Factor VIII Zwitterionic Polymer Conjugates

Abstract

Multi-arm high MW polymers containing hydrophilic groups conjugated to Factor VIII, and methods for preparing said polymers, are provided. [Solution] A composition is provided that includes a conjugate comprising recombinant FVIII (rFVIII) and a zwitterionic polymer, wherein the polymer comprises one or more monomer units and at least one monomer unit comprises a zwitterionic group, and a conjugate molecule comprising rFVIII comprising a light chain and a heavy chain including at least a portion of the B domain, and a zwitterionic polymer.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a non-provisional application and claims the benefit of U.S. Patent Application No. 61 / 875,099, filed September 8, 2014, which is incorporated by reference in its entirety for all purposes. [Background technology]

[0002] Hemophilia A is an inherited blood clotting disorder caused by a deficiency or mutation in factor VIII (FVIII). Factor VIII is a critical component of the intrinsic blood clotting pathway. Factor VIII deficiency causes increased bleeding. Hemophilia A is X-linked and occurs in approximately 1 in 5,000 males.

[0003] Patients with hemophilia A are currently treated with intravenous administration of full-length recombinant human FVIII. Treatment can be prophylactic or as needed following a bleeding injury. The half-life of factor VIII in humans is relatively short, typically about 11 hours. Therefore, effective treatment requires frequent administration, approximately three times per week. However, such frequent administration, typically via intravenous infusion, is undesirable because it requires frequent visits to a clinic or other healthcare provider. Furthermore, such frequent administration can reduce patient compliance with the prescribed dosing regimen.

[0004] Another drawback of current factor VIII therapy is that approximately 25–30% of patients treated with factor VIII develop antibodies to FVIII. Patients with high levels of circulating anti-factor VIII antibodies cannot be successfully treated with current factor VIII therapeutics. These patients require more expensive treatment regimens, including factor VIIa, and immune tolerance therapy.

[0005] The effectiveness of therapeutic drugs can be enhanced by improving their bioavailability and pharmacokinetic properties. One approach to improving bioavailability has been PEGylation. PEGylation involves the addition of polyethylene glycol chains to a drug, typically a protein. PEG conjugates have been attributed with reduced immunogenicity or antigenicity, increased half-life, increased solubility, reduced renal clearance, and reduced enzymatic degradation. As a result of these attributes, PEG conjugates of certain bioactive agents often require less frequent administration, potentially allowing for the use of less active agent to achieve a therapeutic endpoint. Less frequent administration is generally desirable because it reduces the absolute number of painful injections that require inconvenient visits to a healthcare professional.

[0006] Although some success has been achieved with PEG conjugates, "PEGylation" of bioactive agents remains a difficult challenge. As drug developers progress beyond highly potent agonist proteins such as erythropoietin and various interferons, the potential benefits of PEG hydrophilic polymers in increased solubility, stability, and bioavailability do not fully compensate for increased viscosity and immunogenicity.

[0007] PEGylation of FVIII has not been shown to significantly increase the half-life of the conjugate in vivo.

[0008] Therefore, there remains a need for FVIII drugs that have increased in vivo half-lives while retaining sufficient biological activity. Summary of the Invention

[0009] The present invention provides a conjugate comprising recombinant FVIII (rFVIII) and a zwitterionic polymer, wherein the polymer comprises one or more monomer units, and at least one monomer unit comprises a zwitterionic group. Optionally, the zwitterionic group comprises phosphorylcholine. Optionally, the monomer comprises 2-(acryloyloxyethyl)-2'-(trimethylammoniumethyl)phosphate. Optionally, the monomer comprises 2-(methacryloyloxyethyl)-2'-(trimethylammoniumethyl)phosphate (HEMA-PC). The rFVIII may have a partial or complete deletion of the B-domain, or may have an intact B-domain. Optionally, the polymer has 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 arms. Optionally, the polymer has 3, 6, or 9 arms, preferably the polymer has 9 arms.

[0010] Some conjugates, including rFVIII, are such that the polymer portion has a peak molecular weight between 300,000 and 1,750,000 daltons. Some conjugates have a polymer portion with a peak molecular weight between 500,000 and 1,000,000 daltons. Some conjugates have a polymer portion with a peak molecular weight between 600,000 and 800,000 daltons.

[0011] In some conjugates, rFVIII is covalently bound to a polymer. In some conjugates, the polymer is covalently bound to at least one of an amino group, a hydroxyl group, a sulfhydryl group, and a carboxyl group of rFVIII. In some conjugates, the sulfhydryl group is from a cysteine ​​residue in rFVIII. In some conjugates, the cysteine ​​residue is a recombinant cysteine ​​residue. In some conjugates, the recombinant cysteine ​​residue is selected from the group consisting of Y81C, F129C, K377C, H378C, K422C, Q468C, L491C, L504C, K556C, K570C, D1795C, Q1796C, R1803C, K1804C, K1808C, K1810C, T1821C, K1813C, N1864C, T1911C, N2118C, Q2091C, F2093C, and Q2284C, where the residues are numbered from the corresponding residue in SEQ ID NO: 1 ( FIG. 1 ) when the recombinant factor VIII is maximally aligned with SEQ ID NO: 1. In some conjugates, the cysteine ​​residue is naturally present in rFVIII. In some conjugates, the cysteine ​​residue is in the B domain. In some conjugates, the cysteine ​​residue is selected from the group consisting of 1293C, 1373C, 1604C and 1636C, preferably 1604C or 1636C.

[0012] Preferred conjugates have a polymer moiety comprising rFVIII and 3, 6 or 9 arms, preferably 9 arms, with a peak molecular weight between 100,000 and 1,500,000, more preferably 500,000 and 1,000,000 daltons or 600,000 and 850,000 daltons.

[0013] The present invention further provides a conjugate comprising recombinant FVIII (rFVIII) comprising at least a portion of the B domain and a zwitterionic polymer, wherein the polymer comprises one or more monomer units, at least one of the monomer units comprising a zwitterionic group, and the polymer is conjugated to the rFVIII via a cysteine ​​residue in the B domain, with one branched polymer conjugated per molecule of rFVIII. Optionally, the polymer is a branched polymer, optionally having nine branches. Optionally, the polymer is conjugated via a cysteine ​​residue that is one of the two most C-terminal cysteine ​​residues in the portion of the B domain. Optionally, the conjugate has an in vivo half-life in humans of at least 20 hours.

[0014] The present invention further provides a composition comprising a conjugate molecule comprising recombinant FVIII (rFVIII) comprising a light chain and a heavy chain comprising at least a portion of a B domain and a zwitterionic polymer, wherein the polymer comprises one or more monomeric units, at least one of the monomeric units comprising a zwitterionic group, and the polymer is conjugated to the rFVIII via a cysteine ​​residue in the B domain, wherein at least 80, 90, 95, or 99% of the conjugate molecules in the composition have the same portion of the B domain, and one polymer is conjugated per molecule of rFVIII. Optionally, the polymer is branched, optionally with 9 branches. Optionally, the heavy chain comprises at least residues 1-1604 of SEQ ID NO: 1, or at least residues 1-1636 of SEQ ID NO: 1, or at least residues 1-1648 of SEQ ID NO: 1. Optionally, the heavy chain consists of residues 1-1648 of SEQ ID NO: 1. Optionally, at least a portion of the B domain is an intact B domain. Optionally, the polymer is conjugated via a cysteine ​​that is one of the two most C-terminal cysteines in the B domain.

[0015] The present invention further provides a pharmaceutical composition comprising the above conjugate.

[0016] The present invention further provides a method of treating hemophilia, comprising administering to a subject suffering from hemophilia a therapeutically effective amount of the conjugate described above.

[0017] The present invention further provides a method for the prevention of hemophilia in a subject, comprising administering a therapeutically effective amount of Noi The present invention provides a method for administering any of the conjugates or compositions to a subject with hemophilia when the subject is not known to be experiencing external or internal bleeding, wherein the conjugate persists in the blood to promote clotting after subsequent bleeding events. Optionally, the conjugate or composition is administered no more frequently than once a week. Optionally, the conjugate or composition is administered between weekly and monthly. Optionally, the subject has a trough level of FVIII activity greater than 1%, 3%, or 5% of the mean FVIII activity in control subjects without hemophilia. Optionally, the subject has developed antibodies to FVIII from a previous administration of FVIII not conjugated to a polymer. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 shows the amino acid sequence of mature human factor VIII (SEQ ID NO: 1).

[0019] [Figure 2] FIG. 1 shows the domains of human factor VIII and the location of cysteine ​​residues (adapted from Lenting et al., Blood 1998;92:3983-3996). Detailed Description of the Invention

[0020] I. Overview The present invention provides high molecular weight (MW) polymers having hydrophilic groups or zwitterions, such as phosphorylcholine. Also provided are methods and novel starting materials for making high MW polymers. Also provided are conjugates of high MW polymers and functional agents (as defined herein). International Patent Application Nos. PCT / US2011 / 032768 and PCT / US2007 / 005372 are incorporated herein by reference for all purposes.

[0021] II. Definition "Polymer" refers to a series of linked monomers. High MW polymers are prepared from monomers including, but not limited to, acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl-pyridines, vinyl-pyrrolidones, and vinyl esters (such as vinyl acetate). Additional monomers are useful in the high MW polymers of the present invention. When two different monomers are used, the two monomers are referred to as "comonomers," meaning that the different monomers are copolymerized to form a single polymer. A polymer may be linear or branched. When a polymer is branched, each polymer chain is referred to as a "polymer arm." The end of the polymer arm connected to the initiator moiety is the proximal end, and the growing chain end of the polymer arm is the distal end. On the growing chain end of a polymer arm, the polymer arm end group may be a radical scavenger or another group.

[0022] "Initiator" refers to a compound capable of initiating polymerization with the monomers or comonomers of the present invention. The polymerization may be a conventional free radical polymerization, or preferably a controlled / "living" radical polymerization, such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation-termination (RAFT) polymerization, or nitroxide-mediated polymerization. The polymerization may be a "pseudo" controlled polymerization, such as regressive transfer. If the initiator is suitable for ATRP, it contains a labile bond that can undergo homolytic cleavage to form an initiator fragment, I, which is a radical that can initiate radical polymerization, and a radical scavenger, I', that reacts with radicals in the growing polymer chain to reversibly terminate the polymerization. The radical scavenger I' is typically a halogen, but can also be an organic moiety such as a nitrile.

[0023] A "linker" refers to a chemical moiety that connects two groups. A linker may be cleavable or non-cleavable. Cleavable linkers can be hydrolyzable, enzymatically cleavable, pH-sensitive, photolabile, or disulfide linkers, etc. Other linkers include homobifunctional and heterobifunctional linkers. A "linking group" is a functional group that can form a covalent bond consisting of one or more bonds with a bioactive agent. Non-limiting examples include those shown in Table 1.

[0024] As used herein, the term "reactive group" refers to a group that can react with another chemical group to form a covalent bond, i.e., that is covalently reactive under appropriate reaction conditions and typically represents an attachment point for another substance. Reactive groups are moieties, such as maleimides or succinimidyl esters, on the compounds of the invention that can chemically react with a functional group on a different compound to form a covalent bond. Reactive groups generally include nucleophiles, electrophiles, and photoactivatable groups.

[0025] "Functional substance" is defined to include a bioactive agent or a diagnostic agent. "Bioactive agent" is defined to include any substance, drug, compound, or mixture thereof that targets a specific biological location (targeting agent) and / or provides some local or systemic physiological or pharmacological effect that can be demonstrated in vivo or in vitro. Non-limiting examples include drugs, vaccines, antibodies, antibody fragments, scFvs, diabodies, avimers, vitamins and cofactors, polysaccharides, carbohydrates, steroids, lipids, fats, proteins, peptides, polypeptides, nucleotides, oligonucleotides, polynucleotides, and nucleic acids (e.g., mRNA, tRNA, snRNA, RNAi, DNA, cDNA, antisense constructs, ribozymes, etc.). "Diagnostic agent" is defined to include any substance that allows for the detection or imaging of tissue or disease. Examples of diagnostic agents include, but are not limited to, radiolabels, fluorophores, and dyes.

[0026] "Therapeutic protein" refers to a peptide or protein that contains an amino acid sequence that constitutes, in whole or in part, a drug and that can be used for human or veterinary pharmaceutical applications. Many therapeutic proteins are known, including but not limited to those disclosed herein.

[0027] "Phosphorylcholine," also referred to as "PC," is: [ka] (* indicates attachment point) Phosphorylcholine is a zwitterionic group, including salts (such as internal salts) and its protonated and deprotonated forms.

[0028] A "phosphorylcholine-containing polymer" is a polymer that contains phosphorylcholine. A "zwitterion-containing polymer" refers to a polymer that contains a zwitterion.

[0029] Poly(acryloyloxyethyl phosphorylcholine)-containing polymer refers to a polymer that contains 2-(acryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate as a monomer.

[0030] Poly(methacryloyloxyethyl phosphorylcholine)-containing polymers refer to polymers that contain 2-(methacryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate as a monomer.

