Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
  • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
  • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
  • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid

Definitions

• the present invention relates to the preparation of functionalized polymers. More particularly, the present invention relates to reagents and methods for the site-specific chemical modification of target molecules, e.g. biomacromolecules, particularly biologically important polypeptides, and other polydisperse macromolecules such as plastics (e.g., polyethylene or nylon), by means of covending attachment of functionalized polymers, particularly polyal ylene oxide polymers.
• target molecules e.g. biomacromolecules, particularly biologically important polypeptides, and other polydisperse macromolecules such as plastics (e.g., polyethylene or nylon)
• plastics e.g., polyethylene or nylon
• polyethylene glycol-modified polypeptides have reduced immunogenicity and antigenicity and also have a longer lifetime in the bloodstream as compared to the parent polypeptides (Abuchowski and Davis (1981) "Enzymes as Drugs", Holcenberg and Roberts, eds., pp367-383, John Wiley & Sons, N.Y.). These beneficial properties of the modified polypeptides make them very useful in a variety of therapeutic applications, such as enzyme therapy.
• PEG polyethylene glycol
• the hydroxyl end- groups of the polymer must first be converted into reactive functional groups.
• activated PEG Methoxypolyethylene glycol
• mPEG Methoxypolyethylene glycol
• One of the most common form of activated PEG used for preparation of therapeutic enzymes is methoxy-poly (ethylene glycol) succinoyl-N-hydroxysuccinimide ester (Abuchowski et al. (1984) Cancer Biochem. Biophys. 7:175-186).
• methoxy-poly (ethylene glycol) succinoyl-N-hydroxysuccinimide ester Abuchowski et al. (1984) Cancer Biochem. Biophys. 7:175-186.
• ester linkage between the polymer and succinic acid residue has 41813
• a major drawback with all the above methods of protein PEGylation is that the polymer conjugations occur randomly and non-site specifically, typically at amino groups, e.g. lysine side chains and the N-terminal amino group, such that the multiple polymer groups can potentially interfere with biological activity, e.g., substrate binding or receptor binding, and such that preparations of conjugates are typically heterogeneous, particularly in view of the fact that the reactions are random and not usually complete due to the preferential activity or accessibility of the various target residues.
• charge alteration of the target molecule is typically caused by the coupling reaction, particularly in polypeptides upon modification of free amino groups.
• Changes in charge may adversely affect the biological activity of a protein by several mechanisms including the disruption of the tertiary structure and the destruction of active sites. Such changes in charge are typically cumulative as derivatization is carried to completion.
• increasing the number of polymers per molecule of polypeptide using the methods above very often decreases sterically the accessibility of the active site and thus diminishes biological activities possessed by the unmodified polypeptide.
• Polyethylene glycol modified TNF- ⁇ is a typical example where extensive modification resulted in the complete loss of bioactivity (Tsutsumi et al. (1994) Jpn. J. Cane. Res. 85, 9-12).
• the present invention provides methods and compounds for site- specifically, chemoselectively modifying inder mild conditions a target macromolecule, such as proteins, peptides, other organic compounds such as plastics, or surfaces containing macromolecules, with an amino-oxy or aldehyde (or ketone) derivative of an organic polymer, preferably a water organic soluble polymer, more preferably PEG or dextran, via an oxime linkage at a unique site on the target macromolecule.
• Novel amino-oxy derivatives of PEG and other water- soluble polymers referred to as "functionalized" polymers, are provided that are capable of oxime-linkage formation.
• one or more water-soluble polymers may be coupled site-specifically and chemoselectively to a single, pre- chosen site on an individual target macromolecule under mild conditions.
• Another aspect of the subject invention is to provide a process for preparing proteins modified by the site-specific, covalent attachment of the functionalized water-soluble polymer derivatives.
• the present invention provides a functionalized polymer wherein the number of polymer repeating units is an integer between 5 and 2000.
• the present invention also provides a process for preparing the functionalized polymer that contains an oxime-forming group which includes the step of reacting a polymer compound, wherein the number of polymer repeating units is an integer between 5 and 2000, with a compound containing an oxime- forming group, which oxime-forming group is optionally in a deprotectable form, wherein the oxime-forming group is covalently attached to the polymer.
• the present invention further provides a functionalized modified target molecule, preferably a polypeptide, having an oxime-forming reactive group complementary in reactivity to a functionalized polymer of the invention, such that site- specific reactivity and oxime-linkage formation will occur.
• a functionalized modified target molecule preferably a polypeptide, having an oxime-forming reactive group complementary in reactivity to a functionalized polymer of the invention, such that site- specific reactivity and oxime-linkage formation will occur.
• the present invention further provides a modified target macromolecule, i.e. a polymer conjugate, preferably comprising a polypeptide, having bound site-specifically thereto at least one organic polymer wherein the polymer or polymers are covalently bound through a single oxime linkage to a unique site on the target macromolecule.
• a modified target macromolecule i.e. a polymer conjugate, preferably comprising a polypeptide, having bound site-specifically thereto at least one organic polymer wherein the polymer or polymers are covalently bound through a single oxime linkage to a unique site on the target macromolecule.
• the invention also provides a process for preparing a polymer conjugate described above under mild conditions which includes the steps of functionalizing the target macromolecule to provide a first oxime-forming functional group complementary in activity with a second functional oxime-forming group present on a functionalized polymer of the invention, reacting the functionalized target macromolecule with the functionalized polymer such that an oxime bond is formed between the first and second functional groups.
• Multi-polymer-containing amino-oxy-functionalized or aldehyde(or ket ⁇ ne)-functionalized polymer constructs are also provided that allow site-specific, chemoselective ligation under mild conditions of the construct (and thus all of its polymers) to a single site on a target macromolecule via an oxime bond. Families of functionalized polymer constructs are also provided wherein each construct differs in topology but not in molecular weight (average) from the others in the same family.
• Methods for their use include the systematic modification of a target macromolecule to rapidly create a family of target molecules, preferably biologically important proteins, differing in topology but not molecular weight, from which family can be identified macromolecules having desired biological or physical properties, such as enhanced pharmacokinetic behavior.
• the present invention thus provides methods for systematically modifying the Stokes radius of a target macromolecule, which method includes the steps of conjugating a functionalized target macromolecule with a series of functionalized polymers of the invention, preferably the multi-polymer-containing functionalized polymers, in separate reactions, and then determining the effect of conjugation on the Stokes radius.
• the pharmacokinetic behavior of a target macromolecule is systematically modified.
• Kits are provided with appropriate reagents for carrying out the methods of the invention.
• Figure 1 depicts an SDS-PAGE gel under non-reducing conditions showing the migration of conjugates of oxidized 11-8 with different functionalized PEG polymers. In all cases a single derivative was obtained in the conjugate reaction.
• Lanes 1, 3, 5, 7 Result of 20 hour incubation of oxidized IL-8 with functionalized AoA-J ⁇ G 5kD , AoA-PEG 10kD , AoA-PEG 20kDl and AoA-N-(PEG 3kD ) 2 , respectively.
• AoA-N(PEG 3 ⁇ ) 2 is (PEG 3tD -CONH(CH 2 ) 2 ) 2 N-(CH 2 ) 2 -NH-AoA.
• Lanes 2, 4, 6, 8 purified conjugates PEG 5kD -Il-8, PEG 10k _-Il-8, PEG 20lcD -Il-8 and (PEG 3kD ) 2 Tl-8. Lane 9, oxidized 11-8,; Lane 10, protein molecular weight markers: from top to bottom: Phosphorylase B (97.4 kD), bovine serum albumin (66.2kD), ovalbumin (45kD), carbonic anhydrase (31kD), soybean trypsin inhibitor (21.5 kD), lysozyme (14.4kD). "kD” indicates kilo Daltons.
• Figure 2 depicts an SDS-PAGE gel showing migration of 11-8 conjugates obtained with the different functionalized polymers.
• Lane 1 11-8.
• Lanes 2, 3, 4 PEG ikD -Il-8, PEG 10tD -Il-8, PEG 20kD -Il-8.
• Lane 5 Il-8-PEG 20kD -Il-8 (dimer; "dumbbell”).
• Lane 6, 7 (PEG 10kD ) 2 Lys-Il-8, (Lys-PEG ikD ) Il-8.
• Lane 8 Dextran 9kD -II-8.
• Lane 9 Protein molecular weight markers are the same as in Figure 1.
• FIG. 3 depicts an SDS-PAGE gel showing migration of conjugates of functionalized PEG 3kD and PEG 20kD with des-Met-G-CSF. Lane 1: des-Mct-G- CSF.
• Lane 2 result of 24h incubation of oxidized ⁇ fe. ⁇ -Met-G-CSF with AoA- PEG 3kD .
• Lane 3 Purified PEG 3kD - ⁇ tf-Met-G-CSF.
• Lane 4 Coupling of AoA- PEG 20kD t0 oxidized des-Met-G-CSF after 24h incubation.
• Lane 5 Purified VEG 20tD -des-Met-G-CSF.
• Lane 6 Protein molecular weight markers are as in Figure 1.
• FIG. 4 Conjugation of functionalized AoA-PEG 3kD , AoA-PEG 10kD and AoA-PEG 20kD to Interleukin-1 Receptor Antagonist Protein ("E -lra").
• Lane 1 Il-lra.
• Lane 2 Coupling of AoA-PEG l0kD to transaminated Il-lra after 48h incubation at room temperature.
• Lanes 3, 4, 5 Purified conjugates PEG 10kD -Il-lra, PEG 3kD -Il-lra, and PEG 20t ⁇ -Il-lra.
• Lane 6 Protein markers are as in Figure 1.
• Figures 5A, 5B and 5C depict dose-response curves showing inhibition of HL-l/J induced Prostaglandin E2 production by dermal fibroblasts in the presence of decreasing concentrations of Il-lra derivatives.
• Figure 5A is that for PEG (31cD) -Il-lra
• Figure 5B is that for PEG (I0tD) -Il-lra
• Figure 5C is that for PEGpoi- D j-Il-lra.
• the conjugate was prepared as described in Example 8 using the transamination to provide an activated protein.
• Figures 6A and 6B depict pharmacokinetics of PEG 10kD Tl-lra ( Figure 6A) and PEG 20kD -Il-lra (Figure 6B) in rats, compared with that of unmodified Il-lra.
• the results with the two derivatives are plotted separately (dotted lines), each time with the same set of results obtained for unmodified Antril (solid line).
• Blood samples were collected at 3, 10, and 30 minutes and at 1, 3, 7, 12 and 24 hours. Each point represents a single determination with a single animal.
• a slow rise in blood levels after an initial decrease could be shown between 30 and 180 min. Such an unexpected behavior was not observed with other investigated proteins used under the same experimental conditions.
• Figure 7 depicts pharmacokinetics of 11-8, PEG 20fcD -Il-8, Il-8-PEG 20tD - 11-8 and (PEG 20kD ) 2 Lys-Il-8. Blood samples were collected at 3, 7, 15 and 30 minutes, and at 1, 3 and 7 hours. Each point represents the mean value or four animals. The dumbbell and the multiarm constructs appear as the most effective conjugates for improving the lifetime of 11-8 in the bloodstream.
• the subject invention provides novel modifying reagents that are amino-oxy derivatives of polymer, preferably water-soluble polymers such as PEG, i.e., polyethylene glycol.
• the reagents of the subject invention may be used to covalently attach a variety of polymers to target macromolecules of interest.
• target 8 macromolecule can be modified by the attachment of water-soluble polymers without substantially reducing the biological activity of the macromolecule, or reducing the biological activity to a lesser extent than the biological activity would be reduced by the attachment of the same water-soluble polymers to multiple residues located throughout the macromolecule (typically lysine residues for a polypeptide) using activated polymers other than the site-specifically acting compounds of the present invention.
• biological activity includes enzymatic activity, the ability to bind to receptors (including antibodies), the ability to bind ligands, the ability to induce an immune response, and the like.
