-VEGF-A antibody. Optionally, the purified antibody conjugate retains at least 50% of the biological activity relative to an unconjugated anti-VEGF-A antibody. Optionally, the purified antibody conjugate retains at least 90% of the biological activity relative to an unconjugated anti-VEGF-A antibody. Optionally, the purified antibody conjugate has an increased half-life relative to an unconjugated anti-VEGF-A antibody. Optionally, the purified antibody conjugate has at least a 1.5-fold increase in half-life relative to an unconjugated anti-VEGF-A antibody.

Optionally, the method further comprises the step of polymerizing a free radically polymerizable monomer containing phosphorylcholine in a polymerization medium to produce a polymer containing phosphorylcholine, the medium comprising: a radically polymerizable monomer containing phosphorylcholine; a transition metal catalyst M _t ^(q-1)+ , where M _t is a transition metal, q is the highest oxidation state of the metal, and q-1 is the oxidation state of the metal where the metal can act as a catalyst, the transition metal catalyst being supplied as a salt of the form M _t ^(q-1)+ X' _(q-1) , where X' is a counterion or group, or where the transition metal catalyst is supplied in situ by providing a salt of the inactive metal in its highest oxidation state M _t ^q+ X' _q together with a reducing agent that is capable of reducing the transition metal from an oxidized inactive state to a reduced active state; a ligand; and an initiator.

Optionally, the radically polymerizable phosphorylcholine-containing monomer is

,

where R1 is H or C _1-6 alkyl; each of R2, R3, R4 is methyl; and each of X and Y is 2.

Optionally, Mt 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 supplied as a salt of the form _Mt ^(q-1)+ X' _(q-1) . Optionally, M _t ^{(q-1) +} is selected from the group consisting of 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, (SO ₄ ) _1/2 , (PO ₄ ) _1/3 , (R7PO ₄ ) _1/2 , (R7 ₂ PO ₄ ), triflate, hexafluorophosphate, methanesulfonate, arylsulfonate, CN and R7CO ₂ , wherein R7 is H or a straight or branched C _1-6 alkyl group which may be substituted with one to five halogen atoms. Optionally, M _t ^(q-1)+ is Cu ¹⁺ and X' is Br. Optionally, M _t ^(q-1)+ is supplied in situ. Optionally, M _t ^q+ X _q is CuBr ₂ .

Optionally, the reducing agent is an inorganic compound. Optionally, the inorganic compound is selected from the group consisting of a sulfur compound with a low oxidation state, sodium hydrosulfite, sodium sulfite, an inorganic salt containing a metal ion, a metal, hydrazine hydrate, and derivatives of such compounds.

Optionally, the reducing agent is a metal. Optionally, the reducing agent is Cu ⁰ .

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

Optionally, the ligand is selected from the group consisting of 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-pyridyl)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.

Optionally, the initiator has the structure:

where R1 is a nucleophilic reactive group, R2 contains a linker, and R3 contains a polymer synthesis initiator moiety having the structure

,

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 from 1 to 20.

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, alkenylene, alkynyl, alkynylene, cycloalkylene, heterocycloalkyl, heterocycloalkylene, aryl, arylene, aryleneoxy, heteroaryl, amino, amido, and any combination thereof. Optionally, R2 is

,

where X and Y are the same or different and are integers from 1 to 20.

Optionally, each of X and Y is 4. Optionally, R3 additionally includes

,

where R6, R7 and R8 are the same or different and are selected from the group consisting of

,

And

,

where Z is NCS, F, Cl, Br, or I. Optionally, Z is Br. Optionally, each of R6, R7, and R8 is

.

Not necessarily, the initiator has a structure

,

where 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.

Optionally, the method further comprises the step of reacting the polymer with a maleimide reagent to produce a polymer having a terminal maleimide. Optionally, the maleimide compound is

In some embodiments, the embodiments provided herein help to avoid one or more problems associated with other methods or forms of treatment. For example, they can reduce the frequency of intravitreal injection to less than once a month, since such intravitreal injections can be painful and require a clinical setting. In addition, patients with diabetic retinopathy whose vision is relatively unimpaired can be resistant to monthly intravitreal injections and, thus, other forms of treatment can become ineffective. Thus, there is a need for a treatment for diabetic retinopathy with less frequent dosing. In this context, treatment for diabetic retinopathy can be used early in the disease to prevent disease progression and associated vision-threatening events.

In some embodiments, the antibody conjugate comprises (1) an anti-VEGF-A antibody and (2) a polymer comprising phosphorylcholine. The polymer is covalently linked to the antibody via a cysteine outside the variable region of the antibody, and the cysteine is added using recombinant DNA technology.

In some embodiments, the heavy chain of the anti-VEGF-A antibody 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), and the light chain of the anti-VEGF-A antibody 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).

In some embodiments, the polymer conjugated to the antibody has a molecular weight of from about 300,000 to about 1,750,000 Da, as determined by size exclusion chromatography with multi-angle light scattering (hereinafter "SEC-MALS").

In some embodiments, the anti-VEGF-A antibody comprises 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), and the light chain comprises CDRL1: SASQDISNYLN (SEQ ID NO: 12), CDRL2: FTSSLHS (SEQ ID NO: 13), and CDRL3: QQYSTVPWT (SEQ ID NO: 14), and the heavy chain isotype is IgG1. The constant domain of IgG1 has one or more of the following mutations to reduce effector function (EU numbering): E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S.

In some embodiments, any of the methods may employ an anti-VEGF-A antibody that comprises a light chain and a heavy chain, wherein the heavy chain of the anti-VEGF-A antibody 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), and the light chain of the anti-VEGF-A antibody 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).

In some embodiments, the antibody comprises a heavy chain variable region amino acid sequence that comprises SEQ ID NO: 1 and a light chain variable region amino acid sequence that comprises SEQ ID NO: 2.

In some embodiments, the antibody is human IgG1 and the heavy chain constant domain comprises one or more mutations that reduce immune-mediated effector function.

In some embodiments, the antibody is further conjugated to a polymer to form a bioconjugate, wherein the bioconjugate has a molecular weight of from about 450,000 to about 1,900,000 Da.

In some embodiments, the polydispersity index (PI) is equal to or less than 1.5.

In some embodiments, an antibody that binds VEGF-A comprises CDR _H 1, which is CDR _H 1 of SEQ ID NO: 1; CDR _H 2, which is CDR _H 2 of SEQ ID NO: 1; CDR _H 3, which is CDR _H 3 of SEQ ID NO: 1; CDR _L 1, which is CDR _L 1 of SEQ ID NO: 2; CDR _L 2, which is CDR _L 2 of SEQ ID NO: 2; CDR _L 3, which is CDR _L 3 of SEQ ID NO: 2; 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).

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

In some embodiments, a method for producing a conjugated protein comprises reducing one or more cysteines in a protein to form a decapped protein in solution, reoxidizing the decapped protein to reduce at least one disulfide bond in the reduced protein, while ensuring that an engineered cysteine residue in the protein remains in a free thiol form to thereby form a reoxidized decapped protein in solution, and adding at least one excipient to the solution, wherein the excipient reduces polymer-induced protein precipitation. The method further comprises adding a polymer to the solution and conjugating the polymer to the reoxidized decapped protein at the engineered cysteine residue to form the conjugated protein. In some embodiments, the protein is an antibody, a fusion protein comprising an antibody, or a 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 polymer with the reduced protein occurs in an aqueous medium at a pH of 6.0 to 8.5. In some embodiments, the amount of reduced protein is less than the amount of polymer. In some embodiments, the conjugation of the polymer to the protein is carried out at a temperature of 2 to 37 °C. In some embodiments, the method further comprises a method of reacting a solution comprising the conjugated protein with an ion exchange chromatography or hydrophobic interaction chromatography medium, or with an affinity chromatography medium. In some embodiments, the ion exchange chromatography or hydrophobic interaction chromatography medium, or the 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 includes a PEG linker connecting the center of the branch point of the polymer to a maleimide functional group.

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

In some embodiments, an anti-VEGF antibody conjugate is provided that blocks at least 95% of the interaction between a VEGF ligand and a VEGF receptor.

In some embodiments, an anti-VEGF antibody is provided that blocks at least 90% of the interaction between a VEGF ligand and a VEGF receptor.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1 shows the L connection.

Fig. 2 shows the K connection.

Fig. 3 shows the production of OG1802 from R3707.

Fig. 4 shows OG1786.

Fig. 5 shows the preparation of OG1546 from OG1550.

Fig. 6 shows the preparation of OG1784 from OG1546 and OG1563.

Fig. 7 shows the preparation of OG1405 from OG1784.

Fig. 8 shows the preparation of OG 1785 from OG1405.

Fig. 9 shows the preparation of OG1786 from OG1785.

Fig. 10 shows OG1802.

Fig. 11 shows connection E.

Fig. 12 shows some embodiments of the heavy chain of an anti-VEGF-A antibody with some effector function mutations and L443C (EU numbering, which represents position 449 in SEQ ID NO: 1).

Fig. 13 shows some embodiments of the light chain of the anti-VEGF-A antibody (SEQ ID NO: 2).

Fig. 14 shows some embodiments of the heavy chain of bevacizumab (SEQ ID NO: 3).

Fig. 15 shows some embodiments of the light chain of bevacizumab (SEQ ID NO: 4).

Fig. 16 shows some embodiments of the heavy chain of ranibizumab (SEQ ID NO: 5).

Fig. 17 shows some embodiments of the light chain of ranibizumab (SEQ ID NO: 6).

Fig. 18 shows some embodiments of the method for producing an antibody conjugate.

Fig. 19 shows the ion exchange analysis (A280 absorption) of reactions A through G.

Figure 20 shows the effect of different anti-VEGF molecules on biotin-VEGF binding to plate-bound VEGFR ECD-Fc protein and their IC ₅₀ values.

Figure 21 shows the binding affinity of OG1950 to VEGF measured by BIAcore single cycle kinetics.

Fig. 22 shows the binding of OG1950 to Fc gamma receptor I.

Fig. 23 shows the binding of OG1950 to Fc-gamma receptor IIIa.

Fig. 24 shows the binding of QG1950 to the human complement protein C1q.

Fig. 25 shows the results of the proliferation assay (including IC ₅₀ values).

Figure 26 shows the results of single-cycle kinetics of VEGF binding to anti-VEGF agents.

Fig. 27 shows some embodiments of nucleic acid sequences encoding variable regions of heavy and light chains.

Fig. 28 shows the results of screening after incubation of different samples (different excipients) in a polymer solution (OG1802) for 20 hours at a temperature of 2 to 8 °C.

DETAILED DESCRIPTION OF PREFERRED IMPLEMENTATION OPTIONS

The present invention relates to antibodies against VEGF-A. In some embodiments, these antibodies can be conjugated to a moiety that increases the half-life. In some embodiments, the conjugate can be used to treat certain conditions, such as diabetic retinopathy and/or age-related macular degeneration.

The present invention also relates to methods for producing conjugate compositions of antibodies (any type of antibody). In some embodiments, these methods provide reduced formation of aggregates or higher efficiency of formation of the desired antibody conjugate.

These and additional embodiments are disclosed below, following the definitions section.

Definitions

"Neovascular disease" is a disease or illness characterized by altered, dysregulated, or unregulated angiogenesis. Examples of neovascular diseases include neoplastic transformation (eg, cancer) and ophthalmologic neovascular diseases, including diabetic retinopathy and age-related macular degeneration.

"Ophthalmic neovascular" disease is a disease characterized by altered, dysregulated, or unregulated angiogenesis in the patient's eyes. Such disorders include optic disc neovascularization, iris neovascularization, retinal neovascularization, choroidal neovascularization, corneal neovascularization, vitreous neovascularization, glaucoma, pannus, pterygium, macular edema, diabetic retinopathy, diabetic macular edema, vascular retinopathy, retinal degeneration, uveitis, inflammatory retinal diseases, and proliferative vitreoretinopathy.

The term antibody includes intact antibodies and binding fragments thereof. A binding fragment refers to a molecule, other than an intact antibody, that contains a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of binding fragments include Fv, Fab', Fab'-SH, F(ab')2; dimers; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. scFv antibodies are described in Houston JS. 1991. Methods in Enzymol. 203:46-96. Additionally, antibody fragments include single-chain polypeptides having characteristics of a VH domain, namely, the ability to assemble together with a VL domain, or a VL domain, namely, the ability to assemble together with a VH domain into a functional antigen-binding site, thereby providing the antigen-binding property of full-length antibodies.

Specific binding of an antibody to its target antigen(s) means an affinity of at least 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ or 10 ¹⁰ M ^-1 . Specific binding is more readily detectable and distinguishable from non-specific binding, which occurs to at least one unrelated target. Specific binding may result from the formation of bonds between specific functional groups or a specific spatial state (e.g., a "lock and key" type), whereas non-specific binding typically results from van der Waals forces. However, specific binding does not necessarily mean that the antibody or fusion protein binds one and only one target.

The basic structural unit of an antibody is a tetramer of subunits. Each tetramer comprises two identical pairs of polypeptide chains, each pair having one "light" (approximately 25 kDa) and one "heavy" chain (approximately 50 to 70 kDa). The amino-terminal portion of each chain comprises a variable region of approximately 100 to 110 or more amino acids, primarily responsible for antigen recognition. This variable region is initially expressed associated with a cleavable signal peptide. The variable region without the signal peptide is sometimes referred to as the mature variable region. Thus, for example, a mature light chain variable region means a light chain variable region without the light chain signal peptide. However, being referred to as a variable region does not necessarily mean that a signal sequence is present; in fact, signal sequences are cleaved when antibodies or fusion proteins are expressed and secreted. The pair of variable regions of the heavy and light chains defines the binding region of the antibody. The carboxyterminal portion of the light and heavy chains, respectively, defines the constant regions of the light and heavy chains. The constant region of the heavy chain is primarily responsible for effector function. In IgG antibodies, the constant region of the heavy chain is divided into the CH1, hinge, CH2, and CH3 regions. The CH1 region binds to the constant region of the light chain by disulfide and noncovalent bonds. The hinge region provides flexibility between the binding and effector regions of the antibody and provides sites for intermolecular disulfide bonding between the two heavy chain constant regions in the tetrameric subunit. The CH2 and CH3 regions are the major site of effector functions and FcR binding.

Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon and define the antibody isotype as IgG, IgM, IgA, IgD, and IgE, respectively. In the light and heavy chains, the variable and constant regions are joined by a "J" region containing approximately 12 or more amino acids, with the heavy chain also including a "D" region containing approximately 10 or more amino acids (see, Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7) (incorporated by reference in its entirety for all purposes).

The mature variable regions of each light/heavy chain pair form the binding site of the antibody. Thus, an intact antibody has two binding sites, i.e., it is bivalent. In natural antibodies, the binding sites are identical. However, bispecific antibodies can be obtained in which the two binding sites are different (see, for example, 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 JY. 1992. Formation of bispecific antibody by the use of leucine zippers. J Immunol. 148: 1547–1553). All variable regions have the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also called complementarity-determining regions or CDRs. The CDRs from the two chains of each pair are aligned within the framework regions, allowing binding to a specific epitope. From N-terminus to C-terminus, both light and heavy chains comprise the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains. For convenience, the variable heavy chain CDRs may be referred to as CDR _H 1, CDR _H 2, and CDR _H 3; the variable light chain CDRs may be referred to as CDR _L 1, CDR _L 2, and CDR _L 3. The assignment of amino acids to each domain is as defined by Kabat EA, et al. 1987 and 1991. Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD) 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. A widely used numbering system is that proposed by Kabat (Kabat numbering), where corresponding residues between different variable regions of the heavy chain or between different variable regions of the light chain are assigned the same number. Although Kabat numbering can also be used for antibody constant regions, EU numbering is most commonly used, as is the case in the present application. Although specific examples are provided for the illustrative antibodies described herein, it will be understood that upon expression of the protein chains, one to several amino acids at the amino or carboxyl terminus of the light and/or heavy chain, particularly at the C-terminal lysine residue of the heavy chain, may be absent or derivatized proportionally, or throughout the molecules.

The term "epitope" refers to a site on an antigen to which an antibody or the extracellular segment of a decoy binds. An epitope on a protein may be formed from nonessential amino acids or essential amino acids linked by tertiary folding of one or more proteins. Epitopes formed from nonessential amino acids (also known as linear epitopes) are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also known as conformational epitopes) are generally destroyed upon treatment with denaturing solvents. An epitope typically includes at least 3 and usually at least 5 or 8 to 10 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. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

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

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

Competition between antibodies is determined by an assay in which a test antibody inhibits specific binding of a reference antibody to a common antigen (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 2x, 5x, 10x, 20x, or 100x) inhibits binding of the reference antibody by at least 50%. In some embodiments, a test antibody inhibits binding of the reference antibody by 75%, 90%, or 99%, as determined in a competitive binding assay. Antibodies detected in a competition assay (competing antibodies) include antibodies that bind the same epitope as the reference antibody and antibodies that bind an adjacent epitope that is close enough to the epitope bound by the reference antibody to cause steric hindrance.

The term "patient" includes humans and other mammalian subjects receiving either prophylactic or therapeutic treatment.

For the purposes of classifying amino acid substitutions as either conservative or nonconservative, 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 affecting chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids of the same class. Nonconservative substitutions involve the substitution of a member of one of these classes by a member of the other.

The percentage of sequence identities is determined by the antibody sequence being aligned maximally using the Kabat numbering system for the variable region or the EU numbering system for the constant region. After alignment, if a region of a subject antibody (e.g., the entire mature variable region of the heavy or light chain) is compared to the same region of a reference antibody, the percentage of sequence identity between the regions of the subject antibody and the reference antibody is equal to the number of positions occupied by the same amino acid in both the subject antibody region and the reference antibody region divided by the total number of aligned positions of the two regions, with gaps ignored, multiplied by 100 to convert to a percentage. Sequence identities of other sequences can be determined by aligning the sequences using algorithms such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI, using the default skip parameters, or by examining and selecting the best alignment (i.e., the one that results in the highest percent sequence similarity in the comparison window). The percent sequence identity is calculated by comparing the two best aligned sequences in the comparison window, determining the number of positions at which the same residues occur in both sequences to obtain the number of compatible positions, dividing the number of compatible 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 percent sequence identity.

Compositions or methods "comprising" one or more of the stated elements may include other elements not specifically stated. For example, a composition that includes an antibody may contain the antibody alone or in combination with other substances.

The term "antibody-dependent cellular cytotoxicity" or ADCC is a mechanism of cell death induction that depends on the interaction of antibody-laden target cells (i.e., antibody-bound cells) with immune cells that have lytic activity (also called effector cells). Such effector cells include natural killer cells, monocytes/macrophages, and neutrophils. ADCC is initiated by the interaction between the Fc region of the cell-bound antibody and the Fcγ receptors, particularly FcγRI and FcγRIII, on immune effector cells such as neutrophils, macrophages, and natural killer cells. The target cell is eliminated by phagocytosis or lysis, depending on the type of effector cell. The death of the antibody-laden target cell occurs as a result of the activity of the effector cells.

The term opsonization, also known as "antibody-dependent cellular phagocytosis" or ADCP, refers to the process by which antibody-laden cells are engulfed, in whole or in part, by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc region of immunoglobulin.

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

A humanized antibody is a genetically engineered antibody in which the CDRs from a non-human "donor" antibody are grafted onto "acceptor" sequences of a human antibody (see, for example, Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539, Carter, US 6,407,213, Adair, US 5,85,205, 6,881,557, Foote, US 6,888,557). The acceptor sequences of the antibody may be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Thus, a humanized antibody is an antibody having some or all of the CDRs entirely or substantially from a donor antibody, and the variable region and constant region framework sequences, if present, entirely or substantially from human antibody sequences. Similarly, a humanized heavy chain has at least one, two, and typically all three CDRs entirely or substantially from a heavy chain of a donor antibody, and the heavy chain variable region and heavy chain constant region framework sequences, if present, essentially from human heavy chain variable region and constant region framework sequences. Similarly, a humanized light chain has at least one, two, and typically all three CDRs entirely or substantially from a light chain of a donor antibody, and the light chain variable region and light chain constant region framework sequences, if present, essentially from human light chain variable region and constant region framework sequences. In addition to nanobodies and dAb, the humanized antibody comprises a humanized heavy chain and a humanized light chain. A CDR in a humanized antibody is substantially derived from a corresponding CDR in a non-human antibody when at least 85%, 90%, 95% or 100% of the corresponding residues (as determined by Kabat) are identical between the corresponding CDRs. The framework sequences of the antibody chain variable region or the antibody chain constant region are substantially derived from a framework sequence of a human variable region or human constant region, respectively, when at least 85%, 90%, 95% or 100% of the corresponding residues, as determined by Kabat, are identical.