[0031] "Molecular weight" in the context of polymers can be expressed as either number average molecular weight, weight average molecular weight, or peak molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to peak molecular weight. These molecular weight measurements, number average (Mn), weight average (Mw), and peak (Mp), can be measured using size exclusion chromatography or other liquid chromatography techniques. Other methods for determining molecular weight values ​​can also be used, such as end-group analysis or measurements of colligative properties (e.g., freezing point depression, boiling point elevation, or osmotic pressure) to determine number average molecular weight, or light scattering techniques, ultracentrifugation, or viscosity measurements to determine weight average molecular weight. In a preferred embodiment of the present invention, molecular weight is measured by SEC-MALS (size exclusion chromatography-multiangle light scattering). The polymeric reagents of the present invention are typically polydisperse (i.e., the number average molecular weight and weight average molecular weight of the polymer are not equal) and preferably have a low polydispersity value of less than about 1.5, as determined, for example, by gel permeation chromatography. In other embodiments, the polydispersity index (PDI) is more preferably in the range of about 1.4 to about 1.2, even more preferably less than about 1.15, even more preferably less than about 1.10, even more preferably less than about 1.05, and most preferably less than about 1.03.

[0032] As used herein, the phrase "a" or "an" entity refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0033] As used herein, "about" refers to the variation that can be found in measurements made between different instruments, samples, and sample preparations.

[0034] "Protected," "protected type," "protecting group," and "protective group" refer to the presence of a group (i.e., a protecting group) that prevents or blocks reaction of a particular chemically reactive functional group in a molecule under certain reaction conditions. Protecting groups vary depending on the type of chemically reactive group being protected as well as the reaction conditions used and the presence of additional reactive or protecting groups, if any, in the molecule. Suitable protecting groups include those found in the article by Greene et al., "Protective Groups In Organic Synthesis," 3rd Edition, John Wiley and Sons, Inc. New York, 1999.

[0035] "Alkyl" refers to a straight-chain or branched, saturated, aliphatic group having the indicated number of carbon atoms. For example, C1-C6-alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, and the like. Other alkyl groups include, but are not limited to, heptyl, octyl, nonyl, decyl, and the like. Alkyl can contain any number of carbons, 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. Alkyl groups are typically monovalent, but may be divalent, such as when the alkyl group links two moieties.

[0036] The term "lower" referred to above and below in connection with organic groups or compounds, respectively, defines compounds or groups which may be branched or unbranched having up to 7, preferably up to 4 carbon atoms, and (as unbranched) may have 1 or 2 carbon atoms.

[0037] "Alkylene" refers to an alkyl group, as defined above, that links at least two other groups, i.e., a divalent hydrocarbon group. 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 is -(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.

[0038] Substituents for alkyl and heteroalkyl groups (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are -OR', ═O, ═NR', ═N-OR', -NR'R'', -SR', -halogen, -SiR'R''R' in a number ranging from 0 to (2m'+1) where m' is the total number of carbon atoms in the group. R', R'', and R''' can be various groups selected from -OC(O)R', -C(O)R', -COR', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR''C(O)R', -NH-C(NH)=NH, -NR'C(NH)=NH, -NH-C(NH)=NR', -S(O)R', -S(O)R', -S(O)NR'R'', -CN, and -NO. R', R'', and R''' each independently represent hydrogen, unsubstituted (C1-C8) alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted alkyl, alkoxy, or thioalkoxy groups, or aryl-(C1-C4)alkyl groups. When R' and R'' are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is intended to include 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is intended to include groups such as haloalkyl (e.g., -CF3 and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CHOCH3, etc.). Preferably, substituted alkyl and heteroalkyl groups have 1 to 4 substituents, more preferably 1, 2, or 3 substituents. Exceptions include perhaloalkyl groups (e.g., pentafluoroethyl, etc.), which are also preferred and contemplated by the present invention.

[0039] Substituents for alkyl and heteroalkyl groups (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) include, but are not limited to, -OR', ═O, ═NR', ═N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''', - and -N0. R', R'', R''', and R'' each preferably independently represent hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1 to 3 halogens, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compound of the invention includes more than one R group, for example, each R group is independently selected, such as R', R", R'", and R"", when each of these groups is present more than once. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is intended to include groups containing carbon atoms bonded to groups other than hydrogen atoms, such as haloalkyl (e.g., -CF and -CHCF) and acyl (e.g., -C(O)CH, -C(O)CF, -C(O)CHOCH, etc.).

[0040] "Alkoxy" refers to an alkyl group having an oxygen atom connecting the alkoxy to the point of attachment or bonded to two carbons of the alkoxy group. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, and the like. Alkoxy groups can be further substituted with various substituents described herein. For example, alkoxy groups can be substituted with halogens to form "halo-alkoxy" groups.

[0041] The term "carboxyalkyl" refers to an alkyl group (as defined herein) substituted with a carboxy group. The term "carboxycycloalkyl" refers to a cycloalkyl group (as defined herein) substituted with a carboxy group. The term alkoxyalkyl refers to an alkyl group (as defined herein) substituted with an alkoxy group. As used herein, the term "carboxy" refers to carboxylic acids and esters thereof.

[0042] "Haloalkyl" refers to an alkyl as defined above in which some or all of the hydrogen atoms have been replaced with halogen atoms. Halogen (halo) preferably represents chloro or fluoro, but may also be bromo or iodo. For example, haloalkyl includes trifluoromethyl, fluoromethyl, 1,2,3,4,5-pentafluorophenyl, etc. The term "perfluoro" defines a compound or group having all available hydrogens replaced with fluorine. For example, perfluorophenyl refers to 1,2,3,4,5-pentafluorophenyl, perfluoromethyl refers to 1,1,1-trifluoromethyl, and perfluoromethoxy refers to 1,1,1-trifluoromethoxy.

[0043] "Fluoro-substituted alkyl" refers to an alkyl group in which one, some, or all of the hydrogen atoms have been replaced by fluorine.

[0044] A "cytokine" in the context of this invention is a member of a group of protein signaling molecules that can participate in intercellular communication in immune and inflammatory responses. Cytokines are small, water-soluble glycoproteins that typically have a mass of approximately 8-35 kDa.

[0045] "Cycloalkyl" refers to a cyclic hydrocarbon group containing about 3 to 12, 3 to 10, or 3 to 7 ring carbon atoms. Cycloalkyl groups include fused, bridged, and spiro ring structures.

[0046] "Intracyclic" refers to an atom or group of atoms that form part of a cyclic ring structure.

[0047] "Exocyclic" refers to an atom or group of atoms that are attached to, but do not define, the cyclic ring structure.

[0048] "Cyclic alkyl ether" refers to a 4- or 5-membered cyclic alkyl group having 3 or 4 endocyclic carbon atoms and one exocyclic oxygen or sulfur atom (e.g., oxetane, thietane, tetrahydrofuran, tetrahydrothiophene); or a 6- to 7-membered cyclic alkyl group having one or two endocyclic oxygen or sulfur atoms (e.g., tetrahydropyran, 1,3-dioxane, 1,4-dioxane, tetrahydrothiopyran, 1,3-dithiane, 1,4-dithiane, 1,4-oxathiane).

[0049] "Alkenyl" refers to either a straight-chain or branched hydrocarbon of 2 to 6 carbon atoms having 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 carbons. Alkenyl groups are typically monovalent, but can be divalent, such as when the alkenyl group links two moieties.

[0050] "Alkenylene" refers to an alkenyl group, i.e., a divalent hydrocarbon group, as defined above, that links at least two other groups. 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.

[0051] "Alkynyl" refers to either a straight-chain or branched hydrocarbon of 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-hexatriynyl. 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 carbons. Alkynyl groups are typically monovalent, but can be divalent, such as when the alkynyl group links two moieties.

[0052] "Alkynylene" refers to an alkynyl group, i.e., a divalent hydrocarbon group, as defined above, that links at least two other groups. 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.

[0053] "Cycloalkyl" refers to a saturated or partially unsaturated, monocyclic, fused bicyclic, or bridged polycyclic ring assembly containing 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.

[0054] "Cycloalkylene" refers to a cycloalkyl group, i.e., a divalent hydrocarbon group, as defined above, that links at least two other groups. 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.

[0055] "Heterocycloalkyl" refers to a ring system having from 3 to about 20 ring members and from 1 to about 5 heteroatoms (such as N, O, and S). Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si, and P. Heteroatoms can be oxidized, including, but not limited to, -S(O)- and -S(O)-. For example, heterocycles include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, morpholino, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, piperidinyl, indolinyl, quinuclidinyl, and 1,4-dioxa-8-aza-spiro[4.5]dec-8-yl.

[0056] "Heterocycloalkylene" refers to a heterocycloalkyl group, as defined above, that links at least two other groups. The two moieties linked to the heterocycloalkylene can be attached to the same atom or different atoms of the heterocycloalkylene.

[0057] "Aryl" refers to a monocyclic or fused bicyclic, tricyclic, or higher aromatic ring assembly containing 6 to 16 ring carbon atoms. For example, aryl can be phenyl, benzyl, or naphthyl, preferably phenyl. "Arylene" refers to a divalent radical derived from an aryl group. The aryl group can be mono-, di-, or tri-substituted with one, two, or three groups selected from alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy, and oxy-C2-C3-alkylene, all of which are optionally further substituted as defined above; alternatively, it can be 1- or 2-naphthyl; or alternatively, 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 an oxy-C2-C3-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

[0058] Preferred as aryl is naphthyl, phenyl or phenyl mono- or di-substituted by alkoxy, phenyl, halogen, alkyl or trifluoromethyl, especially phenyl or phenyl mono- or di-substituted by alkoxy, halogen or trifluoromethyl, especially phenyl.

[0059] Examples of substituted phenyl groups as R include, 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-(imidazoline) ... and 4-(piperidinyl)-phenyl and 4-(pyridinyl)phenyl optionally substituted by a heterocyclic ring.

[0060] "Arylene" refers to an aryl group, as defined above, that links 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.

[0061] "Arylene-oxy" refers to an arylene group, as defined above, where one of the arylene-linked moieties is linked through an oxygen atom. Arylene-oxy groups include, but are not limited to, phenylene-oxy.

[0062] Similarly, the substituents on the aryl and heteroaryl groups vary and range from 0 to the total number of open valences on the aromatic ring system, including -halogen, -OR', -OC(O)R', -NR'R'', -SR', -R', CN, -NO2, -CO2R', -CONR'R'', -C(O)R', -OC(O)NR'R'', -NR''C(O)R', -NR''C(O)2R', -NR'-C(O)NR''R'''', -NH-C(NH2)=NH, -NR'C(NH2=NH, -NH-C(NH2 )=NR', -S(O)R', -S(O)R', -S(O)NR'R'', -N, -CH(Ph), perfluoro(C1-C4)alkoxy, and perfluoro(C1-C4)alkyl, where R', R'', and R''' are independently selected from hydrogen, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4)alkyl, and (unsubstituted aryl)oxy-(C1-C4)alkyl.

[0063] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring optionally have the formula -TC(O)-(CH2) q -U-, where T and U are independently -NH-, -O-, -CH2-, or a single bond, and q is an integer of 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH2) r Optionally, one of the single bonds in the new ring thus formed may be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CH) s -X-(CH2) t-(wherein s and t are independently an integer of 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.

[0064] "Heteroaryl" refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, of which 1 to 4 are heteroatoms, N, O, or S, respectively. For example, heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any other group substituted, particularly mono- or di-substituted, by, for example, alkyl, nitro, or halogen. Pyridyl refers to 2-, 3-, or 4-pyridyl, advantageously 2- or 3-pyridyl. Thienyl refers to 2- or 3-thienyl. Quinolinyl preferably refers to 2-, 3-, or 4-quinolinyl. Isoquinolinyl preferably represents 1-, 3-, or 4-isoquinolinyl. Benzopyranyl and benzothiopyranyl preferably represent 3-benzopyranyl or 3-benzothiopyranyl, respectively. Thiazolyl preferably represents 2- or 4-thiazolyl, most preferably 4-thiazolyl. Triazolyl is preferably 1-, 2-, or 5-(1,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl.

[0065] Preferably, 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, especially mono- or di-substituted groups.

[0066] As used herein, the term "heteroalkyl" refers to an alkyl group having 1 to 3 heteroatoms (such as N, O, and S). Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si, and P. Heteroatoms can also be oxidized, including, but not limited to, -S(O)- and -S(O)2-. For example, heteroalkyls can include ethers, thioethers, alkyl-amines, and alkyl-thiols.

[0067] As used herein, the term "heteroalkylene" refers to a heteroalkyl group, as defined above, that links at least two other groups. The two moieties linked to the heteroalkylene can be linked to the same atom or different atoms of the heteroalkylene.

[0068] "Electrophile" refers to an ion or atom or collection of atoms, which may be ionic, that has an electrophilic center, i.e., a center that is electron-seeking and can react with a nucleophile. An electrophile (or electrophile) is a reagent that forms a bond with its reaction partner (nucleophile) by accepting both bonding electrons from that partner.