• a “complementary functional reactive group” is defined as one of a pair of functional groups that chemospecifically reacts with the complementary member of the pair.
• amino-oxy-acetyl AoA
• GXL glyoxylyl
• Complementary functional groups used in the invention are groups which can be used without compromising other groups present or other chemistry to be applied.
• amino-oxy-acetyl groups are a most preferred functional group.
• the functionalized polymers may be used for attachment non-site-specifically to appropriately modified target molecules, for example, when multiple residues of a target polypeptide are first modified so that each contains a functional group specifically and complementarity reactive with the functional group on the polymers of the invention.
• the functionalized polymer is attached to the target macromolecule site-specifically by covalent conjugation via an oxime linkage.
• Covalently conjugated or “conjugated” refers to the attachment of polymer to target via a functionalized polymer.
• “Functional” or “functionalized” describes, for the purposes of this invention, the attachment of a oxime-forming reactive group onto a polymer so that the polymer can be site-specifically conjugated to a complementary functionalized target macromolecule.
• the PEG or POG i.e., polyoxyethylated glycerol
• the PEG or POG is activated or functionalized by attaching the reactive group to a terminal hydroxyl groups and then the functionalized polymer is covalently conjugated to an aldehyde group that has been site-specifically introduced into the target molecule.
• the reactive amino acid is typically lysine, and lysine is linked to the reactive group on PEG or POG through its free e-amino group
• the functionalized polymer reagents of the invention are site-specifically attached via a stable oxime linkage to a single, pre-chosen, site on the target molecule.
• a “target macromolecule” as used herein refers to an organic molecule (which includes molecules of biologic origin as well as organic molecules with inorganic components) having a molecular weight of at least 500, more preferably of at least 2000, even more preferably at least 5000, most preferably at least 10,000.
• a “functionalized target macromolecule” is a target macromolecule that has been modified site-specifically to contain an oxime-forming functional group.
• the target macromolecule can be derived from natural, recombinant sources or can be synthetic.
• water-soluble functionalized polymer reagent refers to a water-soluble polymer modified so as to contain a functional group that provides for the site-specific conjugation of the water-soluble polymer to a target macromolecule through an oxime linkage.
• target macromolecules are site-specifically modified to contain a functional group that is complementary in oxime-forming reactivity with the amino-oxy or aldehyde functional group introduced onto the polymer, preferably at one of its termini (or in the case of a bi-functional polymer—at both of its termini).
• a "homogeneous" polymer conjugate composition of the invention refers to a chemical composition in which substantially all of the conjugate molecules have essentially identical chemical structures-each have the identical number and location of attached polymers although there can be a range in the molecular weight of the attached polymer(s).
• the molecular weight range average is typically that as found in commercially available preparations of a polymer used herein for functionalization; preferably the range is that obtained after further size fractionization of commerically available preparation. This is in sharp contrast to a typical polymer conjugate composition in which the individual molecules differ at least in the locations and in the number of attached polymers.
• Compositions of the invention can also be referred to as "self -identical" compositions, as substantially all of the individual molecules of the polymer conjugate are essentially identical to each other.
• substantially all refers to at least 80% of the total conjugate molecules all having the same location and number of polymers attached although each attached polymer may not be of the same size. Increasing degrees of purity, such as 90%, 95%, 98%, 99%, 99.5%, 99.8%, etc., ail the way to 100%, are increasingly preferred meanings of "substantially all.”
• homogeneous polymer conjugate compositions can be comprised of homo- or hetero-polymic, wherein hetero-polymers as defined herein refers to differences in the chemical type or in the MW(av) of polymers bound, each of which can be present at its molecular weight average.
• hetero-polymers as defined herein refers to differences in the chemical type or in the MW(av) of polymers bound, each of which can be present at its molecular weight average.
• the formula of the compounds useful for coupling polymers to complementary functionalized target macromolecules, such as polypeptides, via an oxime linkage is:
• P-X-O-NH 2 Where P represents an organic polymer, preferably water soluble, X represents a spacer group, -O-NH 2 represents amino-oxy.
• Water-soluble organic polymers of interest preferably have hydroxy groups appended to the polymer backbone that are convenient sites for functionalization and may be selected from known water-soluble polymers including but not limited to: (a) dextran and dextran derivatives, including dextran sulfate, P-amino cross linked dextrin, and carboxymethyl dextrin, (b) cellulose and cellulose derivatives, including methylcellulose and carboxymethyl cellulose, (c) starch and dextrines, and derivatives and hydroylactes of starch, (d) polyalklyene glycol and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol, wherein said homopolymers and
• Water-soluble polymer reagents include but are not restricted to polypropylene glycol (“PPG”), polyoxyethylated polyol (“POP”), heparin, heparin fragments, dextran, polysaccharides, polyamino acids including proline, polyvinyl alcohol (“PVA”), and other water-soluble organic polymers.
• PPG polypropylene glycol
• POP polyoxyethylated polyol
• heparin heparin fragments
• dextran polysaccharides
• polyamino acids including proline
• PVA polyvinyl alcohol
• other water-soluble organic polymers include but are not restricted to polypropylene glycol (“PPG”), polyoxyethylated polyol (“POP”), heparin, heparin fragments, dextran, polysaccharides, polyamino acids including proline, polyvinyl alcohol (“PVA”), and other water-soluble organic polymers.
• the water-soluble polymer reagents of the subject invention include amino-oxy derivatives of polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol, wherein said homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, polyvinyl ethyl ethers, and or.jS-Poly [(2-hydroxyethyl)-DL-aspartamide] and other water-soluble organic polymers.
• the water-soluble polymer P is selected from dextran and dextran derivatives, dextrine and dextrine derivatives, polyethylene glycol and derivatives thereof.
• the water-soluble polymer P is selected from polyethylene glycol and derivatives thereof, the monomethyl ether of polyethylene glycol being particularly preferred (so as to avoid cross-linking between proteins).
• Water soluble polyoxyethylated polyols are also useful in the present invention.
• POG polyoxyethylated sorbitol
• polyoxyethylated glucose polyoxyethylated glycerol
• POG polyoxyethylated glycerol
• the glycerol backbone of polyoxyethylated glycerol is the same backbone occurring naturally in, for example, animals and humans in mono-, di-, triglycerides. Therefore, this branching would not necessarily be seen as a foreign agent in the body.
• the POG has a preferred molecular weight in the same range as PEG.
• the structure for POG is shown in Knauf et al. (1988) J. Biol. Chem., 263:15064-15070, and can generally substitute for PEG or its derivatives in the formulas herein.
• polymer P should be non-toxic, and preferably ⁇ on-immunogenic.
• the molecular weight of P may vary considerably. Furthermore, when P is said to have a given molecular weight, that molecular weight may only be approximate, reflecting the average molecular weight of a population of molecules P differing with respect to one another in regards to the number of subunits present in the molecule. In general, P will have a molecular weight of about 200 to 200,000, preferably in the range of about 400 to 50,000, more preferably .1,000 to 60,000, even more preferably in the range of 2,000 to 40,000. Suitable molecular weights for P, when the functionalized polymer are to be coupled to a polypeptide will vary in accordance with the specific protein to modified and the purpose of the modification.
• the spacer group X is optionally present.
• This invention provides functionalized polymers in which the complementary reactive group capable of oxime-linkage formation is an amino-oxy group. Preferred is amino-oxy-acetyl ("AoA").
• additional structure X or spacer group
• the spacer group X represents a non-reacting group comprising substituted or unsubstituted, branched or linear, aliphatic or aromatic groups such as phenyl or C ⁇ -C, 0 alkylene moieties, - o alkyl groups, or a combination thereof, or an amino acid chain (such as a flexible hinge or loop sequence (see for example Argos, J. Mol.
• X comprises - CH 2 - or -CHOH-, or more preferably -CO-CH 2 - or -NH- CO-CH 2 -.
• X is -NH-CO-CH 2 - in the formula for AoA-MPEG: CH 3 -O-(CH 2 CH 2 O)nCH 2 CH 2 -NH-CO-CH 2 -O-NH 2 .
• spacer groups When an amino-oxy group is on the functionalized polymer groups present in the additional connecting structure (spacer groups) adjacent to the amino-oxy function are not critical; however, a requirement of these spacer groups is that they do not interfere with the formation of the oxime linkage between the amino-oxy and its complementary aldehyde group. They should not react in preference to the amino-oxy group with the aldehyde, nor provide steric hindrance to the reaction, nor deactivate the reactive groups. Where the conjugated polymer is to be used for antigenic or immunogenic purposes, it is apparent to one skilled in the art that spacer groups are chosen that are not themselves strongly immunogenic. Where the conjugated polymer is to be used for binding purposes, the preferred spacer group enhances or at least does not interfere with properties such as binding, avidity, product stability or solubility.
• the spacer group X can be chosen to enhance hydrolytic stability of the oxime linkage.
• Increased oxime stability may be obtained with the presence of aromatic groups in X.
• the functionalized polymers are bi-functional, i.e. will have two oxime-forming groups.
• the groups are not complementary in reactivity to each other.
• Preferably two groups are at distal, non- interfering (with each others ability to form an oxime bond) sites on the polymer, more preferably at each of termini.
• dihydroxy polyalkylene glycols are functionalized as taught herein to obtain a polymer having an amino-oxy oxime- forming group at each of its termini.
• the bis-aminooxy polymer is reacted with the site-specifically functionalized target macromolecules of the invention to obtain a "dumbbell" construction where the polymer through oxime linkages acts to link a first functionalized target macromolecule and second functionalized target macromolecule, preferably identical to the first, through each of their site-specific oxime-forming groups.
• Hetero-dimers can be formed when the first and second macromolecules are different. Hetero-dimers can be formed, for example, by sequential reactions of the first and second functionalized macromolecule (for example, by the intermediate step of isolating a polymer conjugate wherein only one of the two polymer reactive groups are linked to a first target macromolecule) or by a single reaction of the polymer with a mixture of the first and second functionalized macromolecule present in the same molar ratio.
• the dumbbell constructs of the present invention, and compositions thereof have the target macromolecules, preferably biologically important polypeptides, linked site- specifically, thus providing constructs, in contrast to previous polymer conjugation methods, of defined structure and homogeneous composition.
• Preferred embodiments of the bi-functional polymer contain two amino-oxy acetyl (AoA) groups. Particularly preferred is AoA-PEG-AoA, where PEG or its derivatives comprise the polymer moiety.
• bi- and multi-poly mer-containing functionalized polymers are provided. Functionalized polymers of these embodiments have the general formulas
• P is an organic polymer as defined herein, m is an integer from 2 to 10, more preferably 2 to 5, X is a spacer group as defined herein, -O-NH 2 is amino- oxy, -C(R)O is aldehyde when R is hydrogen, and L is a multi-valent linking group to which each P (m in number) is separately and covalentiy linked, and wherein the valency of L is at least m+1.
• R is preferably C1-C10, more preferably Cl to C4, linear or branched alkyl group.
• groups present in the X spacer adjacent to the aldehyde or ketone function are not critical; however, a requirement of these groups is that they do not interfere with the formation of the oxime linkage between the aldehyde and its complementary amino-oxy group. They should not react in preference to the aldehyde group with the amino-oxy, nor provide steric hindrance to the reaction, nor deactivate the reactive groups.
• the connecting group does not react with other functions present but if designed to do so then does not do so in an undesirable way (i.e., a way which reduces product homogeneity or activity).
• the spacer group preferably represents a non-reacting group comprising substituted or unsubstituted aliphatic or aromatic groups such as phenyl or C t -C, 0 all ylene moieties, C,-C I0 alkyl groups, or a combination thereof, or an amino acid chain (such as a flexible hinge or loop sequence (see for example Argos, J. Mol. Biol. (1990) 211:943-958), or a nucleotide chain or a sugar chain or a lipid chain or a combination thereof and may contain heteroatoms.