[0113] Although humanized antibodies often include all six CDRs (which may be as defined by Kabat) from a mouse antibody, they can also be produced with fewer of all CDRs (e.g., at least 3, 4, or 5 CDRs from a mouse antibody) (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 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).

A chimeric antibody is an antibody in which the mature variable regions of the light and heavy chains of a non-human (e.g., mouse) antibody are combined with the constant regions of a human light and heavy chain. Such antibodies retain substantially or completely the binding specificity of a mouse antibody and are approximately two-thirds of the human sequence.

A veneered antibody is a type of humanized antibody that retains some and usually all of the CDRs and some of the non-human framework residues of the variable region of a non-human antibody but replaces other framework residues of the variable region that may participate in B- 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), with residues from the corresponding positions of the human antibody sequence. The result is an antibody in which all or substantially all of the CDRs are from the non-human antibody and the variable region frameworks of the non-human antibody are made more human-like by the substitutions. The human antibody may be isolated from a human or otherwise produced by expression of human immunoglobulin genes (e.g., in a transgenic mouse, in vitro, or by phage display). Methods for producing human antibodies include the trioma method of Östberg L, Pursch E. 1983. Human x (mouse x human) hybridomas stably producing human antibodies. Hybridoma 2:361-367; Östberg, US Patent 4,634,664; and Engleman et al., US Patent 4,634,666; the use of transgenic mice comprising human immunoglobulin genes (see, for example, Lonberg et al., W093/12227 (1993); US 5,877,397, US 5,874,299, US 5,814,318, US 5,789,650, US 5,770,429, US 5,661,016, US 5,633,425, US 5,625,126, US 5,569,825, US 5,545,806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91/10741 (1991) and phage display methods (see, for example, Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047, US 5,877,218, US 5,871,907, US 5,858,657, US 5,837,242, US 5,733,743 and US 5,565,332).

"Polymer" refers to a number of monomer groups linked together. A polymer is composed of multiple units of a single monomer (homopolymer) or different monomers (heteropolymer). High molecular weight polymers are prepared from monomers that include, but are not limited to, acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl pyridine, vinyl pyrrolidone, and vinyl esters such as vinyl acetate. Additional monomers are useful in 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. A polymer may be linear or branched. When a polymer is branched, each polymer chain is called a "polymer arm." The end of the polymer arm bound to the initiator moiety is the proximal end, and the growing end of the polymer arm chain is the distal end. At the growing end of a polymer arm chain, the terminal group of the polymer arm may be a radical acceptor or another group.

"Initiator" refers to a compound capable of initiating polymerization using monomers or comonomers. The polymerization may be conventional free radical polymerization or controlled/"living" radical polymerization such as atom transfer radical polymerization (ATRP), reversible addition fragmentation polymerization (RAFT), or nitroxyl stable radical polymerization (NMP). The polymerization may be "pseudo" controlled polymerization such as degenerative transfer. When the initiator is suitable for ATRP, it contains a labile bond that can be homolytically cleaved to form an initiator moiety I, which is a radical capable of initiating radical polymerization, and a radical acceptor I', which reacts with the radical of the growing polymer chain to reversibly terminate the polymerization. The radical acceptor I' is usually a halogen, but may 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 via ATRP.

"Chemical linker" refers to a chemical moiety that links two groups together, such as a half-life enhancing moiety and a protein. The linker may be cleavable or non-cleavable. Cleavable linkers may be hydrolyzable, enzymatically cleavable, pH sensitive, photolabile, or disulfide linkers, among others. 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 with a bioactive agent. Non-limiting examples include those illustrated in Table 1 of WO2013059137 (incorporated by reference).

The term "reactive group" refers to a group that is capable of reacting with another chemical group to form a covalent bond, i.e., is covalently reactive under suitable reaction conditions and typically represents a point of attachment for another substance. A reactive group is a moiety, such as a maleimide or succinimidyl ester, that is capable of chemically reacting with a functional group on another moiety to form a covalent bond. Reactive groups typically include nucleophiles, electrophiles, and photoactivatable groups.

"Phosphorylcholine," also referred to as "PC," refers to:

where * denotes the point of attachment. Phosphorylcholine is a zwitterionic group and includes salts (such as inner salts) and their protonated and deprotonated forms.

"A polymer containing phosphorylcholine" is a polymer that contains phosphorylcholine. "A polymer containing a zwitterion" refers to a polymer that contains a zwitterion.

The polymer containing poly(acryloyloxyethylphosphorylcholine) refers to a polymer containing 2-(acryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate (HEA-PC, shown below in Example 6) as a monomer.

Poly(methacryloyloxyethylphosphorylcholine) containing polymer refers to a polymer containing 2-(methacryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate (HEMA-PC or MPC) as a monomer (see below):

The terms "MPC" and "HEMA-PC" are used here interchangeably.

"Molecular weight" in the context of a polymer may be expressed as number average molecular weight or weight average molecular weight or peak molecular weight. Unless otherwise specified, all references to molecular weight herein refer to peak molecular weight. These molecular weight definitions, number average ( _Mn ), weight average ( _Mw ) and peak ( _Mp ), may be measured using size exclusion chromatography or other liquid chromatographic techniques. Other methods of measuring molecular weight values may also be used, such as using end group analysis or measuring colligative properties (e.g., freezing point depression, boiling point elevation, or osmotic pressure) to determine number average molecular weight, or using light scattering, ultracentrifugation, or viscosity techniques to determine weight average molecular weight. In some embodiments, the molecular weight is measured using SEC-MALS (size exclusion chromatography with multi-angle light scattering). In some embodiments, the polymeric reactants are typically polydisperse (i.e., the number average molecular weight and the weight average molecular weight of the polymers are not equal) and may have low polydispersity values, such as less than about 1.5, based on, for example, the PI value obtained by SEC-MALS measurements. In some embodiments, the polydispersity (PI) values are in the range of about 1.4 to about 1.2. In some embodiments, the PI is less than about 1.15, 1.10, 1.05, or 1.03.

The singular of an object refers to one or more such objects; for example, a compound refers to one or more compounds, or at least one compound. Thus, the terms "one or more" and "at least one" may be used interchangeably herein.

"Approximately" refers to the variation that can be seen in measurements taken between different instruments, samples, and sample preparations.

"Protected,""protectedform," and "protecting group" refer to the presence of a group (i.e., a protecting group) that prevents or blocks the reaction of a particular chemically reactive functional group in a molecule under specified reaction conditions. Protecting groups vary depending on the type of chemically reactive group being protected, as well as the reaction conditions to be used and the presence of additional reactive or protecting groups, if any, in the molecule. Suitable protecting groups include those contained in Greene et al., “Protective Groups In Organic Synthesis,” 3 ^rd Edition, John Wiley and Sons, Inc., New York, 1999.

"Alkyl" refers to a linear or branched, saturated aliphatic radical having the stated number of carbon atoms. For example, C ₁ -C ₆ 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. An alkyl may include any number of carbon atoms, such as 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. An alkyl group is usually monovalent, but may be divalent, such as when an alkyl group links two moieties together.

The term "lower" as used above and below in connection with organic radicals or compounds, respectively, means a compound or radical which may be branched or unbranched with up to and including 7 or up to and including 4 and (as unbranched) one or two carbon atoms.

"Alkylene" refers to an alkyl group, as defined above, linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkylene may be linked to the same atom or different atoms of the alkylene. For example, a straight-chain alkylene may be a divalent radical -( _CH2 ) _n , where n is 1, 2, 3, 4, 5, or 6. Alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene, and hexylene.

Substituents for alkyl and heteroalkyl radicals (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl and heterocycloalkenyl) may be various groups selected from: -OR', =O, =NR', =N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O)R', -C(O)R', -CO ₂ R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O) ₂ R', -NH-C(NH ₂ )=NH, -NR'C(NH ₂ )=NH, -NH-C(NH ₂ )=NR', -S(O)R', -S(O) ₂ R', -S(O) ₂ NR'R'', -CN and -NO ₂ in an amount from zero to (2m' + 1), where m' is the total number of carbon atoms in such radical. R', R" and R"' each independently refers to hydrogen, unsubstituted (C ₁ -C ₈ ) alkyl and heteroalkyl, unsubstituted aryl, aryl substituted by one to three halogen atoms, unsubstituted alkyl, alkoxy or thioalkoxy groups or aryl-(C ₁ -C ₄ ) 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" means 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) _CH2OCH3 , and the like). _In some embodiments, substituted alkyl and heteroalkyl groups have from 1 to ₄ substituents. In some embodiments, substituted acyl and heteroalkyl groups have 1, 2, or 3 substituents. An exception is perhaloalkyl groups (e.g., pentafluoroethyl, and the like).

Substituents of alkyl and heteroalkyl radicals (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl and heterocycloalkenyl) 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', -CO ₂ R', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O) ₂ R'', -NR-C(NR'R''R''')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O) _2R ', -S(O) _2NR'R '', _-NRSO2R ', -CN and _-NO2 in an amount from zero to (2m' + 1), where m' is the total number of carbon atoms in such a radical. each of R', R", R"' and R"" independently refers to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, for example, aryl substituted with one to three halogen atoms, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups or arylalkyl groups. When the compound includes more than one R group, for example, each R group is independently selected, as are each R', R'', R''' and R"" 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" includes, but is not limited to, 1- _pyrrolidinyl and ₄ -morpholinyl. From the above discussion of substituents, one skilled in the art will appreciate that the term "alkyl" refers to groups that include carbon atoms attached 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) _CH2OCH3 , _etc. ).

"Alkoxy" refers to an alkyl group having an oxygen atom that either attaches to the alkoxy group at the point of attachment or is bonded to 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, etc. Alkoxy groups may be further substituted with various substituents as described. For example, alkoxy groups may be substituted with halogens to form a "haloalkoxy" group.

"Carboxyalkyl" means an alkyl group (as defined herein) substituted with a carboxy group. The term "carboxycycloalkyl" means a cycloalkyl group (as defined herein) substituted with a carboxy group. The term "alkoxyalkyl" means an alkyl group (as defined herein) substituted with an alkoxy group. As used herein, the term "carboxy" refers to carboxylic acids and their esters.

"Haloalkyl" refers to an alkyl, as defined above, wherein some or all of the hydrogen atoms are replaced by halogen atoms. Halogen (halo) is chlorine or fluorine, but may also be bromine or iodine. For example, haloalkyl includes trifluoromethyl, fluoromethyl, 1,2,3,4,5-pentafluorophenyl, etc. The term "perfluoro" means a compound or radical that has all of its available hydrogens replaced by 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.

"Fluoro-substituted alkyl" refers to an alkyl group where one, some, or all of the hydrogen atoms are replaced by fluorine.

"Cytokine" is a member of a group of protein signaling molecules that may participate in cell-cell communication in immune and inflammatory responses. Cytokines are typically small, water-soluble glycoproteins with molecular weights of approximately 8 to 35 kDa.

"Cycloalkyl" refers to a cyclic hydrocarbon group that contains from 3 to 12, 3 to 10, or 3 to 7 endocyclic carbon atoms. Cycloalkyl groups include fused, bridged, and spiro ring structures.

"Endocyclic" refers to an atom or group of atoms that form part of a cyclic ring structure.

"Exocyclic" refers to an atom or group of atoms that are attached to but do not define a cyclic ring structure.

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

"Alkenyl" refers to a linear or branched hydrocarbon having from 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 also 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 5 to 6 carbon atoms. An alkenyl group is usually monovalent, but can be divalent, such as when an alkenyl group links two moieties together.

"Alkenylene" refers to an alkenyl group, as defined above, linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkenylene may 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.

"Alkynyl" refers to a linear or branched hydrocarbon containing from 2 to 6 carbon atoms having at least one triple bond. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriinyl. Alkynyl groups can also 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 5 to 6 carbon atoms. An alkynyl group is usually monovalent, but can be divalent, such as when an alkynyl group links two moieties together.

"Alkynylene" refers to an alkynyl group, as defined above, linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkynylene may 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.

"Cycloalkyl" refers to a saturated or partially unsaturated monocyclic fused bicyclic or bridged polycyclic linked ring system 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.

"Cycloalkylene" refers to a cycloalkyl group, as defined above, linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the cycloalkylene may be linked to the same atom or different atoms of the cycloalkylene. Cycloalkylene groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cyclooctylene.

"Heterocycloalkyl" refers to a cyclic system having from 3 ring members to about 20 ring members and from 1 to about 5 heteroatoms such as N, O, and S. Additional heteroatoms may also be useful, including but not limited to B, Al, Si, and P. The heteroatoms may also be oxidized, such as but not limited to -S(O)- and -S(O) _2- . For example, heterocycle includes, but is not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, morpholino, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, piperidinyl, indolinyl, quinuclidinyl, and 1,4-dioxa-8-aza-spiro[4,5]dec-8-yl.

"Heterocycloalkylene" refers to a heterocycloalkyl group, as defined above, linking at least two other groups. The two moieties linked to the heterocycloalkylene may be linked to the same atom or different atoms of the heterocycloalkylene.

"Aryl" refers to a monocyclic or fused bicyclic, tricyclic or higher aromatic linked ring system containing from 6 to 16 carbon atoms in the ring. For example, aryl can be phenyl, benzyl or naphthyl. "Arylene" means a divalent radical derived from an aryl group. Aryl groups can be mono-, di- or trisubstituted with one, two or three radicals selected from alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, aminoalkyl, trifluoromethyl, alkylenedioxy and oxy-C ₂ -C ₃ -alkylene; all of which are optionally further substituted, for example as defined above; or 1- or 2-naphthyl; or 1- or 2-phenanthrenyl. Alkylenedioxy is a divalent substituent attached to two adjacent carbon atoms of phenyl, such as methylenedioxy or ethylenedioxy. Oxy- _C2 - _C3 -alkylene is also a divalent substituent attached to two adjacent carbon atoms of phenyl, such as oxyethylene or oxypropylene. An example of oxy- _C2 - _C3 -alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

In some embodiments, aryl is naphthyl, phenyl, or phenyl mono- or disubstituted with alkoxy, phenyl, halogen, alkyl, or trifluoromethyl, especially phenyl or phenyl mono- or disubstituted with alkoxy, halogen, or trifluoromethyl, and in particular phenyl.

Examples of substituted phenyl groups as R are, for example, 4-chlorophen-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-(imidazol-1-yl)-phenyl, 4-(imidazol-1-ylmethyl)-phen-1-yl, 4-(morpholin-1-yl)-phen-1-yl, 4-(morpholin-1-ylmethyl)-phen-1-yl, 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, optionally substituted in the heterocyclic ring.

"Arylene" refers to an aryl group, as defined above, linking at least two other groups. The two moieties linked to the arylene are linked to different atoms of the arylene. Arylene groups include, but are not limited to, phenylene.

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

Similarly, the substituents for the aryl and heteroaryl groups are different and are selected from: -halogen, -OR', -OC(O)R', -NR'R", -SR', -R', -CN, -NO ₂ , -CO ₂ R', -CONR'R'', -C(O)R', -OC(O)NR'R'', -NR''C(O)R', -NR''C(O) ₂ R', -NR'-C(O)NR''R''', -NH-C(NH ₂ )=NH, -NR'C(NH ₂ )=NH, -NH-C(NH ₂ )=NR', -S(O)R', -S(O) ₂ R', -S(O) ₂ NR'R'', -N ₃ , -CH(Ph) ₂ , perfluoro(C ₁ -C ₄ )alkoxy and perfluoro(C ₁ -C ₄ )alkyl, in an amount from zero to the total number of open valences in the aromatic ring system; and where R', R" and R"' are independently selected from hydrogen, (C ₁ -C ₈ )alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C ₁ -C ₄ )alkyl and (unsubstituted aryl)oxy-(C ₁ -C ₄ )alkyl.

Two substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally substituted with a substituent of the formula -TC(O)-(CH ₂ ) _q -U-, where T and U are independently -NH-, -O-, -CH ₂ -, or a single bond, and q is an integer from 0 to 2. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally substituted with a substituent of the formula -A-(CH ₂ ) _r -B-, where A and B are independently -CH ₂ -, -O-, -NH-, -S-, -S(O)-, -S(O) ₂ -, -S(O) ₂ NR', or a single bond, and r is an integer from 1 to 3. One of the single bonds of the new ring thus formed may optionally be replaced by a double bond. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally substituted with a substituent of the formula -( _CH2 ) _s -X-( _CH2 ) _t- , 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'- . The substituent R' in -NR'- and -S(O) _2NR'- is selected from hydrogen or unsubstituted ( _C1 - _C6 )alkyl.

"Heteroaryl" refers to a monocyclic or fused bicyclic or tricyclic aromatic linked ring system containing from 5 to 16 ring atoms, wherein each of 1 to 4 ring atoms is an N, O or S heteroatom. 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 radicals substituted, especially mono- or disubstituted, for example by alkyl, nitro or halogen. Pyridyl is 2-, 3- or 4-pyridyl, preferably 2- or 3-pyridyl. Thienyl is 2- or 3-thienyl. In some embodiments, quinolinyl is 2-, 3- or 4-quinolinyl. In some embodiments, isoquinolinyl is 1-, 3- or 4-isoquinolinyl. In some embodiments, benzopyranyl, benzothiopyranyl can be 3-benzopyranyl or 3-benzothiopyranyl, respectively. In some embodiments, thiazolyl can be 2- or 4-thiazolyl. In some embodiments, triazolyl can be 1-, 2- or 5-(1,2,4-triazolyl). In some embodiments, tetrazolyl can be 5-tetrazolyl.

In some embodiments, the 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 radicals, especially mono- or disubstituted.

The term "heteroalkyl" refers to an alkyl group having from 1 to 3 heteroatoms, such as N, O, and S. Additional heteroatoms may also be applicable, including, but not limited to, B, Al, Si, and P. Heteroatoms may also be oxidized, such as, but not limited to, -S(O)- and -S(O) _2- . For example, heteroalkyl may include ethers, thioethers, alkylamines, and alkylthiols.

The term "heteroalkylene" refers to a heteroalkyl group, as defined above, linking at least two other groups. The two moieties linked to the heteroalkylene may be linked to the same atom or different atoms of the heteroalkylene.

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

"Nucleophile" refers to an ion or an atom, or a collection of atoms, that may be ionic, that has a nucleophilic center, i.e., a center that seeks an electrophilic center or is capable of reacting with an electrophile. A nucleophile (or nucleophilic reagent) is a reagent that forms a bond with its reaction partner (an electrophile), acting as a donor of both bonding electrons. "Nucleophilic group" refers to a nucleophile after reaction with a reactive group. Non-limiting examples include amino, hydroxyl, alkoxy, haloalkoxy, and the like.

"Maleimido" refers to the pyrrol-2,5-dione-1-yl group having the structure:

which, when reacted with a sulfhydryl (e.g., thioalkyl), forms an -S-maleimido group having the structure

where "•" indicates the point of attachment for the maleimido group and " " indicates the point of attachment of the sulfur atom by the thiol to the rest of the original sulfhydryl group carrier.

For purposes of the present 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 acid other than naturally occurring amino acids. Unnaturally occurring amino acids include, but are not limited to, the D-isomers of naturally occurring amino acids and mixtures of the D- and L-isomers of naturally occurring amino acids. Other amino acids, such as N-alpha-methyl amino acids (e.g., sarcosine), 4-hydroxyproline, desmosine, isodesmosine, 5-hydroxylysine, epsilon-N-methyllysine, 3-methylhistidine, although found in natural proteins, are considered non-natural amino acids found in proteins for the purposes of the present disclosure since they are typically introduced by means other than ribosomal translation of mRNA.

"Linear" in reference to the geometry, architecture, or overall structure of a polymer refers to a polymer having a single polymer arm.

"Branched" in terms of the geometry, architecture, or overall structure of a polymer refers to a polymer having 2 or more polymer "arms" extending from a core structure contained in an initiator. The initiator can be used in an atom transfer radical polymerization (ATRP) reaction. A branched polymer can have two polymer chains (arms), 3 polymer arms, 4 polymer arms, 5 polymer arms, 6 polymer arms, 7 polymer arms, 8 polymer arms, 9 polymer arms, or more. Each polymer arm extends from a polymer initiation site. Each polymer initiation site is capable of being a site for polymer chain growth by the addition of monomers. For example, and without limitation, using ATRP, the polymer initiation site on the initiator is typically an organic halide that undergoes a reversible redox reaction catalyzed by a transition metal compound such as a copper halide. In some embodiments, the halide is bromine.