[0069] "Nucleophile" refers to an ion or atom, which may be ionic, or a collection of atoms, that has a nucleophilic center, i.e., a center that is seeking an electrophilic center or that can react with an electrophile. A nucleophile (or nucleophile) is a reagent that forms a bond with its reaction partner (electrophile) by donating both bonding electrons. A "nucleophilic group" refers to a nucleophile after it has reacted with a reactive group. Non-limiting examples include amino, hydroxyl, alkoxy, haloalkoxy, etc.

[0070] "Maleimides" react with sulfhydryls (e.g., thioalkyls) to form the structure [ka] (The "●" indicates the attachment point of the maleimide group, JPEG0007767260000003.jpg5146" shows the remainder of the original sulfhydryl-bearing group and the attachment point of the sulfur atom of the thiol) The structure forming the -S-maleimide group has: [ka] It refers to a pyrrole-2,5-dione-1-yl group having the formula:

[0071] For purposes of this disclosure, "naturally occurring amino acids" found in proteins and polypeptides are L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamine, L-glutamic acid, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and / or L-valine. An "unnatural amino acid" found in proteins is any amino acid other than those listed as naturally occurring amino acids. Unnatural amino acids include, but are not limited to, D-isomers of the naturally occurring amino acids and mixtures of D and L-isomers of the naturally occurring amino acids. Other amino acids, such as 4-hydroxypurine, desmosine, isodesmosine, 5-hydroxylysine, ε-N-methyllysine, and 3-methylhistidine, are found in naturally occurring proteins but are generally introduced by means other than ribosomal translation of mRNA and, therefore, for the purposes of this disclosure, are considered unnatural amino acids found in proteins.

[0072] "Linear" in reference to the geometry, architecture or overall structure of a polymer refers to a polymer having a single polymer arm.

[0073] "Branched," in reference to the geometry, architecture, or overall structure of a polymer, refers to a polymer having two or more polymer "arms" extending from a core structure contained in the initiator. The initiator may be used in atom transfer radical polymerization (ATRP) reactions. A branched polymer may have two polymer chains (arms), three polymer arms, four polymer arms, five polymer arms, six polymer arms, seven polymer arms, eight polymer arms, nine polymer arms, or more. Each polymer arm extends from a polymer initiation site. Each polymer initiation site can serve as a site for polymer chain growth by the addition of monomers. For example, but not by way of limitation, when using ATRP, the polymer initiation site on the initiator is typically an organic halide that undergoes a reversible oxidation-reduction process catalyzed by a transition metal compound such as cuprous halide. Preferably, the halide is bromine.

[0074] A "pharmaceutically acceptable" composition or "pharmaceutical composition" refers to a composition comprising a compound of the invention and pharmaceutically acceptable excipient(s).

[0075] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to an excipient that can be included in the compositions of the present invention, does not cause significant adverse toxicological effects in patients, and is approved or approvable by the FDA for therapeutic use, particularly in humans. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, saline, lactated Ringer's solution, normal sucrose, normal glucose, etc.

[0076] "Patient" or "subject in need thereof" refers to an organism suffering from or prone to a condition that can be prevented or treated by administration of the pharmaceutical compositions provided herein. Non-limiting examples include humans, other mammals, and non-mammalian animals.

[0077] The conjugate is preferably provided in an isolated form. Isolated means that the target species is at least partially separated from contaminants with which it is naturally associated or used in its preparation, but does not necessarily exclude the presence of other components intended to act in combination with the isolated species, such as pharmaceutical excipients. Preferably, the conjugate is the predominant macromolecular species present in a sample (i.e., it constitutes at least about 50% (on a molar basis) of all macromolecular species present in the composition). Generally, an isolated conjugate constitutes more than 80, 90, 95, or 99% of all macromolecular species present in the composition. Most preferably, the conjugate is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) so that the composition essentially consists of a single macromolecular species. Although the conjugates have the same heavy and light chains, which are considered to be the same species, there may be variations in glycosylation of the protein portion and variations in the number of monomers in the polymer portion attached to different molecules of the conjugate.

[0078] A "therapeutically effective amount" refers to an amount of a combined functional substance or pharmaceutical composition useful for treating, ameliorating, or preventing an identified disease or condition, or for exhibiting a detectable therapeutic or inhibitory effect. Effect can be detected in an individual patient relative to a pre-treatment baseline measurement, or by determining a statistically significant difference in performance between a treated and a control population.

[0079] The "biological half-life" of a substance is a pharmacokinetic parameter that specifies the time required for one-half of the substance to be eliminated from an organism after its introduction into that organism.

[0080] Sequence identity can be determined by aligning sequences using default gap parameters, or by inspection and best alignment (i.e., resulting in the highest percentage of sequence similarity over the comparison window) using algorithms such as BESTFIT, FASTA, and TFASTA from Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, WI. Percentage sequence identity is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where identical residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage sequence identity.

[0081] III. Factor VIII Coagulation factor VIII (FVIII) circulates in plasma at extremely low concentrations and is noncovalently bound to von Willebrand factor (VWF). During hemostasis, FVIII dissociates from VWF and acts as a cofactor for activated factor IX (FIXa)-mediated factor X (FX) activation by enhancing the rate of activation in the presence of calcium and phospholipids or cell membranes.

[0082] FVIII is synthesized as a single-chain precursor of approximately 270-330 kDa with the domain structure A1-A2-B-A3-C1-C2 (Figure 2). When purified from plasma (e.g., "plasma-derived" or "plasmogenic"), FVIII is composed of a heavy chain (A1-A2-B) and a light chain (A3-C1-C2). The molecular mass of the light chain is 80 kDa, while the heavy chain ranges from 90-220 kDa due to proteolysis within the B domain. The domains are depicted in SEQ ID NO: 1 as follows: A1, residues Ala1 to Arg372; A2, residues Ser373 to Arg740; B, residues Ser741 to Arg1648; A3, residues Ser1690 to Ile2032; C1, residues Arg2033 to Asn2172; and C2, residues Ser2173 to Tyr2332. The remaining sequence, residues Glu1649 to Arg1689, is usually referred to as the factor VIII light chain activation peptide. Factor VIII is proteolytically activated by thrombin or factor Xa, which dissociate it from von Willebrand factor to form factor VIIIa, which has procoagulant function. The biological function of factor VIIIa is to increase the catalytic efficiency of factor IXa by several orders of magnitude relative to factor X activation. Thrombin-activated factor VIIIa is 160 kDa A1 / A2 / A3-C1-C2.

[0083] Mature factor VIII is heavily glycosylated and proteolyzed and circulates as a heterodimer with heavy and light chains bound by metal ions. The heavy chain consists of sequence-related domains A1 and A2 and a heavily glycosylated connecting B domain. The light chain consists of A3, C1, and C2 domains. In plasma, factor VIII circulates as a noncovalent complex with von Willebrand factor. The B domain has been shown to be dispensable for factor VIII clotting activity.

[0084] Various in vitro assays have been devised to determine the potential effectiveness of recombinant FVIII (rFVIII) as a therapeutic agent. These assays mimic the in vivo effects of endogenous FVIII. In vitro thrombin treatment of FVIII results in a rapid increase and subsequent decline in procoagulant activity, as measured by in vitro assays. This activation and inactivation occurs concomitantly with specific, limited proteolysis of both the heavy and light chains, altering the availability of different binding epitopes on FVIII, for example, allowing FVIII to dissociate from VWF and bind to phospholipid surfaces, or altering its ability to bind certain monoclonal antibodies.

[0085] The production of recombinant factor VIII by recombinant engineering techniques has been described. See, for example, U.S. Patent Nos. 4,757,006, 5,733,873, 5,198,349, 5,250,421, 5,919,766, and European Patent No. 306,968. The factor VIII gene is located at the tip of the long arm of chromosome X. The human factor VIII gene contains 26 exons spread across 186,000 bp of genomic DNA and encodes a 2,351 amino acid protein, including a 19-amino acid leader sequence. Factor VIII is one of the largest known genes. The mature factor VIII protein is 2,332 amino acids (Swiss Prot P00451). The protein sequence of factor VIII is shown in Figure 1. FVIII is considered recombinant when it is synthesized as a result of genetic engineering other than isolation from a natural source, such as human plasma. Recombinant FVIII may or may not be otherwise altered from plasma-derived FVIII (eg, by truncation or mutation).

[0086] FVIII is subject to numerous known polymorphisms, as described in the SwissProt database. Thus, for example, the aspartic acid residue at position 56 may optionally be a valine according to the present invention. Similarly, the aspartic acid at position 1141 may also be a glutamic acid according to the present invention. All known or discovered allelic and polymorphic variations of FVIII are within the scope of the present invention.

[0087] As used herein, the terms "factor VIII" or "FVIII" refer to any FVIII molecule that exhibits biological activity associated with wild-type FVIII, particularly the promotion of blood clotting. Several assays for FVIII activity are commercially available (see Chandler et al., Am J Clin Pathol 2003;120:34-39). In preferred embodiments of the present invention, the FVIII has at least a portion or all of the B domain (e.g., at least 100, 200, 500, or 900 residues, including at least one cysteine ​​that can be conjugated to a polymer). Preferably, the portion includes the two most C-terminal cysteines (positions 1604 and 1636) of the intact B domain. In one embodiment of the present invention, the FVIII molecule is full-length factor VIII (except that the signal peptide may be deleted). The FVIII molecule is a protein encoded by a DNA sequence capable of hybridizing to DNA encoding factor VIII:C under stringent conditions (e.g., 52°C, 50% formamide, 5xSSC). Such proteins may contain amino acid deletions at various sites between or within the domains A1-A2-B-A3-C1-C2 (see, e.g., U.S. Pat. No. 4,868,112). The FVIII molecule may also be an analog of wild-type FVIII in which one or more amino acid residues have been replaced by site-directed mutagenesis.

[0088] FVIII molecules useful in the present invention include the full-length protein, precursors of the protein, biologically active or functional subunits or fragments of the protein, and functional derivatives thereof, as described herein below. Reference to FVIII is intended to encompass all possible forms of such protein, including forms of FVIII having at least part or all of an intact wild-type B domain sequence and forms in which the B domain is absent.

[0089] In another embodiment of the invention, FVIII moieties with various deletions may be conjugated to the polymers of the invention. The factor VIII molecule according to this embodiment of the invention is a B-domain truncated FVIII factor having the sequence shown, in which the remaining domain lacks amino acids 1-740 and 1649-2332 of SEQ ID NO: 1. Preferably, the factor VIII molecule according to the invention is a recombinant molecule produced in a transformed host cell, preferably of mammalian origin.

[0090] However, the remaining domains (i.e., the three A domains and the two C domains) may differ slightly from the amino acid sequence set forth in SEQ ID NO: 1 (amino acids 1-740 and 1649-2332), e.g., by about 1%, 2%, 3%, 4%, or 5%. In particular, amino acid modifications (substitutions, deletions, or insertions) can be introduced into the remaining domains to modify the binding ability of factor VIII to various other components, e.g., vW factor, LPR, various receptors, other coagulation factors, cell surfaces, etc. Furthermore, factor VIII molecules according to the invention can include other post-translational modifications, e.g., in the truncated B domain and / or one or more of the other domains of the molecule. These other post-translational modifications may be in the form of various molecules conjugated to the factor VIII molecules according to the invention, e.g., polymeric compounds, peptide compounds, fatty acid-derived compounds, etc.

[0091] All factor VIII molecules according to the present invention, whether modified outside the B domain or not, or whether they have other post-translational modifications or not, have factor VIII activity (meaning the ability to function in the coagulation cascade in a similar or equivalent functional manner to FVIII, to induce the formation of FXa through interaction with FIXa on activated platelets, and to support clot formation). Activity can be measured, for example, by clot analysis, endogenous thrombin potential (ETP), or the like. The activity of the factor VIII molecules according to the present invention can be assessed in vitro by techniques well known in the art (e.g., Chandler et al., supra), such as FVIII activity assays (e.g., thrombin potential assays), etc. The factor VIII molecules according to the present invention have at least about 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and even 100% or more of the activity of wild-type human FVIII.

[0092] The B domain of factor VIII spans amino acids 741-1648 of SEQ ID NO: 1. The B domain is cleaved at several different sites, resulting in significant heterogeneity in circulating plasma FVIII molecules. The exact function of the heavily glycosylated B domain is unknown. However, the domain is dispensable for FVIII activity in the coagulation cascade. This apparent lack of function is supported by the fact that B domain-deleted / truncated FVIII appears to have identical in vivo properties to those seen with full-length wild-type FVIII. That said, there are indications that the B domain may have reduced association with cell membranes, at least under serum-free conditions.

[0093] B-domain truncated / deleted factor VIII molecules: Endogenous full-length FVIII is synthesized as a single-chain precursor molecule. Prior to secretion, the precursor is cleaved into heavy and light chains. Recombinant B-domain deleted FVIII can be produced by two different strategies: the B-domain-free heavy and light chains are synthesized separately as two different polypeptide chains (two-chain strategy), or the B-domain deleted FVIII is synthesized as a single precursor polypeptide chain that is cleaved into heavy and light chains in the same way as the full-length FVIII precursor (single-chain strategy).