• substituted or unsubstituted aliphatic or aromatic groups such as phenyl or C t -C, 0 all ylene moieties, C,-C I0 alkyl groups, or a combination thereof, or an amino acid chain (such as a flexible hinge or loop sequence (see for example Argos, J. Mol. Biol. (1990) 211:943-958), or a nucleot
• X is a spacer group comprising -CH 2 -, -CO-, or -CHOH, more preferably -C ⁇ H,-.
• an aldehyde and spacer comprising OHC-CO- or glyoxylyl (“GXL").
• conjugation of the functionalized polymer site-specifically to a target macromolecule results in attachment of two or more polymers site-specifically through a single oxime linkage via the -0-NH 2 or -CHO group attached to the multi-valent L structure.
• Amino-oxy-functionalized polymers are preferred over the aldehyde embodiments in part because of the methods available and those taught herein to introduce an aldehyde (or keto) function on a target macromolecular.
• the bi- or multi-polymer-containing functionalized polymer enables two or more polymers, the same or different, preferably the same, to be attached to a single, pre-chosen site on the target macromolecule.
• L is a trivalent group
• n is 2 (see Example 2 or 10 herein).
• the valence of L is m+1, wherein one valency of L is occupied by the oxime-forming group optionally through X, and the remaining valencies of L are occupied by one or more, i.e. m, polymers.
• the structure of L is not critical nor are the linkages connecting L to the polymers so long as L provides no steric hindrance to the subsequent oxime reaction, nor deactivate the reactive groups.
• Each arm or valency of the linking group L in the functionalized polymer preferably comprises a non-reacting group comprising substituted or unsubstituted aliphatic or aromatic groups such as phenyl or C[-C 10 alkylene moieties, C ⁇ C ⁇ alkyl groups, or a combination thereof, or an amino acid chain (such as a flexible hinge or loop sequence (see for example Argos, J. Mol. Biol. (1990) 211:943-958), or a nucleotide chain or a sugar chain or a lipid chain or a combination thereof and may contain heteroatoms.
• a non-reacting group comprising substituted or unsubstituted aliphatic or aromatic groups such as phenyl or C[-C 10 alkylene moieties, C ⁇ C ⁇ alkyl groups, or a combination thereof, or an amino acid chain (such as a flexible hinge or loop sequence (see for example Argos, J. Mol. Biol. (1990) 211:943-9
• all but one arm or valency of L Prior to conjugation with a polymer, preferably all but one arm or valency of L contains a functional group that can react specifically with a group on the polymer, which preferably is located at a polymer terminus. The remaining valency is protected for later reaction with, or otherwise occupied with, a compound providing the oxime-forming function (said function is in a deprotectable state if desired).
• linker groups are chosen that are not themselves strongly immunogenic.
• the preferred linker group enhances or at least does not interfere with properties such as binding, avidity, product stability or solubility.
• Linking structures can themselves contain valencies occupied with oxime-forming groups such that parallel assembly via oxime formation with a complementary functionalized polymer of the invention is employed to assemble the (P) m L- structure. Accordingly, baseplate structures described in co-pending United States Serial Numbers 08/057,594, 08/114,877, and 08/057,594, and co-pending International application PCT IB94/00093 (which are hereby inco ⁇ orated by reference) are suitable for use as L structures.
• the oxime-forming groups of the baseplates can be replaced with other complementary reactive groups; however, most preferably oxime formation is used for assembly.
• L is pentalysyl peptide (where m is 5) with each lysyl residue containing a covalently attached polymer and the peptide N-terminal contains the oxime-forming group.
• a preferred L structure is derived from a tri-amine compound wherein any two amino groups are each available for coupling to a polymer and the remaining amino group is available for introduction of an oxime-forming group.
• a preferred tri-amine is a compound of the formula NQF S-NH ⁇ , wherein any two amino groups (-NH ⁇ are available for coupling to the polymer and the remaining amino group is available for introduction of an oxime-forming group, and R5 is a non-reacting group comprising substituted or unsubstituted aliphatic or aromatic groups such as phenyl or C ⁇ -C I0 alkylene moieties, C,-C, 0 alkyl groups, or a combination thereof, or an amino acid chain (such as a flexible hinge or loop sequence (see for example Argos, J. Mol. Biol.
• R5 is preferably -CH2-CH2-.
• the three primary amino groups are preferably distal to the nitrogen.
• the tri-amine compound is tris-(2-aminoethyl)amine.
• bi- and multi-polymer functionalized polymers of the invention find particular use in systematically modifying the Stokes radius of a target macromolecule, which in turn, preferably modifies a particularly desirable property of that macromolecule.
• the Stokes radius can be systematically modified, which in turn preferably allows systematic modification of its pharmacokinetic behavior, and ultimately its therapeutic efficacy.
• the functionalized polymers contain PEG or its derivatives as the polymer backbone.
• the functionalized polymer is a mono-functional or a bi-functional polymer that contains PEG or a derivative thereof and has the structure
• Rl-O(R2-0)nR2-R3 where n is an integer between 5 and 2,000, R2 is a lower alkyl group which is straight, branched, substituted, or unsubstituted, and (a) one of Rl and R3 comprises an amino-oxy oxime-forming group and an optional spacer group and the other of Rl and R3 is hydrogen, -CH3, or a protective group, or (b) both Rl and R3 comprise an amino-oxy oxime-forming group and an optional spacer group.
• the R2 of each repeating unit in the polymer can be independently the same as or different from each other.
• the number of repeating units, defined by n is between 5 and 2000, preferably between 10 and 1,000, and more preferably between 50 and 800.
• the polymer has an average molecular weight between about 200 and 100,000, preferably between about 400 and 50,000, and more preferably between about 2000 and 40,000.
• the molecular weight (average) of starting PEG is that available from (but not limited by) commercial sources and includes the group consisting of 5000, 10,000, and 20,000.
• Rl or R3 contains a functional amino-oxy group capable of forming an oxime linkage with a complementary aldehyde or ketone functional group on a target second organic macromolecule.
• the oxime-forming group comprises -O-NH 2 .
• the oxime-forming group comprises -X-O-NH 2 where X is a spacer group defined as herein.
• X is a spacer group comprising -NH-CO-R4- where R4 is a linear, branched, or cyclic lower alkyl, substituted or unsubstituted, preferably CH 2 , and where R4 is directly attached to -O-NH 2 .
• a protective group is non-reactive with respect to the polymer functionalization methods of the invention or the target macromolecules of the invention.
• the protective group has between 1 and 10 carbons, more preferably it is an alkyl group, most preferably it is methyl.
• PEG Prior to functionalization, preferably PEG has at least one hydroxy group more preferably it is a terminal hydroxy group.
• lower alkyl is meant a Cl to CIO, preferably C2 to C4, alkyl group.
• bi- and multi-polymer- containing functionalized polymers containing PEG or its derivatives are provided. These embodiments are those bi- and multi-polymer functionalized polymers as discussed above, but wherein two or more PEG polymers are attached to a single oxime forming group through individual attachment to the linking structure.
• the bi- and multi-PEG functionalized polymers of the invention find particular use in systematically modifying the Stokes radius of a polypeptide, which, in the case of pharmaceutically important polypeptides, preferably allows systematic modification of its pharmacokinetic behavior, and ultimately its therapeutic efficacy.
• the invention relates to preparation and use of a functionalized PEG and dextran: monofunctional MPEG-NH-CO-CH2-0-NH 2 , Dextran-0-CH(CHOH-CH 2 OH)-(CHOH) 2 -CH 2 -NH-(CH 2 ) 2 -NH-CO-CH 2 -ONH 2 , Dextran-0-CH(CHOH-CH 2 OH)-(CHOH) 2 -CH 2 -NH-(CH 2 ) 2 -NH-CO-C 6 H 4 -CHO, bifunctional NH 2 -0-CH2-CO-NH-PEG-NH-CO-CH 2 -0-NH 2 , and multi-PEGylated (PEG) 2 Lys-NH-(CH 2 ) 2 -NH-CO-CH 2 -0-NH 2 , HO-((PEG)Lys) 3 -NH-CO-CH 2 -O-NH 2 , and (PEG-aminoethyl) 2 -N-(
• functionalized polymers as taught herein, containing dextran or its derivatives as the polymer backbone are provided.
• the functionalized polymer is a mono ⁇ functional or a bi-functional polymer that contains dextran or a derivative thereof.
• bi- and multi-polymer functionalized polymers as taught herein, containing dextran or its derivatives are provided. These embodiments are those bi- and multi-polymer functionalized polymers as discussed above, but wherein two or more dextran polymers are attached to a single oxime forming group through individual attachment to the linking structure.
• a polymer as described above having at least one, preferably terminal, reactive group is modified at that reactive group to contain an oxime-forming group.
• Any of numerous modifying chemistries can be used depending on the reactive group available on the polymer. For example, in the preferred case of PEG or its derivatives, a terminal hydroxyl group is available. Should more than one reactive group be present, a form of the polymer in which all but one reactive group (or all but two when a bi ⁇ functional polymer is desired) is protected from reaction can be used a starting material. Modification to an oxime-forming group can occur in sequential steps when convenient.
• the reactive group on the polymer can be converted to another reactive group that is readily acylated with a protected-amino-oxy- containing compound such as Boc-NH-0-CH2-COOSu.
• the amino-oxy group can then be deprotected before use or before storage.
• the most reactive group towards acylating agents is -NH 2 .
• a preferred PEG intermediate is CH3-0-(CH2-CH2-0)n- CH2-CH2-NH 2 , or when a bifunctional polymer is desired, H2N-(CH2-CH2-0)n- CH2-CH2-NH 2 .
• MPEG can be readily converted to CH3-0-(CH2-CH2-O)n-CH2- COOH by the steps of carboxymethylation (Royer and Ananthanmaiah (1979) J. Am. Chem. Soc. 101:3394-3396).
• PEG-NH 2 was prepared via the following series of reactions PEG-OH ⁇ PEG-C1 ⁇ PEG ⁇ N 3 ⁇ PEG-NH 2 according to a procedure already described (Zalipsky et al., (1983), Eur. Polym. J.. 19, 1177- 1183).
• bi- or multi-polymer functionalized polymers are synthesized by first obtaining an L structure of desired multi-valency, usually having one valency protected, and then reacting the protected L-structure with an appropriately activated polymer intermediate typically using linking chemistries known in the art or with a functionalized polymer of the invention via oxime chemistry.
• the product is functionalized according to the invention by deprotection of the protected remaining valency of L followed by subsequent reaction, e.g. acylation in the case of an amino group, with a suitably protected-amino-oxy or protected-aldehyde containing acylating group.
• the final product the bi- or multi-polymer functionalized polymer is obtained.
• L is first derivatized with a suitably protected-amino-oxy or protected-aldehyde containing group, the mono-substituted derivative is isolated, the mono-substituted L derivative is then reacted at each remaining valency with a polymer intermediate (such as one having a COOH when L contains NH 2 or an NH 2 group when L contains COOH) or with a functionalized polymer of the invention (when oxime chemistry is used to assemble P to L).
• a polymer intermediate such as one having a COOH when L contains NH 2 or an NH 2 group when L contains COOH
• MPEG-COOH intermediate polymers which can react with free amino groups on an L-structure, are provided.
• the peptide sequence of an L structure can be synthesized by routine solid phase peptide synthesis ("SPPS"), and while the peptide is still attached to the solid phase PEG-COOH in an activated form, such as the N-hydroxysuccinimide ester, can be added to the nascent peptide chain.
• SPPS solid phase peptide synthesis
• the L structure can consist of a peptide having six reactive groups such as five lysine residues and an N-terminal amine group.
• PEG-COOSu hydroxysuccirrimide ester can react with each of the c-amino groups of the lysine residues (while the N-terminus ⁇ -amino group is left protected).
• N-terminal amine group of the fully acylated peptide is then unprotected and the polymer- containing structure is reacted with a Boc-AoA-containing active ester to introduce the AoA group, which after Boc removal and mild cleavage from the resin, yields a penta-polymer-containing functionalized polymer of the invention.