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

Therapeutic proteins are administered in an effective regimen, which means a dosage, route of administration, and frequency of administration that delays the onset, reduces the severity, inhibits further worsening, and/or improves at least one sign or symptom of the disease. If the patient already has the disease, the regimen may be called a therapeutically effective regimen. If the patient is at increased risk for the disease compared to the general population but is not yet symptomatic, the regimen may be called a prophylactically effective regimen. In some cases, therapeutic or prophylactic efficacy may be observed in an individual patient compared to historical controls or past experience in the same patient. In other cases, therapeutic or prophylactic efficacy may be demonstrated in a preclinical or clinical study in a population of treated patients compared to a control population of untreated patients.

The "biological half-life" of a substance is a pharmacokinetic parameter that determines the time required for half of a substance to be eliminated from a tissue or organism after administration of that substance.

"OG1786" is a 9-arm initiator used to synthesize a polymer with the structure shown in Fig. 30, which shows the trifluoroacetic acid salt form of OG1786. OG1786 can be used as other salts or as a free base.

"OG1801" is an approximately (plus/minus 15%) 750 kDa polymer (either _Mn or _Mp ) prepared using OG1786 as an initiator for the synthesis of ATRP using HEMA-PC monomer.

"OG1802" is OG1801 with added maleimide functionality and is shown in Fig. 36, where each of _n1 , _n2 , _n3 , _n4 , _n5 , _n6 , _n7 , _n8 , and _n9 is an integer (positive) (from 0 to about 3000) such that the overall molecular weight of the polymer is ( _Mw ) 750,000 plus/minus 15% daltons.

Multi-angle light scattering (MALS) is a method of analyzing macromolecules where laser light is incident on the molecule, the oscillating electric field of the light induces an oscillating dipole in it. This oscillating dipole will re-emit light and can be measured with a MALS detector such as the Wyatt miniDawn TREOS. The intensity of the emitted light depends on the magnitude of the dipole induced in the macromolecule, which in turn is proportional to the polarizability of the macromolecule, the larger the induced dipole and therefore the greater the intensity of the scattered light. Therefore, to analyze the scattering of such macromolecules on a solution, their polarizability relative to the surrounding medium (e.g. solvent) should be known. This can be determined from measuring the change Δn in the refractive index of the solution n with the change in molecular concentration Δc by measuring the dn/dc value (equal to Δn/Δc) using a Wyatt Optilab T-rEX differential refractometer. The two molar mass parameters that use the MALS definition are the number average molecular mass (M_n) and the average molecular weight (M_w), where the polydispersity index (PI) is equal to M_w, divided by M_n. SEC also allows the determination of another average molecular weight value, the peak molecular weight M_p, which is defined as the molecular weight of the highest peak in SEC.

IP is used as a measurement of the molecular weight distribution width of a polymer and a bioconjugate, which is obtained by conjugating a discrete protein (e.g., OG1950) with a polydisperse biopolymer (e.g., OG1802). For a protein sample, its polydispersity is close to 1.0 due to the fact that it is a translation product, where each protein molecule in solution must have almost the same length and molar mass. In contrast, due to the polydisperse nature of a biopolymer, where different lengths of polymer chains are synthesized during the polymerization process, it is very important to determine the IP of a sample as one of its quality attributes for a narrow molecular weight distribution.

Size exclusion chromatography (SEC) is a chromatography technique that separates molecules in a solution based on their size. Typically, an aqueous solution is applied to transport the sample through a column that is filled with resins of varying pore sizes. The resin is expected to be inert to the analyte as it passes through the column, and the analytes will be separated from each other based on their unique size and the pore size characteristics of the selected column.

The combination of SEC and MALS or SEC/MALS methods provides an accurate distribution of molar mass and size (RMS radius) rather than based on the values of SEC calibration standards. This type of distribution has many advantages over traditional column calibration methods. Since light scattering and concentration are measured for each elution fraction, molar mass and size can be determined independently of the elution position. This is especially true for species with non-globular macromolecules such as biopolymers (OG1802) or bioconjugates (OG1953); such species typically do not elute in a manner that can be described by the values of the column calibration standards.

In some embodiments, the SEC/MALS assay includes a Waters HPLC system with an Alliance 2695 solvent feed module and a Waters 2996 Photodiole Array UV detector equipped with a Shodex SEC-HPLC column (7.8 x 300 mm). The system is connected online using a Wyatt miniDawn TREOS differential refractometer and a Wyatt Optilab T-rEX. Waters Empower software can be used to control the Waters HPLC system, and Wyatt ASTRA V 6.1.7.16 software can be used to obtain MALS data from the Wyatt miniDawn TREOS, dn/dc data from the T-rEX detector, and mass data using the A280 absorbance signal from the Waters 2996 Photodiole Array detector. SEC can be performed at 1 ml/min in 1×PBS, pH 7.4, after sample injection, the MALS and RI signals can be analyzed by ASTRA software to determine the absolute molar mass (M _p , M _w , M _n ) and polydispersity index (PI). In addition, the calculation also includes the input dn/dc value for the polymer and protein as 0.142 and 0.183, respectively. For OG1953 bioconjugates, the dn/dc value is calculated based on the weighted MW of the polymer and protein of approximately 0.148, using the formula below:

Conjugate dn/dc = 0.142 × [MWpolymer / (MWpolymer + MWprotein)] + 0.183 × [MWprotein / (MWpolymer + MWprotein)],

where the MWpolymer for OG1802 is 800 kDa and the MWprotein for OG1950 is 146 kDa.

General Provisions

The present invention relates to anti-VEGF antibodies and conjugates thereof. In some embodiments, the antibodies themselves are distinct from other anti-VEGF agents and provide superior results compared to other anti-VEGF agents. In some embodiments, the anti-VEGF antibody conjugate exhibits unexpected superiority over other antibodies and/or the expected activity of other antibody conjugates.

Historically, conjugation of a molecule to a protein has often resulted in a reduction in the binding interactions of the protein with its intended target. In some embodiments of the present invention, when conjugating to a site outside the active site, the same level of reduction that might be expected is not necessarily observed. The evidence presented herein shows the opposite of the expected result. In some embodiments, and without being limited by theory, the conjugate may be superior to a single antibody. For example, the interaction of a ligand and its specific receptor is often accompanied by a stereospecific interaction of the ligand and receptor, consistent with the interaction of hydrophilic amino acids on the ligand with hydrophilic amino acids on the receptor and water molecules located at the beginning and center of these interactions. At the same time, this hydrophilic stereospecificity is further enhanced by weakening and/or suppressing non-specific hydrophobic interactions that may typically be mediated/created by hydrophobic-hydrophobic amino acids.

In some embodiments, there is provided an anti-VEGF antibody conjugate that is capable of blocking at least 90% of the interaction between a VEGF ligand ("VEGFL") and a VEGF receptor ("VEGFR"). For example, it can block at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or effectively all of the interaction between VEGFR and VEGFL. In some embodiments, said blocking occurs at saturating concentrations. In some embodiments, there is provided an anti-VEGF antibody conjugate that blocks at least 95% of the interaction between a VEGF ligand and a VEGF receptor. As an example of such superior blocking, see Fig. 20, regarding the ability of OG1953 (and the antibody conjugate presented herein) to block to a higher degree than Lucentis® (ranibizumab) or Avastin® (bevacizumab), or even the OG1950 antibody (unconjugated). Indeed, this result was unexpected in that, although the addition of a polymer to the antibody (to form an antibody conjugate) might be expected to have some or no negative effect on antibody binding/activity, it was unexpected that it actually improved the blocking ability of the antibody.

In some embodiments, the antibodies or conjugates thereof inhibit 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 IC ₅₀ value can be 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 nM or less than any one or more of the preceding values. In some embodiments, the KD value may be 2× ^10-13 , 1× ^10-13 , 1× ^10-12 , 1× ^10-11 , 1× ^10-10 M, or less than any of the preceding values. In some embodiments, the _IC50 value may be 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, or less than any of the preceding values.

In some embodiments, an anti-VEGF antibody is provided that blocks at least 90% of the interaction between a VEGF ligand and a VEGF receptor. For example, it can block at least 91, 92, 93, 94, 95, 96, 97, 98, 99 or substantially all of the interactions between VEGFR and VEGFL. As an example of such superior blocking, see Fig. 20, regarding the ability of OG1950 (and the antibody provided herein) to block to a higher degree than Lucentis® (ranibizumab) or Avastin® (bevacizumab).

In some embodiments, other antibodies, such as Lucentis® (ranibizumab) or Avastin® (bevacizumab), can be conjugated to one or more polymers as described herein, by one or more methods described herein. In some embodiments, any antibody or fragment thereof can be conjugated to one or more polymers as described herein, by one or more methods described herein.

In some embodiments, the antibody comprises a heavy chain amino acid variable region that comprises the sequence of SEQ ID NO: 1 and a light chain amino acid variable region that comprises the sequence of SEQ ID NO: 2. In some embodiments, the antibody is conjugated to one or more polymers provided by the present invention. 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 6 CDRs within SEQ ID NO: 1 and SEQ ID NO: 2, as well as a point mutation 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 includes the following mutations: L234A, L235A, and G237A (EU numbering) and at least one of the following mutations: Q347C (EU numbering) or L443C (EU numbering).

In some embodiments, an antibody is provided that binds VEGF-A. The antibody comprises: CDR _H 1, which is CDR _H 1 of SEQ ID NO: 1, CDR _H 2, which is CDR _H 2 of SEQ ID NO: 1, CDR _H 3, which is CDR _H 3 of SEQ ID NO: 1, CDR _L 1, which is CDR _L 1 of SEQ ID NO: 2, CDR _L 2, which is CDR _L 2 of SEQ ID NO: 2, CDR _L 3, which is CDR _L 3 of SEQ ID NO: 2, 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).

As will be appreciated by one of skill in the art, in light of the present disclosure, any of the antibodies provided herein can be conjugated to any of the polymers provided herein and/or any antibody provided herein can have a cysteine added in such a way that it allows site-specific conjugation to the polymer.

"VEGF" or "vascular endothelial growth factor" is a human endothelial growth factor that affects angiogenesis or the angiogenic process. Specifically, the term VEGF means any member of the class of growth factors that (i) binds to a VEGF receptor such as VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), or VEGFR-3 (FLT-4); (ii) activates VEGF receptor-associated tyrosine kinase activity; and (iii) thereby affects angiogenesis or the angiogenic process.

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

The various effects of VEGF are mediated by the binding of VEGFs, such as VEGF-A (P15692), -B (P49766), -C (P49767), and -D (Q43915), to receptor tyrosine kinases (RTKs). The VEGF family of receptors belong to class V RTKs, and each carries seven Ig-like domains in the extracellular domain (ECD). In humans, VEGF binds to three types of RTKs: VEGFR-1 (Flt-1) (P17948), VEGFR-2 (KDR, Flk-1) (P935968), and VEGFR-3 (Flt-4) (P35916). Unless the context otherwise requires, reference to VEGF means any of VEGF-A, -B, -C, -D and PGF in any of the naturally occurring isoforms or naturally occurring variants or induced variants having at least 90, 95, 98 or 99%, or 100% sequence identity with the naturally occurring form. In some embodiments, such VEGFs are human VEGF. Similarly, reference to VEGFR means any of VEGR-1, R-2 or R-3, including any naturally occurring isoform or naturally occurring variant or induced variant having at least 90, 95, 98 or 99%, or 100% sequence identity with the naturally occurring sequences.

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, specifically all VEGF-A isoforms and bioactive 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 sequence of the heavy and light chains of bevacizumab (DrugBank accession DB00112) is listed as SEQ ID NO: 3 (heavy) and SEQ ID NO: 4 (light).

The variable light chain CDRs of bevacizumab are 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 variable heavy chain CDRs of bevacizumab are CDR _H 1: GYTFTNYGMN, CDR _H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR _H 3: YPHYYGSSHWYFDV. The CDRs are defined according to Kabat, except that the Kabat/Chothia composite numbering system is used for CDRH1. In some embodiments, a cysteine may be added to the bevacizumab sequence and the antibody (and/or variant that includes the 6 CDRs of bevacizumab) may be conjugated to any one or more of the polymers provided herein.

Another anti-VEGF molecule derived from the same murine monoclonal antibody as bevacizumab has been approved for the treatment of wet AMD: ranibizumab (LUCENTIS® (ranibizumab), Genentech/Roche). Ranibizumab is an antibody fragment, or Fab. Ranibizumab was produced by affinity maturation of the variable heavy and light chains of bevacizumab. The sequence of the heavy and light chains of ranibizumab (as published by Novartis) is shown in SEQ ID NOs: 5 and 6, respectively. In some embodiments, a cysteine may be added to the ranibizumab sequence and the antibody (and/or a variant that includes the 6 CDRs of ranibizumab) may be conjugated to any one or more polymers shown herein.

The CDRs of ranibizumab are the same as those of bevacizumab, except where changes were made following affinity maturation: the variable light chain CDRs of ranibizumab are 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 variable heavy chain CDRs of ranibizumab are 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).

In some embodiments, the antibody conjugate is an anti-VEGF-A antibody linked via a cysteine outside the variable region of the antibody to a polymer comprising phosphorylcholine, wherein the cysteine was added using recombinant DNA technology. In some embodiments, the polymer is linked to a single cysteine. In some embodiments, "added using recombinant DNA technology" means that the cysteine residue replaces a non-cysteine amino acid that occurs at the same position in a known or existing antibody, or in an antibody consensus sequence. Thus, for example, when the antibody is IgG1 and the heavy chain has a leucine at EU position 443, the leucine is replaced using recombinant DNA technology with a cysteine (L443C, EU numbering or 449C in SEQ ID NO: 1). Accordingly, the native IgG1 sequence at position EU 347 is Q (glutamine), and Q is replaced with cysteine using recombinant DNA technology to yield Q347C.

In some embodiments, the anti-VEGF-A antibody comprises a light chain and a heavy chain, wherein the heavy chain has an Fc region. In some embodiments, the cysteine is in the Fc region and the anti-VEGF-A antibody is immunoglobulin G (IgG). In some embodiments, the heavy chain of the anti-VEGF-A antibody 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), and position 221 (by sequential counting as 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 position 4 according to Kabat is L.

In some embodiments, the heavy chain isotype of the anti-VEGF-A antibody is IgG1. In some embodiments, the IgG1 constant domain has one or more mutations relative to the 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: (EU numbering) E233X, L234X, L235X, G236X, G237X, A327X, A330X, and P331X, wherein X is any natural or non-natural amino acid. In some embodiments, the mutations are selected from the group consisting of (EU numbering): E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S. In some embodiments, the antibody conjugate comprises the following mutations (EU numbering): L234A, L235A, and G237A.

In some embodiments, the cysteine residue is in the heavy chain of the anti-VEGF-A antibody 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 heavy chain sequence of the anti-VEGF-A antibody is SEQ ID NO: 1 and the light chain sequence of the anti-VEGF-A antibody is SEQ ID NO: 2.

In some embodiments, the phosphorylcholine-containing polymer comprises 2-(methacryloyloxyethyl)-2'-(trimethylammonium)ethyl phosphate (MPC) monomers as follows:

,

Thus, the polymer contains the following repeating units:

;

where n is an integer from 1 to 3000 and the wavy lines indicate the attachment points between monomer units in the polymer.

In some embodiments, the polymer has three or more arms or is synthesized with an initiator that includes 3 or more polymer initiation sites. In some embodiments, the polymer has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 arms or is synthesized with an initiator that includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 polymer initiation sites. More preferably, the polymer has 3, 6, or 9 arms or is synthesized with an initiator that includes 3, 6, or 9 polymer initiation sites. In some embodiments, the polymer has 9 arms or is synthesized with an initiator that includes 9 polymer initiation sites.

In some embodiments, the polymer that is added has a molecular weight of about 300,000 to about 1,750,000 Da (SEC-MAL). In some embodiments, the polymer has a molecular weight of about 500,000 to about 1,000,000 Da. In some embodiments, the polymer has a molecular weight of about 600,000 to about 900,000 Da. In some embodiments, the polymer has a molecular weight of about 750,000 to about 850,000 Da. In some embodiments, the polymer has a molecular weight of about 800,000 to about 850,000 Da. In some embodiments, the polymer has a molecular weight of about 750,000 to about 800,000 Da.

In some embodiments, any of the antibodies described herein may be further conjugated to a polymer to form a bioconjugate. The molecular weight of the bioconjugate (in general, SEC-MALS) can be from about 350,000 to 2,000,000 Daltons, such as from about 450,000 to 1,900,000 Daltons, from about 550,000 to 1,800,000 Daltons, from about 650,000 to 1,700,000 Daltons, from about 750,000 to 1,600,000 Daltons, from about 850,000 to 1,500,000 Daltons, from about 900,000 to 1,400,000 Daltons, from about 950,000 to 1,300,000 Daltons, from about 900,000 to 1,000,000 Daltons, from about 1,000,000 to 1,300,000 dalton, from about 850,000 to 1,300,000 dalton, from about 850,000 to 1,000,000 dalton, and from about 1,000,000 to 1,200,000 dalton.

In some embodiments, the antibody conjugate is purified. In some embodiments, the polymer, which is an aspect of the antibody conjugate, is polydisperse, i.e., the PI of the polymer is not 1.0. In some embodiments, the PI is less than 1.5. In some embodiments, the PI is less than 1.4. In some embodiments, the PI is less than 1.3. In some embodiments, the PI is less than 1.2. In some embodiments, the PI is less than 1.1.

In some embodiments, the antibody conjugate has an anti-VEGF-A immunoglobulin G (IgG) linked to a polymer, wherein the polymer comprises MPC monomers wherein the anti-VEGF-A antibody heavy chain sequence is SEQ ID NO: 1 and the anti-VEGF-A antibody light chain sequence is SEQ ID NO: 2, and wherein the antibody is linked only at position C449 of SEQ ID NO: 1 to the polymer. In some embodiments, the polymer has 9 arms and a molecular weight of about 600,000 to about 1,000,000 Da.

In some embodiments, the antibody conjugate has an anti-VEGF-A immunoglobulin G (IgG) linked to a polymer, wherein the polymer comprises MPC monomers wherein the anti-VEGF-A antibody heavy chain sequence is SEQ ID NO: 1 and the anti-VEGF-A antibody light chain sequence is SEQ ID NO: 2, and wherein the antibody is linked only via C443 (EU numbering or 449C in SEQ ID NO: 1) to the polymer. In some embodiments, the polymer has 9 arms and a molecular weight of about 600,000 to about 1,000,000 Da.

In some embodiments, the antibody conjugate has the following structure:

where each heavy chain of the anti-VEGF-A antibody is designated by the letter H, and each light chain of the anti-VEGF-A antibody is designated by the letter L;

the polymer is linked to the anti-VEGF-A antibody via the sulfhydryl C449 of SEQ ID NO: 1, this linkage is depicted on one of the heavy chains; PC is , where the wavy line indicates the point of attachment to the rest of the polymer; where 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 so that the sum of n1, n2, n3, n4, n5, n6, n6, n7, n8 and n9 is 2500 plus or minus 10%. In some embodiments, n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000. In some 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, a monosubstituted, polysubstituted, or unsubstituted version of the following: saturated C ₁ -C ₂₄ alkyl, unsaturated C ₂ -C ₂₄ alkenyl, or C ₂ -C ₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboxyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano and halogenated alkyl including polyhalogenated alkyl, -CO-OR ₇ , carbonyl -CCO-R ₇ , -CO-NR ₈ R ₉ , -(CH ₂ ) _n -COOR ₇ , -CO-(CH) _n -COOR ₇ , -(CH ₂ ) _n -NR ₈ R ₉ , ester, alkoxycarbonyl, aryloxycarbonyl, where n is an integer from 1 to 6, wherein each of R ₇ , R ₈ and R ₉ is individually selected from the group consisting of a hydrogen atom, a halogen atom, monosubstituted, polysubstituted or unsubstituted versions of the following residues: saturated C ₁ -C ₂₄ alkyl, unsaturated _C2 - _C24 alkenyl or C2- _C24 alkynyl _, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboxyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano and halogenated alkyl including polyhalogenated alkyl, 5-membered ring and 6-membered ring.