[0094] In B-domain-deleted FVIII precursor polypeptides, the heavy and light chain portions are usually separated by a linker. To minimize the risk of introducing immunogenic epitopes into B-domain-deleted FVIII, the linker sequence is preferably derived from the FVIII B domain. The linker must contain a recognition site for a protease that separates the B-domain-deleted FVIII precursor polypeptide into heavy and light chains. In the B domain of full-length FVIII, amino acids 1644-1648 constitute this recognition site. The thrombin site that activates B-domain-deleted FVIII and results in linker removal is located in the heavy chain. Therefore, the size and amino acid sequence of the linker are unlikely to affect its removal from the remaining FVIII molecule upon thrombin activation. B-domain deletion is advantageous for FVIII production. Nevertheless, a portion of the B domain can be included in the linker without reducing productivity. The negative effect of the B domain on productivity was not attributed to any specific size or sequence of the B domain.

[0095] According to the present invention, the term "recombinant factor VIII" (rFVIII) includes any rFVIII (heterologous or natural) obtained via DNA technology, or a biologically active derivative thereof. In certain embodiments, the term encompasses the above-described proteins and nucleic acids encoding the rFVIII of the present invention. Such nucleic acids include, for example, but are not limited to, genes, pre-mRNAs, mRNAs, polymorphic variants, alleles, synthetic and naturally occurring variants. Proteins encompassed by the term rFVIII include, for example, but are not limited to, the above-described proteins and polypeptides, proteins encoded by the above-described nucleic acids, interspecies homologs, and polypeptides encoded by reference nucleic acids or other polypeptides having an amino acid sequence having greater than about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more amino acid sequence identity with the amino acids described herein over a region of at least about 100, about 200, about 300, about 400, or more amino acids. Preferably, the Factor VIII exhibits at least 90, 95, 96, 97, 98 or 99% sequence identity with the entire sequence of the A1, A2, A3, C1 and C2 domains.

[0096] According to a particular embodiment of the present invention, the production of rFVIII comprises any method known in the art for (i) producing recombinant DNA by genetic engineering, (ii) introducing the recombinant DNA into prokaryotic or eukaryotic cells, for example, but not limited to, by transfection, electroporation or microinjection, (iii) culturing said transformed cells, (iv) expressing rFVIII, for example, constitutively or inducibly, and (vi) isolating said rFVIII, for example, from the culture medium or by recovering the transformed cells, to obtain purified rFVIII.

[0097] In a preferred embodiment of the present invention, rFVIII is produced by expression in a suitable prokaryotic or eukaryotic host system characterized by producing a pharmaceutically acceptable rFVIII molecule. Examples of eukaryotic cells include mammalian cells such as CHO, COS, HEK293, BHK, SK-Hip and HepG2.

[0098] In yet another embodiment, a wide variety of vectors are used to prepare rFVIII, selected from eukaryotic and prokaryotic expression vectors. Examples of vectors for prokaryotic expression include plasmids such as, but not limited to, preset, pet, and pad, and promoters used in prokaryotic expression vectors include, but are not limited to, one or more of lac, trc, trp, recA, or araBAD. Examples of vectors for eukaryotic expression include (i) vectors for expression in yeast, such as, but not limited to, pAO, pPIC, pYES, or pMET, using a promoter such as, but not limited to, AOX1, GAP, GAL1, or AUG1; (ii) vectors for expression in insect cells, such as, but not limited to, pMT, pAc5, pIB, pMIB, or pBAC, using a promoter such as, but not limited to, PH, p10, MT, Ac5, OpIE2, gp64, or polh; and (iii) vectors for expression in mammalian cells, such as, but not limited to, pSVL, pCMV, pRc / RSV, pcDNA3, or pBPV, and in one embodiment, viral systems, such as, but not limited to, vaccinia virus, adeno-associated virus, herpes virus, or retrovirus, using a promoter such as, but not limited to, CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and β-actin.

[0099] FVIII molecules may also be conjugated with the polymers of the present invention described herein for other functional substances, including proteins. For example, in one embodiment, the polymer is conjugated to FVIII via free amino groups on the protein using an N-hydroxysuccinimide (NHS) ester. Reagents targeting conjugation to amino groups can randomly react with the ε-amine groups of lysines, the α-amine groups of the N-terminal amino acid, and the δ-amine groups of histidines. Full-length FVIII has 158 lysines, two N-terminal, and 75 histidines. According to embodiments of the present invention, conjugates can be formed using one or more of these sites. However, it is known that for full activity, FVIII requires interactions with multiple partners, such as von Willebrand factor (VWF), coagulation factor X (FX), and activated factor IX (FIXa). Therefore, conjugation of polymers to free amino groups may adversely affect the ability of the conjugated FVIII to affect coagulation.

[0100] In another embodiment, the polymers of the present invention can be coupled to free SH groups using any suitable thiol-reactive chemistry, including but not limited to maleimide chemistry, or the carbohydrate moiety of FVIII can be coupled to a polymeric hydrazide or polymeric amine after prior oxidation. The use of maleimide coupling is a particularly preferred embodiment of the present invention.

[0101] According to a preferred embodiment of the present invention, the polymer may be coupled to any cysteine ​​residue of FVIII using maleimide coupling, provided that sufficient biological activity is retained. Alternatively, any suitable amine or carbohydrate moiety of FVIII may be used for coupling the polymer of the present invention to FVIII, provided that sufficient activity is retained.

[0102] FVIII has four cysteines in the B domain and 19 in other domains. Of the 19 cysteines in B-domain deleted (BDD) FVIII, 16 form disulfides and the other three are free cysteines. A structural model of BDD-FVIII suggests that all three free cysteines are buried and unavailable for reaction with polymers (Baisan et al., 116 Blood 270-279 (2010)). Therefore, according to an embodiment of the present invention, polymers are covalently attached to cysteine ​​residues introduced into FVIII, preferably by site-directed mutagenesis (see Table 1 below for potential sites). See, e.g., EP 2363414. [Table 1] See, for example, Mei, B. et al. (2012) Thrombosis and Hemostasis 116, 270-279.

[0103] According to another aspect of the present invention, the polymer is preferably coupled to a natural cysteine ​​residue in the B domain. Alternatively, according to another aspect of the present invention, a cysteine ​​residue may be added to the B domain via recombinant DNA technology. The polymer can be conjugated to rFVIII via one and only one cysteine ​​residue, or via multiple cysteines. Preferably, the cysteine(s) is / are either of the two most C-terminal cysteines in the B domain, preferably at positions 1604 and 1636 of SEQ ID NO: 1. (When only a portion of the B domain is used, the two most C-terminal cysteines are the C-terminal cysteines of the portion aligned with the two most C-terminal cysteines of the intact B domain.) Attachment of the branched polymer via a single cysteine ​​in any given molecule of rFVIII is advantageous for surrounding rFVIII with the polymer and zwitterionic charge without substantial impairment of any, if any, rFVIII activity. The single cysteine ​​to which the polymer is conjugated may be the same or different in different molecules of rFVIII. Although an understanding of the mechanism is not required to practice the present invention, it is believed that the zwitterionic charge on the polymer surrounding rFVIII essentially anchors a layer of water molecules that are transferred in tandem with rFVIII and protect rFVIII from degradation processes in vivo.

[0104] rFVIII preparations are typically homogeneous due to proteolytic processing at different sites within the B domain, resulting in several bands from the heavy chain on the gel. Conjugation of such preparations of rFVIII resulting in polymerization via one of the two C-terminal cysteines with the polymers of the present invention results in conjugation only with rFVIII molecules in which these two C-terminal cysteines are present. rFVIII molecules with more truncated B domains do not form conjugates to a significant extent. The specificity of conjugation with a single form of the B domain is demonstrated by comparing the bands on a gel before and after conjugation and observing the loss or substantial reduction of only one band prior to conjugation. The conjugated rFVIII can be easily separated from unconjugated rFVIII molecules due to the large difference in molecular weight. As a result, conjugated rFVIII preparations can have much greater homogeneity than typical rFVIII preparations. For example, at least 80, 90, 95, or 99% of the molecules in the formulation can have the same portion of a B domain, e.g., an intact B domain, and one and only one attached polymer (preferably branched) per molecule (although there may be differences in glycosylation between different proteins and differences in length between different polymers). In some formulations, the portion is at least residues SEQ ID NO:1, 1-1604, or 1-1636, or 1-1648 of SEQ ID NO:1. In some formulations, the portion consists of residues 1-1648 of SEQ ID NO:1.

[0105] The B domain of the conjugate containing the conjugate with the polymer may be removed by the endogenous FVIII activation process after administration to a subject. However, the conjugated polymer still plays a role in extending the half-life of the conjugated FVIII until the need for activation occurs. Furthermore, the loss of the polymer during the activation process has the advantage that FVIII can be degraded quickly (compared to FVIII conjugated other than via the B domain) after activation occurs. For this reason, rFVIII conjugated via the B domain is advantageous for the prevention of subjects with hemophilia but who are not known to be experiencing bleeding (internal or external) at the time of administration. Conjugation with a polymer promotes the persistence of the conjugate until the time when it can be determined that the subject is experiencing a bleeding episode.

[0106] At this time, the polymer associated with the B domain is processed from rFVIII, and the remaining rFVIII can promote coagulation and then be inactivated.

[0107] Conjugation of rFVIII to a polymer by this method increases the in vivo half-life of rFVIII in humans by more than 11 hours. For example, the half-life can be 12 to 50 hours. Preferably, the half-life is 20 hours or longer. The half-life is measured as the average value in a population of human subjects without a prior antibody response to human FVIII. Such humans may, but need not, have hemophilia for purposes of measuring the half-life.

[0108] Due to its long half-life, the conjugate can be administered less frequently in prophylaxis than current regimens, for example, at weekly intervals or less. In some prophylaxis regimens, the conjugate is administered between weekly and monthly, for example, weekly, biweekly, or monthly. Despite the reduced administration frequency, subjects receiving the conjugate can have increased FVIII trough levels compared to current methods. In current methods, subjects in prophylaxis regimens spend approximately 18 hours per week at trough levels of FVIII activity less than 1% of the average level in non-hemophilic control subjects. Levels below 1% place subjects at a high risk of acute bleeding. Using this method, subjects can be maintained at trough levels greater than about 1%, 3%, or 5% of the average level of FVIII activity in non-hemophilic control subjects for at least one week, one month, one year, or indefinitely. Activity can be assessed using an in vitro chromogenic assay involving FVIII activation by B-domain processing.

[0109] In prophylactic or other treatments, the conjugates of the invention are suitable for administration to subjects who have previously been treated with (unconjugated as described herein) FVIII and have developed a human antibody response thereto. The polymer portion of the conjugates protects the conjugated FVIII from such antibodies, allowing the FVIII to persist in the blood longer than unconjugated FVIII, and preferably with a half-life essentially the same as that of subjects without antibodies to FVIII.

[0110] With regard to the naturally occurring cysteines in the B domain, an intact B domain is not essential for FVIII activity. The B domain of FVIII begins at amino acid 745 and continues to amino acid 1648. The B domain has four naturally occurring cysteine ​​residues: 1293, 1373, 1604, and 1636. According to the present invention, coupling at one or more of these residues is preferred. Coupling of the polymers of the present invention to residues 1604 and 1636 is particularly preferred.

[0111] The present invention provides a conjugate of the high MW polymer of the present invention and FVIII. According to one aspect of the present invention, there is provided a preferred conjugate, in which FVIII is bound to a zwitterionic polymer, the polymer being composed of one or more monomeric units, at least one of which contains a zwitterionic group. Preferably, the zwitterionic group is phosphorylcholine.

[0112] In a preferred aspect of the invention, one of the monomer units is 2-(acryloyloxyethyl)-2'-(trimethylammoniumethyl)phosphate or 2-(methacryloyloxyethyl)-2'-(trimethylammoniumethyl)phosphate (HEMA-PC). In another preferred embodiment, the polymer is synthesized from a single monomer, preferably 2-(acryloyloxyethyl)-2'-(trimethylammoniumethyl)phosphate or 2-(methacryloyloxyethyl)-2'-(trimethylammoniumethyl)phosphate.

[0113] In a preferred embodiment of the present invention, the FVIII or conjugate is recombinant FVIII (rFVIII). In a preferred embodiment of the present invention, the rFVIII is full-length. In another preferred embodiment of the present invention, the rFVIII is purified from mammalian host cells. In yet another aspect of the present invention, the FVIII comprises a partial or complete deletion of the B domain.

[0114] In yet another embodiment of the invention, the FVIII conjugate preferably has two or more, preferably three or more, polymer arms, and the monomer is HEMA-PC. In another embodiment of the invention, the conjugate preferably has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more polymer arms, and the monomer is HEMA-PC. More preferably, the conjugate has 3, 6, or 9 arms. Most preferably, the conjugate has 9 arms.