• this method finds particular use with synthetic structures (and perhaps certain recombinant products) since these can be designed to exclude additional residues, e.g., amino acid residues, that would require protection during the process and deprotection afterwards.
• synthetic structures and perhaps certain recombinant products
• Boc-Ser(benzyl)-OH or Boc-Ser(t-butyl)-OH in an activated form, such as the N-hydroxysuccinimide ester, can be attached tot he e-amino groups of the lysine residues.
• the N-terminus ⁇ -amino is then deprotected to introduce an amino-oxy group, e.g. AoA, so that after Boc removal a precursor L structure containing e- Ser-pentalysine is obtained.
• a mild oxidizing agent such as periodate at Ph 7
• a mild oxidizing agent such as periodate at Ph 7
• the oxidation reaction can be terminated using any 1,2-diol or l-amino-2-ol or l-ol-2-amino compound having relatively free rotation about the 1,2 bond, such as ethylene glycol.
• the oxidation reaction can be terminated by rapid removal of periodate, for example by reverse phase high performance liquid chromatography (RP-HPLC).
• Target macromolecules can be designed and prepared to site- specifically introduce an oxime-forming group using methods known in the art, including methods found in co-pending United States Serial Numbers 08/057,594, 08/114,877, and 08/057,594, 07/869,061, and 08/241,697, and co-pending International application PCT/IB94/00093 (which are hereby inco ⁇ orated by reference).
• Target macromolecules can be obtained by recombinant methods or isolated from natural sources, and an oxime-forming complementary reactive group, an aldehyde or amino-oxy group, is site-specifically formed at a desired location of the macromolecule.
• polymers are prepared that are reactive site- specifically with polypeptide compounds, or with compounds or materials containing amino acids, in which an ammo acid has been modified to contain an oxime-forming group specifically reactive with the oxime- forming function introduced onto the polymer.
• an ammo acid has been modified to contain an oxime-forming group specifically reactive with the oxime- forming function introduced onto the polymer.
• the N-terminal or C- terminal residue of a polypeptide is so modified using methods presented herein.
• the oxime-forming group is introduced preferably at a C-terminal of the polypeptide by selective enzyme catalyzed reverse proteolysis or at an N-terminal serine or threonine by mild oxidation.
• selective enzyme catalyzed reverse proteolysis or at an N-terminal serine or threonine by mild oxidation.
• a recombinant or natural peptide may have multiple C- or N-termini, such as would occur in a dimer or tetramer, each of which can be functionalized.
• the water-soluble polymer reagents of the invention may be used to modify a variety of polypeptides or similar molecules that have been site-specifically modified to contain a complementary functional group capable of oxime formation with the functional group on the polymer.
• Polypeptides of interest include: antibodies, monoclonal and polyclonal; cytokines, including, M-CSF, GM-CSF, G- CSF, stem-cell growth factor; lymphokines, IL-2, IL-3, growth factors, including, PDGF, EGF; peptide hormones, including, hGH, erythropoietin; blood clotting 30 factors, including, Factor VIII; immunogens; enzymes; enzyme inhibitors; ligands and the like.
• Polypeptides of interest for water-soluble polymer derivatization by the water-soluble polymer amino-oxy or aldehydes include hormones, lymphokines, cytokines, growth factors, enzymes, vaccine antigens, and antibodies.
• Water- soluble polymer derivatization of erythropoietin (EPO), especially human erythropoietin is of particular interest.
• Polypeptides of interest may be isolated from their natural sources, genetically engineered cells, or produced by various in vitro synthesis methods. The following patent applications (which are hereby inco ⁇ orated by reference) report PEGylated modifications of various biologically important proteins: U.S. Pat. No. 4,179337; U.S. Pat. No.
• a peptide, polypeptide, or protein shall mean both naturally occurring and recombinant forms, as well as other non-naturally occurring forms of the peptide or protein which are sufficiently identical to the naturally occurring peptide or protein to allow possession of similar biological or chemical activity.
• peptides can be formed from of non-naturally occurring or non-proteinogenic amino acid residues.
• amino acid residues can be joined via non-amide linkages.
• Peptides or proteins can also contain protecting groups at either terminal that prevent or minimize degradation of the peptide or protein in vivo.
• polypeptides for use as drugs include specific binding proteins, polypeptides recognized by specific-binding proteins, and enzymes.
• specific-binding proteins it is intended antibodies, hormone receptors, lectins, and the like.
• antibodies it is intended to include both polyclonal and monoclonal antibodies with natural immunoglobulin sequences, synthetic antibody derivatives, and the like; antibodies may be modified so as to be joined to any of a variety of labels, fluorescent, radioactive, enzymatic, biotin/avidin or the like.
• Synthetic antibody derivatives include natural immunoglobulin sequences that have been mutated and selected for altered binding specificity, various immunoglobulin gene derived polypeptides, typically single chain, produced by genetically modified bacteria, antibodies modified so as to contain modified constant regions and the like; a review of such synthetic antibody derivatives based on the principles of antibody formation is provided in Winter and Milstein, Nature. 349:293-299 (1991).
• An antibody is a glycoprotein of the globulin type that is formed in an animal organism in response to the administration of an antigen and that is capable of combining specifically with the antigen. These are also referred to as immunoglobulins.
• Antibody fragments can retain some ability to selectively bind with their antigen or hapten.
• antibody fragments include, but are not limited to, Fab, Fab * and (Fab') 2 .
• a native antibody is one which is isolated from an animal or from an animal or hybrid animal (hybridoma) cell line.
• the C-terminus is modified, preferably with enzymes that can direct bifunctional reagents with suitable reactive groups specifically at the C-terminus in polypeptides or proteins (e.g. antibodies).
• the carboxyl terminus of a polypeptide chain is, at least in terms of primary structure, in most cases far from the active site of a protein.
• the amino terminal group is the site-specific target after reaction to activate the N-terminal site on a peptide.
• N-terminal serine (“Ser”) and threonine ("Thr") residues are oxidized in an exceedingly mild reaction with periodate (e.g. 20°C, 26 ⁇ M protein, /41813
• N-terminal Ser reacts about 1000 times as fast as other protein groups (Fields and Dixon, (1968) Biochem. J. 108:883), so that a high degree of specificity is obtained.
• N-terminal Ser or Thr can be introduced by recombinant DNA techniques, or, in appropriate cases, by selecting a source of the protein of interest which has a natural Ser or Thr N-terminus, or by enzymatic cleavage, with for example an aminopeptidase, dipeptidylpeptidase, or proline specific endopeptidase, of non-essential terminal amino acids to expose a Ser or Thr.
• a second preferred reaction is the use of transamination to convert an N-terminal residue of a protein into an active carbonyl function, preferably by reaction with glyoxylate.
• This reaction proceeds under relatively mild conditions (see e.g. Dixon and Fields, (1979) Methods in Enzymology, 25:409-419).
• a wide range of N-terminal residues can be transaminated.
• methionine was transaminated in the specific case of Antril, as described in Example 8.
• the polypeptidyl N-teiminal aliphatic aldehydes or ketones produced by these techniques may be reacted with polymers preferably functionalized with amino-oxy-acetyl groups.
• a protected aminooxyacetyl group was directly attached to polymers already functionalized with either a unique amino group as it was the case with methoxy polyethylene glycol (Example 1) or dextran (Example 5), or two amino groups as in case of H 2 N-PEG-NH 2 (Example 2) for the construction of the dumbbell or to a polyfunctionalized linker, in order to synthesize a multibranched structure which is conjugated to the protein at a single site (Examples 3, 4, 10). Described structures are the following:
• the polymer to be conjugated has to be functionalized with the complementary aldehydic function, e.g. PEG-0-CH 2 -CHO or PEG-NHCO-C,sH 4 -_HO, as described in Example 12, which can be obtained by reacting, for example, amino functionalized PEG with carboxy(benzaldehyde)OSu.
• the complementary aldehydic function e.g. PEG-0-CH 2 -CHO or PEG-NHCO-C,sH 4 -_HO
• a polyaminooxy tag can be introduced at a single site, in order to obtain a multiarm-conjugate, as it is shown in Example 18, with (H 2 N- OCH 2 -CO H(CHj)_ ) 3N and PEG-CHO.
• the site-specific target is a side-chain group of a polypeptide not necessarily non-terminal, preferably an amino acid residue present at a single copy or otherwise preferentially available and sensitive to modification.
• a unique residue may be introduced by recombinant methods.
• the side-chain group may be first modified using method taught herein to put in place a reactive group (aldehyde, keto or AoA) that will subsequently specifically react with a complementary reactive group on a functionalized polymer (e.g. AoA or aldehyde).
• a target macromolecule is modified site-specifically at a location other than at its termini. Modifications to the primary structure itself, by deletion, addition, or alteration of the amino acids inco ⁇ orated into the sequence during translation, can be made without destroying the activity of the protein. Methods for making such modified proteins, known as "muteins", are described in U.S. Pat. No. 4,518,584 issued May 21, 1985, and U.S. Pat. No. 4,752,585, issued June 21, 1985, both are hereby inco ⁇ orated by reference, and are well-known in the art (see Current Protocols in Molecular Biology, ed., Ausubel (1994), Greene Pub. Associates and Wiley-Interscience, J.
• At least one amino acid residue which is not essential to biological activity and is present in the biologically active protein can be replaced with another amino acid which is amenable to subsequent site- specific modification to create a functionalized target macromolecule of the invention.
• Polymers functionalized with a hydrazide group can also react with the specifically introduced aldehydic or keto group, to form a hydrazone linkage. Though this bond is known to be less stable than the oxime bond, especially under acidic conditions, it should be of interest in some special cases where the protein have to be released later in vivo.
• Target macromolecule polypeptides can be produced by a prokaryotic microorganism or a eukaryotic cell that has been transformed with a native or modified polypeptide-encoding DNA sequence, preferably of human origin.
• Variants of naturally occurring polypeptides, wherein substantial identity of amino acid sequences has been maintained i.e., the sequences are identical or differ by one or more amino acid alterations (deletions, additions, substitutions) that do not cause a substantially adverse functional dissimilarity between the mutationally altered protein and native protein are useful herein.
• a target macromolecule modified by a polymer reagent molecule i.e. the subject water- soluble functionalized polymer amino-oxy or aldehyde reagents, so as to be covalently bonded to one or more water-soluble polymers at a single site on the target macromolecule via an oxime linkage.
• a polymer reagent molecule i.e. the subject water- soluble functionalized polymer amino-oxy or aldehyde reagents, so as to be covalently bonded to one or more water-soluble polymers at a single site on the target macromolecule via an oxime linkage.
• polymer conjugates preferably the target molecule is a polypeptide, more preferably a polypeptide of biological importance.
• a polypeptide derivatized with water-soluble polymer PEG or its derivatives via an oxime linkage is provided.
• the subject invention includes functionalized target macromolecules described above modified by reaction with the functionalized polymers molecules described above, wherein one or more polymers are covalently attached via a single, uniquely located, oxime linkage.
• these polymer conjugates are represented by formulas
• P is an organic polymer, preferably water soluble, as described herein
• B represents a functionalized target macromolecule, preferably a polypeptide, as described herein
• m represents an .integer in the range 2 to 10
• X and L and R are as defined herein
• -C(R) N-O- is an oxime bond.
• R is preferably H.
• R can be the side chain of an N-terminal residue of a polypeptide which has been converted to an active carbonyl by transamination.
• m is the range of about 2 to 10, the range of 2 to 5 being particularly preferred.
• B is a polypeptide wherein preferably P is covalently joined site-specifically to a functionalized N- or C- terminal residue on B.
• Individual functionalized target macromolecules can be derivatized by one or more different water-soluble polymers by means of reaction with different embodiments of the polymer compounds of the invention.
• Individual target macromolecules can be modified with multiple water-soluble polymers at a single site when m is greater than one.
• P is a polyalkylene glycol or a dextran derivative.