In some embodiments, the antibody conjugate has the following structure:

where each heavy chain of the anti-VEGF-A antibody is designated by the letter H, and each light chain of the anti-VEGF-A antibody is designated by the letter L;

the polymer binds to the anti-VEGF-A antibody via the sulfhydryl group C443 (EU numbering or 449C in SEQ ID NO: 1), this linkage is depicted on one of the heavy chains; PC is , where the wavy line indicates the point of attachment to the rest of the polymer; where 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 so that the sum of n1, n2, n3, n4, n5, n6, n6, n7, n8 and n9 is 2500 plus or minus 10%. In some embodiments, n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000. In some 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, a monosubstituted, polysubstituted, or unsubstituted version of the following: saturated C ₁ -C ₂₄ alkyl, unsaturated C ₂ -C ₂₄ alkenyl, or C ₂ -C ₂₄ alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboxyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano and halogenated alkyl including polyhalogenated alkyl, -CO-OR ₇ , carbonyl -CCO-R ₇ , -CO-NR ₈ R ₉ , -(CH ₂ ) _n -COOR ₇ , -CO-(CH) _n -COOR ₇ , -(CH ₂ ) _n -NR ₈ R ₉ , ester, alkoxycarbonyl, aryloxycarbonyl, where n is an integer from 1 to 6, wherein each of R ₇ , R ₈ and R ₉ is individually selected from the group consisting of a hydrogen atom, a halogen atom, monosubstituted, polysubstituted or unsubstituted versions of the following residues: saturated C ₁ -C ₂₄ alkyl, unsaturated _C2 - _C24 alkenyl or C2- _C24 alkynyl _, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboxyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano and halogenated alkyl including polyhalogenated alkyl, 5-membered ring and 6-membered ring.

In some embodiments, the antibody conjugate is present in a liquid composition. In some embodiments, the antibody conjugate is combined with a pharmaceutically acceptable carrier.

In some embodiments, an anti-VEGF-A antibody is provided. The anti-VEGF-A antibody heavy chain comprises at least the following CDR sequences: 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). In some embodiments, the anti-VEGF-A antibody heavy chain comprises these CDRs and further comprises a threonine (T) at position 221 (by consecutive counting as in SEQ ID NO: 3). In some embodiments, the light chain of the anti-VEGF-A antibody comprises at least 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). In some embodiments, the anti-VEGF-A antibody has these CDRs and further comprises a leucine (L) at position 4 according to Kabat. In some embodiments, the heavy chain isotype of the anti-VEGF-A antibody is IgG1 and comprises CH ₁ , hinge, CH ₂ , and CH ₃ domains. In some embodiments, the light chain isotype is kappa.

In some embodiments, the IgG1 domain of the anti-VEGF-A antibody has one or more mutations to modulate effector function, such as ADCC, ADCP, and CDC. In some embodiments, the IgG1 mutations reduce effector function. In some embodiments, the amino acids used for the effector function mutations include (EU numbering) E233X, L234X, L235X, G236X, G237X, G236X, D270X, K322X, A327X, P329X, A330X, A330X, P331X, and P331X, wherein X is any natural or non-natural amino acid. 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 heavy chain of the anti-VEGF-A antibody comprises the following mutations (EU numbering): L234A, L235A, and G237A. In some embodiments, the number of effector function mutations relative to the natural human IgG1 sequence is no more than 10. In some embodiments, the number of effector function mutations relative to the natural human IgG1 sequence is no more than 5, 4, 3, 2, or 1. In some embodiments, the antibody reduces Fc gamma binding and/or binding to complement protein C1q such that the ability of the antibody to produce effector function is reduced. This may be particularly useful in ophthalmological indications/diseases.

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).

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

In some embodiments, an anti-VEGF-A antibody comprises a heavy chain constant domain that includes one or more mutations that reduce immune-mediated effector function.

In some embodiments, an anti-VEGF-A antibody is provided. The anti-VEGF antibody comprises a heavy chain that comprises CDR _H 1 comprising the sequence GYDFTHYGMN (SEQ ID NO: 9), CDR _H 2 comprising the sequence WINTYTGEPTYAADFKR (SEQ ID NO: 10), CDR _H 3 comprising the sequence YPYYYGTSHWYFDV (SEQ ID NO: 11), CDR _L 1 comprising the sequence SASQDISNYLN (SEQ ID NO: 12), CDR _L 2 comprising the sequence FTSSLHS (SEQ ID NO: 13), and CDR _L 3 comprising the sequence QQYSTVPWT (SEQ ID NO: 14).

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

The heavy chain of the anti-VEGF-A antibody comprises a cysteine residue added as a mutation using recombinant DNA technology 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 the antibody to the polymer is 1:1; in other words, the conjugate comprises one antibody molecule conjugated to one polymer molecule.

The half-life of anti-VEGF-A antibodies can be increased by attaching "half-life-extending moieties" or "half-life-extending groups". Half-life-extending moieties include peptides and proteins that can be expressed in a frame with a biologic drug (or chemically conjugated as appropriate) and various polymers that can be attached or conjugated to one or more amino acid side chains or terminal functionalities such as -SH, -OH, -COOH, _-CONH2 , _-NH2 , or one or more N- and/or O-glycan structures. Half-life-extending moieties generally function to increase the circulatory half-life of biologic drugs in vivo.

Examples of peptide/protein fragments that increase half-life include an Fc-containing fusion protein (Capon DJ, Chamow SM, Mordenti J, et al. Designing CD4 immunoadhesions for AIDS therapy. Nature. 1989. 337:525–31), a human serum albumin (HAS)-containing fusion protein (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), a carboxy-terminal peptide (CTP)-containing fusion protein (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 fused unstructured repeating peptide sequences (XTEN) (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 (ELPylation) (MCpherson DT, Morrow C, Minehan DS, et al. Production and purification of a recombinant elastomeric polypeptide, G(VPGVG19-VPGV, from Escheriachia coli . Biotechnol Prog. 1992. 8:347-52), a fusion protein containing human transferrin (Prior CP, Lai CH, Sadehghi H et al. Modified transferrin fusion proteins. Patent WO2004/020405. 2004), proline-alanine-serine (PASylation) (Skerra A, Theobald I, Schlapsky M. Biological active proteins having increased in vivo and/or vitro stability. Patent WO2008/155134 A1. 2008), an amino acid homopolymer (HAPylation) (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 a fusion protein containing gelatin-like protein (GLK) (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).

Examples of polymer moieties that increase half-life 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, carboxymethyldextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyphosphazine, polyoxazoline, maleic anhydride with polystyrene, poly(1-hydroxymethylethylene hydroxymethylformal) (PHF), zwitterionic polymer, phosphorylcholine containing polymer, and polymer including MPC, Poly(Gly _x -Ser _y ), hyaluronic acid (HA), Heparosan polymers (HEP), fleximers, dextran, and poly-sialic acids (PSA).

In one embodiment, the half-life enhancing moiety can be conjugated to the antibody via free amino groups of the protein using N-hydroxysuccinimide (NHS) esters. Reagents targeting conjugation to amino groups can randomly react with the ε-amino group of lysines, the α-amino group of N-terminal amino acids, and the Δ-amino group of histidines.

However, the anti-VEGF-A antibodies disclosed herein have many amino groups available for polymer conjugation. Thus, polymer conjugation with free amino groups may negatively affect the ability of the antibody proteins to bind to VEGF.

In some embodiments, the half-life enhancing moiety is coupled to one or more free SH groups using any suitable thiol interactions, including, but not limited to, maleimide interactions, or coupling of polymer hydrazides or polymeric amines to carbohydrate moieties of the antibody after pre-oxidation. In some embodiments, a maleimide coupling reaction is used. In some embodiments, the coupling reaction occurs at naturally occurring or engineered cysteines.

In some embodiments, the polymers are covalently attached to cysteine residues introduced into the anti-VEGF-A antibodies by site-directed mutagenesis. In some embodiments, the 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 WO 2013/093809, US 7,521,541, WO 2008/020827, US 8,008,453, US 8,455,622, and US2012/0213705, incorporated by reference for all purposes. In some embodiments, the cysteine mutations are Q347C (EU numbering) and L443C, related to the human IgG heavy chain by EU numbering.

In some embodiments, conjugates of an antibody and high molecular weight polymers are provided that serve to increase the half-life. In some embodiments, the conjugate comprises an antibody that is linked to a zwitterionic polymer, wherein the polymer is formed from one or more monomer units and wherein at least one monomer unit has a zwitterionic group. In some embodiments, the zwitterionic group is phosphorylcholine.

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.

In some embodiments, some antibody conjugates have 2, 3, or more polymer arms, wherein the monomer is HEMA-PC. In some embodiments, the conjugates have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 polymer arms, wherein the monomer is HEMA-PC. In some embodiments, the conjugates have 3, 6, or 9 arms. In some embodiments, the conjugate has 9 arms.

In some embodiments, the polymer-antibody conjugates have a polymer portion with a molecular weight of 100,000 to 1,500,000 Da. In some embodiments, the conjugate has a polymer portion with a molecular weight of 500,000 to 1,000,000 Da. In some embodiments, the conjugate has a polymer portion with a molecular weight of 600,000 to 800,000 Da. In some embodiments, the conjugate has a polymer portion with a molecular weight of 600,000 to 850,000 Da and has 9 arms. When a molecular weight is given for an antibody conjugated to a polymer, the molecular weight will be obtained by adding the molecular weight of the protein, including any carbohydrate moieties associated therewith, and the molecular weight of the polymer.

In some embodiments, the anti-VEGF-A antibody has a HEMA-PC polymer that has a molecular weight measured _Mw from about 100 kDa to 1650 kDa. In some embodiments, the molecular weight measured _Mw of the polymer is from about 500 kDa to 1000 kDa. In some embodiments, the measured molecular weight of the polymer _Mw is from about 600 kDa to about 900 kDa. In some embodiments, the measured molecular weight of the polymer _Mw is 750 kDa plus or minus 15%.

In some embodiments, the polymer is produced from an ATRP-suitable initiator having one or more polymer initiation sites. In some embodiments, the polymer initiation site has a 2-bromoisobutyrate site. In some embodiments, the initiator has 3 or more polymer initiation sites. In some embodiments, the initiator has 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 polymer initiation sites. In some embodiments, the initiator has 3, 6, or 9 polymer initiation sites. In some embodiments, the initiator has 9 polymer initiation sites. In some embodiments, the initiator is OG1786.

Anti-VEGF-A antibodies can be produced by recombinant expression, comprising (i) producing recombinant DNA by genetic engineering, (ii) introducing recombinant DNA into prokaryotic or eukaryotic cells by, for example and without limitation, transfection, electroporation or microinjection, (iii) culturing the transformed cells, (iv) expressing the antibody, for example constitutively or inducibly, and (v) isolating the antibody, for example from the culture medium or by collecting the transformed cells, in order to (vi) obtain purified antibody.

Anti-VEGF-A antibodies can be produced by expression in a suitable prokaryotic or eukaryotic host system characterized by the production of a pharmacologically acceptable antibody molecule. Examples of eukaryotic cells are mammalian cells such as CHO, COS, HEK 293, BHK, SK-Hip and HepG2. Other suitable expression systems are prokaryotic (e.g. E. coli with the pET/BL21 expression system), yeast (Saccharomyces cerevisiae and/or Pichia pastoris systems) and insect cells.

A wide variety of vectors can be used to produce the antibodies disclosed herein, selected from eukaryotic and prokaryotic expression vectors. Examples of prokaryotic expression vectors include plasmids such as, but not limited to, preset, pet, and pad, wherein promoters used in prokaryotic expression vectors include one or more of, but not limited to, lac, trc, trp, recA, or araBAD. Examples of eukaryotic expression vectors include: (i) for expression in yeast, vectors such as, but not limited to, pAO, pPIC, pYES, or pMET, using 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, using 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, and vectors derived, in one aspect, from viral systems such as, but not limited to, vaccinia virus, adeno-associated viruses, herpes viruses or retroviruses using promoters such as, but not limited to, CMV, SV40, EF-1, UbC, RSV, ADV, BPV and beta-actin.

Method for conjugating proteins with polymers

In some embodiments, a method is provided for producing a conjugate of a therapeutic protein with a half-life-increasing moiety, comprising the step of conjugating a therapeutic protein that comprises a cysteine residue added using recombinant DNA technology with a half-life-increasing moiety having a sulfhydryl-specific reactive group selected from the group consisting of maleimide, vinyl sulfones, orthopyridyl disulfides and iodoacetamides, to provide a conjugate of a therapeutic protein with a half-life-increasing moiety.

In some embodiments, a method for producing an OG1953 antibody conjugate from OG1950 is provided. As shown in FIG. 18, the method comprises reducing the OG1950 protein with a 50-fold molar excess of the reducing agent TCEP (FIG. 18). After reduction, the antibody is oxidized to form a decapped OG1950 antibody, wherein intrachain disulfide bonds are naturally formed in the light and heavy chain of the antibody, but the engineered cysteine in the heavy chain at position L443C (EU or 449C numbering in SEQ ID NO: 1) remains decapped (FIG. 18). OG1950 is then conjugated by adding an excipient and adding a 5-10-fold molar excess of a maleimide biopolymer (FIG. 18). The biopolymer binds to the OG1950 antibody via a covalent thiolester bond (Fig. 18). Following conjugation, the OG19503 antibody conjugate is purified to remove unconjugated antibody and polymer (Fig. 18).

The protein and method described above can also be varied. Thus, in some embodiments, a method for producing a conjugated protein (which does not necessarily have to be an antibody or an anti-VEGF antibody) is provided. The method includes reducing one or more cysteines in a protein to form a decapped protein in solution. After reducing one or more cysteines, the decapped protein is reoxidized to reduce at least one disulfide bond in the reduced protein, thereby leaving an engineered cysteine residue in the protein in a free thiol form to form a reoxidized decapped protein in solution. At least one excipient is then added to the solution. The excipient reduces polymer-induced protein precipitation. After adding the excipient, a polymer is added to the solution that is conjugated to the reoxidized decapped protein at the engineered cysteine residue to form the conjugated protein.

In some embodiments, the molar excess of the reducing agent can be varied to any effective amount. In some embodiments, the molar excess of the reducing agent (which need not be TCEP in all embodiments) can be 10, 20, 30, 40, 50, 60, 70, 80, 90-fold. In some embodiments, any antibody (therapeutic or otherwise) can be used. In some embodiments, the molar excess of the maleimide biopolymer can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15-fold. In some embodiments, the decapped protein is in excess of the polymer. In some embodiments, the amount of reduced protein is less than the amount of polymer. In some embodiments, the amount of recovered protein is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1% of the amount of polymer. In some embodiments, 10-15 times more polymer is used as the protein. In some embodiments, the amount of recovered antibody is greater than the amount of polymer. In some embodiments, the amount of polymer is greater than the amount of recovered antibody.

In some embodiments, the purification step is optional.

In some embodiments, a method for producing an antibody conjugate comprises conjugating an anti-VEGF-A antibody to a polymer comprising phosphorylcholine. In some embodiments, the method comprises the steps of conjugating an anti-VEGF-A antibody to a polymer comprising phosphorylcholine. The anti-VEGF-A antibody comprises an amino acid residue added using recombinant DNA technology. 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 chain or the light chain of the antibody.

In some embodiments, the polymer comprises or consists of a polymer comprising phosphorylcholine. In some embodiments, the polymer comprising phosphorylcholine 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 polymer comprising phosphorylcholine reacts with a cysteine residue on the anti-VEGF-A antibody to produce an antibody conjugate.

In some embodiments, the protein to be conjugated may be an antibody, a fusion protein comprising an antibody, or a binding fragment thereof. In some embodiments, the protein is not an antibody, but is an enzyme, ligand, receptor, or other protein, or mutants or variants. In some embodiments, the native protein comprises at least one disulfide bond and at least one non-native cysteine.

In some embodiments, the excipient may be an acid or a base. In some embodiments, the excipient is a detergent, a sugar, or a charged amino acid. In some embodiments, the excipient helps maintain the protein in solution during conjugation with the polymer. In some embodiments, the excipient is added to the solution containing the protein before the polymer is added to the solution containing the protein.

In some embodiments, the reaction is carried out in aqueous media at a pH of from about 5 to about 9. In some embodiments, the reaction occurs at a pH of from 6.0 to 8.5, from 6.5 to 8.0, or from 7.0 to 7.5.

In some embodiments, the conjugation of the polymer to the protein is carried out at a temperature of 2 to 37 °C. In some embodiments, the conjugation is carried out at a temperature of 0 to 40 °C, 5 to 35 °C, 10 to 30 °C, and 15 to 25 °C.

In some embodiments, the conjugated proteins described in the present invention may be reacted with an ion exchange or hydrophobic interaction chromatography medium, or an affinity chromatography medium for purification (to remove the conjugated protein from the unconjugated protein). In some embodiments, the ion exchange, hydrophobic interaction chromatography medium, and/or affinity chromatography medium separates the conjugated protein from the free polymer and the reoxidized decapped protein.

In some embodiments, the methods disclosed herein and shown in Fig. 18 involve an excipient capable of facilitating and/or maintaining a solubility system. In some embodiments, the method allows a solution to maintain the solubility of the two components intended to interact. That is, the solubility of the protein and the polymer, and then the final conjugate. In some embodiments, without using an excipient, a problem may be that although the protein is soluble, when the biopolymer is added, the solubility of the solution (e.g., the protein) decreases and it falls/drops out of solution. Of course, when the protein drops out, it cannot effectively conjugate with the biopolymer. Thus, an excipient can be used to maintain the solubility of the protein in the presence of the biopolymer, so that the two can combine to form a protein conjugate (or, as shown in Fig. 18, an antibody conjugate). This also allows the solubility of the conjugate to be maintained.

In some embodiments, the polymers disclosed herein may include one or more of the following: a zwitterion, a phosphorylcholine, or a PEG linker connecting the center of the branch point of the polymer to a maleimide functional group. In some embodiments, any of the polymers provided herein may be added to a protein by the methods provided herein.

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

In some embodiments, the method(s) disclosed herein allow for the production and purification of protein and/or antibody conjugates on an industrial scale. In some embodiments, the working volume is at least 1 liter, such as 1, 10, 100, 1000, 5000, 10000 liters or more. In some embodiments, the amount of antibody conjugate produced and/or purified can be 0.1, 1, 10, 100, 1000 or more grams.

In some embodiments, the therapeutic protein may be any of the anti-VEGF-A antibodies disclosed herein comprising a cysteine residue added using recombinant DNA technology. In some embodiments, the anti-VEGF antibody heavy chain comprises the following CDRs: 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). The heavy chain may also comprise a threonine (T) at position 221 (by sequential counting as in SEQ ID NO: 3). In some embodiments, the light chain of the anti-VEGF antibody comprises the following CDRs: 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 light chain of the anti-VEGF-A antibody may also comprise a leucine (L) at position 4 according to Kabat.

In some embodiments, the anti-VEGF-A antibody is 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 (EU numbering): E233, L234, L235, G236, G237, A327, A330, and P331. 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 (EU numbering) L234A, L235A, and G237A.

In some embodiments, the cysteine residue added to the therapeutic protein using recombinant DNA technology should not be involved in the formation of Cys-Cys disulfide bonds. In this regard, therapeutic proteins can be dimeric. For example, an intact anti-VEGF-A antibody has two light chains and two heavy chains. If, for example, a Cys residue is introduced into the heavy chain, the intact antibody will contain two such introduced cysteines at the same positions, and there is a possibility that these cysteine residues will form inter- and intrachain disulfide bonds. If the introduced cysteine residues form Cys-Cys disulfide bonds or have a tendency to do so, then the introduced Cys residue will not be useful for conjugation. It is known in the art to avoid positions in the heavy and light chains that lead to the formation of intrachain disulfide bonds. See, for example, US Patent Application 2015/0158952.

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

In some embodiments, the sulfhydryl-specific reactive group is maleimide.

In some embodiments, the half-life enhancing moiety is selected from the group consisting of polyethylene glycol (PEG), branched PEG, PolyPEG® (Warwick Effect Polymers; Coventry, UK), polysialic acid (PSA), starch, hydroxyethyl starch (HES), hydroxyalkyl starch (HAS), carbohydrate, 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-co-maleic anhydride, maleic anhydride with polystyrene, poly(1-hydroxymethylethylene hydroxymethylformal) (PHF), zwitterionic polymer, phosphorylcholine-containing polymer and polymer containing 2-methacryloyloxy-2'-ethyltrimethylammonium phosphate (MPC).