[0115] In one embodiment of the invention, it is preferred that the polymer-FVIII conjugate has a polymer portion having a molecular weight between 100,000 and 1,500,000 daltons. More preferably, the conjugate has a polymer portion having a molecular weight between 500,000 and 1,000,000 daltons. Even more preferably, the conjugate has a polymer portion having a molecular weight between 600,000 and 800,000 daltons. Most preferably, the FVIII conjugate has a polymer portion having a molecular weight between 600,000 and 850,000 daltons and has nine arms. Here and elsewhere in this application, the total molecular weight for the FVIII-containing polymer is in the range of about 300,000 daltons higher than that indicated for the polymer portion.

[0116] According to an embodiment of the present invention, there is provided a method for synthesizing a zwitterionic polymer-functional agent conjugate, the conjugate having one or more functional agents and one or more polymer arms, each of the polymer arms having one or more monomer types, at least one of the types being zwitterionic. According to an embodiment of the present invention, the method includes the steps of: a. combining an initiator comprising one or more polymer synthesis initiator moieties and a first reactive group with one or more monomer types suitable for polymerization, at least one of the monomer types comprising a zwitterion; reacting the monomer types at the polymer synthesis initiator moieties to form polymer(s) to obtain a polymerization initiator; b. coupling a linker moiety comprising second and third reactive groups with the polymerization initiator to obtain a linker polymerization initiator having unreacted reactive groups; and c. coupling one or more functional agents with the unreacted reactive groups of the linker polymerization initiator to obtain a polymer-functional agent conjugate.

[0117] Prior to the present invention, the initiator molecule or entity had to contain a deprotectable functional group to allow for coupling of a functional substance. An example of such an initiator having a protected maleimide is shown below. [ka] After polymer synthesis, the protected maleimide is thermally deprotected to create a maleimide that can be used to couple functional entities. If one wanted to change the nature of the chemical entity between the maleimide and the polymer initiation site, one would have to synthesize an entirely new initiator.

[0118] When considering the possible scale-up of a polymer synthesis process, a new scale-up synthesis procedure must be developed each time the initiator is modified or changed in any way. Each change in the initiator molecule's properties has a wide range of effects on polymer synthesis. However, according to the present invention, a single initiator moiety can be used for large-scale polymer preparation. Thus, conditions for optimal scale-up polymer synthesis can be developed. Using the claimed invention, such polymers can be adapted to various types of functional materials by "snapping-on" various types of linkers.

[0119] For example, if it is desired to conjugate a larger functional entity to a polymer of the invention, such as an antibody Fab fragment, a longer linker sequence can be snapped onto the polymer. In contrast, smaller functional entities may require a relatively short linker sequence.

[0120] In a preferred embodiment of the method, the initiator has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 sites for polymer initiation. Preferably, the initiator has 3, 6 or 9 sites for polymer initiation.

[0121] According to this aspect of the invention, polymer synthesis initiator The moiety preferably has the following structure: [ka] (X is an NCS or halogen that allows initiation of ATRP or related polymer synthesis schemes, and R is initiator (The remainder of It has.

[0122] According to the present invention, the initiator preferably has the structure: [ka] wherein R1 has a nucleophilic reactive group, R2 comprises a linker, R3 is a polymer synthesis initiator moiety, and s is an integer between 1 and 20. R1 is preferably selected from the group consisting of NH2-, OH- and SH. More preferably, R1 is NH2-.

[0123] According to this aspect of the invention, R2 is preferably alkyl, substituted alkyl, alkylene, alkoxy, carboxyalkyl, haloalkyl, cycloalkyl, cyclic alkyl ether, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkylene, heterocycloalkyl, heterocycloalkylene, aryl, arylene, arylene-oxy, heteroaryl, amino, amido, or any combination thereof.

[0124] More preferably, R2 has the formula: [ka] (wherein m is 1 to 20, and preferably 4). The structure includes:

[0125] According to the present invention, R3 has the formula: [ka] wherein R4, R5 and R6 are the same or different; [ka] (wherein X is NCS, F, Cl, Br or I) selected from the group consisting of Preferably, X is Br.

[0126] In a more preferred embodiment of the present invention, R4, R5 and R6 are each [ka] is. Alternatively, R4, R5 and R6 are each [ka] is. In yet another preferred embodiment, R4, R5 and R6 are each [ka] is.

[0127] According to this aspect of the invention, the monomer is preferably [ka] (wherein R7 is H or C 1~6 is alkyl, ZW is a zwitterion, and t is 1 to 6. Preferably, the zwitterion is phosphorylcholine.

[0128] Even more preferably, the monomer is selected from the group consisting of 2-(methacryloyloxyethyl)-2'-(trimethylammoniumethyl)phosphate (HEMA-PC) and 2-(acryloyloxyethyl)-2'-(trimethylammoniumethyl)phosphate. Most preferably, the monomer is 2-(methacryloyloxyethyl)-2'-(trimethylammoniumethyl)phosphate.

[0129] According to an aspect of the present invention, the steps b The linker portion of the formula is preferably [ka] wherein R8 is [ka] is selected from the group consisting of R9 is [ka] (wherein p is 1 to 12) selected from the group consisting of is an activated ester having the formula:

[0130] Preferably, the linker moiety is [ka] is.

[0131] According to an embodiment of the present invention, the initiator in step a. preferably has the following structure: [ka] (wherein y is an integer of 1 to 50, X is an integer of 0 to 50, and Z is NCS, F, Cl, Br, or I.) Preferably, Z is Br, X is 4, 8 or 12, and Y is 1 to 10. More preferably, Y is 4.

[0132] According to this aspect of the invention, the steps b The linker-polymerization initiator is preferably of the formula: [ka] wherein X is an integer from 1 to 50, Y is an integer from 1 to 50, and the polymer is any polymer synthesized with monomers as defined herein. It has. More preferably, Y is 4, X is 4, 8 or 12, and the monomer is HEMA-pc.

[0133] Preferably, the functional substance is a protein. More preferably, the protein comprises human FVIII. Even more preferably, the FVIII is recombinant FVIII (rFVIII), preferably purified from human host cells. Most preferably, the FVIII has a partial or complete deletion of the B domain.

[0134] According to an aspect of the present invention, a compound of formula: [ka] (wherein y is an integer of 1 to 50, X is an integer of 0 to 50, and Z is NCS, F, Cl, Br, or I.) Preferably, Z is Br, X is 4, 8 or 12, and Y is 1 to 10. More preferably, Y is 4.

[0135] According to another aspect of the present invention, a compound of formula: [ka] (wherein y is an integer from 1 to 50, X is an integer from 0 to 50, and MPC is a poly-MPC arm) A polymer having the formula: is provided. Poly-MPC is prepared by polymerization, for example, ATRP, using 2-(methacryloyloxyethyl)-2'-(trimethylammoniumethyl)phosphate. Preferably, the total molecular weight of the polymer is about 500,000 to about 1,000,000 daltons. More preferably, the total molecular weight of the polymer is about 650,000 to about 850,000 daltons. Even more preferably, the total molecular weight of the polymer is about 750,000 daltons.

[0136] According to this aspect of the invention, X is preferably 4, 8 or 12 and Y is 1 to 10. Even more preferably, Y is 4.

[0137] Such initiators can be used in accordance with the present invention as substrates for polymer synthesis. Preferably, the polymer synthesis is carried out using ATRP or a similar method, for example, AGET (Woodworth et al., Macromolecules, Vol. 31, No. 23, 1998) or ARGET (Macromolecules, 2012, 45(16), pp. 6371-6379 (Simakova, A. et al.)). Any of the monomers described herein can be used in polymer synthesis.

[0138] Pharmaceutical compositions suitable for oral administration can be provided as individual units, such as capsules, liquids, syrups, or suspensions (in aqueous or non-aqueous liquids; or as edible foams or whipped creams; or as emulsions). Suitable excipients for tablets or hard gelatin capsules include lactose, corn starch or its derivatives, stearic acid or its salts. Suitable excipients for soft gelatin capsules include, for example, vegetable oils, waxes, fats, semi-solid or liquid polyols, etc. Excipients that can be used to prepare solutions and syrups include, for example, water, polyols, and sugars. To prepare suspensions, oils (e.g., vegetable oils) can be used to obtain oil-in-water or water-in-oil suspensions.

[0139] Pharmaceutical compositions suitable for nasal administration in which the carrier is a solid include coarse powders, e.g., having a particle size in the range of 20 to 500 microns, administered as in snuff, i.e., by rapid inhalation through the nasal passages from a container of the powder held close to the nose. Compositions in which the carrier is a liquid, suitable for administration as a nasal spray or nasal drops, include aqueous or oily solutions of the active ingredient. Pharmaceutical compositions suitable for administration by inhalation include fine particle dusts or mists that can be generated by various types of metered-dose pressurized aerosols, nebulizers, or inhalers.

[0140] Pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. Excipients that can be used for injection solutions include, for example, water, alcohols, polyols, glycerin, and vegetable oils. The compositions can be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions can also be prepared from sterile powders, granules, and tablets. The pharmaceutical compositions can be substantially isotonic (meaning an osmolality of about 250 to 350 mOsm / kg of water).

[0141] In general, pharmaceutical compositions may contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts (the substance of the present invention itself may be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents, or antioxidants. In addition to the substance of the present invention, pharmaceutical compositions may also contain a therapeutically active agent. The pharmaceutical compositions of the present invention may be used in combination with a pharmaceutically acceptable diluent, adjuvant, or carrier. Such excipients may include, but are not limited to, saline, buffered saline (phosphate buffered saline), glucose, liposomes, water, glycerol, ethanol, and combinations thereof.

[0142] The pharmaceutical compositions can be administered in any effective and convenient manner effective to treat the patient's disease, including, for example, administration by oral, intravenous, subcutaneous, intramuscular, intraosseous, intranasal routes, etc. Therapeutically or prophylactically, the active agent can be administered to the individual as an injectable composition, for example, a sterile aqueous dispersion, preferably isotonic.

[0143] For administration to mammals, particularly humans, the daily dosage of the active agent will be 0.01 mg / kg body weight, typically about 1 mg / kg. In any case, a doctor can determine the actual dosage that is most suitable for an individual, depending on factors including the individual's age, weight, sex, and response. The dosages listed above represent the average case. Of course, there are cases where higher or lower dosages are merited, and such cases are within the scope of the present invention.

[0144] The dosage of the substance of the present invention will vary within wide limits depending on the disease or disorder being treated, the age and condition of the individual being treated, etc., and ultimately the physician will determine the appropriate dosage to be used.

[0145] This dosage may be repeated as appropriate. If side effects occur, the amount and / or frequency of administration may be reduced in accordance with normal clinical practice. In one embodiment, the pharmaceutical composition may be administered once every 1 to 30 days.

[0146] According to a third aspect of the present invention, there is provided a pharmaceutical composition according to the second aspect and another pharmaceutically active agent, the other pharmaceutically active agent being capable of promoting or enhancing the activity of FVIII, for example another blood coagulation factor.

[0147] The pharmaceutical compositions of the present invention may be used alone or in combination with other therapeutic compounds or molecules, such as anti-inflammatory agents, analgesics, or antibiotics. Such administration with other compounds may be simultaneous, separate, or sequential. The components may be prepared in the form of a kit, which may optionally include instructions.

[0148] Preferably, the pharmaceutical compositions and other therapeutic compounds of the present invention are administered directly to a patient in need thereof.

[0149] The present invention also provides kits of parts comprising the pharmaceutical compositions of the present invention and administration vehicles including, but not limited to, capsules for oral administration, inhalers for pulmonary administration, and injectable solutions for intravenous administration.

[0150] According to a fourth aspect of the present invention there is provided a method of treating a blood coagulation disorder comprising administering a composition of the present invention to a patient in need thereof, and therefore this aspect of the invention also includes the use of such a composition in said method.

[0151] Blood clotting disorders can be characterized by loss of function of blood clotting factors or the production of autoantibodies. Examples of blood clotting disorders include hemophilia A and acquired hemophilia A.

[0152] As used herein, the term "treatment" includes any regime that can benefit humans or non-human animals. Treatment of "non-human animals" extends to treatment of domestic animals (including horses), companion animals (e.g., cats and dogs), and livestock / farm animals (including sheep, goats, pigs, cattle, and members of the equine family). Treatment may be for any pre-existing condition or disease, or may be preventative (prophylactic treatment). Treatment may be for genetic or acquired diseases. Treatment may be for acute or chronic conditions.