• Salts of any of the macromolecules described herein, e.g., polypeptides, water-soluble polymers and derivatives thereof, will naturally occur when such molecules are present in (or isolated from) aqueous solutions of various pHs. All salts of peptides and other macromolecules having the indicated biological activity are considered to be within the scope of the present invention. Examples include alkali, alkaline earth, and other metal salts of carboxylic acid residues, acid addition salts (e.g., HC1) of amino residues, and zwitterions formed by reactions between carboxylic acid and amino residues within the same molecule.
• homogeneous polymer conjugate compositions can be comprised of homo- or hetero-polymers.
• a heterobi- or heteromulti-polymer functionalized-polymer is attached to a target macromolecule not all of the polymer molecules attached at the single site will be identical with respect to chemical type or MW(av), nevertheless the composition can be homogeneous as defined herein.
• step-wise assembly of polymers onto a linking structure (see co- pending United States Serial Numbers 08/057,594, 08/114,877, and 08/057,594, and co-pending International application PCT/LB94/00093, which are hereby inco ⁇ orated by reference) allows the introduction of different polymers at each step to create a homogeneous composition of a hetero-polymer.
• L linking structure
• multi-polymer functionalized polymers have the reverse complementary structures, i.e. amino-oxy linking structure and aldehydic functionalized polymer, from those described above.
• a additional polymer conjugate embodiment of the invention is a "dumbbell" construction wherein two target macromolecules, either the same or different, are each attached via oxime linkages to an intervening polymer, preferably through the termini of the polymer.
• a preferred embodiment comprises PEG or its derivatives as the intervening polymer.
• oximation reaction will occur site-specifically between the two reagents such that the polymer will be grafted to the target macromolecule by an oxime linkage.
• an oximation reaction can occur between GXL-functional group introduced on a target macromolecule and its complementary AoA group introduced on a polymer at pH 4.6 to form the oxime-conjugate. Oximes form over a wide range of pH values and form rapidly at pH values less than about pH 5.
• the extent of oxime formation can be monitored by RP-HPLC and the reaction can be terminated by preparative RP-HPLC.
• the molecular weight of the resulting compound can be determined by gel electrophoresis or matrix-assisted laser deso ⁇ tion ionization mass spectrometry.
• an AoA-target macromolecule can be used, and an oximation reaction can occur between the AoA-group and a polymer having an aldehyde group.
• oximation is run at pH below 4.6. Lower pH can be advantageous for the solubility of some peptides.
• a pH of 2.1 is preferred for increasing the solubility of some peptides.
• oximation occurs much faster at pH 2.1 than at pH 4.6.
• One skilled in the art can determine the pH versus solubility profile of a polymer and a target for oxime-conjugate formation and choose an appropriate pH for a specific oximation reaction, taking into account pH stability of the molecules during the period of the oximation reaction. Oximation due to chemoselective ligation of the complementary chemical groups results rapidly and essentially quantitatively in the formation of a homogenous preparation of a oxime conjugate of defined structure when the AoA and GXL pair are used.
• Oxime-forming complementary chemically reactive groups can be attached in either a protected or an unprotected form.
• Methods to attach an oxime-forming complementary chemically reactive group to a target macromolecule include attachment through a chemically reactive side chain group.
• an oxime-forming complementary chemically reactive group can be attached to a cysteine-containing target macromolecule via the S atom by alkylation or disulfide formation. Then upon oximation to a functionalized polymer having an aldehydic function the target macromolecule is attached via its Cys residue through a thioether link (or disulfide bond) and an oxime link to the polymer.
• Preferred alkylating compounds are alkyl halides having an attached AOA group.
• ком ⁇ онент having a BOC protected amino-oxy group preferably an AoA group.
• Preferred are Br-CH2-CO-NHCH2CH2NH-CO-CH2-0-NH-Boc, where the AoA group is protected and can be removed prior to an oximation step, and Br-CH2-CO-NHCH2CH2NH-CO-CH2-0-NH 2 .
• Another alkylating reagent is Br- CH 2 CH 2 CH 2 NH-COCH 2 ONH-Boc.
• the bromoacetyl group is much more reactive for alkylation of the thiol group of, for example, Cys residues. Less preferred is the iodoacetyl group because it sometimes is too reactive and may be lost by photolysis.
• alkylating groups in addition to the bromoacetyl group, include the maleoyl group.
• linkers for protein modification using this group are exemplified as AOA-Lys(maleoyl-beta-alanyl)-OH and maleoyl-beta- alanyl-NHCH 2 CH 2 NH-COCH 2 ONH 2 .
• the maleoyl group is useful for making macromolecular conjugates, it is known to have serious stability problems (hydrolytic opening of the ring) and so is less suitable for making homogeneous polyoximes.
• alkylation involving the maleoyl group gives a linker which is more rigid and bulky than the link formed by alkylation with the bromoacetyl group, and is thus more visible to the immune system.
• a preferred linker for in vivo application is one against which an immune response is not /41813
• Examples of compounds for attachment of a oxime-forming complementary chemically reactive groups to the side chain of cysteine through a disulfide bond are those containing a 2-pyridyl-S-S- radical.
• Preferred examples are 2-pyridyl-S-S-CH2CH2NH-CO-CH2-0-NH-Boc and 2-pyridyl-S-S-CH2CH2NH-CO-CH2-0-NH 2 .
• the resulting Cys-containing derivatives possess an aminooxyacetyl (or protected aminooxyacetyl) group attached through a disulfide bond.
• the modification disclosed herein is useful for connecting functionalized polymers to a polyaldehyde baseplate via a Cys side-chain through disulfide and oxime bonds.
• target macromolecules e.g. peptides
• disulfide reduction a process which is known to occur in the body.
• the oxime-linked polymer-conjugates and the polyoxime based multi- polymer functionalized polymers of the invention have several novel characteristics.
• One novel characteristic is that the preparations are homogeneous.
• the oxime- linked compounds are stable in aqueous solution or semi-aqueous solution, and can be prepared at temperatures from -3°C to 50°C, but most advantageously at room temperature.
• the oxime-linked compounds of this invention have utilities related to the specific biological reactivity and specific chemical and physical reactivities of their individual component parts.
• the functionalized polymer of the invention (be it a mono-functional polymer, a bi-functional polymer, a bi-polymer functionalized polymer, or a multi- polymer functionalized polymer) is obtained, it is conjugated with a functionalized target macromolecule via an oximation reaction.
• An oxime linkage is formed from the complementary functional groups that had been introduced onto the polymer and target macromolecule.
• the grafting of the polymer to the target molecule occurs site-specifically as directed by the selected placement of the oxime-forming functional group on the target macromolecule.
• the method of conjugation is performed in the following manner.
• a functionalized polymer solution is combined with a functionalized target macromolecule solution at a final acidic pH.
• the oximation reaction is allowed to proceed to formation of the desired conjugate.
• the oximation reaction can occur over a wide range of pHs, preferably acidic to about pH2, more preferably less than about pH 5, particularly when rapid formation is desired. More preferably, the pH is less than 4, most preferably about 3.6.
• the oximation reaction is compatible with lower pH values when it is necessary for mamtaining target molecule stability or solubility.
• the reaction temperature is preferably room temperature, but can be adjusted to meet the specific needs for stability and solubility of the target macromolecule.
• the reaction time is between 10 minutes and 72 hours, more preferably between 3 hours and 48 hours, and most preferably between 6 hours and 24 hours.
• the functionalized polymer is in molar excess to the functionalized target macromolecule, preferably between about 3-fold to 20-fold molar excess, more preferably between about 4-fold to 15-fold, and most preferably between about 5- fold to 10-fold molar excess.
• the functionalized polymer is preferably diluted in aqueous, preferably buffered, solution at acidic pH.
• Acetate, 0.1M, pH4.6 is a preferred solution.
• a chaotropic agent is present in order to aid in accessibility of the functional group of the target macromolecule to the functionalized polymer.
• a preferred agent should be inert to the oximation reaction, inert to both the polymer and target molecule, and will not prevent the return of biological activity (if it was affected by the chaotropic agent) when removed from the final product.
• a preferred agent is guanidine hydrochloride ("GuHCl”), preferably at about 1 to 8 molar concentration in the final reaction, more preferably about 4 to 6 molar concentration.
• the conjugation reaction can be performed in the presence of an organic solvent.
• the solvent, and its concentration, should not irreversibly interfere with the biological activity of interest of the target macromolecule.
• Suitable solvents are well-known in the field. For example, for certain poorly soluble peptides, up to 50% acetonitrile can be used for coupling.
• the polymer conjugate can be purified from the reaction mixture.
• purification methods that are known to those of ordinary skill in the art such as size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, preparative isoelectric focusing, etc.
• One particularly preferred method is to combine a size separation method with charge separation method, for example, size exclusion chromatography followed by ion exchange, see U.S. Ser. No. 253,708, which is hereby inco ⁇ orated by reference in its entirety.
• the size separation method is size exclusion chromatography which discriminates between molecules based on their hydrodynamic radius. Hydrodynamic radius is defined as the effective molecular radius of a particle in an aqueous environment.
• the charge separation method is ion exchange chromatography which discriminates between molecules based on differential affinity of charged ions or molecules in solution for inert immobile charged substances.
• the size exclusion chromatography method and the ion exchange chromatography method comprise contacting a mixed solution of polymer conjugates with either column in the appropriate buffers and under the appropriate conditions.
• the method of conjugation includes a "one-pot" synthesis.
• a functionalized polymer solution can be simultaneously mixed with both a periodate solution and a target macromolecule having an N- terminal residue of serine or threonine, which is highly susceptible to functionalization as described herein, under mild reaction conditions. Formation of the desired polymer conjugate, with the polymer site-specifically attached via an oxime linkage to the N-terminal amino acid of the target macromolecule will occur without the need for additional manipulation.
• the pH of the "one-pot" reaction is from about 2 to 9, preferably from about 3 to 7, more preferably about 4.5 to 7, most preferably about 6 to 7, with a preferred embodiment at about pH 6.5.
• Both the polymer and periodate are in molar excess to the target molecule.
• the periodate is preferably in the molar excess range of 2-fold to 5-fold, more preferably 2- to 3- fold. Reaction times of less than about 3 hours are achieved.
• the polymer conjugate is isolated as described herein.
• the reaction temperature is between preferably about 0°C to avoid freezing to below whatever temperature might denature the target macromolecule, generally below about 100°C to avoid denaturation, and in the case of most protein about 50° C. More preferably the reaction is at about 15 to 25 °C, even more preferably about 20°C.
• the molar ratio of functionalized polymer to target is a value preferably in the range between about 3-fold to 50-fold, more preferably 4-fold to 30-fold, even more preferably about 5- fold to 25-fold.
• the molar ratio of target molecule to periodate is preferably in the range of about 1-fold to about 6-fold, more preferably about 2-fold to 4-fold.
• Il-8:NaI0 4 :AoA was approximately 1:4:10.
• fractions can be screened against various standards.
• Preferred screening methods include SDS-PAGE, isoelectric focusing, bioactivity, and pharmacokinetics.
• fractions may be further purified.
• the polymer/protein conjugate mixture can be fractionated with the size exclusion chromatography column, the fractions collected, then run on an SDS-PAGE gel to determine which fractions contain the desired polymer/protein conjugate. Then, the fractions of interest may be further purified by contact with the ion exchange chromatography, the fractions collected, and analyzed by isoelectric focusing to determine which fractions have the desired polymer/protein conjugate.
• the polymer conjugate mixture Before the polymer conjugate mixture is subjected to chromatography, it can be initially prepared by removing impurities. For example, salts and chaotropic agents can be removed with preparatory columns, or can be dialyzed against appropriate buffers.
• polymer conjugate Once the polymer conjugate is purified it can be tested for bioactivity using methods known in the art.
• protein and other organic target macromolecules may be chemically modified by conjugation to water-soluble organic polymers such as polyethylene glycol (PEG).