In some embodiments, the half-life enhancing moiety is a zwitterionic polymer. In some embodiments, the zwitterion is phosphorylcholine, i.e., a polymer comprising phosphorylcholine. In some embodiments, the polymer is comprised of MPC units.

In some embodiments, the MPC polymer has three or more arms. In some embodiments, the MPC polymer has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 arms. In some embodiments, the MPC polymer has 3, 6, or 9 arms. In some embodiments, the MPC polymer has 9 arms. In some embodiments, the polymer is synthesized with an initiator comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more polymer initiation sites.

In some embodiments, the MPC polymer has a molecular weight of about 300,000 to 1,750,000 Da. In some embodiments, the MPC polymer has a molecular weight of about 500,000 to 1,000,000 Da or about 600,000 to 900,000 Da.

In some embodiments, the method of producing a conjugate of a therapeutic protein and a half-life enhancing moiety comprises the additional step of reacting the therapeutic protein with a thiol reducing agent under conditions that produce a reduced cysteine sulfhydryl group. As discussed above, it is preferred that the cysteine residue added by recombinant DNA technology is non-paired, i.e., does not participate in or is not substantially involved in intrachain Cys-Cys disulfide bonds. However, Cys residues that do not participate in such Cys-Cys disulfide bonding and are free for conjugation are known to react with free cysteine in the culture medium to form disulfide adducts. See, for example, WO 2009/052249. Thus, the derivatized cysteine will not be available for conjugation. In order to free the newly added cysteine from the disulfide adduct, the protein is treated with a reducing agent, such as dithiothreitol, after purification. However, such treatment with a reducing agent will reduce all cysteine residues in the therapeutic protein, including native cysteines, many of which are involved in inter- and intra-chain Cys-Cys disulfide bonds. Native Cys-Cys disulfides typically play a key role in protein stability and activity and should be reformed. In some embodiments, all native (e.g., inter- and intra-) Cys-Cys disulfides are reformed.

To reform native inter- and intrachain disulfide residues, after reduction to remove disulfide cysteine adducts, the therapeutic protein is exposed to oxidizing conditions and/or oxidizing agents for a predetermined period of time, such as overnight. In some embodiments, overnight exposure to atmospheric air may be used to reform native disulfide bonds. In some embodiments, an oxidizing agent is used to reduce native disulfides. In some embodiments, the oxidizing agent is selected from the group consisting of aqueous CuSO ₄ and dehydroascorbic acid (DAC). In some embodiments, the oxidizing agent is DAC. In some embodiments, the range of DAC used is from 5 to 30 equivalents. In some embodiments, the range is from 10 to 20 equivalents. In some embodiments, the range is 15 equivalents.

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 ₄ ), sodium cyanoborohydride (NaCNBH ₃ ), β-mercaptoethanol (BME), cysteine hydrochloride, and cysteine. In some embodiments, the thiol reducing agent is TCEP.

In some embodiments, the concentration of the thiol reducing agent is from 1 to 100 times molar excess relative to the concentration of the therapeutic protein. In some embodiments, the concentration of the thiol reducing agent is from 20 to 50 times 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 to oxidize the therapeutic protein.

In some embodiments, the method of conjugating a therapeutic protein to a half-life enhancing moiety comprises an additional step of purifying the therapeutic protein conjugate after conjugation. In some embodiments, the therapeutic protein conjugate is purified by a method selected from the group consisting of ion exchange chromatography, hydrophobic chromatography, size exclusion chromatography, and affinity chromatography, or combinations thereof.

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.

In some embodiments, the therapeutic protein conjugate has an increased half-life compared to an unconjugated therapeutic protein. In some embodiments, the therapeutic protein conjugate has at least a 1.5-fold increase in half-life compared to an unconjugated therapeutic protein. In some embodiments, the therapeutic protein conjugate has at least a 5-fold increase in half-life compared to an unconjugated therapeutic protein.

In some embodiments, the zwitterionic polymer in the method of conjugating a therapeutic protein to a half-life enhancing 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, wherein the medium comprises: a radically polymerizable zwitterionic monomer; a transition metal catalyst M _t ^(q-1)+ , where M _t is a transition metal, q is the highest oxidation state of the metal, and q-1 is the lowest oxidation state of the metal, wherein the metal catalyst is supplied as a salt of the form Mt ^(q-1)+ X' _(q-1) , where X' is a counterion or group, or the transition metal catalyst is supplied in situ by providing a salt of the inactive metal in its highest oxidation state M _t ^q+ X' _q together with a reducing agent that is capable of reducing the transition metal from an oxidized inactive state to a reduced active state; a ligand; and an initiator.

In order 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 results in cycling of the transition metal catalyst between the higher oxidation state and the lower oxidation state as the polymer chains cycle from chain end growth to decay. See, e.g., U.S. Patent 7,893,173.

In some embodiments, the radically polymerizable zwitterionic monomer is selected from the group consisting of

,

where R1 is H or C _1-6 alkyl, ZW is a zwitterion, and n is an integer from 1 to 6.

In some embodiments, the radically polymerizable monomer is

,

where R1 is H or C _1-6 alkyl, R2, R3, R4 are the same or different and are 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, each of R1, R2, R3 and R4 is methyl, and each of X and Y is 2.

In some embodiments, the radically polymerizable monomer is

,

wherein R1 is H or C _1-6 alkyl, R2 and R3 are the same or different and are H or C _1-4 alkyl, R4 is PO ₄ -, SO ₃ -, or CO ₂ -, and X and Y are the same or different and are integers from 1 to 6. In some embodiments, R1, R2, and R3 are methyl, R4 is PO ₄ -, and each of X and Y is 2.

In some embodiments, the monomer is

,

wherein R1 is H or C _1-6 alkyl, R2, R3 and R4 are the same or different and are H or C _1-4 alkyl, R5 is PO ₄ -, SO ₃ - or CO ₂ -, 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 methyl, R5 is PO ₄ -, and X and Y are 2.

In some embodiments, the transition metal Mt 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-based catalyst is supplied as a salt of the form Mt ^(q-1)+ X' _(q-1) . M _t ^{(q-1) +} is selected from the group consisting of 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, (SO ₄ ) _1/2 , (PO ₄ ) _1/3 , (R7PO ₄ ) _1/2 , (R7 ₂ PO ₄ ), triflate, hexafluorophosphate, methanesulfonate, arylsulfonate, CN and R7CO ₂ , where R7 is H or a linear or branched C _1-6 alkyl group which may be substituted with one to five halogen atoms. In some embodiments, M _t ^(q-1)+ is Cu ¹⁺ and X' is Br.

In some embodiments, M _t ^(q-1)+ is supplied in situ. In some embodiments, M _t ^q+ X _q is CuBr ₂ . In some embodiments, the reducing agent is an inorganic compound. In some embodiments, the reducing agent is selected from the group consisting of a low oxidation state sulfur compound, sodium hydrosulfite, an inorganic salt containing a metal ion, a metal, 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 .

In some embodiments, the reducing agent is an organic compound. In some embodiments, the organic compound is selected from the group consisting of alkyl thiols, mercaptoethanol or carbonyl compounds that can be easily enolized, ascorbic acid, acetylacetonate, camphosulfonic acid, hydroxyacetone, reducing sugars, monosaccharides, glucose, aldehydes, and derivatives of such organic compounds.

In some embodiments, the ligand is selected from the group consisting of 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-pyridyl)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.

In some embodiments, the initiator has the structure:

where R1 is a nucleophilic reactive group, R2 comprises a linker, and R3 comprises a polymer synthesis initiator moiety having the structure

,

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 from 1 to 20.

In some embodiments, Z is Br, and each of R4 and R5 is methyl. In some embodiments, R1 is selected from the group consisting of _NH2- , OH-, and SH-.

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

,

where X and Y are the same or different and are integers from 1 to 20. In some embodiments, each of X and Y is 4.

In some embodiments, R3 is

,

where R6, R7 and R8 are the same or different and are selected from the group consisting of

And

,

wherein Z is NCS, F, Cl, Br, or I. In some embodiments, Z is Br and each of R6, R7, and R8 is

.

In some embodiments, the initiator has the structure:

,

where A and B are the same or different and are integers from 2 to 12, and Z is any halide, such as Br. In some embodiments, A and B are each 4.

In some embodiments, the method further comprises the step of reacting the polymer with a maleimide reagent to produce a polymer having a terminal maleimide. In some embodiments, the maleimide compound is

Method of treatment

In some embodiments, a method of treating or preventing an ophthalmological disease is provided, comprising the step of administering a therapeutic protein selected from the group consisting of an anti-VEGF-A antibody (and conjugates thereof). In some embodiments, any one or more of the antibodies or antibody conjugates provided herein can be used to treat and/or prevent an ophthalmological disease. The method comprises administering to a subject any one or more of the antibodies or antibody conjugates provided herein.

In some embodiments, a method of treating or preventing an ophthalmological disease is provided. The method comprises administering an effective dose of any antibody and/or antibody conjugates described herein to a subject in need thereof. In some embodiments, the disease may be age-related macular degeneration (AMD) or diabetic macular edema (DME). In some embodiments, the disease may be wet AMD.

In some embodiments, the ophthalmological disease is selected from one or more groups 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), branch retinal vein occlusion (BRVO), corneal neovascularization, retinal neovascularization, retinopathy of prematurity (ROP), subconjunctival hemorrhage, and hypertensive retinopathy. Optionally, the ophthalmological disease is diabetic retinopathy.

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

In some embodiments, one or more compositions provided herein may reduce the high-burden consequences of intravitreal injections of anti-VEGF agents for the treatment of wet (proliferative) age-related macular degeneration (AMD). Practical outcomes in patients with wet AMD have lagged behind the clinical outcomes demonstrated in phase 3 clinical trials, such as the MARINA and ANCHOR trials with Lucentis® (ranibizumab) and the VIEW 1 and VIEW trials with Eylea® (aflibercept). An anti-VEGF therapeutic protein with a longer residence time, so that it can be administered less frequently and therefore better tolerated by patients, may provide practical outcomes closer to the phase 3 clinical outcomes for a larger number of patients.

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

The therapeutic proteins (e.g., both antibodies and antibody conjugates) described herein can be administered by expressing such polypeptides in vivo in a patient, i.e., gene therapy.

There are two main methods for producing nucleic acid (optionally contained in a vector) in patient cells: in vivo and ex vivo. For in vivo delivery, the nucleic acid is administered directly to the patient, typically at the site where the therapeutic protein is needed, i.e., where the biological activity of the therapeutic protein is required. For ex vivo treatment, cells are removed from the patient, the nucleic acid is introduced into the isolated cells, and the modified cells are administered to the patient either directly or, for example, encapsulated in porous membranes that are implanted into the patient (see, for example, U.S. Patents 4,892,538 and 5,283,187). Numerous methods exist for introducing nucleic acids into viable cells. The methods vary depending on whether the nucleic acid is transferred into cultured cells in vitro or transferred in vivo into the cells of the intended host. Methods suitable for transferring nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, transduction, cell fusion, diethylaminoethyldextran, calcium phosphate precipitation method, etc. Transduction involves the association of a replication-defective, recombinant viral (including retroviral) particle with a cellular receptor, followed by the introduction of nucleic acids carried by the particle into the cell. Typically, the vector used for ex vivo gene delivery is a retrovirus.

In some embodiments, methods for in vivo nucleic acid transfer include transfection with 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, for example, DOTMA, DOPE, and DC-Chol, see, for example, Tonkinison et al., Cancer Investigation, 14(1): 54-65 (1996)). In some embodiments, vectors used in gene therapy are viruses, which include adenoviruses, AAV, lentiviruses, or retroviruses. A viral vector, such as a retroviral vector, comprises at least one transcriptional promoter/enhancer or locus defining element(s) or other elements that control gene expression by other means, such as alternative splicing, nuclear RNA export, or post-translational modification of a messenger. In addition, a viral vector, such as a retroviral vector, comprises a nucleic acid molecule that, when transcribed in the presence of a gene encoding a therapeutic protein, is operably linked thereto and acts as a translation initiation sequence. Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus employed (if not already present in the viral vector). In addition, such a vector typically includes a signal sequence for secretion of the PRO polypeptide from the host cell into which it is placed. In some embodiments, the signal sequence for this purpose is a mammalian signal sequence. In some embodiments, the signal is a native signal sequence for a therapeutic protein. Optionally, the vector construct may also contain a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence. As an example, such vectors typically include a 5'LTR, a tRNA binding site, a packaging signal, a second strand DNA synthesis start point, and a 3'LTR or a portion thereof. Other vectors that are non-viral may be used, such as cationic lipids, polylysine, and dendrimers.

In some situations, it is desirable to provide a source of nucleic acid using an agent that targets target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc. In the case of liposomes, proteins that bind to a cell surface membrane protein associated with endocytosis can be used to target and/or facilitate uptake of, for example, capsid proteins or fragments thereof for a particular cell type, antibodies to proteins that undergo internalization in the cycle, and proteins that target intracellular localization and increase intracellular half-life. The method of receptor-mediated endocytosis is 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 labeling and gene therapy protocols, see Anderson et al., Science, 256: 808–813 (1992). See also WO 93/25673 and references cited therein.

Suitable gene therapy and methods for producing retroviral particles and structural proteins can be found, for example, in U.S. Patent 5,681,746.

In some embodiments, a method for treating or preventing an ophthalmological disease in a mammal is provided, comprising administering a nucleic acid molecule that encodes a therapeutic protein selected from the group consisting of an antibody against VEGF-A. In some embodiments, the nucleic acid is shown in Fig. 27.

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

Therapeutic proteins can be included in a pharmaceutical composition with a pharmaceutically acceptable excipient. Pharmaceutical compositions intended for oral administration can be presented as discrete units such as capsules, as solutions, syrups or suspensions (in aqueous or non-aqueous liquids, or as an edible foam or mass, 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, soft or liquid polyols, etc. Excipients that can be used to obtain solutions and syrups 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.

Pharmaceutical compositions can be adapted for nasal administration, where the excipient is a solid substance, including a coarse powder having a particle size of, for example, from 20 to 500 microns, which is administered in a manner similar to taking tobacco, i.e. by rapid inhalation through the nasal passage from a container of powder held close to the nose. Suitable compositions in which the excipient is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient. Pharmaceutical compositions adapted for administration by inhalation include fine dust particles or aerosols, which can be obtained by means of various types of metered-dose pressurized aerosols, nebulizers or insufflators.

Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solution which may contain antioxidants, buffers, bacteriostats and solutes which render the composition substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Excipients which can be used for injection solutions include, for example, water, alcohols, polyols, glycerol and vegetable oils. The compositions can be presented in single-dose or multi-dose containers, for example, in sealed ampoules and vials, and can be stored in a lyophilized state requiring only the addition of a sterile liquid, for example, water for injection, immediately before use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets. The pharmaceutical compositions may be substantially isotonic, which means an osmolality of about 250 to 400 mOsmol/kg water.

The pharmaceutical compositions may contain preservatives, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, coloring agents, odorants, salts (the substances themselves may be presented as a pharmaceutically acceptable salt), buffers, coating agents, or antioxidants. They may also contain therapeutically active agents in addition to the substance. The pharmaceutical compositions may be used in combination with one or more pharmaceutically acceptable excipients. Such excipients may include, but are not limited to, saline, buffered saline (such as phosphate-buffered saline), dextrose, liposomes, water, glycerol, ethanol, and combinations thereof.

Antibodies and pharmaceutical compositions containing them can be administered in an effective mode for the treatment or prevention of a patient's disease, including, for example, oral, intravitreal, intravenous, subcutaneous, intramuscular, intradermal, intranasal, topical, intraperitoneal, and intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration, or other routes of administration. In therapy or as a prophylactic agent, the active agent 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.

For administration to mammals, and especially humans, it is expected that the dosage of the active agent is from 0.01 mg/kg body weight, usually about 1 mg/kg. The physician can determine the actual dosage most suitable for an individual, which depends on factors including the age, weight, sex and response of the individual, the disease or disorder being treated, and the age and condition of the individual receiving treatment. The above doses are approximate for the average case. Of course, there may be cases where higher or lower doses are worth considering. In some embodiments, the dosage may be from 0.5 to 20 mg/eye, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 mg.

Such dosage may be repeated as often as necessary (e.g. weekly, biweekly, monthly, bimonthly, quarterly, twice a year, annually). If side effects develop, the amount and/or frequency of dosage may be reduced in accordance with normal clinical practice. In one embodiment, the pharmaceutical composition may be administered once every one to thirty days. In one embodiment, the pharmaceutical composition may be administered twice every thirty days. In one embodiment, the pharmaceutical composition may be administered once a week.

Antibodies and pharmaceutical compositions can be used alone or in combination with other compounds, such as therapeutic compounds or molecules, for example, anti-inflammatory drugs, analgesics or antibiotics. Such administration with other compounds can be simultaneous, separate or sequential. The components can be prepared as a kit, which can contain instructions, if necessary.

The antibodies and pharmaceutical compositions disclosed herein can be used to treat or prevent disease, particularly ophthalmological diseases or conditions, as described herein.

Thus, the conjugates are typically prepared and administered by ocular, intraocular, and/or intravitreal injection, and/or periscleral injection, and/or subretinal injection, and/or sub-Tenon's injection, and/or suprachoroidal injection, and/or subconjunctival, and/or topical administration as eye drops and/or ointment. Such antibodies and compositions can be delivered in a variety of ways, such as into the vitreous as a device and/or a depot that provides slow 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 a 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).

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

Therapeutic antibodies and related conjugates are typically placed in a container having a sterile access port, such as a bag of intravenous solution or a vial with a stopper pierced by a hypodermic needle. Such compositions may also be supplied as prefilled syringes.

A "stable" composition is a composition in which the protein or protein conjugated to a polymer of another half-life enhancing moiety substantially retains its physical stability and/or chemical stability and/or biological activity upon storage. By "stable" is also meant a composition that has little or no signs of instability, including aggregation and/or deamidation. For example, the provided compositions may remain stable for at least two years when stored, as specified, at a temperature of 5 to 8 °C.

Various analytical methods for measuring protein stability are available in the art and are discussed, for example, in Peptide and Protein Drug Delivery, 247-301 (Vincent Lee ed., New York, N.Y., 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 compositions are stable upon storage for at least 6 months, 12 months, 12 to 18 months, or 2 or more years.

A protein, such as an antibody or fragment thereof, "retains its physical stability" in a pharmaceutical composition if it shows no evidence of aggregation, precipitation, deamidation and/or denaturation when visually inspected for color and/or clarity, or when measured by ultraviolet light scattering or size exclusion chromatography.

A protein "retains its chemical stability" in a pharmaceutical composition if the chemical stability at the time is such that the protein is believed to retain its biological activity. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. A chemical alteration can include a size modification (e.g., truncation), which can be assessed, for example, by size exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and/or matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS). Other types of chemical alteration include a charge change (e.g., resulting from deamidation), which can be assessed, for example, by ion exchange chromatography. An antibody "retains its biological activity" in a pharmaceutical composition if the biological activity of the antibody at the time is no more than 10% (within the limits of assay error) of the biological activity exhibited at the time of preparation of the pharmaceutical composition, as determined in an antigen binding assay, for example.

A protein-polymer conjugate "retains its chemical stability" when the chemical bond between the protein and the polymer remains intact, e.g., is not hydrolyzed or otherwise degraded. The protein portion of the conjugate retains its chemical stability as described above.

By "isotonic" is meant that the composition of interest has substantially the same osmotic pressure as human blood or vitreous humor for intravitreal injection. Isotonic compositions typically have an osmotic pressure of approximately 250 to 400 mOsmol. Isotonicity can be measured, for example, using a vapor osmometer or a freezing point osmometer.

As used herein, the term "buffer" refers to a buffer solution that resists changes in pH due to its acid-base conjugates. In some embodiments, the pH of the buffer is from about 3.0 to about 8.0; for example, from about 4.5 to 8; or from about 6 to about 7.5; or from about 6.0 to about 7.0, or from about 6.5 to about 7.0, or from about 7.0 to about 7.5; or from about 7.1 to about 7.4. A pH of any point between the above ranges is also contemplated.

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

In some embodiments, the PBS buffer consists of at least _Na2HPO4 , _KH2PO4 , and NaCl to provide an appropriate pH. In some embodiments, the buffer may contain other pharmaceutical excipients such as KCl and other _salts , detergents, and/or preservatives to _provide storage stability of the solution.