[0153] Nucleophilic groups on proteins, including antibodies, that can be used for conjugation with polymers according to embodiments of the present invention include, but are not limited to, (i) N-terminal amine groups, (ii) side chain amine groups, e.g., lysine, (iii) side chain thiol groups, e.g., cysteine, and (iv) sugar hydroxyl or amino groups where the protein is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and can react with electrophilic groups on (i) activated esters (such as NHS esters, HOBt esters, haloformates, and acid halides); (ii) alkyl and benzyl halides (such as haloacetamides); and (iii) polymer-attached linker moieties and linker reagents containing aldehyde, ketone, carboxyl, and maleimide groups to form covalent bonds. Many proteins, including antibodies, have cysteine ​​thiol groups that can potentially be used for conjugation. Many cysteine ​​residues are in the form of reducible interchain disulfides, i.e., cysteine ​​bridges. Cysteine ​​residues in the disulfide form are generally not available to react with reagents such as maleimides. Cysteine ​​residues can be free or unpaired. However, free residues are often available in various These residues are found "capped" by one or more reagents in the medium and are similarly unavailable for conjugation. Cysteine ​​residues may be rendered reactive for conjugation with linker reagents such as maleimides by treatment with a reducing agent such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP) so that the protein is fully or partially reduced. Thus, each cysteine ​​bridge theoretically forms two reactive thiol nucleophiles. In the case of a free cysteine, one thiol nucleophile is formed by reduction. Depending on the conditions used, reduction with TCEP or DTT can result in loss of proper protein folding with a concomitant loss of activity. However, activity can be restored by allowing the protein to refold under appropriate conditions.

[0154] For example, additional nucleophilic groups can be introduced into antibodies through modification of lysine residues by reacting them with 2-iminothiolane (Traut's reagent), resulting in conversion of the amine to a thiol. Reactive thiol groups may also be introduced into proteins by introducing one, two, three, four, or more cysteine ​​residues (e.g., by preparing mutants containing one or more non-wild-type cysteine ​​amino acid residues). [Example]

[0155] Initiator synthesis Example 1 Preparation of a 3-arm "snapping" initiator A TFA / amine salt initiator (Compound B) having the following structure was synthesized as follows. [ka]

[0156] First, the following structure: [ka] The BOC-protected three-arm initiator, Compound A, having the formula (I), was prepared as follows: A 25 mL round-bottom flask was charged under nitrogen with tert-butyl 2-[2-(2-aminoethoxy)ethoxy]ethylcarbamate (66 mg, 0.26 mmol, 1.2 equivalents) and (2,2,2-tri(2-bromo-2-methyl-propionyloxymethyl)ethoxy)acetic acid (prepared as described in PCT / US2012 / 060301 for product 4.5, incorporated herein by reference) (142 mg, 0.22 mmol, 1.0 equivalents), followed by N,N-dimethylformamide (2 mL), and then N,N-diisopropylethylamine (0.19 mL, 1.1 mmol, 5.0 equivalents). The flask was cooled to 0 °C using an ice bath. To this was added propylphosphonic anhydride solution (50% by weight in ethyl acetate, 0.16 mL, 0.26 mmol, 1.2 equiv.) over 1 minute. The reaction was warmed to room temperature and stirred for 1.5 hours. The reaction was quenched by the addition of water and then partitioned with water and ethyl acetate. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with water, saturated aqueous sodium bicarbonate, water, 0.5 M aqueous citric acid, and water, then dried (sodium sulfate), filtered, and concentrated in vacuo. The residue was added to a silica gel column (60 mL) and eluted with 70% ethyl acetate and 30% hexanes. Tubes containing the product were pooled and concentrated in vacuo to give 150 mg (0.17 mmol, 77%) of compound A. 1H NMR(400MHz CDCl3):δ=Need to put in data 1.44(s,9H,OC CH3 ), 1.96(s,18H,CC (CH3)2 Br), 3.31 (q, J=4.8Hz, 2H, OCNH CH2 CH2O),3.5-3.6(m,12H),3.99(s,2H,O CH2 C), 4.32(s, 6H, C CH2 OC=O), 5.0(br s, 1H, CH2 NH C=OO), 6.8(br s, 1H, CH2 NHC=OC), LC-MS (ES, m / z): [M+H]+ calculated for C30H51Br3N2O12+H = 871.1; found 871.8.

[0157] Compound A was deprotected to give compound B as follows: 20 mL round bottom flask To a solution of compound A (120 mg, 0.14 mmol, 1 equiv.) under nitrogen was added compound A (120 mg, 0.14 mmol, 1 equiv.), dichloromethane (2 mL), followed by trifluoroacetic acid (2 mL, 26.9 mmol, 192 equiv.). The reaction was stirred at room temperature for 30 minutes. The reaction was diluted with hexanes dichloromethane (20 mL) and concentrated in vacuo. The reaction was diluted with hexanes (50 mL) and concentrated in vacuo (twice) to give 2.2 g (2.73 mmol, (including residual dichloromethane)) of compound B. 1H NMR(400MHz CDCl3):δ=1.94(s,18H,CC (CH3)2 Br), 3.2(br, 2H, OCNH CH2 CH2O),3.5-3.8(m,12H),3.99(s,2H,O CH2 C), 4.34(s, 6H, C CH2 OC=O), 7.11(br t, 1H, CH2 NH C=O), 7.99 (br, 3H, NH3+ ). LC-MS (ES, m / z): [M+H]+ calculated for C25H43Br3N2O10+H = 771.1; found 771.6.

[0158] Example 2 Preparation of a 6-arm "snapping" initiator A TFA / amine salt initiator (compound F1) was synthesized having the following structure: [ka]

[0159] As a first step in preparing F1, compound C was synthesized having the following structure: [ka] 100mL round bottom flask To a solution of 1-tosyl-11-(3,4,7-triaza-4,6,10-triphenyl-adamantan-1-ylmethoxy)-3,6,9-trioxaundecane (prepared as described in PCT / US2012 / 060301 for product 2.2) (4.0 g, 5.5 mmol, 1.0 equiv.), di(tert-butyl)imidodicarbonate (1.43 g, 6.6 mmol, 1.2 equiv.), potassium carbonate (1.9 g, 13.7 mmol, 2.5 equiv.), potassium iodide (0.137 g, 0.82 mmol, 0.15 equiv.), followed by acetonitrile (25 mL) was added under nitrogen using a reflux condenser. The reaction was stirred at room temperature for 5 minutes and then at 60° C. for 30 hours. The reaction was quenched by the addition of water (25 mL) and tert-butyl methyl ether (125 mL). The organic layer was separated, and the aqueous layer was extracted with tert-butyl methyl ether (75 mL). The combined organic layers were washed with saturated aqueous sodium chloride (20 mL), then dried (sodium sulfate), filtered, and concentrated in vacuo. The residue was added to a silica gel column (195 g, 6.5 cm × 12 cm) and eluted with 20% tert-butyl methyl ether in 80% hexane up to 100% tert-butyl methyl ether. The tubes containing the product were pooled and concentrated in vacuo to give 3.7 g (4.79 mmol, 87%) of compound C.

[0160] 1H NMR (400MHz DMSO-d6): δ=1.42(s,18H,C[C (CH3)3 ]2), 2.72(s, 2H, C CH2 N, isomer), 2.88 (s, 2H, C CH2 N, isomer), 3.2-3.6 (m, 20H), 5.25 (s, 2H, N CH Ph, isomer), 5.70 (s, 1H, N CH Ph, isomer), 7.3-7.8 (m, 15H, Phenyl ).

[0161] Next, compound D was synthesized, having the following structure: [ka] 500mL round bottom flask To a solution of compound C (2.7 g, 3.49 mmol, 1.0 equiv.), lithium hydroxide monohydrate (0.73 g, 17.5 mmol, 5 equiv.), tetrahydrofuran (20 mL), methanol (8 mL), followed by water (8 mL) were added under nitrogen using a reflux condenser. The reaction was stirred at 60° C. for 6 hours. The reaction was concentrated in vacuo and then partitioned by adding water (75 mL) and ethyl acetate (100 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (50 mL). The combined organic layers were washed with saturated aqueous sodium chloride (30 mL), then dried (sodium sulfate), filtered, and concentrated in vacuo. The residue was applied to a silica gel column (110 g, 5.5 cm × 10.5 cm) and eluted with 50% hexane in 50% tert-butyl methyl ether up to 100% tert-butyl methyl ether. The tubes containing the product were pooled and concentrated in vacuo to give 1.38 g (2.05 mmol, 59%) of compound D.

[0162] 1H NMR (400MHz DMSO-d6): δ=1.36(s,9H,C[C (CH3)3 ]2), 2.72(s, 2H, C CH2 N, isomer), 2.88 (s, 2H, C CH2 N, isomer), 3.1-3.4 (m, 20H), 5.25 (s, 2H, N CH Ph(isomer)), 5.70 (s, 1H, N CH Ph(isomer), 6.73 (t, J=6.0Hz, 1H, O=C NH CH2), 7.3-7.7(m, 15H, Phenyl ).

[0163] The next step in the preparation of F1 is to obtain a compound having the following structure: [ka] The synthesis of compound E having the following structure was carried out. 100mL round bottom flaskTo this was added compound D (2.96 g, 4.4 mmol, 1.0 equiv.), diethyl ether (20 mL), followed by water (16 mL). The flask was cooled to 0 °C using an ice bath. To this was added hydrobromic acid solution (48 wt % in water) (1.64 mL, 14.5 mmol, 3.3 equiv.). The reaction was rapidly stirred at 0 °C for 1 hour. The organic layer was separated, and the aqueous layer was returned to the reaction flask at 0 °C, to which diethyl ether (20 mL) was added, and stirring was continued for 15 minutes. The organic layer was again separated, and the aqueous layer was returned to the reaction flask at 0 °C, to which diethyl ether (20 mL) was added, and stirring was continued for 10 minutes. The organic layer was separated, and the aqueous layer was adjusted to pH 4.5 by the addition of 1 M aqueous sodium hydroxide solution. Water was removed by azeotroping with acetonitrile under vacuum to give 2.5 g (3.85 mmol, 87%) of compound E as a white solid.

[0164] 1H NMR (400MHz DMSO-d6):δ=1.36(s,9H,OC (CH3)3 ), 3.05-3.58(m,24H, CH2 ), 6.8(t,1H,O=C NH CH2), 8.0(br s, 9H, CH2 NH2*HBr ).

[0165] LC-MS (ES, m / z): calculated for [M+H]+C18H40N4O6+H=409.3; found 409.6.

[0166] The next step in the preparation of compound F1 is to obtain a compound having the following structure: [ka] The synthesis of compound F having the following structure was carried out. A 200 mL round-bottom flask was charged with bis-2,2-[(2-bromoisobutyryl)hydroxymethyl]propionic acid (prepared as described in Example 7 of U.S. Patent Application No. 13 / 641,342, incorporated herein by reference) (2.32 g, 5.37 mmol, 3.3 equivalents) and Compound E (1.06 g, 1.63 mmol, 1.0 equivalent), followed by dimethylformamide (15 mL), and then diisopropylethylamine (3.4 mL, 19.5 mmol, 12 equivalents). A solution of propylphosphonic anhydride (50 wt % in ethyl acetate, 3.7 mL, 5.87 mmol, 3.5 equivalents) was added to the reaction mixture under nitrogen. The reaction mixture was stirred for 60 minutes. The reaction was quenched by the addition of water (1 mL), loaded onto a preparative HPLC column, and eluted with 50% acetonitrile (containing 0.1% trifluoroacetic acid) to 95% acetonitrile (containing 0.1% trifluoroacetic acid) in water. Tubes containing the product were pooled, concentrated under vacuum, frozen, and placed on a lyophilizer. This yielded 640 mg (0.39 mmol, 24%) of compound F.

[0167] Finally, the tBOC protecting group was removed to give the final initiator, F1, with the structure shown above. flask To the flask was added product 174-44 (600 mg, 0.36 mmol) and dichloromethane (3.6 mL). The flask was cooled to 0 °C using an ice bath. To this was added trifluoroacetic acid (3.6 mL). The reaction was stirred at room temperature for 45 minutes. The reaction was diluted with hexane and then concentrated under vacuum. The reaction was diluted with hexane and concentrated under vacuum. The residue was dissolved with acetonitrile (3 mL), diluted with water (1.5 mL), frozen, and placed on a freeze dryer. This afforded 537 mg (0.32 mmol, 89%) of compound F1 as an oil.

[0168] Example 3 Preparation of 9-arm "snapping" initiator Compound L A TFA / amine salt initiator (compound L) having the following structure was synthesized as follows. [ka]

[0169] First, compound K was synthesized, which has the following structure: [ka] In a 200 mL round-bottom flask under nitrogen, compound J (1.9 g, 2.67 mmol, 3.3 equiv.) [ka] and Compound E (0.525 g, 0.81 mmol, 1.0 equiv) (see above), followed by dimethylformamide (10 mL) and then diisopropylethylamine (2.5 mL, 14.6 mmol, 18 equiv). The flask was cooled using an ice bath to 0° C. To this was added propylphosphonic anhydride solution (50 wt % in ethyl acetate, 2.5 mL, 4.04 mmol, 5 equiv) over approximately 6 minutes.

[0170] The reaction was warmed to room temperature and stirred for 15 minutes. The reaction was quenched by adding 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 was used without further purification to yield 2.0 g (0.80 mmol, 99%) of compound K.