• PEG polyethylene glycol
• the production of such protein conjugates is of interest because of the desirable properties conferred by the attachment of the water-soluble polymers. These desirable properties include increased solubility in aqueous solutions, increased stability during storage, reduced immunogenicity, increased resistance to enzymatic degradation, compatibility with a wider variety of drug administration systems, and increased in vivo half-life.
• polypeptides are especially of interest when the polypeptide is to be used as a therapeutic agent injected into the body or when the polypeptide is to be used in assays, usually immunoassays, for the detection and/or quantification of a compound of interest.
• the utility of the functionalized polymers of the invention extends to preparation of polymer-conjugates of low molecular weight peptides and other materials that contain or are modified to contain a functional group complementary to that on the polymer.
• Non-medical uses of functionalized polymers, particularly PEGylated proteins include the preparation of polypeptides for assays, e.g. immunoassays,
• the functionalized polymers can be attached to a solid phase, such as the surface of a silicon chip, a tissue culture plate, cell or membrane, or a synthetic or natural resin.
• a solid phase such as the surface of a silicon chip, a tissue culture plate, cell or membrane, or a synthetic or natural resin.
• the functionalized polymers are useful to monitor the appearance (or disappearance) of a functional group on a target macromolecule or solid phase.
• the polymer conjugate After the polymer conjugate is produced and purified it may be inco ⁇ orated into a pharmaceutical composition when target macromolecules believed to be therapeutically effective for human and veterinary uses, such as cancer therapy and the treatment of infectious diseases have been used.
• a therapeutic agent is any molecule, which, when administered to an animal, prevents or alleviates a disease or arrests or alleviates a disease state in the animal.
• Therapeutic agents may include, but are not limited to, antitumor antibiotics, antiviral proteins, radioisotopes, pharmaceuticals or a toxin.
• the polymer conjugate can be formulated in a nontoxic, inert, pharmaceutically acceptable aqueous carrier medium.
• a "pharmaceutically acceptable carrier” means any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution; water; or emulsion, such as an oil/water emulsion; potentially including various types of wetting agents.
• the polymer conjugate can be formulated in a nontoxic, inert, pharmaceutically acceptable aqueous carrier medium, preferably at a pH ranging from 3 to 8, more preferably ranging from 6 to 8.
• the sterile polymer conjugate composition will comprise protein dissolved in an aqueous buffer having an acceptable pH upon reconstirution.
• the polymer conjugate can be formulated with a number of excipients such as amino acids, polymers, polyols, sugar, buffers, preservatives, other proteins, etc.
• excipients such as amino acids, polymers, polyols, sugar, buffers, preservatives, other proteins, etc.
• specific examples include: octylphenoxy polyethoxy ethanol compounds; polyethylene glycol monostearate compounds; polyoxyethylene sorbitan fatty acid esters; sucrose; fructose; dextrose; maltose; glucose; dextran; mannitol; sorbitol; inositol; galactitol; xylitol; lactose; trehalose; bovine or human serum albumin; citrate; acetate; Ringer's and Hank's solutions; saline; phosphate; cysteine; arginine; carnitine; alanine; glycine; lysine
• composition it is parenterally administered to the subject by methods known in the art.
• Administered means providing the subject with an effective amount of the compound or pharmaceutical composition.
• Methods of administration to an animal are well known to those of ordinary skill in the art and include, but are not limited to, oral, intravenous, transdermal, and parenteral administration. Administration may be effected continuously or intermittently throughout the course of other treatments. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the compound or composition for treatment, the pu ⁇ ose of therapy and the animal or patient being treated.
• This composition may contain other compounds that increase the effectiveness or promote the desirable qualities of the particular target macromolecule portion.
• the composition must be safe for administration via the route that is chosen, sterile and effective. To maintain the sterility and to increase the stability of a polymer conjugate, the composition can be iyophilized and reconstituted prior to use.
• the formulation is suitable for parenteral administration to humans or animals in therapeutically effective amounts. These amounts may be determined by the in vivo efficacy data obtained after preclinical testing for: animal models of the disease state of interest or in vitro assays generally accepted as correlating with in vivo efficacy. It is also at interest to supply the water-soluble functionalized polymer reagents of the invention in the form of a kit, so as to provide for the convenient and reproducible derivatization of target macromolecules of interest. Kits of interest may contain solutions comprising the water-soluble functionalized polymer reagent of the invention, buffers, reaction indicator compounds, instruction, protein concentration measurement reagents, e.g., for Bradford assays, and the like. Reagent solutions will preferably be supplied in premeasured amounts.
• Kits can contain reagents for site-specifically introducing a complementary functional group onto a target macromolecule of interest, preferably under mild conditions.
• reagents can include a functionalized polymer of the invention that allows the functionalization reaction to be monitored (as by formation of a polymer conjugate), peptidases for N-terminal cleavage, proteases for C-terminal site-specific conjugation, linker groups containing protected amino-oxy groups, periodate, molecular weight markers (as in the form of polymer conjugates of known molecular weight) and optionally any buffers or solvents for carrying out functionalization of a target macromolecule.
• Kits can contain a series of individual solutions (or powdered form) polymer-conjugates containing polymers of known composition, molecular weight and configuration (whether mono-polymer, bi-polymer or multi-polymer) attached to target macromolecules of known molecular weight and composition that can be used as standards, for example to estimate completion and/or yield of conjugation reactions or to provide molecular weight standards.
• Polymer-conjugate embodiments of the invention can be used in improved kits for diagnostic pu ⁇ oses or as improved reagents for assays, for example, in binding assays such as immunoassays.
• polymer conjugate compositions bearing antigen peptides provide increased detection sensitivity in solid-phase immunoassays.
• the larger, bi-valent or multivalent polymer conjugates can more readily, adhere to surfaces such as the multiwell plates used in immunoassays.
• Polymer conjugates, particularly multi-polymer-containing polymer conjugates find use in in vitro assays that use a signal amplification step for detection of an analyte, as for example in a branched DNA ("bDNA”) based assay.
• bDNA branched DNA
• Amplification is achieved by the attachment of multiple polymers (rather than a single polymer) to a single analyte molecule, wherein each polymer contributes to a detectable signal in a subsequent assay step.
• Targeting of the polymer conjugate to the analyte is readily provided, including for example by use of a linking L group that comprises an analyte binding group or by use of a hetero-polymer construct of the invention wherein at least one polymer on the construct provides analyte binding.
• multi-polymer-containing polymer conjugates of the invention particularly those in which the polymer comprises a specific nucleotide sequence as a repeating unit that is subsequently detected by hybridization to a labeled second oligonucleotide, wherein the label can be radioactive, fluorescent, enzyme- linked, or the like as is known in the art.
• the functionalized polymer can itself be derivatized to contain a detectable marker enzyme-link, or other reporter group.
• Also provided herein are methods of systematically modifying the Stokes radius of an organic target macromolecule including the steps of (a) obtaining a site-specifically-functionalized target macromolecule comprising a first oxime- forming group, (b) obtaining a series of functionalized organic polymers of the invention that differ from each other in the series in topology but not molecular weight(average) and that include a second oxime-forming group complementary reactive to the first oxime-forming group on the target macromolecule, and then (c) conjugating the functionalized target macromolecule separately with each functionalized polymer via a chemoselective, site-specific oximation, preferably under mild conditions as taught herein, to obtain a series of conjugated polymers.
• the steps (a) and (b) are performed in any order. If desired one can identify a change in Stokes, radius for each conjugated polymer in the series, preferably by size separation methods.
• the method can further include correlating the change in Stokes radius with a change in a biological or physical property of interest of a target macromolecule.
• the method includes identifying a change in a biological or physical property of interest, e.g. pharmacokinetic behavior, /41813
• the second oxime- forming group is -0-NH 2 .
• the in vivo blood clearance behavior of a macromolecule must in part be dependent on its Stokes radius.
• the elution volume of a macromolecule from a gel filtration column relates theoretically to the Stokes radius, not to its molecular weight.
• Mobility of a macromolecule when subject to gel electrophoresis can reflect changes in the Stokes radius of a macromolecule.
• polymers varying in molecular weight will migrate as a linear function of the log of molecular weight (and where the charge to mass ratio for each polymer is approximately the same).
• functionalized polymers of varying topology are provided that allow one to impart a desired change in the Stokes radius of a target macromolecule, more preferably create a family of molecules by systematic introduction of a series of changes in Stokes radius.
• a series of conjugated target macromolecules can be rapidly prepared in which either a mono-polymer, a bi-polymer or a multi-polymer-containing functionalized polymer construct is site-specifically, chemoselectively attached at a unique site, wherein each attached functionalized polymer construct adds the same molecular weight and polymer composition but in a differing topology as the others to the target.
• the mobility of each polymer conjugate is su ⁇ risingly different.
• Kits contain a series of functionalized polymers for attachment to a target of interest, wherein the series comprises polymer constructs of similar molecular weight but varying in the number of polymers present in the construct.
• a series can comprise constructs of a 40 kD MW(av) polymer, and multi-polymer-containing construct of two 20 kD MW(av) polymers, four 10 kD MW(av) polymers, and five 8 kD MW(av) polymers.
• the present process is advantageous because the attachment of polymer(s) to a target is predictable and selective.
• a further advantage of the subject invention is that macromolecules, e.g. polypeptides, modified by the reagent compounds retain a greater degree of their biological activity than when the same target is modified to the same degree by joining water-soluble polymers by employing non-oxime and non-site-specific chemistries.
• the subject invention provides for modified targets that possess the advantages associated with the conjugation of water-soluble polymers while minimizing the loss of biological activity associated with the modification. Consequently, the targets that are more highly derivatized by the use of multi- polymer functionalized polymers, and thus possessing the advantages associated with the higher degree of derivatization, can be produced that have the same level of biological activity as polypeptides derivatized by water-soluble polymers to a lesser extent.
• An additional advantage of the present invention over other methods of coupling water-soluble polymers to proteins e.g., the use of active carbamates, such active esters can react with nucleophiles other than primary amines such as hydroxyl groups, phenolic groups, and sulfhydryl groups on a protein, and the use of imidates that selectively react with the primary amino group of lysine residues, is that cross-reactivity is avoided because of the highly selective nature of the particular chemistry of the invention.
• the complementary reactive groups that interact to form an oxime linkage between polymer and target are higlily specific.
• the oximation reactions taught herein provided complete or essentially quantitative yield of the reaction product. Such complex molecule formation occurs under very mild conditions. Rapidity is particularly su ⁇ rising under dilute conditions which are often useful to minimize inter-molecular aggregation or reactions.
• the oximation reaction can occur unattended, such that self-assembly of the polymer-conjugate takes place.
• Polymer conjugates are easily purified by virtue of the essentially quantitative yield and because trace intermediates and the final product typically differ substantially (i.e. , by the presence or absence of at least one polymer unit) so that methods for their separation are readily chosen and applied.
• Oxime linkages have superior hydrolytic stability over a range of physiological conditions compared to hydrazones or the like. Oxime linkages are not commonly subject to enzymatic hydrolysis. Thus polymer conjugates have the advantage of being particularly suited to applications where integrity and stability of a complex is desirable. As demonstrated in the examples, the oximation reaction is very mild and thus is suitably advantageous for preparing biological macromolecules retaining biological activity or function. The oxime chemistry dispenses with the need to have reversible chemical protection of subunits. A great flexibility is provided herein for site-specific modification of both polymers and targets to create reactive groups capable of forming oxime linkages.
• Polymer-conjugates can be designed to improve solubility of peptides as well as present peptides to receptors or antibodies or the immune system of an animal in multi-valent and/or constrained forms.
• Polymer-conjugates formed from synthetic or recombinant polymers and target macromolecules have the additional advantage of being virus free.
• the functionalized polymers of the invention and the methods provided herein allow a polymer to be conjugated to polypeptide and protein derivative by a condensation reaction between an aldehyde and an amino- oxy compound.
• the complementary reaction is between a aldehyde or ketone and an amino-oxy-acetyl. In all cases an oxime bond is formed.