A "preservative" is a compound that can be included in a composition to substantially reduce bacterial activity therein, thereby facilitating, for example, the preparation of a multi-use formulation. Examples of potential preservatives include octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkyl benzyl dimethyl ammonium chlorides in which the alkyl groups are long chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl, and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

In some embodiments, compositions that are required to be safe for human use or for animal testing must have sufficiently low levels of endotoxin. "Endotoxin" is a lipopolysaccharide (LPS) derived from the cell membrane of gram-negative bacteria. Endotoxin consists of a hydrophilic polysaccharide moiety covalently linked 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 endotoxin, i.e., its toxicity. Endotoxins are lost in large quantities during bacterial cell death, as well as during growth and division. They have high heat resistance and are not destroyed under normal sterilization conditions. Extreme heat or pH treatment is required, for example, 180 to 250 °C and more than 0.1 M acid or base (Petsch D, Anspach F. 2000. Endotoxin removal from protein solutions. J Biotechnol. 76: 97-119). Obviously, such conditions are harmful to biological drugs.

In the biotechnology and pharmaceutical industries, endotoxins can be found both during production and in final products. Since bacteria can grow in energy-poor environments including water, saline, and buffers, endotoxin levels will increase unless precautions are taken. Injection of endotoxin into animals or humans causes a wide range of pathophysiological effects including endotoxin 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.

Pyrogenic reactions and shock are induced in mammals by intravenous administration of endotoxin at low concentrations (1 ng/ml) (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 a pharmaceutical and biological product is 5 endotoxin units (EU) per kg body weight per hour according to all pharmacopoeias (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 unit of measurement of the biological activity of an endotoxin. For example, 100 pg of standard EC-5 endotoxin and 120 pg of endotoxin from Escherichia 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). Achieving this threshold level has always been a problem 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).

Of particular concern is the presence of endotoxin in drugs delivered by intravitreal injection. Intravitreal injection of a drug (penicillin) was first reported in 1945 by Rycroft. Rycroft BW. 1945. Penicillin and the control of deep intra-ocular infection. British J Ophthalmol 29 (2): 57–87. The vitreous provides a chamber into which high levels of drug can be introduced and maintained for relatively long periods of time. The concentration of drug that can be achieved by intravitreal injection is much higher than that achieved by topical administration or systemic administration (e.g. intravenous).

One of the most dangerous complications that can potentially occur with intravitreal injections is endophthalmitis. Endophthalmitis is divided into two classes: infective and sterile. Infective endophthalmitis is usually caused by bacteria, fungi, or parasites. Symptoms of infective endophthalmitis include severe pain, loss of vision, and redness of the conjunctiva and underlying episclera. Infectious endophthalmitis requires prompt diagnosis and treatment. Treatment options include intravitreal antibiotic injection and primary vitrectomy in some cases. Enucleation is the removal of the blind and painful eye. See, for example, Christy NE, Sommer A. 1979. Antibiotic prophylaxis of postoperative endophthalmitis. Ann Ophthalmol 11 (8): 1261–1265.

In contrast, sterile endophthalmitis does not involve exposure to 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. To ensure a diagnosis of sterile endophthalmitis, vitreous cultures must be negative. Marticorena J, Romano V, Gomez-Ulla F. 2012 “Sterile Endophthalmitis after Intravitreal Injections” Med Inflam. 928123.

Intravitreal injection of endotoxin-contaminated biologics has been found to result in sterile endophthalmitis. Marticorena, et al. Bevacizumab (Avastin) is approved by the Food and Drug Administration (FDA) for the treatment of glioblastoma and metastatic colorectal cancer, advanced nonsquamous non-small cell lung cancer, and metastatic renal cell carcinoma. 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 and must be prepared by a pharmacist. Clusters of sterile endophthalmitis have been observed, which are thought to result from inadvertent contamination of bevacizumab with endotoxin during preparation by the pharmacist.

Given the severe clinical results of intravitreal injection of endotoxin, the total amount of endotoxin that can be given to a patient by intravitreal dosing is extremely limited. In some embodiments, a solution is provided comprising an antibody or antibody conjugate having an endotoxin level that does not exceed 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, 1, 0.5, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01 EU/mL.

Two widely used FDA-approved tests for the presence of endotoxin are the rabbit pyrogen test and the horseshoe crab amoebocyte lysate (LAL) test (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 rise in temperature of a rabbit following injection of a test solution. However, the use of the rabbit pyrogen test has declined significantly over the years due to expense and long turnaround time. Much more common is the LAL test. LAL is prepared from horseshoe crab blood and clots upon exposure to endotoxin.

One of the simplest LAL assays is the LAL gel clot test. Essentially, the LAL coagulation activity assay is combined with serial dilutions of the sample being tested. The 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 a LAL gel. At some point, a negative reaction is observed. The amount of endotoxin in the original sample can be estimated from the dilution assay.

Other LAL tests have also been developed, including the turbidimetric LAL assay (Ong KG, Lelan JM, Zeng KF, Barrett G, Aourob M, Grimes CA. 2006. A rapid highly-sensitive endotoxin detection system. Biosensors and Bioelectronics 21:2270–2274) and the chromogenic LAL assay (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). The turbidimetric and chromogenic assays are much more sensitive and quantitative than the simple gel-clot dilution test.

In some embodiments, a method is provided for reducing the amount of endotoxin in a composition comprising an antibody disclosed herein. The method comprises the steps of 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 eluent having an antagonist; contacting the affinity chromatography eluent 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.

The above method for reducing the amount of endotoxin or another method or process disclosed herein may be performed in the steps in the order described above, or may optionally be performed by changing the order of the steps or even repeating one or more steps. In one embodiment, the method for reducing the amount of endotoxin in a composition is performed in the steps in the described order. In some embodiments, the steps of contacting with the affinity chromatography resin, washing and eluting are repeated in the same order more than once, before contacting the affinity chromatography eluent with the ion exchange resin. The method may also include a filtration step, for example with a 0.1, 0.22 or 0.44 micron filter, which may be performed on one or more of the eluents removed after each resin binding step.

In some cases, the steps of contacting the composition with an affinity chromatography resin, washing, and eluting the antibody from the affinity chromatography resin may be repeated more than once before contacting the first eluent with the ion exchange resin. In one embodiment, the affinity chromatography resin comprises a recombinant protein A ("rProteinA") resin.

One example of a suitable recombinant Protein A resin is rProteinA Sepharose FF® resin (Amersham, Piscataway, N.J.). In another embodiment, a suitable affinity chromatography resin will include a protein G chromatography resin. In other embodiments, a suitable affinity chromatography resin includes a mixed protein A and protein G resin. In other embodiments, a suitable affinity chromatography resin includes a hydrophobic charge induction chromatography resin including a 4-mercaptoethylpyridine ligand, such as a MEP HyperCel® resin (BioSepra, Cergy, Saint Christophe, France).

In some embodiments, the ion exchange resin comprises an anion exchange resin. As will be appreciated by one skilled in the art, ion exchangers can be based on various materials relative to the matrix, as well as the attached charged groups. For example, the following matrices can be used, in which said materials can be more or less cross-linked: MacroCap Q (GE Healthcare Biosciences, Piscataway, NJ), agarose-based (e.g., Sepharose CL-6B®, Sepharose Fast Flow®, and Sepharose High Performance®), cellulose-based (e.g., DEAE Sephacel®), dextran-based (e.g., Sephadex®), silica-based and a synthetic polymer. For an anion exchange resin, the charged groups covalently attached to the matrix can be, for example, diethylaminoethyl, quaternary aminoethyl, and/or quaternary ammonium. In some embodiments, the anion exchange resin comprises a quaternary amine group. An exemplary anion exchange resin that comprises a quaternary amine group for binding an anti-M-CSF antibody is Q Sepharose® resin (Amersham, Piscataway, N.J.).

In other aspects, if the levels of endotoxin are higher than desired after the composition has been subjected to the aforementioned anion exchange chromatography step, the composition can alternatively be exposed to a cation exchange resin. In some embodiments, any endotoxin in the composition should have differential binding to the ion exchange resin than the protein of interest in order to ensure purification of the protein from the endotoxin. In this regard, the endotoxin is negatively charged and typically binds to the anion exchange resin. If both the protein and the endotoxin bind to the anion exchange resin, purification of one from the other can be accomplished by using a salt gradient to elute both into different fractions. Relative binding of the protein to a particular resin can also be accomplished by changing the pH of the buffer relative to the pI of the protein. In some embodiments, cation exchange chromatography is the only ion exchange chromatography used.

In some embodiments, if the endotoxin levels are too high after the anion exchange resin, the composition can be further subjected to a second ion exchange step, such as by contacting the compositions with a cation exchange resin and then washing, then eluting from the ion exchange resin. In some embodiments, the cation exchange resin contains a sulfonic acid group for binding. Exemplary cation exchange resins include SP Sepharose® resin FF (Amersham, Piscataway, N.J.) Poros XS (CEX) (Life Technology, Grand Island, New York).

In some embodiments, after the antibody protein solution is obtained with the specified level of endotoxin, several steps are performed before the final protein is obtained. In some embodiments, the half-life enhancing moiety is conjugated to the protein. The conjugate is then converted to the final dosage form that is administered to patients. In some embodiments, the conjugate is purified again on an ion exchange resin, which may be a cation exchange resin. In other embodiments, the protein is obtained. In all cases, routine laboratory techniques should be used to prevent endotoxin contaminants from entering the protein sample or the protein-polymer conjugate.

EXAMPLES

Example 1. Method 1 for obtaining OG1802.

The first method for preparing OG1802 is as follows. First, a TFA/amine salt initiator (compound L) having the structure shown in Fig. 1 was prepared as follows.

First, compound K, having the structure shown in Fig. 2, was prepared as follows. Compound J (OG1563) (1.9 g, 2.67 mmol, 3.3 equiv) was placed in a 200 mL round-bottomed flask under nitrogen atmosphere.

and compound E (0.525 g, 0.81 mmol, 1.0 equiv) (see Fig. 11), then dimethylformamide (10 mL), then diisopropylethylamine (2.5 mL, 14.6 mmol, 18 equiv). The flask was cooled to 0 °C using an ice bath. A solution of propylphosphonic anhydride (50 wt % in ethyl acetate, 2.5 mL, 4.04 mmol, 5 equiv) was added to the mixture over approximately 6 minutes.

The reaction mixture was warmed to room temperature and stirred for 15 min. The reaction mixture 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, which was used without further purification, afforded 2.0 g (0.80 mmol, 99%) of compound K.

^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): [(M+2H-boc)/2]+ Calculated for (C84H136Br9N7O33+2H-Boc)/2 = 1196.6; Found 1196.6.

The following compound L (Fig. 1) was prepared as follows: to a 100 mL round bottom flask under nitrogen atmosphere were added compound K (2.0 g, 0.8 mmol), dichloromethane (10 mL), and then trifluoroacetic acid (5 mL). The reaction mixture was stirred at room temperature for 30 min. The reaction mixture was concentrated in vacuo. The reaction mixture was diluted with dichloromethane (10 mL) and concentrated in vacuo. The residue was dissolved with acetonitrile (10 mL), filtered through a syringe filter (Acrodisc CR25, PN 4225T) and loaded onto a preparative HPLC column and eluted with 60% acetonitrile in water (with 0.1% trifluoroacetic acid) to 98% acetonitrile (with 0.1% trifluoroacetic acid). The tubes containing the product were pooled, concentrated in vacuo, frozen and placed in a lyophilizer. This afforded 990 mg (0.4 mmol, 50% over 2 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, both CH2 NH C=O and NH3 +).

LC/MS (ES, m/z): [(M+2H)/2]+ Calculated for (C84H136Br9N7O33+2H)/2 = 1196.6; Found 1197.4.

Compound L was then used as an initiator to prepare the MPC polymer. The initiator is typically prepared as a stock solution in DMF at approximately 100 mg/mL. The initiator and ligand (2,2'-bipyridyl) were added to a Schlenk flask. The resulting solution was cooled to 78 °C using dry ice/acetone and degassed under vacuum for 10 min. The flask was filled with argon and the catalyst (CuBr unless otherwise noted), stored under argon, was added to the Schlenk flask (the molar ratio of atomic bromine on the initiator/catalyst (CuBr)/ligand was maintained at 1/1/2). The solution immediately turned dark brown. The Schlenk flask was sealed and immediately purged using a short vacuum/argon cycle. The HEMA-PC solution was prepared by mixing a certain amount of monomer, prepared in a glove box kept under nitrogen, with 200-resistant degassing ethanol. The monomer solution was added dropwise into a Schlenk flask (via a cannula) (and homogenized with gentle stirring). The temperature was maintained at 78 °C. A thorough vacuum was applied to the reaction mixture for at least 10-15 min until bubbles ceased to emerge from the solution. The flask was then filled with argon and warmed to room temperature. The solution was stirred and as the polymerization proceeded the solution became viscous. After 3-8 hours or overnight the reaction mixture was quenched by direct exposure to air to oxidize the Cu(I) to Cu(II), the mixture turned blue-green in color and 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 and then freeze-dried to give an anhydrous white powder. Table 1.1 below provides polymer data for the polymer using compound L as an initiator.

Table 1.1

Theor. M _W
(kDa) Polymer ID No. Initiator M _n (kDa) M _p (kDa) IP 500 130 L 490 530 1,1 750 150 L 645 750 1,1

Then, the Mal-PEG4-PFP maleimide ester was fixed (as shown in Fig. 29) to the 750 kDa polymer mentioned above to provide OG1802. In a 20 mL tube, the R3707 polymer (750 kDa polymer prepared using L as an initiator, 515 mg) was placed and dissolved using ethanol (4.0 mL) after stirring for 40 minutes. To this was added a 1% solution of 4-methylmorpholine in acetonitrile (22 μL). In a separate tube, Mal-PEG4-PFP (1.97 mg) was dissolved in acetonitrile (1.0 mL) and this solution was added to the polymer solution over a period of about 2 minutes at room temperature and the resulting solution was stirred overnight. The reaction mixture was diluted with 0.1% aqueous trifluoroacetic acid (2 mL) (pH approximately 5) followed by water (approximately 12 mL), filtered through a syringe filter (Acrodisc Supor, PN 4612), and distributed evenly into 3 Amicon membrane dialysis centrifuge tubes (30,000 NMOM). The tubes were diluted and mixed with water (5 mL each) and centrifuged (3200 rpm) for 25 min. The filtrate was removed for analysis, and the retentate was diluted and mixed with water (approximately 10 mL/tube). Centrifugation was repeated 5 more times, after which the retentate was removed and placed in a tube. The Amicon membrane tubes were washed with water (2 x approximately 2 mL each tube) and combined with the retentate. The retentate solution was filtered through a syringe filter (Acrodisc Supor, PN 4612), frozen, and placed in a lyophilizer to yield 485 mg as a white powder.

Example 2. Getting the OG1786 initiator

OG1786 is a nine-arm polymer initiator used as a precursor in the preparation of OG1802. Each arm terminates in 2-bromoisobutyrate, which is capable of initiating polymerization under ATRP. OG1786 is a salt of trifluoroacetic acid (TFA), as shown in Fig. 30. OG1786 was prepared as follows. First, OG1550 is reacted with TFA (trifluoroacetic acid) to obtain OG1546, as shown in Fig. 31.

To a 1 L round bottom flask equipped with a magnetic stirrer and addition funnel were added OG1550 (14.8 g), methyl tert-butyl ether (MTBE) (350 mL), and water (30 mL). The mixture was stirred to dissolve OG1550, 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 min. After complete addition, the mixture was stirred for an additional 15 min, then removed from the ice bath and allowed to warm to room temperature. The mixture was stirred (after removal from the ice bath) for an additional 4 to 5 hours until TLC showed approximately 5% starting material remaining and the pH of the aqueous solution was 3 to 4 (pH indicator paper).

The mixture was separated. The MTBE layer was washed with water (30 mL). The aqueous layers were combined, then the aqueous solution was extracted with MTBE (150 mL). The second MTBE phase was washed with water (30 mL). The combined aqueous layers were washed with a third portion of MTBE (100 mL). The third MTBE phase was washed with water (25 mL). The aqueous layers were again combined (approximately 250 mL, pH approximately 4, determined with pH paper).

The product was collected by lyophilization. This yielded 11.5 g of a white solid. This material is extremely hygroscopic and is best handled under nitrogen. The product was confirmed by LC/MS.

The resulting OG1546 was then reacted with OG1563 to yield OG1784 (as shown in Fig. 32).

To a 250 mL flask equipped with a magnetic stirrer under nitrogen atmosphere was added OG1546 (hygroscopic, 9.0 g) followed by N,N-dimethylformamide (110 mL). The mixture was stirred at room temperature until OG1546 was completely dissolved (about 15 min), then OG1563 (29.9 g) was added and the mixture was stirred for another 3 min until OG1563 was dissolved. The resulting solution was cooled in an ice bath and N,N-diisopropylethylamine (37.6 mL) was added over 3 min, followed by propylphosphonic anhydride (T3P), 50% in ethyl acetate (34.5 mL) dropwise over 5 min (the addition of T3P is exothermic). After the addition of T3P was complete, the flask was removed from the cooling bath and allowed to reach room temperature. Samples were then processed at 5-minute intervals for LC/MS analysis. The reaction mixture showed a very light yellow/brown color.

After 20 min, the reaction mixture was cooled again in an ice bath and 5 ml of water was added. The mixture was then removed from the cooling bath and another 50 ml of water was added in portions, then 50 ml of 0.5 M citric acid was added, followed by isopropyl acetate (300 ml). The mixture was separated. The aqueous phase (approximately 300 ml) was extracted with additional isopropyl acetate (150 ml). The aqueous phase was AQ1 for the HPLC test. The combined organic phases were washed with aqueous citric acid (115 ml, 65 mM, which was a mixture of 15 ml of 0.5 M citric acid plus 100 ml of water) and the aqueous phase was AQ2 (pH approximately 3). The organic phase was washed with water/saturated sodium chloride (100 ml/25 ml) and the aqueous phase was AQ3 (pH approximately 3). The organic phase was finally washed with saturated sodium chloride (100 mL) and the aqueous phase was AQ4. Neither AQ fraction contained significant product (data not shown). The organic phase was confirmed to contain product by LC/MS. The product was dried over sodium sulfate (80 g), filtered and washed with isopropyl acetate (75 mL) and concentrated on a rotary evaporator to give a tan oil (33.2 g). The crude product was stored overnight under nitrogen.

The next day, the crude product was brought to room temperature, then dissolved in acetonitrile/water (46 mL/12 mL) and filtered using a HPLC filter (Cole-Parmer PTFE 0.2 μm, product number 02915-20). The filtrate was divided into three equal parts and purified three times.

The filtrate was loaded onto a RediSep Rf Gold C18 column (275 g, SN 69-2203-339, Lot# 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 relevant fractions were checked by HPLC. Fractions considered sufficiently pure were pooled (from all three runs) and concentrated (bath temperature maintained at approximately 20 °C) on a rotary evaporator, then partitioned between dichloromethane (100 ml) and water (5 ml)/saturated sodium chloride solution (25 ml). The aqueous extract was extracted twice with dichloromethane (2 x 30 ml). The combined organic phases were dried over sodium sulfate (35 g), filtered, washed with DCM (30 mL), and concentrated. The product and purity were confirmed by LC/MS. The practical yield and purity of R5172 and R5228 are presented in Table 2.1.

Table 2.1

OG1784 part R5172 R5228 OG1546 used 5.3 g 9.0 g OG1563 used 17.6 g 29.9 g Practical solution 53% 58% Purity (a/a 210 nm) 99.3% 100.0%

OG1405 was then prepared from OG1784 as shown in Figure 33. To a 500 mL round bottom flask equipped with a magnetic stirrer was added OG1784 (20.9 g), followed by dichloromethane (50 mL), then trifluoroacetic acid (20 mL). The mixture was stirred at room temperature and HPLC analysis showed complete deprotection within 23 min. The mixture was concentrated on a rotary evaporator, redissolved in dichloromethane (25 mL) and reconcentrated, then redissolved in acetonitrile (25 mL) and reconcentrated. The product was confirmed by LC/MS. The above material (OG1405, 34.5 g, estimated 21.0 g quantitative yield) was used as the crude oil in the next step. No purification was required.

OG1405 was then reacted with OG1402 to give OG1785 as shown in Fig. 34. In a 500 mL flask equipped with a magnetic stirrer, OG1402 (5.5 g) was placed under nitrogen atmosphere, followed by the addition of acetonitrile (70 mL), then N,N-diisopropylethylamine (26.3 mL) and the T3P solution (see above) (7.9 mL). The solution was stirred at room temperature for 30 min, then cooled in an ice bath and a solution of OG1405 (crude oil as above, 34.5 g) in acetonitrile (70 mL) was added. The mixture was warmed to room temperature. After 20 min, the reaction mixture was cooled in an ice bath and quenched with water (5 mL). The mixture was then concentrated in vacuo to half the volume using a rotary evaporator. Samples were taken for LC/MS.