[0171] 1H NMR (400MHz 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=5.6Hz, 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=6.8Hz, 3H, CH2 NH C=O), 7.84 (t, J=6.0 Hz, 3H, CH2 NH C=O).

[0172] LC-MS (ES, m / z): calculated for [(M+2H-boc) / 2]+(C84H136Br9N7O33+2H-Boc) / 2=1196.6; found 1196.6.

[0173] Compound L was then synthesized as follows: flask To the reaction mixture under nitrogen, compound K (2.0 g, 0.8 mmol), dichloromethane (10 mL), followed by trifluoroacetic acid (5 mL) were added. The reaction was stirred at room temperature for 30 minutes. The reaction was concentrated under vacuum. The reaction was diluted with dichloromethane (10 mL) and concentrated under vacuum. The residue was dissolved with acetonitrile (10 mL), filtered through a syringe filter (Acrodisc CR25, PN 4225T), loaded onto a preparative HPLC column, and eluted with 60% acetonitrile in water (with 0.1% trifluoroacetic acid) up to 98% acetonitrile (with 0.1% trifluoroacetic acid). The tubes containing the product were pooled, concentrated under vacuum, frozen, and placed on a lyophilizer. This yielded 990 mg (0.4 mmol, 50% over two steps) of compound L as a white powder.

[0174] 1H NMR(400MHz 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 CH2NH 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 +).

[0175] LC-MS (ES, m / z): calculated for [(M+2H) / 2] + (C84H136Br9N7O33 + 2H) / 2 = 1196.6; found 1197.4.

[0176] Example 4 Longer Spacer 9-Arm Initiator Preparation of Snapping Initiator Compound O A TFA / amine salt initiator (Compound O) having the following structure was synthesized as follows. [ka]

[0177] First, compound M was synthesized, which has the following structure: [ka] A 20 mL vial was charged with compound L (410 mg, 0.164 mmol, 1.0 equiv.) (see above) and α-t-butyloxycarbonylamino-ω-carboxyocta(ethylene glycol) (97.5 mg, 0.18 mmol, 1.1 equiv.), followed by N,N-dimethylformamide (2 mL) and then N,N-diisopropylethylamine (0.171 mL, 0.982 mmol, 6 equiv.). The flask was cooled to 0 °C using an ice bath. To this was added a propylphosphonic anhydride solution (50 wt% in ethyl acetate, 0.205 mL, 0.327 mmol, 2 equiv.) over approximately 1 minute. The reaction was warmed to room temperature and stirred for 30 minutes. The reaction was quenched by adding water (10 mL), saturated aqueous sodium bicarbonate (10 mL), and ethyl acetate (40 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (25 mL). The combined organic layers were washed with saturated aqueous sodium bicarbonate (10 mL), 0.5 M aqueous citric acid (10 mL), water (10 mL), and saturated aqueous sodium chloride (10 mL), then dried (sodium sulfate), filtered, and concentrated in vacuo. The residue was used without further purification to give Compound M (0.5 g, 0.172 mmol, 105%).

[0178] LC-MS (ES, m / z): calculated for [(M+2H-boc) / 2]+(C103H173Br9N8O42+2H-Boc) / 2=1408.2; found 1408.9.

[0179] 100mL round bottom flaskTo the reaction mixture under nitrogen, compound M (0.5 g), dichloromethane (4 mL), followed by trifluoroacetic acid (3 mL) were added. The reaction was stirred at room temperature for 15 minutes. The reaction mixture was concentrated under vacuum. The residue was dissolved with acetonitrile (3 mL), filtered through a syringe filter (Acrodisc CR25, PN 4225T), loaded onto a preparative HPLC column, and eluted with 50% acetonitrile (with 0.1% trifluoroacetic acid) in 50% water (with 0.1% trifluoroacetic acid) up to 90% acetonitrile (with 0.1% trifluoroacetic acid). The tubes containing the product were pooled, concentrated under vacuum, frozen, and placed on a lyophilizer. This yielded 101 mg of compound O (21% over two steps).

[0180] 1H NMR (400MHz DMSO-d6): δ=1.90(s,54H,CC(CH3)2Br),2.3(br t,8H,CCH2CH2NH and CH2CH2C=O),3.0(m,8H,CCH2NH and OCH2CH2NH),3.1-3.6(m,64H,OCH2C),3.87(s,6H,O=CCH2O),4.27(s,18H,CCH2OC=O),7.6-7.8(m,10H,both CH2NHC=O and NH3+).

[0181] LC-MS (ES, m / z): calculated for [(M+2H) / 2] + (C98H165Br9N8O40 + 2H) / 2 = 1408.2; found 1408.3.

[0182] Example 5 Preparation of Longer Spacer 9-Arm "Snapping" Initiator Compound P A TFA / amine salt initiator (Compound P) having the following structure was synthesized as follows: [ka] A 20 mL vial was charged with compound L (430 mg, 0.172 mmol, 1.0 equiv.) (see above) and α-t-butyloxycarbonylamino-ω-carboxyocta(ethylene glycol) (154 mg, 0.215 mmol, 1.25 equiv.), followed by N,N-dimethylformamide (2 mL) and then N,N-diisopropylethylamine (0.18 mL, 1.03 mmol, 6 equiv.). The flask was cooled to 0 °C using an ice bath. To this was added propylphosphonic anhydride solution (50 wt. % in ethyl acetate, 0.215 mL, 0.343 mmol, 2 equiv.) over 1 minute. The reaction was warmed to room temperature and stirred for 30 minutes. The reaction was quenched by adding water, saturated aqueous sodium bicarbonate, and ethyl acetate. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with saturated aqueous sodium bicarbonate, 0.5 M aqueous citric acid, water, and saturated aqueous sodium chloride, then dried (sodium sulfate), filtered, and concentrated in vacuo. The residue was used without further purification to give 0.6 g (0.194 mmol) of compound N shown below.

[0183] LC-MS (ES, m / z): calculated for [(M+2H-boc) / 2]+(C111H189Br9N8O46+2H-Boc) / 2=1496.3; found 1497.2. [ka] 100mL round bottom flaskTo this was added, under nitrogen, compound N (0.6 g), dichloromethane (4 mL), followed by trifluoroacetic acid (3 mL). The reaction was stirred at room temperature for 15 minutes. The reaction was concentrated under vacuum. The residue was dissolved with acetonitrile (3 mL), filtered through a syringe filter (Acrodisc CR25, PN 4225T), loaded onto a preparative HPLC column, and eluted with 50% acetonitrile (with 0.1% trifluoroacetic acid) in 50% water (with 0.1% trifluoroacetic acid) up to 90% acetonitrile (with 0.1% trifluoroacetic acid). The tubes containing the product were pooled, concentrated under vacuum, frozen, and placed on a lyophilizer. This yielded 200 mg (0.064 mmol, 37% over two steps) of compound P.

[0184] 1H NMR(400MHz DMSO-d6):δ=1.90(s,54H,CC (CH3)2 Br), 2.3(br t,8H,C CH2 CH2NH and CH2 CH2 C=O), 3.0(m, 8H, C CH2 NH and OCH2 CH2 NH), 3.1-3.6(m, 84H, O CH2 C), 3.87(s, 6H, O=C CH2 O), 4.27(s, 18H, C CH2 OC=O), 7.6-7.8 (m, 10H, both CH2 NH C=O and NH3 +).

[0185] LC-MS (ES, m / z): calculated for [(M+2H) / 2] + (C106H181Br9N8O44 + 2H) / 2 = 1496.3; found 1496.6.

[0186] Polymer synthesis Example 6 Preparation of Zwitterionic Polymers The initiator is typically prepared as a stock solution of approximately 100 mg / mL in DMF. The initiator and ligand (2,2'-bipyridyl) were introduced into a Schlenk tube. The resulting solution was cooled to -78 °C using a dry ice / acetone mixture and degassed under vacuum for 10 minutes. The tube was filled with argon, and the catalyst (CuBr, unless otherwise indicated) was introduced into the Schlenk tube, maintained under argon (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 tube was sealed and immediately purged by applying short cycles of vacuum / argon. A solution of HEMA-PC was prepared by mixing a specified amount of monomer, prepared in a glovebox maintained under nitrogen, with 200 proof-strength degassed ethanol. The monomer solution was added dropwise (via a cannula) to the Schlenk tube (and homogenized by light; stirring: not required). The temperature was maintained at -78°C. A full vacuum was applied to the reaction mixture for at least 10-15 minutes until bubbling from the solution ceased. The tube was then backfilled with argon and allowed to warm to room temperature. The solution was stirred, and as polymerization proceeded, it became viscous. After standing for 3-8 hours or just overnight, the reaction was quenched by direct exposure to air to oxidize Cu(I) to Cu(II). The mixture turned blue-green 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 precipitated with tetrahydrofuran or dialyzed against water, followed by lyophilization to yield a free-flowing white powder. Table 2 shows representative polymers made according to the present invention. [Table 2] [Table 3] JPEG0007767260000040.jpg220129JPEG0007767260000041.jpg205130

[0187] Example 7 Deprotection of Protected Maleimides It was observed that protected maleimide biopolymers tend to shift Mp to higher values ​​after thermal deprotection when the biopolymer powder was heated at 120 °C for 90 min. This makes biopolymer preparation more difficult, as the amount of Mp upshift depends on the biopolymer (Mp, structure, etc.). Alternative methods for deprotection are needed.

[0188] Initial experiments were performed in water in a closed capillary loop. Furan release was demonstrated during thermal deprotection of aqueous biopolymer solutions in an oven at 120 °C. No upshift in Mp was observed after thermal deprotection in aqueous solution.

[0189] This procedure was also repeated for the biopolymer dissolved in ethanol. It was confirmed that thermal deprotection in ethanol solution completely eliminated the Mp upshift. Different heating methods, such as oven or oil bath, were tested, and only minor differences were observed as long as the heating time and temperature were kept the same. The duration of heating must be optimized to avoid biopolymer degradation while simultaneously ensuring most of the furan-protected maleimide bipolymer is deprotected. The procedure was terminated and the ethanol solution of the biopolymer was used in a pressure reactor (capable of maintaining a pressure of 70 PSI).

[0190] A typical procedure begins with a clean, dry glass reactor. The biopolymer is dissolved in ethanol to form a clear, transparent solution. The concentration of the biopolymer is typically between 50 mg / mL and 150 mg / mL, usually around 100 mg / mL. This is a good balance between minimizing ethanol consumption and avoiding a highly viscous polymer solution.

[0191] The clear biopolymer solution should be transferred to a clean pressure reactor, purged with N for 3-5 min, and then tightly capped. The mass of the reactor + bipolymer solution should be recorded before and after thermal deprotection so that any leaks can be detected.

[0192] The pressure reactor containing the biopolymer solution (to be deprotected) is placed in an oven set at 120° C. for 2 hours. After deprotection, the pressure reactor is removed from the oven and allowed to cool. The deprotected biopolymer solution can be purified by solvent precipitation, spray drying, or freeze drying.

[0193] Preparation of conjugable polymers Example 8 Preparation of Maleimide-Conjugable 3-Arm Polymers A maleimide-conjugable polymer (B3) having the following structure was prepared as follows: [ka] In a 20 mL vial, polymer identification number 100 (Table 2) (280 mg, 0.00123 mmol, 1.0 equiv.) was dissolved with water (2 mL). To this was added 0.5 M aqueous sodium phosphate (0.2 mL). In a separate vial, 3-maleimidopropionic acid, NHS-ester (1.5 mg, 0.00548 mmol, 4.5 equiv.) was dissolved in tetrahydrofuran (0.6 mL). The NHS ester solution was added to the polymer solution over approximately 2 minutes at room temperature, and the resulting solution was stirred for 75 minutes. The reaction mixture was diluted with 4:1 water:tetrahydrofuran (4 mL) and placed in Amicon centrifugal membrane dialysis tubing (30,000 mwco), and the tubing was placed in a centrifuge (3000 rpm) for 30 minutes. The filtrate was removed for analysis, while the retentate was diluted and mixed with 4:1 water:tetrahydrofuran (6 mL) and the tube was placed in a centrifuge (3000 rpm) for 30 minutes. The filtrate was removed for analysis, while the retentate was diluted and mixed with water (8 mL) and placed in a centrifuge (3000 rpm) for 30 minutes. The filtrate was removed for analysis, while the retentate was diluted and mixed with water (8 mL). The centrifugation procedure was repeated three more times, after which the retentate was removed and placed in a vial. The Amicon membrane tube was rinsed with water (2 x approximately 2 mL), which was combined with the retentate, frozen, and placed in a freeze dryer. This yielded 262 mg (93%) of B3 as a white powder.