• the present invention provides further in vitro use of polymers, such as PEG.
• a functionalized polymer can be used to "tag" a target molecule and thus enable the molecule's subsequent detection and or quantitation in a number of ways. Most simply, the attached polymer allows one to perform a simple size separation that will separate the polymer tagged-target molecule from other molecules in a mixture. For example, one can readily follow the modification of a target molecule for the production of desired functional sites as measured by the ability to react with the reactive functional group on the functionalized polymer, which is in rum detected as the appearance of the polymer-tagged target. It is now readily apparent that different physicochemical properties of organic polymers can be taken advantage of in this way simply by changing the polymer.
• a slightly hydrophobic polymer would allow separation based on hydrophobicity, or one can use a polymer binding column that then selects for or against the polymer-conjugate as desired.
• the polymer can chosen, or modified, so that it can be directly detected. This imparts the advantage that the polymer may contain multiple detectable sites (or repeating units), such that each site present in the polymer binds or is recognized by a detection system, thus resulting in the amplification of detection signal.
• Branched DNA containing reagents exemplify the case wherein each polymer unit, in this case a specific nucleotide sequence or repeating sequence, is detected by specifically binding a second measurable reagent (Urdea (1994) Bio/Technology 12: 926-928).
• a second measurable reagent Urdea (1994) Bio/Technology 12: 926-928.
• the appearance of the reactive keto group at the N-terminus of a target protein could not be easily assessed. Disappearance of the N-terminal amino group could not be assessed by either mass spectrometry since only the copper adduct could be identified, or by cellulose acetate electrophoresis.
• the appearance of the reactive keto group was easily assessed by its ability to react with a functionalized PEG derivative of the invention as detected, for example, by simple size separation in an SDS-PAGE system.
• MPEG 5kD -N 3 (3.5g) was dissolved in absolute ethanol (100ml), 10% Pd/C (0.3g) added and the mixture was hydrogenated in a low pressure hydrogenation apparatus over night. The catalyst was filtered and ethanol evaporated in vacuo. The polymer was recrystaUized from toluene/hexane; yield 3g (86%).
• MPEG 5kD -NH 2 (lg, 0.2mmol) and Boc-NH-0-CH 2 -COOSu (0.5mmol) were dissolved in DMSO (4ml), the apparent pH adjusted to 8-9 with N- methylmo ⁇ hoUne and the mixture stirred over night.
• MPEG-NH 2 acylation was controUed by the quantitative ninhydrin procedure (Sarin et al. 1981 Anal. Biochem. 117:147-157) on a lOul aUquot of the reaction mixture.
• the modified MPEG was then recovered by dUution with 5 volumes of water, foUowed by dialysis against water, dialysis and lyophilization.
• Boc group was removed by dissolving the product in 10ml of TFA for lhr at room temperature. TFA was removed under vacuo, the material was taken up in water, extensively dialyzed against water and finaUy lyophiUzed; yield 0.93g (93%).
• EXAMPLE 2 Functionalization of Methoxypolyethylene Glycol: Synthesis of NH 2 -0-CH 2 CONH-PEG-NHCO-CH 2 -0-NH 2 r"AoA-NH-PEG-NH-AoA'"). Boc-NH-0-CH 2 -COOSu (86mg, 0.3mmol) was dissolved in 1ml dry DMSO, added to a solution of lg (50umol) of NH 2 -PEG 2 okD" NH 2 in 4ml of the same solvent and the apparent pH adjusted to 8-9 with N-methylmo ⁇ holine. The mixture was stirred at room temperature overnight and acylation controUed by the standard ninhydrin analysis.
• Z-Lys(Z)-NH-(CH 2 >2-NHCO-CH2-0-NH-Boc was synthesized by adding 1.1 g Boc-NH-0-CH 2 -COOSu (3.8mmol) to 0.9g of Z-Lys(Z)-NH-(CH 2 ) 2 -NH 2 (1.9mmol) dissolved in 3ml of dry DMSO and the apparent pH adjusted to 8-9 with N-methylmo ⁇ holine. The solution was stirred during 5h at room temperature, dUuted with 10 volumes of 0.1 % TFA and the product purified on a preparative HPLC column 250x 25mm i.d.
• the Z group was cleaved by catalytic hydrogenation.
• the material was dissolved in absolute ethanol (50ml), acidified with lOOul CH3COOH, 10% Pd/C (O.lg) added and the mixture hydrogenated in a hydrogenation apparatus overnight.
• the catalyst was filtered and the solvent removed by rotary evaporation.
• the product was purified on the same column than previously used with a linear gradient from 0 to 50 %B over 25min. After lyophiUzation (weight, 600mg; yield, 95%) the product was characterized by ESMS: calcd M+H, m/z 360.2; found m/z 361.8).
• Lys-NH-(CH 2 ) 2 -NHCO-CH 2 -0-NH-Boc was acylated with PEG 5kD - COOH, and PEG 10kD -COOH, and PEG 20KD-C00H.
• the solution was then dUuted with 5 volumes of water, dialyzed against distiUed water, filtered and appUed on a DEAE Sephadex A25 column equiUbrated in water.
• the acylated Unker was eluted in the first fraction with distiUed water and was lyophiUzed.
• the Boc group was then cleaved by dissolving the product in 10ml of TFA for lhr at room temperature. TFA was removed under vacuo, the material was taken up in water, extensively dialyzed against water and finaUy lyophiUzed (yield, 300mg, 90%), to get the foUowing derivative: PEG 5kD -Lys(PEG 5kD )-NH-(CH 2 ) 2 - NHCO-CH 2 -0-NH 2 .
• a comparable linker was obtained by acylation of the lysine derivative with PEG 10kD -COOH, and PEG 20KD -COOH.
• Z-(Lys(Boc))5-OH was used as starting material.
• the reaction mixture was dUuted with distilled water, dialyzed and the soluble fraction further purified by ion exchange chromatography on a DEAE-A25 an CM-C25 Sephadex column, and lyophiUzed.
• the Z-group was cleaved by a 2h HBr treatment and the product was then acylated with Boc-AoA- OSu. FinaUy, the AoA function of the linker was deprotected by TFA treatment. The extent of functionalization could be evaluated by determination of reactive aminooxy groups with TNBS at 495nm, and was found to be 80%.
• a unique reactive group was introduced in dextran kD and 39kD) by modification of the reducing sugar by reductive aminaticin. (lg, 25umol) was dissolved in anhydrous DMSO (4.5ml) with gentle heating (45 °C) and aUowed to return to 25 °C. Ethylene diamine (500ul, 7.5mmol) and crushed 4-A molecular sieves (200mg) were added. The flask was flushed with N 2 and then sealed and incubated for 24h at 37°C. NaBH ⁇ (80mg, 2.1mmol) was added, the flask flushed with N 2 and the solution further incubated for 24h at 37°C.
• the viscous solution was then dUuted with 5 volumes of water, the pH adjusted to 5.0 by the addition of glacial acetic acid, extensively dialyzed against distiUed water and finaUy lyophUized to afford 800mg (80% recovery on the based on starting dextran).
• the yield of functionalization was estimated from the absorbance of the 2,4,6-trinitrobenzene sulfonic acid derivative at 420nm (Fields, R., 1972, Methods Enzymol., 25B, 464-468).
• NH 2 -derivatized ((800mg, 20umol) was dissolved in DMSO (5ml), Boc-NH-0-CH 2 -COOSu (19mg, 60umol) dissolved in the same solvent (1ml) added, the apparent pH adjusted to 8-9 with N-methyimo ⁇ holine and the solution stirred overnight at room temperature.
• the solution was dUuted with 5 volumes of water, extensively dialyzed against distilled water and lyophiUzed.
• the end-modified dextran was quantitatively acylated as controUed by the TNBS test.
• the material was dissolved in 20ml TFA during lh for deprotection, TFA removed under vacuo, dUuted with water, extensively dialyzed and freeze-dried.
• the polymers functionalized with a single aminooxy group to their already introduced terminal amino group are able to react, preferably under acidic conditions, most preferably pH 3.0-5.0, with a carbonyl function, for example one located at or site-specificaUy introduced at the N-terminus of a protein by, for example, periodate oxidation or transamination (as dictated by the particular N- terminal sequence of the protein), to form a stable oxime bond.
• a carbonyl function for example one located at or site-specificaUy introduced at the N-terminus of a protein by, for example, periodate oxidation or transamination (as dictated by the particular N- terminal sequence of the protein), to form a stable oxime bond.
• Site-specific placement of a carbonyl group at the N-terminus of a protein is demonstrated in the Examples below. 11-8, G-CSF and Il-lra were used to Ulustrate this novel approach for site specific conjugation of aminooxy functionalized synthetic polymers.
• EXAMPLE 6 Site-Specific Modification of NH 2 -Terminal Residue of a Protein: Modification and Conjugation of IL-8.
• IL-8 The N-terminal serine residue of the short form of IL-8 (72 residues; 2-5 mg/ml) was oxidized with a 10 fold excess of sodium periodate in a 1 % NH4HCO3 buffer at pH 8.3, in the presence of 50 molar excess of methionine during lOmin at room temperature.
• IL-8 does not contain any methionine these standard conditions have been developed to eliminate aU risk of methionine oxidation during periodate treatment (Gaertner et al. 1992 Peptides 1992, Schneider and Eberle, eds, pp239-240, ESCOM, Leiden, The Netherlands).
• the reaction was stopped by the addition of 2000 excess ethylene glycol over periodate and further incubated for 15min at room temperature, and the protein was finaUy dialyzed against a 0.1M AcONa buffer, pH 4.6. Dialysis tubing was previously boUed in 1 % sodium bicarbonate during 30min. Oxidation was confirmed by ESI-MS (calcd., m/z 8351.3; found m/z 8351.3 ⁇ 1.4). The oxidized protein was concentrated up to 3-4mg/ml and was used in that form for the site specific attachment of the functionalized polymers to the N-terminus of the protein backbone.
• the conjugation product was purified by ion exchange chromatography on a Pharmacia MonoS column equiUbrated in 25mM AcONa buffer, pH 4.7 using a linear gradient from 0 to 2M NaCl over 20min foUowed by reverse phase HPLC on an analytical C8 column using a Unear gradient from 30 to 65% B over 35 min ( Figure 2).
• the iodination reaction was terminated by addition of 15 l of 2mM sodium bisulfite and 10 ⁇ l of 50 (v/v) potassium iodide.
• the radiodinated protein was separated from free 12S l on a GF5 column (Pierce) equilibrated in PBS and previously washed with the same buffer containing 0.1 % bovine serum albumin. The specific radioactivity obtained was in the range of 5-10 mCi/mg protein.
• I-labeUed 11-8 or its derivative was injected as a bolus in the taU vein of female Wistar rats (10 ⁇ g/kg) and samples of blood coUected as described in example 8.
• EXAMPLE 7 Site-Specific Modification and Conjugation of NH 2 -Terminal Residue of a Protein: Modification and Conjugation of rh-G-CSF.
• G-CSF was concentrated to 5mg/mL in a Tris 50mM, 0.3 % sodium lauryl sarcosinate, pH 8.0 buffer and then incubated with aminopeptidase (enzyme/substrate ratio, 1/20) for 20h at 37°C, in the presence of lOmM MgCl , 2.5mM PMSF and benzamidine, and 2mg/mL aprotinine. AU these inhibitors were added to prevent any cleavage of the polypeptidic chain by any contaminating proteolytic enzyme present in the commercial aminopeptidase preparation.
• Aminopeptidase was then inactivated by adding EDTA at a lOmM concentration and the solution dialyzed at 4°C versus Tris 20mM, pH 8.0 and against water and the material finally purified by reversed phase HPLC on a 250x10mm i.d. NucleosU C8 column, using a flow rate of 3mL/min and a Unear gradient of 50-80% solvent B over 15min.
• the modified protein was characterized by ESI-MS.