More water (50 mL) was added followed by 0.5 M citric acid (75 mL) and isopropyl acetate (175 mL). The mixture was separated after 5 min. The aqueous extract 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), then washed finally with saturated sodium chloride (25 mL). They were then dried over sodium sulfate (124 g), filtered and washed with isopropyl acetate (30 mL), and concentrated on a rotary evaporator to a brown oil (27.3 g). Samples were taken for LC/MS analysis.

The oil was dissolved in acetonitrile/water (3:1, 15 mL/5 mL), filtered through an HPLC filter disc (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.

Portions were 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 with a gradient of solvent A:water/solvent B:acetonitrile. The appropriate fractions were combined and partitioned between dichloromethane (150 mL) and water (5 mL)/saturated sodium chloride solution (25 mL). The aqueous extract was extracted twice with dichloromethane (2 × 50 mL). The combined organic phases were dried over sodium sulfate (60 g), filtered and washed with dichloromethane (40 mL), and concentrated. The structure and purity were confirmed by various analyses including LC/MS: OG1785 was isolated as a foamy solid (R5329, 19.0 g, 83% yield, 95.1% purity (a/a 210 nm), stored under nitrogen at 4°C.

The tert-butyloxycarbonyl protecting group on OG1785 was then removed using trifluoroacetic acid (TFA) to yield OG1786, as shown in Fig. 35.

Example 3. Obtaining polymer OG1801

The OG1801 polymer is prepared initially from the OG1786 initiator. OG1801 has an amino functionality that is more stable (than the maleimide) during polymer preparation. A modified version of ATRP is used to prepare the OG1801 polymer, in which copper species (Cu(I)) are generated in situ by adding metallic copper to Cu(II). The starting materials and reagents required in the reaction are calculated based on the input batches of OG47 monomer (HEMA-PC) and the target molecular weight ( _Mw ).

50 g of OG47 monomer were weighed in a glove box and 200 mL of degassed EtOH was added to dissolve the monomer at room temperature; samples were collected for monomer concentration analysis. Cu(II), Bpy, Cu(0) were weighed in a 500 mL flask; argon was purged while adding the monomer solution to the flask; the flask was stoppered and evacuated for 25 min until bubbles disappeared. The reaction mixture gradually changed color from light green to dark green, then to light brown; approximately 200 mg of OG1786 initiator was weighed in a glove box and dissolved in approximately 2000 μL of DMF at room temperature to obtain 100 mg/mL of stock solution; samples were collected for initiator concentration and purity analysis; the initiator solution was added to the flask under argon atmosphere. The reaction solution turned dark brown and thickened over time; the system was sealed and the reaction was allowed to proceed for 2 days.

OG1801 was then prepared for maleimide addition and the catalyst (copper) was removed as follows: A normal phase silica packed RediSep® Rf column was used to remove the catalyst. The column size was selected based on the amount of copper in the reaction mixture. For example, for a 50 g batch of OG1801, a 330 g column (No. 69-2203-330, column size 330 g, CV = 443 mL) was used. Teflon tubing was used for all compounds since EtOH was the elution solvent.

After copper removal, all fractions were transferred to a round bottom flask in batches and the EtOH was evaporated to dryness using a rotary evaporator at 45 to 50 °C under reduced pressure. At this stage, the volume of EtOH collected from the condensate was controlled to ensure that more than 90% of the EtOH was removed. The polymer was dissolved in 250 mL of water for injection and filtered using a 0.2 μm filter. This yielded a clear, light yellow polymer solution of approximately 150 mg/mL. The solution was stored at 2 to 8 °C for up to 3 months before use.

Example 4. Obtaining polymer OG1802

The starting materials and reagents required for the reaction were calculated based on batch input OG1801. The linker was 3-maleimidopropionic acid, NHS ester. 30 mL of 0.5 M sodium phosphate (in water for injection, pH 8) was added to 50 g of the polymer solution (approximately 150 mg/mL). Stirring was done for 1 min; the pH was determined to be 8.0 using pH paper. 204.8 mg of the linker was weighed and dissolved in 4.1 mL of DMF to give a 50 mg/mL stock solution. The linker solution was added dropwise at 815 μL per minute to the polymer solution with rapid stirring. 4095 μL of the linker solution was added over 5 min. The reaction was carried out at room temperature for 30 min. 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 liter (0.2 µm) vacuum filter.

OG1802 (see Fig. 36) was then purified as follows: Milipore cross-flow cassettes were used to purify the polymers in an aqueous system. Concentration of the polymer solution to 250 mL (approximately 200 mg/mL) was initiated. Fresh WFI was added from the reservoir and the flow rate of fresh WFI was adjusted to match the permeate (approximately 2 μL/min). The UF/DF was left at 2-8 °C overnight. Typically 2.5 L of WFI (10 times the volume ratio of the polymer solution) was used. A sample of the retentate was collected for purity analysis. The target purity was greater than 98%. The polymer solution was filtered using a 0.2 μM 1 L filter bottle. The polymer solution was stored at 2 to 8 °C for 3 months prior to conjugation.

Example 5. Alternative phosphorylcholine polymers

The HEA-PC polymer was prepared as described below. HEA-PC (2-(acryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate), which is an acrylate, unlike the HEMA-PC methacrylate described above, has the following structure:

HEA-PC was polymerized with the initiator presented in Example 1 as compound L.

Table 5.1

Reactant Name Quantity M _w Initiator L connection (as above) 1.65 mg 2505,5 Monomer HEA-PC 0.461 g 281.24 Catalyst Cu(I) Bromide 1.2 mg 143.45 Ligand Tris[2-(dimethylamino)ethyl]amine
(Me6TREN) 2.73 mg 230.39 Solvent A N,N-dimethylformamide (DMF) 21.85 µl 73.09 Solvent B Water 0.7 ml 18.02 Solvent C Methanol 0.7 ml 32.04

A 200 mg/mL initiator stock solution was prepared by dissolving 2.2 mg initiator in 11 μL dry DMF and 200 mg/mL ligand solution by dissolving 4.6 mg Me6TREN in 23 μL dry DMF. 8.25 μL of the initiator stock solution and 13.6 μL of the ligand were added to a test tube. Degassed at -78 °C for 5 min, then backfilled with argon and 1.2 mg CuBr was added. Degassed and backfilled 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 solution inside the reactor at -78 °C. The vial was rinsed with 200 μl of methanol and added to the reactor at −78 °C, followed by 0.5 ml of distilled water, then another 200 μl of water. Degassed thoroughly until no bubbles were visible and any heterogeneity had disappeared (solids dissolved or disappeared). Filled with 4 psi (27.57 kPa) of argon and allowed to react at room temperature for one hour. The reaction mixture was now viscous. The reaction was allowed to proceed for approximately one hour. A solution of bipyrindine 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. Conversion was estimated to be 94% by H NMR.

The following day, the polymer was dialyzed and subjected to SEC/MALS analysis using a Shodex SB806M_HQ column (7.8 x 300 mm) in 1 x PBS pH 7.4 at 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 (pre-dialysis IP is 1.12, _Mn is 695 kDa, _Mp is 778 kDa). Maleimide functionality was then added to the polymer to allow it to be conjugated to the protein.

The Mal-PEG4-PFP maleimide ester (see Example 1 above) was then placed onto the HEA-PC polymer as presented in Example 1. The resulting maleimide-functionalized HEA-PC polymer was then conjugated with sulfhydryl groups as described herein for the HEMA-PC polymers.

The polymer PC acrylamide was also prepared using the monomer 2-(acrylamyl)ethyl-2-(trimethylammonium)ethyl phosphate (Am-PC), which has the following structure:

Am-PC was used for polymerization using a 3-arm initiator (TFA salt) having the structure:

The synthesis of the Am-PC polymer was carried out as follows:

Table 5.2

Reactant Name/Identity Quantity M _W Initiator 3-beam initiator (as above) 2.2 mg 885.35 Monomer Am-PC 0.5 g 280.26 Catalyst (I) Cu(I) Bromide 1 mg 143.45 Catalyst (II) Cu(II) Bromide 0.2 mg 223.35 Ligand tris[2-(dimethylamino)ethyl]amine
(Me6TREN) 3.94 mg 230.39 Solvent A N,N-dimethylformamide (DMF) 31.7 µl 73.09 Solvent B Water 1 ml 18.02 Solvent C Methanol 1 ml 32.04

A 200 mg/mL ligand stock solution was prepared by dissolving 9 mg Me6TREN in 45 μL dry DMF. Add 19.7 μL of the stock solution to the reaction vessel. A 200 mg/mL initiator stock solution was prepared by dissolving 6.5 mg of compound in 32.5 μL DMF. Add 11 μL of the initiator stock solution to the ligand as above. Degas for 5 min. Add 1 mg CuBr. A 200 mg/mL CuBr ₂ stock solution was prepared by dissolving 4 mg CuBr ₂ in 20 μL DMF. Add 0.5 g of the monomer (AmPC) to 1 mL of methanol (slow dissolution/viscous solution), then 1 μL of the CuBr ₂ stock solution. Add the monomer solution dropwise to the reaction mixture as above. Wash with 1 ml of water. The reaction mixture was thoroughly degassed (freeze-thaw). The reaction was left to proceed for 24 hours.

The Am-PC polymer was then dialyzed. The molecular weight of the above polymer was determined by SEC/MALS: M _n was 215 kDa, M _p : 250 kDa, IP was 1.17. The conversion determined by 1H NMR was 94%. Maleimide functionality was added to the Am-PC polymer as discussed above for HEMA-PC and HEA-PC. The maleimide functionalized Am-PC polymer was conjugated to the protein as described above.

Example 6. Reverse Ellman analysis for calculating free maleimide in a compound.

After addition of the maleimide functionality to the OG1801 polymer to form OG1802 (as above), the Ellman assay was used to determine the amount of maleimide functionality (i.e., conjugated) in the sample. A thiol converts Ellman's reagent (DTNB) to TNB-, then to TNB2- in water at neutral and alkaline pH, which produces a yellow color (measured at 412 nm). A standard curve was established with cysteine. Since maleimide reacts with thiol, the assay actually measured the remaining thiol (cysteine). Inhibition was calculated as (original thiol minus thiol remaining after addition of maleimide polymer) divided by (original thiol) and expressed as a percentage.

Reagents used in the assay: A standard curve was prepared using cysteine from 62.5 μM to 2 μM. Polymer stock solutions were prepared by dissolving the powder in 1×PBS pH 7.4 (reaction buffer) and mixing thoroughly. Equal molar solutions of polymer and cysteine were mixed and allowed to react at 27 °C for 30 min. 150 μM DTNB solution was added to the cysteine standards and the polymer/cysteine reaction mixture and color developed at 27 °C for 5 min. Optical density (OD) at 412 nm was read on a Spectramax plate reader and percent inhibition was calculated using Softmax Pro software and the cysteine standard curve.

Example 7. A protein sequence of an antibody (OG1950) comprising a heavy chain of an anti-VEGF-A antibody with the L443C mutation (EU numbering, or 449C in SEQ ID NO: 1) and a light chain of an anti-VEGF-A antibody

An anti-VEGF-A antibody with the L443C mutation (EU numbering) having the sequence shown below in SEQ ID NO: 1 (Fig. 12) (heavy chain) was cloned. The light chain of the anti-VEGF-A antibody having the sequence shown in SEQ ID NO: 2 (Fig. 13) was cloned.

Example 8a. Cleaning and decapping OG1950

The heavy and light chains of OG1950 were cloned into expression plasmids and transfected into CHO cells. The cells were grown in appropriate media and harvested for analysis. OG1950 was purified using the methods described above. The cysteine residue of OG1950 at position 443 (L443C (EU numbering)) is normally "capped" or oxidized by chemicals in the cell culture medium and is not available for conjugation. Therefore, purified OG1950 was subjected to a decapping process to remove the cap and allow conjugation of the free (i.e., not involved in Cys-Cys disulfide bonds) cysteine residue to the maleimide functionality of the polymer. Decapping was performed by mixing purified OG1950 protein with 30-fold molar excess of TCEP (3,3',3''-phosphanetriyltripropanoic acid) reducing agent for 1 h at 25 °C. The TCEP reduction reaction was monitored by DNS-PAGE. After denaturation, OG1950 protein was washed with UF/DF using a Pellion XL ultrafiltration cassette with 20 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM TCEP decapping buffer. Then, TCEP reagent was removed in the same UF/DF system with 20 mM Tris pH 7.5, 150 mM NaCl. Then, the reduced OG1950 was refolded with air (atmospheric oxygen), after which DNS-PAGE was performed as analysis.

Example 8b. Cleaning and decapping OG1950

The heavy and light chains of OG1950 were cloned into expression plasmids and transfected into CHO cells. The cells were grown in appropriate media and harvested for analysis. OG1950 was purified using the methods described above. The cysteine residue of OG1950 at position 443 (L443C (EU numbering)) is normally "capped" or oxidized by chemicals in the cell culture medium and is not available for conjugation. Therefore, purified OG1950 was subjected to a decapping process to remove the cap and allow conjugation of the free (i.e., not involved in Cys-Cys disulfide bonds) cysteine residue to the maleimide functionality of the polymer. Decapping was performed by mixing purified OG1950 protein with 30-fold molar excess of TCEP (3,3',3''-phosphanetriyltripropanoic acid) reducing agent for 1 h at 25 °C. The TCEP reduction reaction was monitored by DNS-PAGE. After reduction, OG1950 protein was washed by ultrafiltration/diafiltration (UF/DF) system using Pellicon XL ultrafiltration cassette with 30 kDa MWCO membrane from Millipore with 20 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM TCEP buffer to remove the cap and excess TCEP. Residual TCEP reagent was removed in the same UF/DF system with 20 mM Tris pH7.5, 150 mM NaCl. The reduced OG1950 was then reoxidized with dHAA at ambient temperature for 1 h followed by UF/DF to remove dHAA to form decapped OG1950. The decapping status was monitored by DNS-PAGE analysis.

Example 9. Conjugation of OG1950 to MPC polymer

Decapped OG1950 was conjugated to the OG1802 polymer. An excess of OG1802 (10-20-fold molar excess) was used. Conjugation was monitored by DNS-PAGE and carried out to completion. The OG1950 conjugate was purified by cation exchange chromatography and buffer exchanged with UF/DF. The polymer-OG1950 conjugate was purified by chromatography as described above.

Example 10. Kinetics of OG1950 SPR binding

This example illustrates the binding of OG1950 to VEGF-165 in BIAcore™ single cycle kinetics.

The interaction of SPR OG1950 with human VEGF-165 was analyzed in a BIAcore™ T200 system (GE Healthcare) equipped with a Protein A chip (GE Healthcare). The single cycle kinetics method was performed. 25 μg/mL of antibody was maintained in HBS-EP buffer ⁺ (0.01 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 0.15 M sodium chloride, and 0.05% polysorbate 20) with a 25 s pulse at a flow rate of 10 μL/min. Then, recombinant human VEGF-165 (R&D Systems) was applied at different concentrations with a 60 s pulse at a flow rate of 30 μL/min and a terminal dissociation time of 1000 s. The experiment was carried out at 25 °C. The surface was regenerated with 10 mM glycine, pH 1.7, for 1 min at a flow rate of 50 μl/min. Binding kinetics analysis was performed using Biacore T200 evaluation software with responses globally fit to a 1:1 interaction. The results are summarized in Table 10.1 and Fig. 21.

Table 10.1. Kinetics of OG1950 binding to VEGF-165

ka (1/Mс) kd (1/s) KD(M) OG1950 3.35E+05 6.77E-08 2.02E-13

OG1950 exhibits less than 1 pM KD in a 1:1 binding model (beyond the sensitivity of the instrument).

Example 11. Conjugation and purification of OG1953 using cation exchange chromatography

OG1950 protein expression and protein recovery: OG1950 protein was expressed in the GSCHOK1 mammalian expression system followed by purification using a Protein A affinity column. The purity of the purified OG1950 using the Protein A column was >90% based on size exclusion chromatography and DNS-PAGE. The engineered specific cysteine residue of OG1950 was accessible for thiol conjugation with the OG1802 biopolymer. The thiol reactive chemical group of OG1802 reacted to form a stable covalent bond, which formed the bioconjugate OG1953. For this, 1 mg of OG1950 was completely reduced with the reducing agent tris-(2-carboxyethyl)phosphine hydrochloride (TCEP), followed by removal of TCEP by buffer exchange using a 30 kDa concentrator-centrifuge. The fully reduced OG1950 protein was then reoxidized to ensure that all expected protein disulfide bonds were formed, except for the engineered cysteine residue (decapped OG1950), which remained in the reduced form for conjugation to the biopolymer via a specific thiol interaction.

Conjugation: Conjugation 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). Different additives were evaluated in different conjugation reactions as presented in Table 11.1 to compare their effect on conjugation efficiency. All reactions were performed in a fixed volume and incubated at 4 °C for 20 h. Table 11.1 and Fig. 19 show the ion exchange analysis (A280 uptake) and fractionation of conjugation reactions A through G. Reaction A included buffer only; reaction B included OG1950 antibody only; Reaction C included OG1950 antibody plus OG1802 polymer; Reaction D included OG1950 antibody plus OG1802 polymer plus sucrose; Reaction E included OG1950 antibody plus OG1802 polymer plus trehalose; Reaction F included OG1950 antibody plus OG1802 polymer plus glutamic acid; Reaction G included OG1950 antibody plus OG1802 polymer plus aspartic acid. Reactions B–G were started with 100 μg OG1950 protein with a fixed total reaction volume. Upon reaction completion, equal volumes of Reactions B–G were injected for ion exchange analysis. A comparison of conjugation efficiencies is shown in Fig. 19. Peak 1 (P1) represents excess biopolymer (OG1802) that is not conjugated to the protein and cannot bind to the ion exchange column and therefore elutes as an unbound fraction in each reaction; Peak 2 (P2) represents the polymer-antibody bioconjugate in each reaction; Peak 3 (P3) represents free antibody that is not conjugated to the polymer in each reaction.

Table 11.1

Samples (P2)
OG1953 conjugate (Au × seconds) (P3)
OG1950 protein (AU × seconds) Conjugation efficiency
P2/[P3 B-reactions] A Control buffer NA NA NA B Control protein OG1950 0 410129 NA C Biopolymer OG1950 plus OG1802 37334 125884 9 % D C plus Sucrose 198064 110356 48% E C plus Trehalose 202682 139313 49% F C plus Glutamic acid 30401 220577 7% G C plus Aspartic acid 29054 175266 7%

As can be seen from the above results, different excipients allow significantly higher conjugation efficiency. Without being limited by theory, such excipients that help maintain the solubility of ingredients help conjugation efficiency.

Cation exchange assay of OG1953 conjugation reaction: After completion of the conjugation reaction, 5 μL of each reaction mixture was diluted 3 times 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 bind and elute mode, where the reaction mixture was first diluted to reduce the salt concentration so that both the conjugate and unreacted protein would bind to the column, followed by elution with a salt gradient, where increasing NaCl concentration resulted in the conjugate (OG1953) and unconjugated free protein (OG1950) eluting at different retention times due to the change in differential charge. The results of the IEX assay, as shown in Fig. 19 show that the OG1953 conjugate (peak P2) is very well separated from the unreacted free polymer (OG1802), as represented by peak P1, and the unreacted free protein (OG1950), as represented by peak P3.

Increased purification of OG1953 conjugate for activity analysis. Conjugation reactions D and E were combined and further separated by IEX, where the eluted peak (P2) conjugate fractions were collected and concentrated for activity analysis.

Example 12. Effect of anti-VEGF molecules on biotin-VEGF binding to VEGFR using ELISA.