[0194] Example 9 Preparation of Maleimide 6-Arm Conjugable Polymer A maleimide-conjugable 6-arm polymer (F4) having the following structure was synthesized as follows: [ka] In a 20 mL vial, polymer identification number 110 (Table 2) (502 mg, 0.00205 mmol, 1.0 equiv.) was dissolved with water (4 mL). To this was added 0.5 M aqueous sodium phosphate (0.4 mL). In a separate vial, 3-maleimidopropionic acid, NHS-ester (2.45 mg, 0.0092 mmol, 4.5 equiv.) was dissolved in tetrahydrofuran (1 mL). The NHS ester solution was added to the polymer solution over 2 minutes at room temperature, and the resulting solution was stirred for 100 minutes. The reaction mixture was diluted with 4 mL of 4:1 water:tetrahydrofuran and equally distributed between two Amicon centrifugal membrane dialysis tubes (30,000 mwco). The tubes were then placed in a centrifuge (3000 rpm) for 30 minutes. The filtrate was removed for analysis, while the retentate was diluted and mixed with 4:1 water:tetrahydrofuran (6 mL) and the tubes were placed in a centrifuge (3000 rpm) for 30 minutes. The filtrate was removed for analysis, while the retentate was diluted and mixed with water (8 mL each) and placed in a centrifuge (3000 rpm) for 30 minutes. The filtrate was removed for analysis, while the retentate was diluted and mixed with water (8 mL / tube). The centrifugation procedure was repeated three more times, after which the retentate was removed and placed in a vial. The Amicon membrane tubes were rinsed with water (2 x approximately 2 mL per tube), and the combined retentate was frozen and placed in a freeze dryer. This yielded 459 mg (91%) of polymer F4 as a white powder.

[0195] Example 10 Preparation of a 6-arm conjugable polymer A maleimide-conjugable 6-arm polymer (S) having the following structure was synthesized as follows: [ka] Polymer identification number 120 (Table 2) (500 mg, 0.00091 mmol, 1.0 equiv.) was placed in a 20 mL vial and stirred for 10 minutes before dissolving with ethanol (4 mL). To this was added a 1% solution of 4-methylmorpholine in acetonitrile (0.030 mL, 0.00273 mmol, 3 equiv.). In a separate vial, product 176-55 (2.65 mg, 0.00455 mmol, 5 equiv.) was dissolved in acetonitrile (1 mL), and this solution was added to the polymer solution at room temperature over approximately 1 minute. An additional aliquot of acetonitrile (1 mL) was added, and the resulting solution was stirred for 18 hours. The reaction mixture was diluted with 0.1% aqueous trifluoroacetic acid (2 mL) (approximately pH 6), followed by water (approximately 14 mL), filtered through a syringe filter (Acrodisc Supor, PN4612), and evenly distributed into three Amicon centrifugal membrane dialysis tubes (30,000 mwco). The tubes were diluted with water (approximately 5 mL each), mixed, and centrifuged (3000 rpm) for 30 minutes. The filtrate was removed for analysis, while the retentate was diluted with water (approximately 10 mL per tube) and mixed. The centrifugation procedure was repeated five more times, after which the retentate was removed and placed in a vial. The Amicon membrane tubes were rinsed with water (2 x approximately 2 mL per tube), and this was combined with the retentate. The retentate solution was filtered through a syringe filter (Acrodisc Supor, PN4612), frozen, and placed in a freeze dryer. This yielded 469 mg (0.00085 mmol, 93%) of polymer S as a white powder.

[0196] Example 11 Preparation of Maleimide 9-Arm Conjugable Polymer A maleimide-conjugable 9-arm polymer (Q) having the following structure was synthesized as follows: [ka] Conjugable polymer Q was prepared as follows: In a 20 mL vial, polymer identification number 160 (Table 2) (540 mg, 0.0007 mmol, 1.0 equiv.) was placed and dissolved with water (4 mL). To this was added 0.5 M aqueous sodium phosphate (0.4 mL). In a separate vial, 3-maleimidopropionic acid, NHS-ester (0.93 mg, 0.0035 mmol, 5 equiv.) was dissolved in tetrahydrofuran (1 mL). The NHS ester solution was added to the polymer solution over approximately 2 minutes at room temperature, and the resulting solution was stirred for 30 minutes. The reaction mixture was diluted with water (approximately 15 mL), filtered through a syringe filter (Acrodisc Supor, PN4612), and evenly distributed into three Amicon centrifugal membrane dialysis tubes (30,000 mwco). The tubes were diluted with water (approximately 5 mL each), mixed, and centrifuged (3000 rpm) for 30 minutes. The filtrate was removed for analysis, while the retentate was diluted and mixed with water (approximately 10 mL / tube). The centrifugation procedure was repeated five more times, after which the retentate was removed and placed in a vial. The Amicon membrane tubes were rinsed with water (2 x approximately 2 mL per tube), which was then combined with the retentate. The retentate solution was filtered through a syringe filter (Acrodisc Supor, PN4612), frozen, and placed in a freeze dryer. This yielded 508 mg (94%) of Polymer Q as a white powder.

[0197] Example 12 Preparation of Maleimide 9-Arm Conjugable Polymer A maleimide-conjugable 9-arm polymer (R) having the following structure was synthesized as follows: [ka]

[0198] This was prepared using the same technique as described for conjugable polymer Q.

[0199] Example 13 Preparation of Wild-Type Factor VIII for Conjugation Mammalian-expressed wild-type (FVIII-WT) is known to have all cysteine ​​residues oxidized to form disulfide bridges using thiol-reactive polymers containing reactive groups such as maleimide or iodoacetamide, or, in the case of the free cysteines present in the B domain, blocked (capped) by metabolic products from the medium that prevent the unpaired free cysteine ​​from becoming available for conjugation. These capping moieties can be removed using reducing agents such as TCEP or DTT, followed by removal of the reducing agent and refolding of the protein.

[0200] FVIII-WT was formulated at a concentration of 0.5 mg / mL in 50 mM MOPS pH 7, 10 mM CaCl2, 200 mM NaCl, 1% sucrose, and 0.01% Tween 80. A 150x molar excess of TCEP solution was added and incubated at 4°C for 1 hour. A Sephadex G25 desalting column was used to remove TCEP. The G25 column was equilibrated with formulation buffer, the TCEP-reduced sample was loaded, and collected fractions were analyzed by SDS-PAGE. Protein-containing fractions were pooled and incubated overnight at 4°C to allow protein refolding (regeneration of disulfide pairs by oxidation), while unpaired cysteines remained in the free sulfhydryl form (uncapped). Alternatively, TCEP removal was achieved using an anion exchange (e.g., Q Sepharose FF) column, in which the TCEP-reduced sample was diluted to lower the salt concentration and then loaded onto the QFF column, followed by a wash step with low-salt MOPS buffer and elution with a step gradient of NaCl. Under these conditions, the protein eluted at approximately 300 mM NaCl. Protein fractions were pooled for conjugation, as described below. The ion exchange method for TCEP removal is preferred over the desalting column approach because it is more amenable to scale-up.

[0201] Analysis of the TCEP-treated form by SDS-PAGE analysis showed two predominant bands: (1) a high-MW band migrating at approximately 180 kDa, representing the heavy chain plus B domain (HC-BD); and (2) a low-MW band migrating at approximately 80 kDa, representing the light chain (LC). The sample was also analyzed by gel filtration using a Superose 6 column. The column was equilibrated with 20 mM Tris pH 7.5, 10 mM CaCl, 200 mM NaCl, 10% ethanol, 1% sucrose, and 0.001% Tween 80, and subsequently injected with different FVIII samples, including (1) TCEP-treated, refolded FVIII-WT; and (2) native FVIII-WT for comparison. The elution profiles of both samples at 280 nm each showed a predominant single peak at the expected retention time.

[0202] Example 14 Conjugation of FVIII-WT with high molecular weight zwitterionic polymers The revealed free cysteine ​​thiols in the TCEP-treated form of FVIII-WT were used for conjugation with various maleimides and the above-mentioned iodo-acetamide functionalized polymers, which differed in molecular weight, structure, and linker length as shown in Table 4. The conjugation reaction mixture contained 50 mM MOPS pH 7, 10 mM CaCl, 200 mM NaCl, 0.01% Tween 80, and 20 mM Tris pH 8, 200 mM The reaction mixture contained approximately 0.5 mg / mL of FVIII-WT protein in a 5-100x molar excess of maleimide polymer dissolved in NaCl, 10 mM CaCl2, and 0.01% Tween 80. The reactions were allowed to proceed overnight at 4°C and subsequently analyzed for conjugation efficiency by SDS-PAGE under both non-reducing and reducing conditions. Results showed the disappearance of the single heavy chain-B domain (HC-BD) band, but not the simultaneous appearance of a truncated form of the domain and a higher molecular weight band at the top of the gel, indicating the presence of newly formed conjugate. The conjugation efficiency of each reaction (calculated as the percentage of the remaining HC-B domain band compared to the no-polymer control) is shown in Table 4. [Table 4] JPEG0007767260000048.jpg135152 Table 5 below shows the activity of conjugates formed from 9-branched hema-PC polymers from initiators O or P in Table 3. [Table 5]

[0203] Conjugation reactions were performed using 1 mg of TCEP-treated FVIII-WT and a 50x molar excess of the conjugable polymer used in #17 (Table 4) and the protocol described above. Conjugation efficiencies of >90% were determined using SDS-PAGE as before. The conjugate band remained under reducing conditions.

[0204] The conjugate was purified using cation exchange chromatography using MacroCap SP resin. The conjugation reaction was diluted with 50 mM MOPS, pH 7, 10 mM CaCl. l2 The eluate was diluted 10-fold in 0.01% Tween 80 and loaded onto 3 mL of resin packed in a 5 mL drip column. Column flow was achieved by gravity, and the unbound fraction was collected. The column was flushed and washed with a combined 21 column volumes (CV) of wash buffer containing 20 mM NaCl. The bound protein was then eluted with wash buffer containing different NaCl concentrations, including 100, 150, 200, 250, and 500 mM NaCl. At least 5 CV of eluate was collected for each NaCl concentration. Fractions were subjected to SDS-PAGE analysis to determine the NaCl concentration at which the protein eluted. Preliminary analysis showed that the free protein eluted at 150 mM salt, and the conjugate eluted at 100 mM salt. This was confirmed by analytical gel filtration on a Superose 6 column, which gave a single peak for the conjugate and free protein. The conjugate pool was concentrated and sterile filtered using a 0.2 μm SpinX centrifugal filter to give a final concentration (as to the protein) of 2.16 mg / mL with a final process yield of 40%.

[0205] The activity of the conjugate, measured using the COAMATIC Factor FVIII Assay Kit, was equivalent to FVIII-WT.

[0206] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, certain changes and modifications can be made within the scope of the appended claims. Furthermore, each reference provided herein is incorporated by reference in its entirety for all purposes to the same extent as if each reference were individually incorporated by reference. To the extent that the content of any cited reference, including website or accession numbers, may change over time, the description is intended to be effective as of the filing date of this application. Unless otherwise apparent from the context, any step, element, aspect, or feature of an embodiment can be used in combination with any other.

Claims

1. A conjugate comprising a functional agent and an amphoteric polymer having the formula: 【Chemistry 1】 (Wherein, s is 1 to 20, R1 is 【Chemistry 2】 and * is bound to the functional substance; R2 is a combination of alkoxy and amido, and R3 is, 【Transformation 3】 wherein R4, R5, and R6 are the same or different, and 【Chemistry 4】 wherein Z is a polymer, 【Transformation 5】 (Wherein R7 is H or C １～６ is alkyl, ZW is a zwitterion, and t is 1 to 6. (synthesized with a monomer selected from the group consisting of selected from the group consisting of

2. The conjugate of claim 1 , wherein R2 comprises a structure of the following formula: 【Transformation 6】 (wherein m is an integer from 1 to 20).

3. The R2 is 【Transformation 7】 (wherein X is an integer from 0 to 50, and Y is an integer from 1 to 50). The conjugate of claim 2, wherein

4. The conjugate of any one of claims 1 to 3, wherein the zwitterion is phosphorylcholine.

5. The conjugate of any one of claims 1 to 3, wherein the monomer is 2-(methacryloyloxyethyl)-2'-(trimethylammoniumethyl)phosphate (MPC) or 2-(acryloyloxyethyl)-2'-(trimethylammoniumethyl)phosphate.

6. The conjugate of claim 5, wherein the monomer is 2-(methacryloyloxyethyl)-2'-(trimethylammoniumethyl)phosphate (MPC).

7. The R2 is 【Transformation 8】 wherein Y is an integer from 1 to 10; and X is 4, 8, or 12. The conjugate of claim 6, wherein

8. 8. The conjugate of claim 7, wherein Y is 4.

9. The conjugate of any one of claims 1 to 8, wherein the polymer has a molecular weight of about 500,000 to about 1,000,000 daltons.

10. 10. The conjugate of claim 9, wherein the polymer has a molecular weight of about 650,000 to about 850,000 daltons.

11. 10. The conjugate of claim 9, wherein the polymer has a molecular weight of about 800,000 daltons.

12. 10. The conjugate of claim 9, wherein the polymer has a molecular weight of about 750,000 daltons.

13. 2. The conjugate of claim 1 having a structure represented by the formula: 【Chemistry 9】 where X is an integer from 0 to 50, Y is an integer from 1 to 50, and * is the point where the polymer is attached to a functional material.

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