• the ESI mass spectrum exhibited two series of multi-charged ions, the major one corresponding to des-Met -rhG-CSF (calcd., m/z 18820.8; found, m/z 18821.7 ⁇ 2.1) and the minor one to Met -rh-G-CSF ( calcd., m/z 18951.9, found m/z 18956.1 ⁇ 5.5).
• Oxidation of des-Mtt -G-CSF was dissolved in a 0.1M sodium phosphate buffer, pH 6.5 at 5mg/ml in the presence of 6M guanidine chloride, and oxidation performed with a 5-fold excess of periodate over the polypeptide. After lOmin of incubation at room temperature in the dark, unreacted periodate was destroyed with a 1000- fold excess of ethylene glycol. Oxidation was confirmed by ESI-MS (calcd., m/z 18775.3; found, m/z 18777.9 ⁇ 2.7).
• both PEG 3kD and PEG 20t -G-CSF could be isolated by reverse phase HPLC, but in the case of PEG 5kD -G-CSF, this step was preceded by hydrophobic interaction chromatography on a polypropyl aspartamide column, using a Unear gradient from 20mM sodium phosphate, 1,2M Na;,S0 4 , pH 6.3 to 50 mM sodium phosphate, 5% (v.v) isopropanol, pH 7.4 over 15 min. foUowed by a 25 min Unear gradient to 25% of a third buffer containing 25 mM sodium phosphate, 50% MeOH pH 7.1, with a flow rate of 0.6ml/min.
• EXAMPLE 8 Site-Specific Modification and Conjugation of NH 2 -Terminal Residue of a Protein: Modification by Transami ⁇ ation and Conjugation of IL-l-ra.
• the protein conjugates were purified by hydrophobic interaction chromatography using a poly-propylapartamide column (200x4mm, 5um, 1000A, PolyLC Inc., Columbia, MA), foUowed by a gel filtration on a BioSep-SecS-2000 Phenomenex column (600x7.8mm) and ion exchange chromatography on a Mono Q column.
• mice Female Wistar rats obtained from Iffa-Credo (L'Arbresle, France), weighing between 150g and 200g, were used. Approximately 50 ⁇ g of :2J I-labeled protein were injected as a bolus in the taU vein and samples of blood coUected from the tail at selected times (as near as possible to 3, 10, 30 min, 1, 3, 7 and 24h). The samples were weighed to determine their exact volume, and their radioactivity measured. The curves of relative blood levels for Il-lra, PEG 10kD -Il-lra and PEG 20kD -H-lra given intravenously to the rats are compared in Figures 6A and 6B.
• the apparent first T, ⁇ of Antril was very short, and the apparent second T, ⁇ , though much longer, did not therefore have as strong an influence on the area under the pharmacokinetic curve.
• the apparent first T, A value for both the derivatives were significantly longer than that for the unmodified protein.
• a manual fit to these curves gave apparent first T 1/4 values of approximately 3 min for authentic Il-lra, 14 min. for PEG, 0kD -Il-lra, and 20 min. for PEG 20tD -Il-lra. No detectable differences were observed in the apparent second T, ⁇ amongst aU three proteins, and in all animals less than 2 % of the initially observed radioactivity remained in circulation after 24h.
• EXAMPLE 9 Site-Specific Modification and Conjugation of NH 2 -Terminal Residue of a Protein: Modification by Enzymatic Cleavage/Periodate Oxidation and Conjugation of IL-l-ra.
• the protein was therefore incubated 20h at 37°C at a 30mg/ml concentration in a 50mM sodium phosphate buffer, pH 7.0 with an enzyme/substrate ratio of 1/10. The protein was then isolated by gel filtration on the
• Three series of consecutive peaks corresponding to different analogues could be identified: the major one (16874.5 ⁇ 5.4D) corresponding to the expected product £fe_(Met-Arg-Pro)Il-lra (calculated value,
• EXAMPLE 10 Functionalization of Methoxypolyethylene Glycol: Synthesis of Multivalent Linker (TEG-aminoethylVN-fCH -NH-CO-CH.-O-NH,. This multivalent linker is comparable to that described in Example 3, and was obtained by acylation of Tris-(2-aminoethyl)amine with one equivalent of Boc-NH-0-CH 22 -CONSu in DMSO. The monosubstituted derivative was isolated by HPLC. characterized by ESIMS (calcd. M+H, m/z 319.4, found m/z 320.6).
• the material was solubilized in lOmM HCl and lyophiUzed, and further acylated with PEG 3kD -COOH (1.2 molar excess over remaining amino groups) in DMF, in the presence of equimolar amounts of hydoxybenzotriazole and DCC.
• the solution was stirred overnight at room temperature and the fuUy acylated derivative was purified after extensive dialysis against distiUed water by ion exchange chromatography on DEAE-A25 and CMC-25 and finaUy deprotected by treatment to get the foUowing derivative:
• J.L-8 was mixed at the same time with both periodate and an aminooxy compound (N ⁇ -Aminooxyacetyl, N'LC-biotinyl- lysine), and after an appropriate reaction period, with no work-up whatsoever, the wanted product— IL-8 specificaUy biotinylated at its N-terminus-was identified.
• the reaction progressed at pHs 8, 6.5 and 4.6.
• the AoA-LC biotinyl Lysine was made from N ⁇ (BOC-AoA)Lysine and N-hydroxysuccinimido ester of LC (long chain) biotin (which was obtained from the Pierce Chemical Co ⁇ ).
• N ⁇ (BOC-Ao A) Lysine was made by a two step process as foUows.
• N-a-tertButyloxycarbonylaminooxyacetyl, N-e- tri ⁇ uoroacetyl-Lysine was obtained by adding to 371 mg of ⁇ -e-trifluoroacetyl- lysine ( ⁇ ovaBiochem, 4448 Laefelfingeu, Switzerland) suspended in 3 mL of DMSO was added, a suspension in 1 mL of DMSO of 576 mg of the ⁇ - hydroxysuccinimido ester of aminooxyacetic acid, prepared as described previously Pochon, et al. (1989) Int. J. Cancer 43:1188-1194), modified as described (VUaseca et al. (1993) Bioconjugate Chem.
• the mixture was then further dUuted with 32 mL water, cooled to 0°C, and brought to an apparent pH of 3.0 (glass electrode) with acetic acid.
• the solution was then divided in two, and each half placed on a Chromabond 1000 mg (Machery-Nagel, D ⁇ ren, 52348, Germany) equiUbrated with 0.1 % aqueous TFA.
• Chromabond 1000 mg (Machery-Nagel, D ⁇ ren, 52348, Germany) equiUbrated with 0.1 % aqueous TFA.
• Each Chromabond was then washed with 20 mL of the same solution, and eluted with 4 mL of a mixture of 0.1 % TFA:acetonitrile, 4:6 (v/v).
• Acetonitrile was removed from the eluates in a current of filtered air, and the remaining Uquid removed by vacuum centrifugation.
• the trifluoroacetyl group was removed from the e-amine of the lysine by adding 3 mL of water to each of the dried down eluates, cooling to 0°C, then adding 330 ⁇ L of piperidine. The mixture was maintained in an ice bath with occasional agitation for 3 h. The reaction was stopped by the careful addition of 500 ⁇ L of glacial acetic acid, and solutions could be stored frozen at this point. To continue the procedure, each mixture was dUuted to 10 mL with water and the pH adjusted with acetic acid to pH 3.0 (glass electrode) if necessary.
• the AoA-LC biotinyl Lysine was made by first dissolving 15.3 mg of the ⁇ (BOC-AoAJLysine in 478 ⁇ l of DMSO and adding 5/.1 N-ethylmo ⁇ holine. To this were added 53.2 rag of the biotin compound N-hydroxysuccinimido ester of LC (long chain) biotin dissolved in 478 ⁇ l DMSO. If either component was reluctant to dissolve before mixing, all went into solution afterwards. After 18 hours at room temperature any excess active ester was destroyed by adding 956 ⁇ l 41813
• the dried fraction was dissolved in 200 ⁇ l CH 3 CN plus 1800 *1 0.196 TFA.
• the solution was clarified if necessary by centrifugation and 1 ml was injected at a time on the C8 analytical column described above.
• the column had been equiUbrated with 20% of the second solvent described above, and this was brought to 40% in the first five minutes after injection. The value was then taken to 80% over the next 100 minutes.
• the wanted product was the principal peak on this latter part of the gradient. The peak was dried down. Mass spectrometry showed the expected mass.
• the BOC group was removed by dissolving 1 mg in 200 ⁇ l TFA. After 45 minutes at 20° the solution was dried down and used without a further purification.
• EXAMPLE 13 Site-Specific Modification and Conjugation of Cvsteine Residue of a Protein: Modification and Conjugation of rh-G-CSF at Cvsteinel7.
• Boc-NH-0-CH 2 -COHN-(CH 2 ) 2 -NH-CO-CH 2 -Br The Boc group was removed and the linker rapidly added at a 20mM concentration to rh-G-CSF solubilized in a 0.1M phosphate buffer (Na), pH 7.0, 5mN EDTA and 6M GuHCl. After 30 min. incubation, the solution was acidified.
• the alkylated G- CSF was purified by reverse phase HPLC and characterized by mass spectrometry (calcd. m/z 18971.8, found, m/z 18978.2+7.8).
• the protein was then resolubUized in acetate buffer (0.1M Na), pH 4.6 containing 6M GnHCl and reacted with a PEG- CHO (from Example 12 above).
• the extent of polymer conjugation was comparable to that obtained in the two-step coupling at the N-terminus described in the previous example. If the alkylation with the preceding linker is performed at higher pH values (e.g. pH 8.0, pH 9.0) histidine and methionine residues wiU also be modified.
• EXAMPLE 14 Site-Specific Modification and Conjugation of a Protein: Modification and Conjugation of rh-G-CSF.
• a smaU bivalent tag containing a group reactive with a functionaUzed polymer of the invention, can be added to the protein in a large excess and wiU therefore react rapidly with the created glyoxylyl function in the protein.
• the functionaUzed polymer is added to achieve conjugation to the protein via the tag. This two-step approach is useful, for example, when one wishes to avoid or minimizes both steric problems and those that might be caused by side reactions.
• a illustrative smaU, bivalent tag is a bisaminooxy tag.
• H 2 N-0- CH 2 C0-Lys(C0-CH 2 -0-NH 2 ) was synthesized by the attachment of Boc-AoA-ONSu to Lys-OMe, foUowed by NaOH treatment and TFA deprotection (calcd. M+H, 293:1; found M+H 293.9).
• a functionalized polymer having a reactive carbonyl group was used for conjugation.
• Tris(2-aminoethyl)amine used in Example 10, was acylated with 3 equivalent of Boc-NH-0-CH 2 -COOSu in DMSO.
• the fuUy substituted derivative was isolated by HPLC, characterized by ESIMS (calcld. M & M, m/z 665.5, found m/z 665.3).
• the material was deprotected by lh TFA treatment and TFA removed by rotary evaporation.
• the material was resolubiUzed in water and lyophUized then reacted in a 15 molar excess (to avoid crosslinking) with oxidized II-8 in 0.1M AcoNa, pH 3.6.
• the conjugation product (bearing two free AoA groups) was isolated by HPLC and characterized by ESIMS (calcld. M & H, m/z 8698.6, found m/z 8696.0 ⁇ 0.5).
• This conjugate can be reacted with PEG-CHO to yield a two-polymer armed derivative, i.e. a bi-polymer-containing functionaUzed polymer.
• multiarm i.e. multi-polymer-containing functionalized polymer
• structures can be constructed, especiaUy if the aim of the polymer conjugation is to decrease immunogenicity of the protein.
• a linker bearing 3 to 10 arms, preferably 4 to 6, would be more appropriate.
• Such construction can be obtained by linking, in a first reaction, to the protein a peptidic tinker of this structure:
• the serine residues of the introduced linker are oxidized, and then reacted as described in preceding examples, with PEG-AoA functionaUzed polymers, to covalently attach n number of chains to the protein.

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