The ability of OG1950, OG1953 and other anti-VEGF molecules to inhibit biotin-VEGF-165 binding to VEGFR was tested by ELISA assay. 96-well ELISA plates were coated with 1 μg/ml recombinant VEGF R1-Fc protein (R&D systems lot# 321-FL-050). Plates were incubated overnight at room temperature. Plates were then washed and blocked with blocking buffer (1% BSA in 1× PBS, pH 7.4) for ≥90 min with gentle shaking. 3-fold dilution series of samples were prepared. Starting at 400 nM, samples were mixed with biotin-VEGF (R&D systems lot# order# 06). The final concentration of biotin-VEGF was 4 ng/ml (100 pM). The upper concentration of the sample dilution series was 85 μg/mL. After dilution, samples were incubated at room temperature for ≥30 min and then washed 3 times. After washing, 100 μL of the sample/biotin-VEGF mixture was transferred to each well. The plates were incubated for ≥90 min at room temperature with gentle shaking. After incubation, 100 μL of a 1:1500 diluted SA-HRP (R&D systems lot # A7906) was added to each well. The incubated plates were protected from light for approximately 20 min. The plates were then washed 3 times. After washing, 100 μL of TMB substrate (R&D systems lot # DY998) was added. The plates were incubated and protected from light for approximately 30 min. Color development was monitored. Color development was stopped by adding 50 µl of stop solution. The plates were read at 450 nm. The results are shown in Fig. 20.

These results demonstrate that the OG1953 antibody/conjugate achieved maximal inhibition of biotin-VEGF binding to VEGFR compared to OG1950 (unconjugated antibody) as well as the commercially available VEGF inhibitors Lucentis® (ranibizumab) and Avastin® (bevacizumab). In a typical VEGF receptor-VEGF ligand binding assay, addition of OG1953 resulted in 97% to 98% inhibition of VEGF ligand binding to VEGF receptor. This compares to the addition of OG1950, which results in 75 to 87% inhibition of VEGF ligand-VEGF receptor binding, and the addition of Lucentis® (ranibizumab) or Avastin® (bevacizumab), each of which only results in 68 to 78% inhibition of VEGF ligand-VEGF receptor binding. Furthermore, previous studies have shown that the addition of either OG1950, Lucentis® (ranibizumab), or Avastin® (bevacizumab) at 400 nM concentrations does not result in 100% inhibition of VEGF ligand-VEGF receptor binding.

Fig. 20 also shows that another anti-VEGF antibody conjugate Bio Conjugate (anti-VEGF BC) achieves maximal inhibition of VEGF binding to the VEGF receptor compared to the antibody (anti-VEGF) alone. Together, these results demonstrate that (i) the OG1953 antibody conjugate is more potent at inhibiting VEGF ligand binding to the VEGF receptor compared to currently available VEGF inhibitors and (ii) conjugation of VEGF antibodies at a site outside the active site region may result in enhanced inhibition of VEGF ligand binding to the VEGF receptor.

In some embodiments, the present invention provides anti-VEGF antibody conjugates that exhibit superior (or at least equal) blocking ability compared to an anti-VEGF antibody alone.

Example 13. Method for determining the binding of OG1950 to Fc gamma receptor I and IIIa.

Binding kinetics experiments were performed at 25 °C using a BIAcore T200. Anti-his antibody was immobilized on a CM5 chip. Histidine-tagged FcγRI and FcγRIIIa at a concentration of 0.5 μg/mL prepared in HBS-EP buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% Tween-20) were injected independently for 60 s at a flow rate of 5 μL/min in the active flow cell only. The antibody-Avastin® (bevacizumab) conjugate, used as a positive control, was then injected through the control and active flow cells using 60 s injections at 30 μL/mL using a single cycle kinetics approach. Antibody concentrations ranging from 0.48 to 300 nM were used for FcγRI and from 7.8 to 2000 nM for FcγRIIIa. After each run, the flow cells were regenerated with a 60 s injection of 10 mM glycine pH 1.7 at a flow rate of 50 μg/mL. Data were double reference subtracted using subtraction of both the flow cell and control runs. Analysis was performed using BIA evaluation software. The results are shown in Figures 22 and 23. The results show that OG1950 did not show any significant binding to Fc gamma receptor I and IIIa in this assay.

Example 14. Method for determining the binding of OG1950 to human complement protein C1q

Complement was assessed using a C1q binding ELISA. The antibody panel was titrated 1:2 from a top concentration of 10 μg/mL in 1×PBS overnight at 4°C. Plates were then blocked after a 2-hour block in 1% BSA. Purified human C1q was then applied at 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 Fig. 24. The results show that C1q has greater binding affinity for Avastin® (bevacizumab) relative to OG1950 at antibody concentrations ranging from 10 μg/mL to 0.625 μg/mL.

In some embodiments, OG1950 has less than 10% binding to Avastin. In some embodiments, OG1950 has less binding to C1q than Avastin® (bevacizumab).

Example 15. Effect of anti-VEGF agents on VEGF-stimulated HRMVEC proliferation

Human retinal microvascular endothelial cells (HuRMVEC) proliferation assays were performed as follows: cells were maintained in complete CSC medium (cell systems, #4Zo-500) supplemented with 2% CultureBoost (cell systems, #4CB-500) and 0.2% Bac-off (cell systems, #4ZO-643) and seeded in 96-well plates in assay medium (serum-free medium (cell systems, #4Z3-500-S) with 5% FBS) at a density of 10,000 cells per well. VEGF inhibitors were first added at the indicated concentrations to each well. After 30 min, VEGF 165 (R&D systems, #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 read at OD450 nM.

The results showed that both independent OG1953 samples (OG1953A and OG1953B) exhibited potent inhibition of HuRNVEC proliferation. Both the maximum inhibition and IC ₅₀ values of OG1953 were comparable to that of OG1950 antibody alone in this assay. The maximum inhibition of OG1953 was also significantly better than that of Avastin® (bevacizumab) and Eylea® (aflibercept). The results (including IC ₅₀ values and their comparison with Lucentis® (ranibizumab), Eylea® (aflibercept), and Avastin® (bevacizumab)) are shown in Fig. 25.

Example 16. Single cycle kinetics (SCK) of VEGF binding to anti-VEGF agents captured on a protein A chip at 25 degrees

Binding kinetics were performed at 25 °C using a BIAcore T200 on Avastin® (bevacizumab), OG1950, and OG1953. Briefly, anti-VEGF agents were captured on a protein A (GE) chip. 1 μg/mL OG1950 and Avastin® (bevacizumab) were passed at a flow rate of 25 μL/min for 2 minutes. 10 μg/mL OG1953 was passed at 10 μL/min for 10 minutes. VEGF (recombinant, R&D systems) was passed over the captured antibodies for a contact time of 240 seconds each at 0.56 nM, 1.67 nM, 5 nM, 15 nM, and 45 nM for single cycle kinetics and dissociated for 30 minutes. Analysis was performed using BiaEvaluation software (GE). All sensorgrams were double-reference subtracted and fitted using a 1:1 Langmuir binding model. The rejection of these anti-VEGF agents may be underestimated in this experiment due to dissociation between anti-VEGF agents and protein A capture.

The results are shown in Fig. 26, including the calculated values of KD, ka and kd.

Example 17. Screening of excipients to prevent IgG1 precipitation caused by OG1802 polymer

When using the standard conjugation method, the OG1950 conjugation reaction mixture appeared turbid and a precipitate was immediately present upon stirring. Further investigation revealed that the precipitate was the protein itself, which in turn resulted in reduced conjugation efficiency observed by DNS-PAGE or ion exchange analysis, as the protein was lost to precipitation rather than participating in the conjugation reaction.

Initial troubleshooting experiments identified conditions that did not produce a clear reaction solution including (1) decreasing the polymer molar excess ratio from greater than 10 to less than 5; (2) pre-adjusting the pH of the reaction solution to be more acidic (e.g., pH 5) or basic (e.g., pH 8.5) from the standard neutral pH range (e.g., pH 6.5 to 7.5); (3) testing other IgG1 protein samples with the same or different isoelectric points (pI) compared to OG1950; (4) replacing the sample storage buffer with 1× PBS, pH 7.4 or 20 mM Tris, pH 7.4, 100 mM NaCl; and (5) pre-adjusting the OG1802 solution with 20 mM Tris, pH 7.4.

It is known that proteins carry a net surface charge that contributes to the solubility of the protein in aqueous solution. Amino acids that are hydrophilic include arginine, lysine, aspartic acid, and glutamic acid. At a neutral pH of 7, the side chains of these amino acids carry positive charges for arginine and lysine and negative charges for aspartic acid and glutamic acid. Changing the pH of a solution can modulate the intrinsic solubility of a protein, so some of the above-mentioned error-correcting experiments, such as (2), used this approach. Theoretically, the solubility of proteins in aqueous solution differs depending on the degree of hydrophobicity or hydrophilicity of the surface. Proteins with surfaces that are more hydrophobic will precipitate readily. The addition of ions (such as NaCl or another salt) creates an electron shielding effect that cancels out some of the activity between the water particles and the protein, decreasing solubility as the proteins bind to each other and begin to aggregate. In the current situation, it has been suggested that the biopolymer directly or indirectly modulates the protein surface charge and/or exposed surfaces in a manner that promotes intermolecular hydrophobic interactions, leading to protein precipitation.

Excipients that have been identified to modulate protein solubility include the following categories (i) detergents, including neutral detergent (e.g., 0.1-1% polysorbate 20 or Tween 20) or charged detergent (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 analogue, or 1-100 mM arginine) (v) polyethylene glycol (e.g., 0.03-1 mM PEG8000); and (vi) an organic solvent (e.g. 20% ethanol).

In a further design of experiment (DOE), various excipients from each of the above categories were selected based on their compatibility with pharmaceutical preparation for human injectable use. In addition, a highly acidic pH of 4 and a basic pH of 9 were also included in the evaluation. An IgG1 protein standard that did not contain an engineered cysteine was selected for the evaluation to minimize the potential interference of such an unpaired cysteine residue complicating the observation of precipitates. Table 17.1 shows such a matrix. The shaded code/rows represent conditions that yield a clear solution, while the unshaded rows represent conditions that yield a solution with varying degrees of turbidity or with precipitate.

Table 17.1

code Excipient Working buffer solution 1 OG1898_R5782_5xR7473 (control) 20 mM Tris pH 7.4, 100 mM NaCl 2 5 mg/ml_R5782_5xR7473_pH4 sodium acetate 3 5 mg/ml_R5782_5xR7473_pH9 sodium carbonate 4 5 mg/ml_R5782_5xR7473_1% Tween20 Tris 20 mM NaCl 5 5 mg/ml_R5782_5xR7473_0.1% Tween20 Tris 20 mM NaCl 6 5 mg/ml_R5782_5xR7473_1% SDS Tris 20 mM NaCl 7 5 mg/ml_R5782_5xR7473_0.1% SDS Tris 20 mM NaCl 8 5 mg/ml_R5782_5xR7473_6% Sucrose Tris 20 mM NaCl 9 5 mg/ml_R5782_5xR7473_6% Trehalose Tris 20 mM NaCl 10 5 mg/ml_R5782_5xR7473_0.03 mM Aspartic acid Tris 20mM NaCl 11 5mg/ml_R5782_5xR7473_1mM Aspartic acid Tris 20 mM NaCl 12 5 mg/ml_R5782_5xR7473_0.03 mM Glutamic acid Tris 20 mM NaCl 13 5 mg/ml_R5782_5xR7473_1 mM Glutamic acid Tris 20 mM NaCl 14 5 mg/ml_R5782_5xR7473_1 mM Arginine Tris 20 mM NaCl 15 5 mg/ml_R5782_5xR7473_100 mM Arginine Tris 20 mM NaCl 16 5 mg/ml_R5782_5xR7473_1 mM Urea Tris 20 mM NaCl 17 5 mg/ml_R5782_5xR7473_100 mM Urea Tris 20 mM NaCl 18 5 mg/ml_R5782_5xR7473_0.03 mM PEG8000 Tris 20 mM NaCl 19 5 mg/ml_R5782_5xR7473_1 mM PEG8000 Tris 20 mM NaCl 20 5 mg/ml_R5782_5xR7473_20% EtOH Tris 20 mM NaCl 21 5 mg/ml_OG1931_R8399_5xR7473_CTL Tris 20 mM NaCl 22 5 mg/ml_OG1931_R8399_5xR7473_1 mM Aspartic acid Tris 20 mM NaCl 23 5 mg/ml_OG1931_R8399_5xR7473_1 mM Arginine Tris 20 mM NaCl 24 8 mg/ml_OG1321_R7587_5xR7473_CTL Tris 20 mM NaCl 25 5 mg/ml_R5782_5xR7473_1 mM Lysine Tris 20 mM NaCl 26 5 mg/ml_R5782_5xR7473_100 mM Lysine Tris 20 mM NaCl

The resulting precipitated solutions (or non-precipitated solutions) are shown in Fig. 28.

All patent applications, websites, other publications, accession numbers, and the like, whether referenced above or below, are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual element was specifically and individually indicated and thereby incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number on the effective filing date of the present application shall be construed. The effective filing date shall mean the earlier of the actual filing date or the filing date of a priority application related to the accession number, as applicable. Likewise, if different versions of a publication, website, or the like are published at different times, the earliest published version on the effective filing date of the present application shall be construed, unless otherwise indicated. Any feature, step, element, embodiment, or aspect disclosed herein may be used in combination with any other unless otherwise specifically indicated. Although certain embodiments have been described in detail by way of illustration and example for the purpose of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

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SEQUENCE LIST

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Perlroth, Victor D.

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Phe Asn Arg Gly Glu Cys

210

<210> 7

<211> 235

<212> DNA

<213> Artificial Sequence

<220>

<223> Heavy chain

<400> 7

atgaaagctg tggtgctggc cgtggctctg gtcttcctga cagggagcca ggctgaggtg 60

cagctggtgg aatccggcgg aggcctggtc cagcctggcg gatccctgag actngtcctgt 120

gccgcctccg gctacgactt cacccattac ggcatgaact gggtccgaca ggcccctggc 180

aagggcctgg aatgggtcgg atggatcaac acctacaccg gcgagcccac ctacg 235

<210> 8

<211> 702

<212> DNA

<213> Artificial Sequence

<220>

<223> Light chain

<400> 8

atgggatgga gctgtatcat cctcttcttg gtggcaacag ctacaggcgt gcactccgac 60

atccagctga cccagtcccc ctccagcctg tccgcctctg tgggcgacag agtgaccatc 120

acctgttccg ccagccagga catctccaac tacctgaact ggtatcagca gaagcccggc 180

aaggccccca aggtgctgat ctacttcacc tcctccctgc actccggcgt gccctccaga 240

ttctccggct ctggctccgg caccgacttt accctgacca tctccagcct gcagcccgag 300

gacttcgcca cctactactg ccagcagtac tccaccgtgc cctggacctt cggccagggc 360

accaaggtgg aaatcaagcg gaccgtggcc gctccctccg tgttcatctt cccaccctcc 420

gacgagcagc tgaagtccgg aaccgcctcc gtcgtgtgcc tgctgaacaa cttctacccc 480

cgcgaggcca aggtgcagtg gaaggtggac aacgccctgc agagcggcaa ctcccaggaa 540

tccgtcaccg agcaggactc caaggacagc acctactccc tgtccagcac cctgaccctg 600

tccaaggccg actacgagaa gcacaaggtg tacgcctgcg aagtgaccca ccagggcctc 660

agctccccag tgaccaagtc cttcaaccgg ggcgagtgct ag 702

<210> 9

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Antigen binding protein components

<400> 9

Gly Tyr Asp Phe Thr His Tyr Gly Met Asn

1 5 10

<210> 10

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Antigen binding protein components

<400> 10

Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys

1 5 10 15

Arg

<210> 11

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Antigen binding protein components

<400> 11

Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val

1 5 10

<210> 12

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Antigen binding protein components

<400> 12

Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 13

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Antigen binding protein components

<400> 13

Phe Thr Ser Ser Leu His Ser

1 5

<210> 14

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Antigen binding protein components

<400> 14

Gln Gln Tyr Ser Thr Val Pro Trp Thr

1 5

<---

Claims (45)

1. An anti-VEGF-A antibody comprising a heavy chain and a light chain, wherein: 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), and 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, wherein the IgG1 constant domain comprises one or more of the following mutations to modulate effector function (EU numbering): E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S. 2. The antibody according to claim 1, comprising the following mutations (EU numbering): L234A, L235A and G237A. 3. The antibody of claim 1, wherein the heavy chain sequence of the anti-VEGF-A antibody is SEQ ID NO: 1, and the light chain sequence of the anti-VEGF-A antibody is SEQ ID NO: 2. 4. The antibody according to claim 1 or 2, which is IgG. 5. A pharmaceutical composition for the treatment of an ophthalmological neovascular disease, comprising an antibody according to any one of claims 1-4 and a pharmaceutically acceptable carrier. 6. A method for treating or preventing an ophthalmological disease, comprising administering to a subject in need of treatment or prevention of an ophthalmological disease an antibody according to any one of claims 1-4 or a pharmaceutical composition according to claim 5, wherein the ophthalmological 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), branch retinal vein occlusion (BRVO), corneal neovascularization, retinal neovascularization, retinopathy of prematurity (ROP), subconjunctival hemorrhage and hypertensive retinopathy. 7. The method of treatment or prevention according to claim 6, wherein the disease is diabetic retinopathy. 8. Anti-VEGF-A antibody comprising: a heavy chain comprising SEQ ID NO: 1; and a light chain comprising SEQ ID NO: 2. 9. An antibody conjugate having anti-VEGF activity comprising the antibody of claim 8 conjugated to a polymer to form a bioconjugate, wherein the bioconjugate has a molecular weight of from about 350,000 to 1,900,000 daltons. 10. An antibody that binds to VEGF-A, comprising: CDR _H 1, which is CDR _H 1 of SEQ ID NO: 1; CDR _H 2, which is CDR _H 2 of SEQ ID NO: 1; CDR _H 3, which is CDR _H 3 of SEQ ID NO: 1; CDR _L 1, which is CDR _L 1 of SEQ ID NO: 2; CDR _L 2, which is CDR _L 2 of SEQ ID NO: 2; CDR _L 3, which is CDR _L 3 of SEQ ID NO: 2; at least one of the following mutations (EU numbering): L234A, L235A, and G237A; and at least one of the following mutations (EU numbering): Q347C or L443C. 11. The antibody according to claim 10, which is human IgG1. 12. The antibody of claim 10 or 11, wherein the antibody comprises all three of the following mutations (EU numbering) L234A, L235A and G237A, and wherein the antibody comprises L443C (EU numbering). 13. An antibody conjugate having anti-VEGF activity comprising an antibody according to any one of claims 1-4 and a polymer comprising phosphorylcholine, wherein the polymer is covalently linked to the antibody via a non-native cysteine outside the variable region of the antibody. 14. The antibody conjugate of claim 13, wherein the cysteine is in the heavy chain and is C443 (EU numbering). 15. An antibody conjugate according to claim 13 or 14, wherein the polymer containing phosphorylcholine is formed from 2-(methacryloyloxyethyl)-2'-(trimethylammonium)ethyl phosphate (MPC) monomers as follows: 16. The antibody conjugate of claim 15, wherein the polymer has three or more arms or is synthesized with an initiator comprising 3 or more polymer initiation sites. 17. The antibody conjugate of claim 15, wherein the polymer has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 arms or is synthesized with an initiator comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 polymer initiation sites. 18. The antibody conjugate of claim 17, wherein the polymer has 2, 3, 6 or 9 arms or is synthesized with an initiator comprising 2, 3, 6 or 9 polymer initiation sites. 19. The antibody conjugate of claim 18, wherein the polymer has 9 arms or is synthesized with an initiator that includes 9 polymer initiation sites. 20. The antibody conjugate of claim 19, wherein the polymer has a molecular weight of from about 300,000 to about 1,750,000 Da, as measured by size exclusion chromatography with multi-angle light scattering (hereinafter "SEC-MALS"). 21. The antibody conjugate of claim 20, wherein the polymer has a molecular weight of from about 500,000 to about 1,000,000 Da. 22. The antibody conjugate of claim 21, wherein the polymer has a molecular weight of from about 750,000 to about 850,000 Da. 23. An antibody conjugate according to any one of claims 13-22, which is purified. 24. The antibody conjugate of claim 23, wherein the polymer is polydisperse. 25. The antibody conjugate of claim 24, wherein the polymer has a polydispersity index (PI) of less than about 1.2. 26. An antibody conjugate according to claim 13 or 14, having the following structure: Where each heavy chain of an anti-VEGF-A antibody is designated by the letter H, and each light chain of an anti-VEGF-A antibody is designated by the letter L; the polymer is linked to the anti-VEGF-A antibody via the C443 sulfhydryl group (EU numbering), with the linkage depicted on one of the heavy chains; PC is where the wavy line indicates the point of attachment to the rest of the polymer, where 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 equal to 2500 plus or minus 15%.